Showing papers on "Keratan sulfate published in 1983"

Vitreous: an inhibitor of retinal extract-induced neovascularization.

Abstract: One of the major problems in assessing neovascularization in mammalian experimental animal models is the immunologic response of the host to stimuli from nonautologous species. Hence, crude bovine vitreous and retinal extracts may produce a complex immune reaction when tested in the rabbit. To circumvent this problem, the chicken chorioallantoic membrane (CAM) assay is most appropriate. In this study the CAM assay for angiogenesis has been modified to study antiangiogenic substances. The modified assay is described in detail and used to demonstrate for the first time the inhibition by adult bovine vitreous of neovascularization induced by extracts of adult bovine retina. In addition to vitreous, three common glycosaminoglycans (keratan sulfate, chondroitin sulfate C, and hyaluronic acid) were assayed for antiangiogenic activity. The results indicate that vitreous inhibition of retinal extract-induced neovascularization is dose dependent, while the sulfated glycosaminoglycans tested had no antiangiogenic activity. A commercial preparation of bovine vitreous hyaluronic acid exhibited a slight, but not statistically significant, inhibitory activity. When vitreous extracts were digested with hyaluronidase, no loss of antiangiogenic activity occurred. These results suggest that the inhibitor of angiogenesis from adult vitreous is probably not a common glycosaminoglycan. The results are consistent with the hypothesis that antiangiogenic substance(s) in vitreous and angiogenic components from retina may act as natural antagonists in controlling the process of retinal neovascularization.

The glycosaminoglycans in menisci in experimental and natural osteoarthritis

Abstract: The glycosaminoglycans in the menisci of beagles 5--7 years old were analyzed at various times after osteoarthritis was induced by sectioning the anterior cruciate ligament of one knee; the unoperated knee served as control. In the first month after induction, there were signs of inflammation in the operated joint. After 1 week, the water content was elevated and the glycosaminoglycan content (per dry weight) was reduced. The content of keratan sulfate decreased more than that of chondroitin sulfate, but the hyaluronic acid content did not change consistently. The relative proportions of chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate remained unchanged. After 3--18 months, the glycosaminoglycan levels reverted to normal, and there was some evidence that after 15--18 months, they were elevated above normal. These results, together with results obtained from single examples of mild and severe osteoarthritis in working foxhounds, suggest that, in contrast to articular cartilage, the meniscus is capable of some regeneration in response to injury.

Bovine tracheal cartilage proteoglycans. Variations in structure and composition with age.

Abstract: The variation with age in structure and composition of proteoglycans from bovine tracheal cartilage was studied from fetal life to 12 years of age. Cartilage content of proteoglycans as related to collagen was found to increase with age. The extractability of the proteoglycans decreases markedly with age. Extracted proteoglycans were characterized and change during growth and maturation as follows: 1. They contain fewer chondroitin sulfate chains. The chains are of constant size; but at higher ages they are more highly sulfated, primarily with 6-sulfate groups. 2. They contain an increasing number of constant size keratan sulfate chains, while the number of O-linked oligosaccharides (structurally related to the keratan sulfate linkage region to protein) decreases. 3. They become smaller, at least partially, as a result of an increasing proportion of a smaller size proteoglycan. 4. They have a higher protein content.

Histochemical properties of cartilage proteoglycans.

Abstract: Proteoglycan interaction with alcian blue at different concentrations of magnesium chloride was studied both in vitro and in histological sections of paraffin-embedded tissues. Our experiments indicate that a) proteoglycans with different contents of chondroitin sulfate and keratan sulfate, prepared under nondegradative conditions, are not distinguishable on the basis of the critical electrolyte concentrations at which staining is abolished; b) the state of aggregation of proteoglycans only very slightly affects the alcian blue affinity of the macromolecules at different concentrations of magnesium chloride; c) the interaction of proteoglycans with other components of the connective tissue matrix is an important factor in determining the strength of binding of alcian blue to the polyanionic macromolecules in histological sections. These factors should be considered in interpreting histochemical data obtained by staining tissue sections with alcian blue at different concentrations of magnesium chloride. Proteoglycans, like glycosaminoglycans, are only weakly periodic acid-Schiff-positive.

