Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.36 (hyaluronidase)
4,606 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.
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PMID:An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates. 262 58

Rabbit corneal proteoglycans were labeled by intrastromal injection of 3H-glucosamine and 35S-sulfate 1 and 2 weeks after partial-thickness radial scalpel incisions. Proteoglycans were extracted with guanidine-HCl and purified by ion exchange chromatography. Wounding caused a marked decrease in the total incorporation of labeled precursors into proteoglycans. The labeled proteoglycans were more readily extracted with guanidine-HCl after wounding. Labeled proteoglycans from wounded corneas had a larger molecular size on gel filtration chromatography than did proteoglycans from control corneas, a result of an increased amount of keratan sulfate in the large molecular size fractions. Analysis of labeled glycosaminoglycan (GAG) from guanidine-extracted proteoglycans and from the corneal tissue after guanidine-HCl extraction showed an increase in the relative amount of heparan sulfate and keratan sulfate after wounding, and a decrease in relative amount of dermatan sulfate. The 35S:3H ratio of heparan and dermatan sulfates increased after wounding, and that of keratan sulfate decreased, suggesting changes in sulfation. Degradation of labeled dermatan sulfate with hyaluronidase and with periodate revealed a 2-fold increase in iduronic acid content and 2-4-fold increase in hyaluronidase-resistant dermatan sulfate in the wounded corneas. Reduction in proteoglycan content, reduced sulfation of keratan sulfate, and accumulation of a high-sulfate, high-iduronic acid dermatan sulfate are previously reported properties of proteoglycan in scar tissue from perforating corneal wounds. Demonstration of these properties in proteoglycan after wounds similar to radial keratotomy incisions suggests that deposition of scar tissue can result from wounds which do not damage Descemet's membrane.
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PMID:Proteoglycans of rabbit corneas with nonperforating wounds. 292 15

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 cartilage proteoglycan monomer. Monoclonal F1.2 does not react with the keratan sulfate species in human and fetal calf serum and can therefore detect the production of keratan sulfate-bearing proteoglycan by chondrocytes cultured in serum-containing media.
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PMID:Monoclonal antibodies reactive with keratan sulfate-bearing tryptic fragments of bovine nasal cartilage proteoglycan. 295 51

Among the most important events in connective tissue physiology are the nucleation, growth and calcification of collagen fibrils. It has been speculated that all are associated with, or even controlled by collagen-proteoglycan interactions. We therefore developed methods for investigating these associations in tissues, particularly for understanding their significance for type I collagen, the commonest form of collagen in the body, especially predominant in bone. Using an electron-dense dye, Cupromeronic blue, in the 'critical electrolyte concentration' mode, and digestion by hyaluronidase, chondroitinase ABC or keratanase, supported by biochemical analyses, we found that dermatan sulphate proteoglycan of soft connective tissue (skin, tendon, cornea) was regularly and orthogonally arrayed at the fibril surface, at the d or e band. Keratan sulphate proteoglycan in the cornea associates orthogonally at the a and c bands. Bone, demineralized by a non-aqueous technique which retains proteoglycans in the tissue, does not contain orthogonal arrays; the interfibrillar proteoglycan filaments are oriented parallel to the fibril axis. The main proteoglycan in bone is chondroitin sulphate-rich. There are thus four separate specific binding sites on type I collagen fibrils, each one associating with one particular proteoglycan, and apparently no other. This implies that there are two corresponding binding sites in each proteoglycan. Available evidence shows that there are two species of small dermatan sulphate and keratan sulphate proteoglycans. It is suggested that each species is specific for its own band (a, c, d or e). Hyaluronate and chondroitin sulphate are probably mainly interfibrillar, acting in a space-filling capacity.
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PMID:Proteoglycan-collagen interactions. 381 15

The cartilages from the hip joints of 13 normal and 15 osteoarthritic humans were analyzed for glycosaminoglycan content and distribution. The GAGs were separated by elution with CPC on a short cellulose column by the technique of Svejcar and Robertson after digestion of the tissue with pronase and papain. The eluates were identified by a variety of methods including determination of molar ratios, N-acetyl-hexosamine determinations after hyaluronidase treatment and thin-layer chromatography of unhydrolyzed and hydrolyzed GAGs. From the data obtained, it was demonstrated that cartilage from arthritic patients showed a significant increase in the concentration of chondroitin 4-sulfate and a significant decrease in keratan sulfate, with only slight changes in the total amount of GAG present. Calculations of the molar ratios showed variation in the sulfation with chondroitin 4-sulfate appearing in the "supersulfated" state in the arthritic cartilage. The data lead to speculation regarding the process of osteoarthritis, and it is concluded that the changes seen are more likely to represent an altered pattern of synthesis rather than selective degradation. Since the changes suggest a younger cartilage, a theory is advanced that the chondrocyte responds to the chronic stress of osteoarthritis by modulation to a chondroblastic phase.
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PMID:The glycosaminoglycans of normal and arthritic cartilage. 425 96

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.
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PMID:Ultrastructural cytochemistry and immunocytochemistry of proteoglycans associated with epiphyseal cartilage calcification. 613 41

