Showing papers on "Keratan sulfate published in 1987"

Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain.

Abstract: Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KS-PGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was absorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind. Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 3T3 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF.(ABSTRACT TRUNCATED AT 400 WORDS)

The immunologic detection and characterization of cartilage proteoglycan degradation products in synovial fluids of patients with arthritis.

Abstract: Antibodies were used in radioimmunoassays with gel chromatography to detect the hyaluronic acid-binding region, core protein, and keratan sulfate of human cartilage proteoglycan in the synovial fluids of patients with rheumatoid arthritis, juvenile rheumatoid arthritis, and osteoarthritis. All fluids contained proteoglycan that was mainly included on Sepharose CL-4B; this result indicates cleavage of proteoglycan (which is normally excluded). The hyaluronic acid-binding region was the smallest and most commonly detected fragment. It was relatively free of keratan sulfate and core protein, and it could sometimes bind to hyaluronic acid. Other larger fragments containing core protein and/or keratan sulfate were detected in every fluid.

Localization of glycosaminoglycans in human and canine menisci and their attachments.

Abstract: The semilunar meniscal body, horns and peripheral attachment were dissected from human and canine knee joints. The concentrations of hyaluronate, chondroitin sulfate, dermatan sulfate and keratan sulfate differed markedly among the tissues. The meniscal body was fibrocartilaginous but showed marked histological microheterogeneity with hyalinized areas intermixed with fibrous areas and contained mostly chondroitin sulfate and keratan sulfate. On the other hand, the fibrous peripheral attachment contained mostly dermatan sulfate and hyaluronate and almost no keratan sulfate and the ligamentous horns contained mostly dermatan sulfate and chondroitin sulfate. Human menisci contained more dermatan sulfate and less chondroitin sulfate than did canine menisci. From morphometric measurements of canine menisci, an estimate was made of the amount of peripheral tissue which had been included with the body when dissections were based on gross appearance and how much this might influence analysis of the composition. T...

Epitopes of proteoglycans eliciting an anti-proteoglycan response in chronic immune synovitis.

Abstract: This study details the immune response to cartilage proteoglycan in experimental chronic IgG-induced immune synovitis. Antibodies reactive with purified rabbit proteoglycan monomer were observed in nine of nine rabbits with immune synovitis. IgG-immunized but nonsynovitic control animals with no pathology showed no antibody response. A panel of murine monoclonal antibodies with defined specificity towards rabbit proteoglycan were utilized to characterize the epitope specificity of the immune synovitis polyclonal anti-proteoglycan response. One murine monoclonal antibody, 6C11, inhibited the binding of the polyclonal antisera to proteoglycan in all nine animals with significant (greater than 40%) inhibition in six of nine rabbits. Further inhibition studies utilizing DEAE-cellulose-resolved proteoglycan tryptic peptides revealed that peptides poor in chondroitin sulfate were strong inhibitors of binding of the polyclonal antibodies to the proteoglycan substrate. In particular, keratan sulfate-containing tryptic peptides were most inhibitory on a per weight basis. These results indicate that, in chronic IgG-induced immune synovitis, anti-proteoglycan antibodies elicited are heterogeneous with regard to specificity, but a relatively large proportion predominantly recognized a portion of the proteoglycan molecule containing core protein and associated keratan sulfate.

Catabolin/interleukin-1 regulation of cartilage and chondrocyte metabolism.

Abstract: Catabolin/interleukin-1 effects on metabolism were studied in bovine nasal cartilage organ culture and articular chondrocyte cell culture. Keratan sulfate (KS) and hyaluronic acid (HA) were determined by an ELISA; prostaglandin E2 by RIA, sulfated glycosaminoglycan using dimethylmethylene blue and proliferation by incorporation of tritiated thymidine. Gel filtration of untreated 4-day organ culture media indicated that large sulfated and KS-containing proteoglycans were released and eluted in the void volume. Catabolin/interleukin-1 increased release of sulfated glycosaminoglycans and these were of lower molecular weight with an altered distribution of KS. Catabolin/interleukin-1 treatment of chondrocytes caused a decrease in KS production and proliferation but an increase in HA and in prostaglanding E2 production. Alterations of the chondrocyte metabolism by catabolin/interleukin-1 causing proteoglycan matrix degradation and modulation of chondrocyte glycosaminoglycan biosynthesis and proliferation may play a role in cartilage erosion and failure to repair in arthritic diseases.