Matrix Synthesis in High Density Cultures of Bovine Epiphyseal Plate Chondrocytes

Abstract: Bovine epiphyseal plate chondrocytes were cultured by a method combining both suspension culture and high density monolayers. The matrix synthesized by the cultured cells was analyzed at fifteen days for glycosaminoglycan, proteoglycan, and collagen content.In the cell culture product glycosaminoglycan distribution was: 65% chondroitin-6-sulfate, 18% chondroitin-4-sulfate, 15% keratan sulfate, and less than 2% dermatan sulfate. Essentially all the radioactive sulfate in labelled specimens was present in high molecular weight aggregates. The collagen which was synthesized co-migrated with Type II collagen standard. Parallel analysis showed the matrix of cultured cells to be similar to that of intact epiphyseal plate tissue.This study demonstrates the ability to grow epiphyseal plate chondrocytes in a cell culture system which allows matrix synthesis similar to that seen in vivo

Type ii collagen‐induced arthritis. A Morphologic and Biochemical Study of Articular Cartilage

Abstract: Articular cartilage was obtained from type II collagen-induced arthritic rat joints. Transmission electron microscopy showed a gradual degeneration of chondrocytes, disorganization of the collagenous extracellular matrix, and formation of microscars. Biochemical analyses indicated that type II collagen was the only collagen present and that it was normal in regard to hydroxylation of lysine and glycosylation of hydroxylysine. Analyses of the proteoglycan in the extracellular matrix revealed a 50% loss of chondroitin sulfate and keratan sulfate.

Ultrastructural cytochemistry and immunocytochemistry of proteoglycans associated with epiphyseal cartilage calcification.

Abstract: Proteoglycans (PGs) are closely associated with cartilage calcification. We have examined the hypertrophic zone of rat epiphyseal cartilage, in which calcification is occurring, using the high-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycosaminoglycans, an immunoferritin method specific for chondroitin sulfate A, and the tannic acid-ferric chloride (TA-Fe) method to stain cartilage matrix granules (MGs) presumed to be PG monomers. HID-TCH-SP produced stain deposits with a diameter of 11.2 +/- 3.2 nm (mean +/- SD; n = 200) in the MGs. However, HID-TCH-SP staining was not discernible in membrane-limited matrix vesicles (MVs). In areas of advanced calcification, partially disrupted MVs and globular bodies (GBs), derived in part from disrupted and/or degenerated MVs, contained a few too many small HID-TCH-SP stain deposits. Further down the epiphyseal cartilage, intact MVs markedly decreased and the GBs, containing many small HID-TCH-SP stain deposits, significantly increased in number. These GBs were found exclusively in the longitudinal septa rather than in the transverse septa. After enzyme digestion with testicular hyaluronidase, small (7.2 +/- 1.2 nm in diameter) stain deposits remained in the MGs and GBs, presumably localized to keratan sulfate. Immunoferritin localizing chondroitin sulfate strongly stained MGs, whereas MVs and GBs lacked staining. TA-Fe staining of glycoconjugates in the GBs demonstrated a striking decrease in the diameter of MGs associated with calcification in the GBs as compared with those in the noncalcifying area around the GBs. These results indicate that the GBs containing needle-like apatite crystals in morphologic preparations represent sites of chondroitin sulfate degradation. Testicular hyaluronidase-resistant sulfated glycosaminoglycans presumed to be keratan sulfate and partially degraded PGs selectively remain within the GBs as a probable requisite for expansion of the initial calcification in MVs.

Corneal glycosaminoglycan synthesis in long-term organ culture.

