Inhibition of hyaluronan uptake in lymphatic tissue by chondroitin sulphate proteoglycan.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.     

Inhibition of the contraction of collagen gels by extracts from human dermis.

Extracts from the human dermis were prepared and evaluated with respect to their ability to influence fibroblasts to contract collagen gels in vitro. The extract which had the most inhibitory effect on fibroblasts to cause contraction of collagen gels was extract D. It also inhibited fibroblast growth. Inhibition of contraction was not simply related to fibroblast cell numbers and the data suggests a specific effect upon the ability of fibroblasts to cause contraction. Other extracts were without significant effect.     

Endocytosis and degradation of chondroitin sulphate by liver endothelial cells.

Intravenously administered chondroitin sulphate, chemically labelled by [3H]acetylation of partially deacetylated polysaccharide, was taken up and degraded by the non-parenchymal cells of the liver. Studies using primary monolayer cultures of pure Kupffer cells, liver endothelial cells and parenchymal cells revealed that [3H]chondroitin sulphate was taken up and degraded by the liver endothelial cells only. Binding studies at 4 degrees C with [3H]chondroitin sulphate and 125I-chondroitin sulphate proteoglycan indicated that the glycosaminoglycan and the proteoglycan are recognized by the same binding sites on the liver endothelial cells. The ability of hyaluronic acid to compete with the labelled ligands for binding suggested that the binding site is identical with the recently described hyaluronate receptor on the liver endothelial cells [Smedsrød, Pertoft, Eriksson, Fraser & Laurent (1984) Biochem. J. 223, 617-626]. Fluorescein-labelled chondroitin sulphate proteoglycan accumulated in perinuclear vesicles of the liver endothelial cells, indicating that the proteoglycan is internalized and transported to the lysosomes. The finding that [3H]chondroitin sulphate and 125I-chondroitin sulphate proteoglycan were degraded by the liver endothelial cells to low-molecular-mass radioactive products suggested that both the polysaccharide chain and the core protein were catabolized by the cells.     

Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels

The effects of hyaluronic acid (HA) on the proliferation and chondroitin sulfate (CS) synthesis of chondrocytes embedded in collagen gels were examined. Articular cartilage was isolated from the humerus, femur, and tibia of 21 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gels and cultured in Dulbecco's modified Eagle's medium (DMEM) with various doses of HA for 4 weeks. Histological and biochemical evaluations were performed at postculture weeks 1, 2, 3, and 4. For biochemical evaluations, isomers such as chondroitin 6-sulfate (delta(di)-6S) and chondroitin 4-sulfate (delta(di)-4S) synthesized by cultured chondrocytes were determined by high performance liquid chromatography (HPLC) combined with fluorometry. Morphological and histological studies demonstrated that HA-treated chondrocytes in collagen gel proliferated profusely while maintaining their phenotype. At postculture week 4, 0.1 mg/ml of HA induced an eightfold increase in cell counts compared with HA pretreatment values, or 1.5-fold more than control group. Synthesis of delta(di)-6S (delta(di)-6S content/cell) in groups treated with 0.01 and 0.1 mg/ml of HA significantly increased, while gel accumulation rates in groups treated with 0.1 and 1.0 mg/ml of HA scored significantly higher values than other groups. In collagen gel culture, HA enhanced the proliferation and delta(di)-6S synthesis of chondrocytes while maintaining their phenotype. In clinical application, since the supply of autologous chondrocytes for transplantation is not unlimited, the HA-treated culture method may be useful for increasing the number of chondrocytes and thus improving the quality of implants.     

Initiation of chondroitin sulphate synthesis by beta-D-galactosides. Substrates for galactosyltransferase II.

beta-Galactosides were found to initiate chondroitin sulphate chain synthesis in chick-embryo cartilage in vitro and thereby relieve inhibition by cycloheximide of [3H]-acetate incorporation into chondroitin sulphate. beta-Galactosides with an apolar aglycan group such as phenyl O-beta-galactoside were active, whereas those with a charged or polar aglycan group such as pyridine 3-O-beta-galactoside or those with sulphur instead of oxygen in the glycosidic linkage (phenyl beta-thiogalactoside) were not. beta-Galactosides also serve as substrates for microsomal galactosyltransferase activity from chick-embryo cartilage. Phenyl O-beta-galactoside and pyridine 3-O-beta-galactoside were effective substrates for this enzyme, but phenyl S-beta-thiogalactoside and pyridine 2-S-beta-thiogalactoside were only slightly active. This galactosyltransferase was shown to be a separate enzyme from galactosyltransferase I, which catalyses transfer of galactose from UDP-galactose to beta-xylosides. It is proposed that the enzyme catalysing this reaction is galactosyltransferase II, responsible for transfer of the second galactose residue of the chondroitin sulphate linkage oligosaccharide. No transfer of glucuronic acid from UDP-glucuronic acid to beta-galactosides, catalysed by the microsomal preparation could be detected.     