Keratan sulfate levels in sera of patients bearing cartilage tumors

Abstract: Previous studies on chondrosarcoma proteoglycans have suggested that keratan sulfate content and average keratan sulfate chain length may be inversely related to the degree of cartilage tumor malignancy. The present study extended this work to an analysis of serum keratan sulfate in chondrosarcoma patients. Keratan sulfate levels of 24 adult patients bearing cartilage tumors were observed to be higher than that for age- and sex-matched controls. In nine of ten patients, keratan sulfate level exhibited a significant decrease following removal of the tumor. Analysis of five general hospital patients before and after surgery did not detect such a change. The data were suggestive of an inverse relationship between tumor histopathologic grade and presurgical serum keratan sulfate concentration. Finally, despite the wide range of serum values within each histopathologic tumor group, sequential serum keratan sulfate measurements may be useful in monitoring patients for secondary growth of chondrosarcoma tumors after primary excision.

67Ga accumulation in inflammatory lesion and its mechanism: Comparison with malignant tumor

Abstract: The accumulation of 67Ga in inflammatory lesions increased with time after injection of turpentine iol and reached a plateau 5 days later. At that time the uptake in the lesions was larger than any other tissue, after ten days the lesion uptake decreased. In experiments using rats which had been kept for 5 days after subcutaneous injection of turpentine oil, the accumulation of 67Ga in inflammatory lesions increased with time until six days after administration of 67Ga-citrate. It is clear from this study that 67Ga is avidly accumulated in areas where the subcutanous tissue is infiltrated with neutrophils and macrophages, that it is not accumulated at the sites in which neutrophils are crowded, that nuclear material, mitochondria, lysosomes and microsomes do not play a major role in 67Ga accumulation in the lesion and that the main binding acid mucopolysaccharide in the lesion is acid mucopolysaccharide which is none of the following: keratan sulfate, heparan sulfate, heparin, or chondroitin sulfate A, B or C. It is presumed that the main 67Ga binding acid mucopolysaccharide is keratan polysulfate (or other oversulfated acid mucopolysaccharides).

Monoclonal antibodies reactive with keratan sulfate-bearing tryptic fragments of bovine nasal cartilage proteoglycan.

Abstract: Monoclonal antibody-producing cell lines were derived from BALB/c mice immunized with a testicular hyaluronidase digest of tryptic fragments of bovine nasal cartilage proteoglycan. Sera and hybridoma culture supernatants were screened by solid-phase immunoassay for reactivity against a chondroitinase ABC digest of the same proteoglycan fragment fraction. Antibody specificity was determined by competitive inhibition with purified proteoglycan fragment subfractions and their enzymatically modified derivatives. Two monoclonal antibodies were produced which reacted with keratan sulfate-rich fragments from bovine nasal and human articular cartilage proteoglycan. One, monoclonal LC8.13, is directed against keratan sulfate itself, but differs from 5-D-4, a previously described monoclonal antibody to keratan sulfate, in its lesser reactivity with keratanase-treated fragments. The second, monoclonal F1.2, appears to be directed against a conformation-dependent determinant on the core protein of this segment of the...

Amino acid sequence of a peptide from keratan sulfate II-core protein linkage regions.