Abstract: types. Physical or enzymatic damage to the cultured corneas resulted in marked changes in the ratio of labeled GAG types secreted. Physical damage to the epithelial or endothelial surfaces and/or mild trypsin treatment of the corneas before culture resulted in a stimulation of galactosaminoglycan secretion and a consequent decrease in the relative abundance of keratan sulfate. The presence of soy trypsin inhibitor prevented these changes. Treatment of the corneas with collagenase resulted in a decrease in all GAG secretion, particularly keratan sulfate. When corneas were removed from chilled, enucleated eyes and rinsed with solutions of soy trypsin inhibitor before incubation, the GAG secreted in culture had a similar ratio of keratan sulfate to galactosaminglycan as GAG labeled in vivo. This ratio was maintained 1 week in culture. These results suggest that secretion of the characteristic connective tissue of the corneal stroma by keratocytes is dependent on interaction between the cells and components of the stromal extracellular matrix. Invest Ophthalmol Vis Sci 24:208-213, 1983 In adult animals, corneal connective tissue is secreted by the stromal keratocytes, flattened mesenchymal cells located between the stromal lamellae. For short periods of time in organ culture, keratocytes continue to secrete characteristic stromal connective tissue components. 1 After 48 hours in organ or cell culture, marked changes take place in the nature of these secretions. A well-characterized aspect of this change is the alteration in corneal glycosaminglycan synthesis. There are two major corneal types of glycosaminoglycan (GAG), keratan sulfate and galactosaminglycan (a chondroitin sulfate-dermatan sulfate hybrid). In vivo, more than half of the total GAG secreted by keratocytes is keratan sulfate, but after 48 hours in organ or cell culture, this level decreases to 10% or less of the total. 23 Keratocytes grown in

A comparison of the proteoglycans produced by rabbit articular chondrocytes in monolayer and spinner culture and those of bovine nasal cartilage.

Abstract: The structural and immunological properties of the glycosaminoglycans and the core proteins of bovine nasal cartilage proteoglycan and the proteoglycans produced by rabbit articular chondrocytes in spinner and monolayer culture were compared. Culture medium with 35SO4- or 3H-serine-labeled proteoglycan was mixed with bovine nasal cartilage 4M guanidine-HCl extract and digested with trypsin. The proteoglycan fragments were then isolated by DEAE-cellulose chromatography and fractionated by dissociative CsCl density gradient centrifugation. Approximately 90% of the 35SO4 incorporated into proteoglycan by the cultured chondrocytes was in chondroitin sulfates and about 5% in keratan sulfate. Although there was considerable overlap in the Sepharose 4B elution of the tryptic proteoglycan fragments of highest buoyant density, some monolayer-produced proteoglycan fragments eluted earlier and some spinner-produced proteoglycan fragments eluted later than the proteoglycan fragments from bovine nasal cartilage. These differences in apparent fragment size could relate to differences in glycosaminoglycan chain length, since the glycosaminoglycans released by treatment with alkali from monolayer-produced proteoglycan in part eluted from Sepharose 4B earlier and those from spinner-produced proteoglycan in part eluted later than the chondroitin sulfate chains released from bovine cartilage proteoglycan. After digestion with chondroitinase ABC, 3H-serine-labeled high density tryptic proteoglycan fragments from monolayer and spinner culture yielded Sepharose 6B elution profiles which were similar to each other but did not coincide with the peaks of carbazole reactivity found with similarly treated fragments of bovine nasal cartilage proteoglycan. Cross-reactivity was demonstrated by radioimmunoautography between bovine cartilage and rabbit chondrocyte proteoglycan fragments restricted to gradient fractions of low buoyant density, but immunological cross-reactivity was not found for the antigens associated with the keratan sulfate-rich and chondroitin sulfate-bearing tryptic fragments of bovine nasal cartilage proteoglycan. These studies indicate that the proteoglycan core proteins produced by rabbit articular chondrocytes in monolayer and spinner culture are, in part, different from the core protein of bovine nasal cartilage proteoglycan and that the three proteoglycans differ in the length of some of their chondroitin sulfate chains.

N-acetylglucosamine 6-sulfate residues in keratan sulfate and heparan sulfate are desulfated by the same enzyme.

