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Biomedical subjects

J E Silbert

Publications and source records attributed to J E Silbert.

At least 19 recordsLinked to original sources
PubMed1992

Effects of detergent on the sulphation of chondroitin by cell-free preparations from chick-embryo epiphyseal cartilage.

The effects of the non-ionic detergent Triton X-100 on 6-sulphation of two species of endogenous nascent proteochondroitin by a chick-embryo cartilage microsomal system was examined. Sulphation of the larger (Type I) species with adenosine 3'-phosphate 5'-phosphosulphate was slightly diminished when Triton X-100 was present, whereas sulphation of the smaller (Type II) species was slightly enhanced. An ordered rather than random pattern of sulphation was obtained for the smaller proteoglycan, but with a considerably lower degree of sulphation than that of the larger proteochondroitin. These differences were consistent with other differences between these two species as described previously. Sulphation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system with Triton X-100 present produced ordered rather than random sulphation patterns. When a 100,000 g supernatant fraction was utilized for sulphation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and ordered sulphation was still obtained. When hexasaccharide was used, sulphation of multiple N-acetylgalactosamine residues of the individual hexasaccharides resulted. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulphation of multiple N-acetylgalactosamine residues on the individual hexasaccharide molecules was even greater, so that tri-sulphated products were found. This suggests that ordered rather than random sulphation of chondroitin with these enzyme preparations is due to enzyme-substrate interaction rather than to membrane organization.

Animals↗
PubMed1992

Effects of brefeldin A on the localization of chondroitin sulfate-synthesizing enzymes. Activities in subfractions of the Golgi from chick embryo epiphyseal cartilage.

Membranes from brefeldin A-treated and untreated chick embryo epiphyseal cartilage were fractionated separately by equilibrium sucrose density gradient centrifugation. Fractions were assayed for Gal I transferase, Gal II transferase, Gal ovalbumin transferase, chondroitin polymerization on endogenous acceptors, GalNAc transfer to exogenous chondroitin hexasaccharide, and sulfate transfer to exogenous chondroitin. Gal I transferase and Gal II transferase activities were found in heavier cis- and medial-Golgi fractions, but with distributions different from each other. Brefeldin A had no effect on either their distribution or their total activity. Gal ovalbumin transferase activity in fractions from untreated cartilage was found as a dual peak in medial- and trans-Golgi areas. The latter peak was diminished in the fractions from the brefeldin A-treated cartilage, whereas the former peak was correspondingly increased. A similar dual medial- and trans-Golgi distribution for chondroitin polymerization on endogenous acceptors was seen with fractions from untreated cartilage. This was modified in fractions from brefeldin A-treated cartilage with a complete loss of synthesis in the trans-Golgi peak and a slight increase in synthesis in the medial-Golgi peak. However, the distribution of GalNAc transferase activity using exogenous chondroitin hexasaccharide indicated that considerable chondroitin-synthesizing activity still remained in these trans-Golgi fractions. This demonstrated that brefeldin A had caused a block in movement of endogenous proteochondroitin acceptors to the trans-Golgi site of synthesis. Sulfotransferase activity was also found in a dual distribution similar to that of the chondroitin polymerization and GalNAc transferase, with a small reduction in activity in the trans-Golgi fractions of brefeldin A-treated cartilage. Thus, treatment of cartilage with brefeldin A resulted in the loss of considerable trans-Golgi chondroitin sulfate-synthesizing enzyme activity and a block in the transport of one form of proteochondroitin precursor to the trans-Golgi membranes.

Animals↗
PubMed1992

Effects of brefeldin A on the synthesis of chondroitin 4-sulfate by cultures of mouse mastocytoma cells.

Mouse mastocytoma cells were cultured with brefeldin A in medium containing [35S]sulfate and [3H]glucosamine in order to determine the effects of this fungal metabolite on the formation of chondroitin 4-sulfate by these cells. There was a marked reduction in the incorporation of [35S]sulfate into the glycosaminoglycan which was approximately equal to the reduction in the incorporation of [3H]hexosamine into the same molecule. The chondroitin 4-sulfate chain size was greatly diminished, while the number of chains appeared to remain relatively constant, indicating that the brefeldin A partially disrupted the polymerizing system, but had little effect upon movement of the nascent proteochondroitin to the site for chondroitin polymerization and sulfation.