The distribution of sulphate residues in the chondroitin sulphate chain

1. Electrophoresis of chondroitin sulphate, before and after partial degradation with testicular hyaluronidase, revealed charge heterogeneity of the degraded but not of the intact polymer. 2. Hyaluronidase-treated chondroitin sulphate was fractionated by gel chromatography. Two subfractions which were essentially monodisperse with regard to molecular weight (values of 8600 and 4800, respectively) were separated further by chromatography on Dowex 1. The resulting subfractions differed considerably with respect to their sulphate/disaccharide molar ratios. 3. Amino acid and neutral-sugar analyses of the Dowex 1 subfractions showed that the less sulphated fragments contained the carbohydrate-protein linkage region, whereas the high-sulphated fragments essentially lacked this constituent. It was concluded that chondroitin sulphate contains relatively less sulphate in the vicinity of the carbohydrate-protein linkage region than in the more peripheral portion of the polysaccharide chain.     

Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta.

Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 X 10(6) (PG-25), 0.30 X 10(6) (PG-35) and 0.88 X 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.     

The mechanism of initiation of chondroitin sulphate synthesis by beta-D-galactosides

Embryonic chicken cartilage was incubated in vitro with phenyl-beta-[6-3H]galactoside and cycloheximide. Free chondroitin sulphate chains initiated by galactoside were isolated and degraded to yield 3-O-beta-D-glucuronosyl D-galactose (GlcA-Gal) derived from the sequence, GlcA-Gal-Gal-Xyl-Ser, which links the polysaccharide to protein. This enabled the distribution of 3H into specific galactose residues within the linkage oligosaccharide to be determined. Most of the 3H label (65%) was released as free galactose although 35% was recovered as GlcA-Gal. Similar experiments performed with unlabelled phenyl-beta-galactoside and [14C]galactose yielded 14C-labelled GlcA-Gal as a major product. We conclude that beta-galactosides initiate chondroitin sulphate synthesis primarily by serving as substrates for galactosyl transferase II.     

Secondary structure of chondroitin sulphate in dimethyl sulphoxide

The transient phase of histone H1 phosphorylation was studied by the quenched-flow method. A 'minimal' kinetic scheme of the above process was proposed. A formal kinetic analysis was given to a four-step mechanism of the reaction. Computer simulation of the transient-phase kinetics of H1 phosphorylation and the steady-state kinetics of phosphate transfer from the enzyme phosphoform to histone permitted us to estimate all kinetic constants of the proposed mechanism.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

A distinct terminal structure in newly synthesized chondroitin sulphate chains.

A method was developed for the analysis of non-reducing terminal structure of radiolabelled chondroitin sulphate chains with the aid of N-acetylgalactosamine 4-sulphatase ('terminal 4-sulphatase'), N-acetylgalactosamine 6-sulphatase ('terminal 6-sulphatase'), beta-glucuronidase and beta-N-acetylhexosaminidase. Studies with this method on the non-reducing terminal structure of [35S]sulphate- and [3H]glucose-labelled chondroitin sulphate chains from rat and chick-embryo cartilages showed that the presence of a high proportion of 4-sulphated hexosamine residues is a common feature of the termini of newly synthesized chondroitin sulphate chains. Of the non-reducing terminal 4-sulphated hexosamine residues, about 14% (chick embryo) or 46% (rat) contained an additional sulphate group at position 6. The internal portion of the chondroitin sulphate chains, in contrast, contained little or no 4,6-bis-sulphated hexosamine residue, suggesting that 4,6-bis-sulphated structure may play a role in biosynthetic control at the level of chain termination.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Summary Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

Proteoglycan-collagen interactions in intervertebral disc. A chondroitin sulphate proteoglycan associates with collagen fibrils in rabbit annulus fibrosus at the d-e bands

Rabbit annulus fibrosus and nucleus pulposus were analysed for hydroxyproline, chondroitin sulphate, keratan sulphate and dermatan sulphate. Tissue proteoglycans were stained for electron microscopy with Cupromeronic blue, used in the critical electrolyte concentration mode, with and without prior digestion by chondroitinase AC or ABC, hyaluronidase or keratanase. Collagen bands, a-e were demonstrated with UO2++. A chondroitin sulphate proteoglycan was found orthogonally associated with loosely packed collagen fibrils in annulus fibrosus at the d and e bands. The close metabolic and structural analogies with the dermatan sulphate proteoglycans previously shown to be located at collagen d-e bands in tendon, skin, etc. (Scott and Haigh (1985) Biosci. Rep. 5:71-81), are discussed. Tightly packed annulus collagen fibrils were surrounded by axially oriented proteoglycan filaments, mostly without specific locations.