Abstract: We have prepared a series of oligosaccharides to assess the substrate specificity of exo sulfatase activity in cultured human skin fibroblasts toward N-acetylglucosamine-6-sulfate residues present in keratan sulfate (KS) and heparan sulfate (HS). Non-reducing end alpha-GlcNAc-6-SO4 residues (derived from HS) were desulfated by a specific sulfatase that when deficient leads to the accumulation of HS and the expression of mucopolysaccharidosis type IIID (Sanfilippo D). Under the in vitro conditions studied there are two pathways for the degradation of oligosaccharides containing non-reducing end beta-GlcNAc-6-SO4 residues (derived from KS). In one pathway beta-N-acetylglucosaminidase produces GlcNAc-6-SO4 which is then desulfated. In the other pathway the beta-GlcNAc-6-SO4 residue is desulfated and then cleaved by the action of an beta-N-acetylglucosaminidase activity. There was no detectable beta-N-acetylglucosaminidase activity in fibroblasts from a Tay-Sachs patient to produce GlcNAc-6-SO4 from beta-GlcNAc-6-SO4 residues in KS of oligosaccharides. There was approximately 10% of this normal beta-N-acetylglucosaminidase activity in fibroblasts from a Sandhoff patient, suggesting the A and S forms may be involved in this reaction. Desulfation of GlcNAc-6-SO4 residues in KS, HS and the monosaccharide GlcNAc-6-SO4 was considerably reduced or not detected in fibroblasts from a Sanfilippo D patient. As KS was not detected in the urine of a Sanfilippo D patient we propose that KS degradation in these patients proceeds by the action of a beta-N-acetylglucosaminidase activity to produce GlcNAc-6-SO4 which is not further degraded.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the carbohydrate-protein linkage region in bovine corneal keratan sulfate. II. Structural studies on linkage region-enriched neutral glycopeptides.

Abstract: In order to elucidate the chemical structure of the carbohydrate-protein linkage region of bovine corneal keratan sulfate, glycopeptides which had been highly enriched in the linkage region under mild conditions were subjected to glycosidase digestion in combination with methylation analysis. After removal of the peripheral N-acetylglucosamine and galactose residues by exhaustive glycosidase digestion, the residual glycopeptide (GP-A) was found to contain 0.6 mol of fucose, 3 mol of mannose, and 2 mol of N-acetylglucosamine per mol. GP-A was then serially digested with exoglycosidases, and the released sugars and the composition of the residual glycopeptides isolated by gel filtration were analyzed by gas-liquid chromatography. Further, the dansyl derivative of GP-A was digested with endo-beta-N-acetylglucosaminidase D and the products were analyzed to explore the location of the fucose residue. The results obtained, combined with those from methylation analysis of all the glycopeptides, revealed that the carbohydrate structure of the linkage region and its environs of corneal keratan sulfate is as follows. (Formula: See Text).

Characterization of keratan sulfate isolated from liver affected by Morquio syndrome.

Abstract: Chemical structures of keratan sulfate (KS) isolated from the liver affected by Morquio syndrome type A (classical type) were investigated. In the KS from Morquio syndrome liver, the molar ratios of hexose, total sulfate, N-sulfate and sialic acid to hexosamine were 5.07, 0.90, 0.18 and 0.08, respectively, and about 10% of hexosamine consisted of galactosamine. The KS resulted in a production of oligosaccharides of relatively larger size after digestion with keratanase, as compared with bovine corneal KS. These findings strongly suggest that KS accumulated in the liver affected by Morquio syndrome may be derived from bony KS.

Fluorescence Spectral Studies on the Specific Interaction between Sulfated Glycosaminoglycans and Potato Lectin

Abstract: On excitation at 280 nm, Solanum tuberosum agglutinin (STA) produced a fluorescence emission spectrum with a maximum at 347 nm, which is typical of tryptophanyl residues. The fluorescence emission maximum was shifted toward shorter wavelength and quenched by the addition of specific sugars. The changes were analyzed precisely and quantitatively by measuring the fluorescence difference spectra. This method required far smaller amounts of samples than UV-difference spectroscopy to obtain the binding parameters. The binding constants of chitin sulfate, keratan sulfate, and highly sulfated keratan sulfate were calculated to be 1.8 X 10(5) M-1, 3.4 X 10(5) M-1, and 5.2 X 10(5) M-1, respectively. The results indicate that consecutive and non-consecutive, alternating (1 leads to 4)-N-acetyl-beta-D-glucosaminyl residues in the glycosaminoglycans can interact freely with STA in spite of the existence of the sulfate groups at C-6 of the N-acetyl-D-glucosaminyl residues and extra sulfate groups at C-6 of the galactosyl residues linked with N-acetyl-6-O-sulfo-beta-D-glucosamine.