Animals↗
PubMed1991

Formation of two species of nascent proteochondroitin in separate loci of a microsomal preparation from chick-embryo epiphyseal cartilage.

The potential relationship of an intact membrane organization to the synthesis of chondroitin was examined before and after modification of a chick-embryo cartilage microsomal system with the non-ionic detergent Triton X-100. Incubations with labelled UDP-GlcA and UDP-GalNAc indicated that Triton X-100 had little effect on the amount of chondroitin synthesized to form one species of large proteochondroitin (Type I). However, Triton X-100 had a marked stimulatory effect on the formation of another smaller species of proteochondroitin (Type II). Presence of this detergent during chondroitin polymerization also resulted in chains that were slightly smaller. Neither of the two proteochondroitin species were collagenase-sensitive, nor did they contain dermatan-like regions. Thus in these respects they were unlike the small proteochondroitins (PG-Lb or PG-Lt) that have been found in chick-embryo cartilage. They also differed greatly in size from these small proteoglycans as well as from the large aggregatable proteochondroitin (PG-H) from the same source. Synthesis of the larger (Type I) proteochondroitin species was not affected by prior treatment of the microsomes with chondroitin ABC lyase at concentrations sufficient for elimination of synthesis of most of the smaller (Type II) proteochondroitin species. Use of chondroitin ABC lyase subsequent to synthesis of the chondroitin also resulted in preferential degradation of the smaller species. Thus there were differences in formation and limitation in access of the chondroitin ABC lyase to the two species, consistent with other differences described previously. These results indicate that there are separate loci within the microsomal membranes for synthesis of the two species.

Animals↗
PubMed1991

Subfractionation of chick embryo epiphyseal cartilage Golgi. Localization of enzymes involved in the synthesis of the polysaccharide portion of proteochondroitin sulfate.

Membranes from chick embryo epiphyseal cartilage were fractionated by equilibrium sucrose density gradient centrifugation and assayed for galactosyl xylose transferase, chondroitin polymerization and sulfation as well as the marker enzymes glucose-6-phosphatase, NADH cytochrome c reductase, galactosyl ovalbumin transferase, and sialyltransferase. The order of distribution of chondroitin sulfate synthesis from dense to light membranes correlated with the established sequence of events for its synthesis. The linkage region enzyme, viz. galactosyl xylose transferase, distributed with NADH cytochrome c reductase in an earlier and heavier cis compartment. Chondroitin polymerization and sulfation had a dual distribution similar to the galactosyl ovalbumin transferase and sialyltransferase in separate later and lighter medial and trans compartments, or in an extended medial or trans compartment. The galactosyl xylose transferase had a distribution distinctly different from that of the galactosyl ovalbumin transferase indicating that these distinct enzymes showed no cross-reactivity with their respective acceptor substrates. The dual distribution of chondroitin sulfate synthesis was consistent with our previous demonstration of the two nascent proteochondroitin populations produced by microsomal preparations from the same source. The results indicated separate subcellular locations for synthesis of the two forms.

Animals↗
PubMed1991

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

Aggrecans↗
PubMed1990

Relationship of sulfation to ongoing chondroitin polymerization during biosynthesis of chondroitin 4-sulfate by microsomal preparations from cultured mouse mastocytoma cells.

