The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.     

The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans.

Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.     

The synthesis of proteoglycans by human T lymphocytes

We have examined the proteoglycans produced by highly-purified cultures of human T-lymphocytes. The proteoglycans were metabolically labelled with [35S]sulphate and analysed in cellular and medium fractions using DEAE-cellulose chromatography, gel filtration and specific enzymatic and chemical degradations. The results showed that the T cells synthesized a relatively homogeneous, proteinase-resistant chondroitin 4-sulphate proteoglycan that accumulated in the culture medium during a 48 h incubation period. The cellular fraction contained a significant amount of free chondroitin sulphate chains that were not secreted into the medium. These polysaccharides were formed by intracellular degradation of proteoglycan in a chloroquine-sensitive process, indicating a requirement for an acidic environment. In contrast to chondroitin sulphate derived from proteoglycan, chondroitin sulphates synthesized on the exogenous primer, beta-D-xyloside, were mainly secreted by the cells. beta-D-Xylosides caused an 8-fold stimulation in the synthesis of chondroitin sulphate, but decreased the synthesis of proteoglycan by about 50%. These proteoglycans contained shorter chondroitin sulphate chains than their normal counterparts. The results indicate that although proteoglycans are mainly secretory components in human T-cell cultures, a specific metabolic step leads to the intracellular accumulation of free glycosaminoglycans. Separate functions are likely to be associated with the intracellular and secretory pools of chondroitin sulphate.     

The sulphation of chondroitin sulphate in embryonic chicken cartilage

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [(35)S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1.5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3'-phosphate 5'-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.     

The relation of RNA synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

Addition of actinomycin D (or cordycepin, an alternative inhibitor of RNA synthesis) to cartilage cultures resulted in a first-order decrease in the rate of incorporation of [35S]sulphate into proteoglycan (half-life = 7.5 +/- 1.1 h). Addition of 1.0 mM-benzyl beta-D-xyloside relieved the initial inhibition of glycosaminoglycan synthesis induced by actinomycin D; however, after a lag of about 10 h the rate of xyloside-initiated glycosaminoglycan synthesis also decreased with apparent first-order kinetics (half-life = 7.1 +/- 1.8 h), which paralleled the decrease in the rate of core-protein-initiated glycosaminoglycan synthesis. The hydrodynamic size of the proteoglycans formed in the presence of actinomycin D remained essentially constant (Kav. 0.21-0.23), whereas the constituent glycosaminoglycan chains were larger than those formed by control cultures, which suggested that the core protein was substituted with fewer but larger glycosaminoglycan chains. Proteoglycans formed in the presence of beta-D-xyloside were significantly smaller (Kav. approximately 0.33) than those synthesized by control cultures, and were further diminished in size after exposure of cultures to actinomycin D. Glycosaminoglycan chains synthesized by these same cultures on to both core-protein and xyloside acceptors were also smaller than those of control cultures. The decrease in synthesis observed after exposure to actinomycin D was not reflected by any significant decrease in the activities of several glycosyltransferases involved in chondroitin sulphate synthesis (galactosyltransferase-I, galactosyltransferase-II, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II).     

Control of chondroitin sulphate biosynthesis. beta-D-Xylopyranosides as substrates for UDP-galactose: D-xylose transferase from embryonic-chicken cartilage.

Embryonic-chicken epiphyseal cartilage was incubated in vitro with a variety of beta-xylosides and the amount of [3H]acetate incorporation into chondroitin sulphate was determined under conditions when normal protein core production was inhibited by cycloheximide. The ability of the different beta-xylosides to relieve thea cycloheximide-mediated inhibition of chondroitin sulphate synthesis was influenced by the nature of the aglycan group of te xyloside. beta-Xylosides with apolar and uncharged aglycan groups were most effective and produced a severalfold stimulation of chondroitin sulphate biosynthesis. beta-Xylosides with charged aglycan groups were less effective initiators of chondroitin sulphate synthesis. The rate of galactose transfer from UDP-galactose to each of the beta-xylosides, catalysed by a cell-free microsomal preparation from embryonic cartilage, was measured. This study showed that the nature of the aglycan group of the beta-xyloside was a factor determining the capacity of the xyloside to act as an acceptor for galactosyltransferase I, the enzyme that catalyses the first galactose transfer reaction of chondroitin sulphate synthesis. The aglycan group of the xyloside also appeared to influence other steps leading to chondroitin sulphate chain initiation in vitro.     

The effect of beta-D-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors.

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3',5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.     