Abstract: Keratan sulfate II was prepared from the proteolytic digest of pig nucleus pulposus proteoglycan. The polysaccharide chains containing the fragment peptides of the core protein at their reducing terminal were subjected to anhydrous HF-solvolysis reaction and one of the glycopeptides from the keratan sulfate II-core protein linkage regions was isolated. The amino acid sequence of the peptide was deduced to be Ala-Pro-Ser-Pro-Gly, which is different from those reported for the attachment sites of chondroitin sulfate on core proteins from various sources. The results provided the first solid amino acid sequence for the keratan sulfate II-core protein linkage regions and suggested that the amino acid sequence of the core protein might determine the distribution of chondroitin sulfates and keratan sulfates along the core protein of the proteoglycan molecule.

The Normal Variation in Structure and Composition of Canine Hip Articular Cartilage Proteoglycans

Abstract: The composition of the weight bearing portion of the femoral head articular cartilage from both sides of greyhounds 11–32 months of age was studied with regard to variability of proteoglycans. Extractability of proteoglycans was very similar for all samples. Other parameters, i.e. contents of proteoglycans in the cartilage, hyaluronic acid content of the proteoglycan fraction, as well as contents of keratan sulfate and chondroitin sulfate, size of proteoglycans and ability of proteoglycans to aggregate with hyaluronic acid, were similar in samples from the right and left side of each dog. The variability from one dog to another was large and not related to age. The data show that in experimental studies of joint disease the contralateral joint can be used as a control to a treated joint, while the large interindividual variability makes comparison difficult between individuals.

Immunodetection and characterization of the degradation of cartilage proteoglycans in vitro and in vivo.

Abstract: The large aggregating cartilage proteoglycans can be detected immunologically using antibodies directed against the hyaluronic acid-binding region, core protein, keratan sulfate, chondroitin sulfate, and hyaluronic acid. With radioimmunoassays we have detected proteoglycans released from adult human articular cartilage in vitro and in vivo. In vitro cleavage appears to occur mainly adjacent to the hyaluronic acid binding region with the release of the rest of the molecule. Two distinct populations have been observed, the larger of which is chondroitin sulfate-rich and the smaller keratan sulfate-rich. Although they are a little smaller and probably aggregate less with hyaluronic acid, these populations correspond in size and composition to two similar populations detected in situ in healthy adult human articular cartilage. In vivo studies of synovial fluid have revealed that the proteoglycan fragments are smaller and the most commonly detected fragment is the hyaluronic acid binding region. This suggests that in vivo further degradation occurs, probably due to degradation mediated by the inflamed synovium and polymorphonuclear leucocytes present in the synovial fluid of inflamed joints.

Heterogeneity of bovine corneal proteokeratan sulfate.

Abstract: Proteokeratan sulfate was extracted and purified from bovine corneal stroma and then characterized by chemical and biochemical analyses. It was fractionated into several fractions by affinity chromatography on a concanavalin A-Sepharose column or by hydrophobic chromatography on a phenyl-Sepharose column. These fractions differed widely from one another in carbohydrate content, though no significant differences of their amino acid compositions were observed. One fraction (ca. 25%, on a dry weight basis) tightly bound to a concanavalin A-Sepharose column, compared with another fraction (ca. 65%) weakly bound to the same column, was poor in galactose and N-acetylglucosamine, but contained mannose in a high proportion. Fractions (ca. 30%) tightly bound to a phenyl-Sepharose column, in contrast to the one (ca. 66%) weakly bound, had low carbohydrate contents, like the fraction tightly bound to a concanavalin A-Sepharose column. Additionally, the fractions tightly bound to these affinity columns exhibited strong inhibitory actions on erythrocyte-concanavalin A agglutination. To obtain further details of the carbohydrate moiety of the proteokeratan sulfate, an attempt was made to separate and characterize peptidokeratan sulfate and Asn-linked oligosaccharide derived from some proteokeratan sulfate fractions. The present work revealed that the proteokeratan sulfate contains keratan sulfate and high mannose-type oligosaccharide in an approximate chain number ratio of 3.5:1.0, the keratan sulfate content varies widely and the oligosaccharide content increases with decrease of the keratan sulfate content, and the protein core is homogeneous at least with respect to the amino acid composition.