A microsomal preparation from chondroitin 4-sulfate-synthesizing cultured mouse mastocytoma cells was incubated with UDP-[3H]GalNAc, UDP-GlcA, and 3'-phosphoadenylylphosphosulfate (PAPS) for 30 s at 10 degrees C and with UDP-[14C]GlcA, UDP-GalNAc, and PAPS for 4 h at 37 degrees C for synthesis of 3H- and 14C-labeled chondroitin/chondroitin sulfate. The latter incubation provided more than 100 times as much product as did the short incubation at 10 degrees C. Upon chromatography of the isolated labeled glycosaminoglycans on a Sepharose CL-6B column, most of the [14C]glycosaminoglycan from the 4 h, 37 degrees C incubation was excluded from the column, indicating that this nascent glycosaminoglycan had been polymerized fully. In contrast, most of the [3H]glycosaminoglycan from the 30 s, 10 degrees C incubation was mostly retarded upon cochromatography on this same column, indicating that the nascent glycosaminoglycan was still growing in size. The labeled fractions representing chondroitin/chondroitin sulfate of varying sizes were analyzed for degree of sulfation by degradation with chondroitin ABC lyase followed by paper electrophoresis of the products. Results indicated that the [14C]chondroitin/chondroitin sulfate formed in the 4-h incubation was 60-70% sulfated. Incomplete chains of [3H]chondroitin/chondroitin sulfate formed in the 30-s incubation were also sulfated as much as 20-25%. As the size of the [3H]chondroitin/chondroitin sulfate increased, there was a concomitant increase in sulfation. These results demonstrate that in this microsomal system sulfation takes place while the nascent chondroitin glycosaminoglycan chains are still actively growing in length, although the sulfation lags somewhat behind the polymerization. This not only indicates a common membrane location for both polymerization and sulfation of chondroitin but also demonstrates that the sulfation of chondroitin by these mastocytoma cells may occur during the process of glycosaminoglycan polymerization rather than subsequent to completion of the glycosaminoglycan chains.

Animals↗
PubMed1990

Glycosaminoglycans of bovine aorta endothelial cells: identification and localization by use of a platelet factor 4-fluorescein probe.

We utilized platelet factor 4 (PF4) conjugated to fluorescein to stain the proteoglycans of permeabilized fixed bovine aorta endothelial cells in monolayer culture. Treatment of the monolayers with chondroitin ABC lyase and/or a preparation from Flavobacterium heparinum was used to remove chondroitin sulfate and/or heparan sulfate before staining, with resultant separate identification and partial localization of these glycosaminoglycans. When PF4-fluorescein was utilized with untreated control monolayers, fairly uniform reticular, perinuclear, and cell surface fluorescence was seen. After treatment with chondroitin ABC lyase, fluorescence was retained only on the cell surface. In contrast, treatment with the F. heparinum preparation resulted in the loss of all cell surface fluorescence. Use of both glycosaminoglycan lyases together resulted in loss of essentially all the fluorescence. The cell surface heparan sulfate observed by fluorescence after removal of cell surface chondroitin sulfate appeared to be unevenly distributed, with a heavier accumulation at one pole of each cell. This technique offers a specific method for identification and partial localization of cell surface heparan sulfate.

Animals↗
PubMed1989

Biosynthesis of chondroitin sulfate. Organization of sulfation.

The potential relationship of an intact membrane organization for the synthesis of chondroitin and chondroitin 4-sulfate was examined after modification of a mouse mast cell microsomal system with the nonionic detergent, Triton X-100. The results indicated that Triton X-100 had no effect on the rate of polymerization but had a slight effect on the size of glycosaminoglycan chains. An "all or nothing" pattern of sulfation of newly formed chondroitin was obtained in both the presence and the absence of Triton X-100, and this pattern did not change whether sulfation was initiated concurrent with or subsequent to polymerization. Sulfation of exogenous [14C]chondroitin and exogenous proteo[3H]chondroitin by the microsomal system required Triton X-100 but still produced an all or nothing pattern rather than a random sulfation pattern. When a 100,000 x g supernatant fraction was utilized for sulfation of [14C]chondroitin or proteo[3H]chondroitin, Triton X-100 was not needed, and a partial sulfation pattern was obtained. However, it was similar to the all or nothing pattern in that it still produced two populations, with some chains nonsulfated and others approximately 50% sulfated. When chondroitin hexasaccharide was used with 3'-phosphoadenylylphospho[35S]sulfate, multiple GalNAc residues of the individual hexasaccharides were found to be sulfated. This was relatively independent of Triton X-100 or the concentration of the hexasaccharide acceptors. With soluble enzyme, sulfation of multiple GalNAc residues on the individual hexasaccharide molecules was even greater, so that trisulfated products were found. These results suggest that efficient sulfation of chondroitin is related to enzyme-substrate interaction more than to membrane organization.

Animals↗
PubMed1989

Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources.

