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:3.1.6.4 (chondroitinase)
2,039 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adult, canine intervertebral disc cells were isolated with a sequential digestion of pronase and bacterial collagenase. The nonchondrodystrophoid nucleus pulposus exhibits two populations of cells: large notochordal cells and smaller chondrocyte-like cells. The cells from the transition zone and anulus fibrosus are uniform in size, ranging from 17 to 21 microns. The isolated cells were encapsulated in alginate beads and cultured in Ham's F-12 medium containing 5% heat-inactivated fetal bovine serum. Alginate bead formation requires calcium ions and can be reversed with a suitable chelator, thus releasing viable cells. We observed that 58% of the newly synthesized proteoglycans formed large-molecular-weight aggregates with hyaluronic acid. The proteoglycans contained low amounts of keratan sulfate (KS) (less than 5% of the total glycosaminoglycans synthesized). The chondroitin sulfates (CS) consisted of 51-67% as 6-O-sulfate and 29-39% as 4-O-sulfate, with the remainder (4-10%) present as 4,6-sulfate for all three zones of the disc. The majority of cells synthesized significant amounts of matrix as evidenced by Alcian Blue staining. By immunohistochemical analysis, the matrix contained chondroitin 6-sulfate as demonstrated by monoclonal antibodies to the unsaturated disaccharides remaining on the proteoglycan core after chondroitinase ABC digestion. Keratan sulfate was also present in the majority of the matrices around cells. These results emphasize the similarity of the newly synthesized proteoglycans secreted by cells grown in alginate beads to those synthesized by the neonate disc. These experiments also demonstrate the usefulness of this method as a microculture technique for disc cells.
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PMID:Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres. 138 73

1. We have isolated, chemically and immunologically characterized versican and decorin from bovine gingiva. 2. Versican was of large molecular weight and the molecular size of the core protein was estimated to be greater than 200 kDa. 3. The glycosaminoglycan chains were susceptible to chondroitinase ABC and N-linked oligosaccharides were present on the protein core of the molecule. 4. Immunological studies provided evidence that a hyaluronic acid binding region was present in the core protein of versican. 5. The overall structure was similar to that of versican isolated from bovine sclera. 6. Decorin had a molecular weight of 102 kDa and its glycosaminoglycan chain was completely digested by specific glycosidases. 7. The partially deglycosylated core protein had a molecular weight of 55 kDa and N-linked oligosaccharides were present on the molecule.
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PMID:Isolation and characterization of bovine gingival proteoglycans versican and decorin. 139 83

A sensitive chemiluminescence high-performance liquid chromatographic method has been developed for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate as their unsaturated disaccharide-dansylhydrazine derivatives involving an effective sample clean-up system. The dansylhydrazones of the unsaturated disaccharides derived from the hyaluronic acid, chondroitin sulphate and dermatan sulphate by chondroitinase ABC and/or chondroitinase ACII, were separated by reversed-phase chromatography using a mixture of 0.1 M sodium acetate buffer (pH 6.0) and 80% acetonitrile on a column (250 mm x 4.0 mm I.D.) packed with amide-80 silica beads (5 microns diameter). For post-column elution in the chemiluminescence system, 1 mM bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate and 3mM hydrogen peroxide in acetonitrile were used. The detection limit of each glycosaminoglycan was 100 fmol. The method was applicable to the determination of the levels of hyaluronic acid, chondroitin sulphate and dermatan sulphate in rat peritoneal mast cells.
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PMID:Chemiluminescence high-performance liquid chromatography for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate. 142 67

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.
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PMID:Intra- and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia. 143 Oct 58

The disaccharides constituting chondroitinase-digestible glycosaminoglycan (GAG) in the skin lesions of patients with systemic sclerosis were determined using high-performance liquid chromatography (HPLC). In scleroderma there was an increase in the amount of delta Di-4S(DS), the main disaccharide unit of dermatan sulphate, and a decrease in delta Di-HA, the disaccharide unit of hyaluronic acid, as compared with normal skin from a similar site. The distribution pattern of the main disaccharides constituting chondroitin sulphate and dermatan sulphate in scleroderma differed from that in scars or scleredema.
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PMID:Disaccharide analysis of the skin glycosaminoglycans in systemic sclerosis. 153 59

After immunization of mice with partially-purified heparan sulfate proteoglycan (HSPG) isolated from rat glomeruli, a monoclonal antibody (mAb JM-403) was obtained, which was directed against heparan sulfate (HS), the glycosaminoglycan side chain of HSPG. In ELISA it reacted with isolated human glomerular basement membrane (GBM) HSPG, HS and hyaluronic acid, but not with the core protein of human GBM HSPG, and not with chondroitin sulfate A and C, dermatan sulfate, keratan sulfate and heparin. Furthermore, it did not bind to laminin, collagen type IV or fibronectin. Specificity of JM-403 for HS was also suggested by results of inhibition studies, which found that intact HSPG and HS, but not the core protein, inhibited the binding of JM-403 to HS. In indirect immunofluorescence on cryostat sections of rat kidney, a fine granular to linear staining of the GBM was observed, along with a variable staining of the other renal basement membranes. Pretreatment of the sections with heparitinase completely prevented the binding of mAb JM-403, whereas pretreatment with chondroitinase ABC or hyaluronidase had no effect. The precise binding site of mAb JM-403 was investigated by indirect immunoelectron microscopy. It revealed a diffuse staining of the whole width of the GBM. One hour after intravenous injection of JM-403 into rats, the mAb was detected along the glomerular capillary wall in a fine granular pattern, which shifted towards a more mesangial localization after 24 hours. No binding was observed anymore by day 15. Intravenous injection induced a dose-dependent, transient and selective proteinuria that was maximal immediately after the injection.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats. 159 46