Bovine nasal cartilage proteoglycan monomer which had been digested with chondroitinase ABC to form the keratan sulfate-protein core was injected into rabbits. High titer antiserum was obtained as judged by the binding of 125I-labeled keratan sulfate-protein core. Native proteoglycan did not inhibit the interaction of the antiserum with labeled keratan sulfate-protein core. However, the disaccharide obtained from chondroitin 4-sulfate by the action of chondroitinase ABC, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, inhibited the interaction 50% at a concentration of 500 microM. The corresponding 6-sulfated and nonsulfated disaccharides at the same concentration gave 15% and 10% inhibition, respectively. Chondroitinase-digested proteoglycan from Swarm rat chondrosarcoma was strongly inhibitory, while the hyaluronidase-digested chondrosarcoma proteoglycan exhibited no detectable inhibition. Evidently, the antiserum raised against chondroitinase ABC-digested bovine nasal cartilage proteoglycan contains antibodies which recognize the unsaturated uronic acid residue linked to N-acetylgalactosamine 4-sulfate. These antibodies will be valuable for identifying and quantitating chondroitin 4-sulfate-containing proteoglycans in tissues.
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PMID:Immunological determinants of proteoglycans. Antibodies against the unsaturated oligosaccharide products of chondroitinase ABC-digested cartilage proteoglycans. 615 67

The role of osteoclasts or chondroclasts in degradation and synthesis of complex carbohydrates was investigated using the high iron diamine-thiocarbohydrazide-silver proteinate method (HID-TCH-SP) for sulfated glycoconjugates and the periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained the calcified cartilage matrix, the osteoclast ruffled border, vacuoles and heterophagosomes but not the Golgi apparatus and primary lysosomes. The size and number of HID-TCH-SP stain deposits progressively decreased from the calcified cartilage matrix to the ruffled border (p less than 0.001). Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits averaging 13 nm. in diameter in the extracellular matrix, cytoplasmic vacuoles, and heterophagosomes of osteoclasts. Only sparse stain deposits averaging 8 nm. in diameter and presumed to be keratan sulfate remained in these sites after enzyme digestion. Osteoclast heterophagosomes contained the highest concentration of hyaluronidase-resistant material suggesting delayed degradation of keratan sulfate at this site. PA-TCH-SP strongly stained collagen fibrils in the calcified cartilage matrix. Reactive collagen fibrils were also observed in extracellular channels but only rarely were identifiable collagen fibrils observed in cytoplasmic vacuoles. A progressive decrease in the diameter of PA-TCH-SP reactive collagen fibrils was observed between the calcified cartilage matrix and the ruffled border region (p less than 0.001). PA-TCH-SP stained cisternae of rough endoplasmic reticulum, Golgi saccules, and primary lysosomes consistent with the synthesis and packaging of glycoprotein enzymes at these sites. These results indicate that the dissolution of sulfated glycoconjugates requires osteoclastic engulfment of degraded material and subsequent intracellular digestion, whereas the dissolution of collagen fibrils appears to be completed extracellularly.
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PMID:Extracellular and intracellular digestion of complex carbohydrates by osteoclasts. 617 27

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.
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PMID:Vitreous: an inhibitor of retinal extract-induced neovascularization. 618 31

Several monoclonal antibodies which recognize different antigenic determinants of chondroitin sulfate proteoglycan were used to study chondroitin sulfate proteoglycan biosynthesis in chicken chondrocyte cultures. The intracellular sites of synthesis and processing and extracellular deposition in matrix were localized by double immunofluorescence reactions. One rat monoclonal antibody, S103L , which recognizes an antigenic determinant of the core protein of the chicken cartilage chondroitin sulfate proteoglycan monomer, was used to identify both extracellular chondroitin sulfate proteoglycan and intracellular compartments containing chondroitin sulfate proteoglycan precursors. Intracellular staining with S103L was localized to perinuclear regions, and, in some chondrocytes, to a few other cytoplasmic vesicles as well. When chondrocytes were not fed for several days, intracellular chondroitin sulfate proteoglycan precursors were accumulated in larger compartments distributed throughout the cytoplasm. Polyclonal chondroitin sulfate proteoglycan antibodies displayed similar staining characteristics. In contrast, several of the monoclonal antibodies, including the rat monoclonals S11D and P100D , and the mouse monoclonals 1-B-5, 3-B-3 and 9-A-2, did not recognize native chondroitin sulfate proteoglycan, but reacted only with chondroitinase ABC-digested (and/or hyaluronidase-digested) chondroitin sulfate proteoglycan. These antibodies were particularly useful in the demonstration of the extracellular codistribution of chondroitin sulfate proteoglycan with either type II collagen or fibronectin. In other experiments, the monoclonal antibodies to chondroitin sulfate proteoglycan served to demonstrate that the perinuclear subset of intracellular compartments is uniquely involved in the addition of chondroitin sulfate oligosaccharides to the chondroitin sulfate proteoglycan core protein. Lastly, using the mouse monoclonal 5-D-4, which recognizes keratan sulfate determinants, the perinuclear region was identified as the site for keratan sulfate addition. Results suggest heterogeneity of keratan sulfate synthesis at the level of individual chondrocytes, even for cells apparently containing equivalent amounts of intracellular chondroitin sulfate proteoglycan.
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PMID:Immunofluorescence studies of chondroitin sulfate proteoglycan biosynthesis: the use of monoclonal antibodies. 620 57


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