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine and [35S]-sulfate for varying periods of time. Incorporation of 3H into macromolecules appearing in the medium was linear after approximately 45 min, and incorporation of 35S was linear after approximately 30 min. The amounts of 35S-proteoglycan formed by each of the cultures during 5-h incubations were compared and were found to be fairly similar for the six lines, varying from 0.08 to 0.14 nmol sulfate/microgram DNA. Isolated 3H,35S-glycosaminoglycans were then treated with chondroitin ABC lyase to characterize the location and degree of sulfation. Results indicated a considerable variation in completeness of chondroitin/dermatan sulfation and in proportions of 6-sulfation to 4-sulfation among the various lines. However these variations did not seem to be related to whether the cells were from normals or diabetics. 3H,35S-Labeled disaccharides were isolated and ratios of 3H to 35S determined in order to calculate the [3H]glucosamine dilution by endogenous glucosamine derived from glucose or other sources during the period of incubation. Dilutions varied widely from 160- to 635-fold among the different cell lines, but the variations did not seem to be related to whether the cells were from normals or diabetics.

Adult↗
PubMed1989

Increased proteoglycan synthesis following the differentiation of F9 embryonal carcinoma cells: formation of a differentiation-specific proteoheparan sulfate.

We have examined changes in proteoglycan synthesis by F9 embryonal carcinoma cells after the cells have been treated with retinoic acid or retinoic acid plus cholera toxin. Retinoic acid is known to stimulate the differentiation of this cell type to a primitive endoderm-like cell characterized by the production of basement membrane components such as type IV collagen, laminin and proteoglycans. We have now demonstrated that proteoglycan synthesis and secretion were further stimulated when cholera toxin was added in addition to retinoic acid. Moreover, media of these fully differentiated cells was found to contain a different species of proteoheparan sulfate not produced by stem cells or retinoic acid-treated cells. This proteoheparan sulfate had a high density upon CsCl gradient centrifugation. The protein core of this proteoheparan sulfate was estimated by SDS gel electrophoresis to be approximately 15,000 daltons.

Animals↗
PubMed1988

Chlorate: a reversible inhibitor of proteoglycan sulfation.

Bovine aorta endothelial cells were cultured in medium containing [3H]glucosamine, [35S]sulfate, and various concentrations of chlorate. Cell growth was not affected by 10 mM chlorate, while 30 mM chlorate had a slight inhibitory effect. Chlorate concentrations greater than 10 mM resulted in significant undersulfation of chondroitin. With 30 mM chlorate, sulfation of chondroitin was reduced to 10% and heparan to 35% of controls, but [3H]glucosamine incorporation on a per cell basis did not appear to be inhibited. Removal of chlorate from the culture medium of cells resulted in the rapid resumption of sulfation.

Animals↗
PubMed1988

Sulphation by cultured cells. Cysteine, cysteinesulphinic acid and sulphite as sources for proteoglycan sulphate.

Bovine aortic smooth-muscle cells, bovine aortic endothelial cells, and IMR-90 human embryonic lung fibroblasts were tested to determine their ability to use cysteine or cysteine metabolites as a source of sulphate (SO4). Cells were incubated in SO4-depleted medium containing [3H]glucosamine plus 0.2 mM-cystine, 0.3 mM-cysteinesulphinic acid or 0.3 mM-sulphite (SO3). The [3H]chondroitin sulphate produced by the different cells was found to vary considerably in degree of sulphation under these conditions. One line of smooth-muscle cells utilized cysteine effectively as a SO4 source and thus produced chondroitin sulphate which was highly sulphated. IMR-90 fibroblasts produced partly sulphated chondroitin sulphate under these conditions, while another smooth-muscle cell line could not utilize cysteine, but could utilize cysteinesulphinic acid as a partial SO4 source. In contrast with the above cells, endothelial cells could not use cysteine or cysteinesulphinic acid as a source of SO4 and produced chondroitin with almost no SO4. All of the cells were able to utilize SO3. Incubation of the cells in the SO4-depleted medium containing [35S]cysteine confirmed that only the first line of smooth-muscle cells could convert significant amounts of [35S]cysteine to 35SO4. Furthermore, the addition of 0.4 mM inorganic SO4 did not inhibit the production of SO4 from cysteine by these cells.