Groups of purebred beagles and greyhounds of similar ages (1.5-2.5 years) were used for the study. Intervertebral disc proteoglycans (PGs) were radiolabelled in vivo (with [35SO4(2-)], 24 hours and 60 days prior to euthanasia, when lumbar discs were dissected into nucleus pulposus (NP) and annulus fibrosus (AF). Aliquots of each disc region were separately analysed for total PG content as hexuronate. The remaining tissue was subjected to extraction with 4.0 M GuHCl. High buoyant density PGs were isolated from these extracts by CsCl density gradient ultracentrifugation. The hydrodynamic size and aggregatability of the 24-hour, 60-day-old, and resident PG populations were determined by Sepharose CL2B chromatography in the presence or absence of excess hyaluronic acid. While the hydrodynamic sizes of the newly synthesized (24-hour) disc PG preparations appeared to be similar, the 60-day-old greyhound disc PGs were found to be larger than the corresponding beagle disc PG populations. However, the keratan sulphate-core protein complexes prepared by chondroitinase ABC digestion of the newly synthesized (24 hour) disc PGs showed that the greyhound disc preparations were also larger than those from beagle discs. Approximately 80% of the newly synthesized PGs from beagle and greyhound discs were capable of aggregating with hyaluronic acid, however, this was reduced to 55% for the 60 day-old PGs in NP and AF and even less for the resident PG populations (as determined by hexuronate analysis). Significantly, PG aggregation was lower in the greyhound NP and AF preparations than in the corresponding PGs isolated from the beagle disc.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A comparison of the high buoyant density proteoglycans isolated from the intervertebral discs of chondrodystrophoid and non-chondrodystrophoid dogs. 160 37

In this report we describe a system capable of resolving all of the known unsaturated disaccharides derived from the chondroitin sulphates, dermatan sulphate and hyaluronic acid by chondroitinase digestion. This system is superior to others in that the non-sulphated and mono-, di- and tri-sulphated disaccharides can be separated with good resolution in approximately 40 min in an isocratic solvent. The system employs an amino-cyano silica gel column (Whatman Partisil 5 PAC, 25 cm) and is eluted with an isocratic solvent consisting of 48% (v/v) acetonitrile, 14% (v/v) methanol and 38% (v/v) aqueous buffer. This aqueous buffer contains 0.5 M Tris-HCl, 0.1 M boric acid, 23.4 mM sulphuric acid, pH 8.0. UV absorption is monitored at 229 nm and for most disaccharides as little as 150 ng can be reliably determined. The addition of boric acid to the eluent is essential for good resolution of all components and the addition of low concentrations of sulphuric acid is used to control the elution times of various components. The system was applied to the analysis of glycosaminoglycan standards and excellent agreement with previous compositional analyses was obtained.
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PMID:High pressure liquid chromatographic identification of hyaluronic acid and chondroitin sulphate disaccharides. 179 40

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies. 190 83

We detected glomerular anionic sites in fixed, LR Gold-embedded ultra-thin tissue sections using cationic colloidal gold. Manual and computer-assisted quantitation were compared, and the influence of pH and glycosaminoglycan-degrading enzymes on site expression was examined. Both quantitation methods produced similar results. Alteration of pH within a narrow range (pH 2.5-3.0) markedly affected the staining pattern. At pH 2.5, epithelial and endothelial glycocalyx and regular sites restricted to the lamina rara externa were stained. At pH 3.0 and above, glycocalyx was unstained but intracellular and nuclear staining was present; glomerular basement membrane (GBM) and mesangial matrix sites were abundant. After chondroitinase ABC or hyaluronidase digestion, GBM staining was eliminated at pH 2.0 and reduced at pH 7.0 (p less than 0.001), suggesting that degraded sites are associated with chondroitin sulfate or hyaluronic acid. By contrast, prolonged heparitinase I digestion was ineffective at either pH. Digestion of purified substrates revealed crossreactivity of heparitinase towards chondroitin sulfate and of chondroitinase towards hyaluronic acid. Since tissue sites were reduced by chondroitinase but not heparitinase, we suggest that degradation is due to hyaluronidase activity of chondroitinase and the anionic sites are associated with hyaluronic acid. However, the influence of pH indicates that lamina rara externa sites are structurally distinct from other GBM anionic sites.
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PMID:Detection of glomerular anionic sites in post-embedded ultra-thin sections using cationic colloidal gold. 190 27


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