Cells, Cultured↗
PubMed1988

Sulfation of chondroitin. Specificity, degree of sulfation, and detergent effects with 4-sulfating and 6-sulfating microsomal systems.

Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage, and from chondroitin 4-sulfate-producing mouse mastocytoma cells, were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine to form non-sulfated proteo[14C]chondroitin. Aliquots of the incubations were then incubated with 3'-phosphoadenylylphosphosulfate (PAPS) in the presence or absence of various detergents. In the absence of detergents, there was good sulfation of this endogenous proteo[14C]chondroitin by the original microsomes from both sources. Detergents, with the exception of Triton X-100, markedly inhibited sulfation in the mast cell system but not in the chick cartilage system. These results indicate that sulfation and polymerization are closely linked on cell membranes and that in some cases this organization can be disrupted by detergents. When aliquots of the original incubation were heat inactivated, and then reincubated with new microsomes from chick cartilage and/or mouse mastocytoma cells plus PAPS, there was no significant sulfation of this exogenous proteo[14C] chondroitin with either system unless Triton X-100 was added. Sulfation of exogenous chondroitin and chondroitin hexasaccharide was compared with sulfation of endogenous and exogenous proteo[14C]chondroitin. Sulfate incorporation into hexasaccharide and chondroitin decreased as their concentrations (based on uronic acid) approached that of the proteo[14C]chondroitin. At the same time, the degree of sulfation in percent of substituted hexosamine increased. However, the degree of sulfation did not reach that of the endogenous proteo[14C]chondroitin. Hexasaccharide and chondroitin sulfation were stimulated by the presence of Triton X-100. However, in contrast to the exogenous proteo[14C]chondroitin, there was some sulfation of hexasaccharide and chondroitin in the absence of this detergent. These results indicate that the intact microsomal system was not accessible to the larger substrates, and that even with detergents exogenous substrates were not sulfated as effectively as newly formed proteo[14C]chondroitin in an intact microsomal system. When the proteo[14C]chondroitin formed by the chick cartilage microsomal system was incubated together with the mast cell microsomal system and PAPS, sulfation only occurred at the 4-position. When the proteo[14C]chondroitin formed by the mouse mast cell microsomal system was incubated together with the chick cartilage microsomal system and PAPS, sulfation only occurred at the 6-position.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Aortic endothelial cell proteoheparan sulfate. I. Isolation and characterization of plasmamembrane-associated and extracellular species.

Proteoheparan sulfate biosynthesis was studied in cultured bovine aortic endothelial cells by means of pulse and pulse-chase experiments and subcellular fractionations. Three proteoheparan sulfate species were found in the medium. The major species, which the authors have called HS I, appeared in the medium only after an initial lag period and was also found associated with the plasma membrane. The other two (HS II and HS III) appeared in small amounts in the medium at early time points. At later times these were not readily observed because the large amounts of HS I present in the medium. The major medium species, HS I, appeared to be composed of approximately four heparan sulfate chains of approximately 35,000 daltons and a core protein of approximately 55,000 daltons apparent molecular weight. HS I appeared to be homogeneous by gel filtration on Sepharose CL 2B and 6B and elution from DEAE Sephacel, electrophoresis on Nu-Sieve agarose, and CsCl density centrifugation. After digestion with heparinase the core protein appeared to be homogeneous by S-200 Sephacel chromatography. HS I was also found associated with plasma membrane fractions of the cultured bovine aortic endothelial cells, and antisera raised against it stained epithelial and endothelial cells in patterns consistent with a cell surface localization. Of the other two species found in the medium, one (HS II) also appeared to be a component of the cell layer. This species appeared to contain approximately four heparan sulfate chains of approximately 20,000 daltons apparent molecular weight. Antisera raised against a similar molecule produced by HR 9 cell cultures stained basement membranes intensely, supporting the subcellular matrix localization of this molecule. The third species (HS III) was detected in culture medium only and apparently contained two heparan sulfate chains of approximately 20,000 daltons apparent molecular weight. These results support the concept of multiple endothelial cell proteoheparan sulfate species which exhibit differences in structure and localization and possibly diverse specialized functions.

5'-Nucleotidase↗