Altered proteoglycan synthesis by micromelial limbs induced by 6-aminonicotinamide. Appearance of abnormal forms of cartilage-characteristic proteoglycan (PG-H).

Since administration of 6-aminonicotinamide (10 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs, biosynthesis of cartilage-characteristic proteoglycan-H (PG-H) as an important index of limb chondrogenesis was examined in day-7 normal and micromelial hind limbs by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]glucosamine and either [35S]sulphate or [35S]methionine, followed by analyses of labelled PG-H by glycerol density-gradient centrifugation under dissociative conditions, showed a marked reduction in the PG-H synthesis. (2) PG-H synthesized by the micromelial limbs was much lower than that synthesized by the normal limbs in the biosynthetic ratio of chondroitin sulphate to keratan sulphate and glycoprotein-type oligosaccharide, although no significant difference was observed in the immunological properties of these proteoglycans. (3) The degree of sulphation of chondroitin sulphates of PG-H was lowered in the micromelial limbs as judged by the increase of unsulphated disaccharide (delta Di-OS) released by chrondroitinase ABC digestion, although there were no significant differences between the normal and the micromelial limbs in the average molecular size (Mr = 38,000) of labelled chondroitin sulphates of PG-H. (4) Addition of beta-D-xyloside, an artificial initiator for chondroitin sulphate synthesis, to the micromelial limbs in culture recovered the incorporation of labelled glucosamine into chondroitin sulphate to that comparable with the normal control with beta-D-xyloside, although the incorporation of [35S]sulphate was lower in the micromelia than in the control with beta-D-xyloside. These results suggest that the reduction in the biosynthesis of the PG-H as well as the production of altered forms of PG-H induced by 6-aminonicotinamide during a critical period of limb morphogenesis may be an important factor for the micromelia.     

Effect of cycloheximide, beta-D-xylosides and beta-D-galactosides on heparin biosynthesis in mouse mastocytoma.

Heparin biosynthesis has been investigated with mouse mastocytoma in vitro. Minced tumour tissue catalysed the incorporation of [35S]sulphate and [3H]glucosamine into heparin and to a smaller extent into chondroitin sulphate. Addition of cycloheximide caused an inhibition (greater than 80%) of incorporation of each labelled precursor into both polysaccharides. Addition of benzyl beta-D-xyloside relieved the inhibition of incorporation into chondroitin sulphate and restored it to more than threefold that of the control incubation. The effect of beta-D-xyloside on incorporation into heparin was less marked although a consistent small increase of incorporation into this polysaccharide was observed. beta-D-Xyloside did, however, cause a marked incorporation of 35S and 3H labels into material of low molecular weight, which appeared to comprise heparin-like fragments. It is proposed that these fragments arise through a breakdown of the usual process of heparin biosynthesis.     

Protein–polysaccharide of chicken cartilage and chondrocyte cell cultures

The nature of the matrix produced by embryonic chicken chondrocytes in cell culture was studied, and compared with adult and embryonic chicken cartilage. Adult chicken cartilage contains a protein-polysaccharide easily extracted with EDTA-sodium chloride at 4 degrees C. Purification of this macromolecule on Bio-Gel P-300 and Bio-Gel A-50m yielded a progressively more homogeneous species in the ultracentrifuge. It contained mostly chondroitin 4-sulphate, some chondroitin 6-sulphate, and keratan sulphate. Embryonic chicken cartilage was previously shown to contain mostly chondroitin 4-sulphate, some chondroitin 6-sulphate and essentially no keratan sulphate. The matrix produced in chondrocyte cell cultures was shown to contain a protein-polysaccharide with alkali-labile linkages of chondroitin 4-sulphate to the protein core. A fraction was isolated from the matrix with many properties of keratan sulphate.     

Immunoelectron microscopic studies of glycosaminoglycans in the metaphyseal bone trabeculae of growing rats

Summary The types and distribution of glycosaminoglycans (GAGs) were studied immunocytochemically in osteoid, mineralized bone matrix, and cartilage matrix of growing rat metaphyseal bone after aldehyde fixation and EDTA demineralization, using four monoclonal antibodies (mAbs 1-B-5, 2-B-6, 3-B-3 and 5-D-4). These mAbs specifically recognize epitopes in non-sulphated chondroitin (C0-S); chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and C0-S; and keratan sulphate (KS) respectively. In osteoid, all mAbs except 1-B-5 weakly stained matrix material on and between collagen fibrils, and moderately stained organic material corresponding to bone nodules, which are known sites of mineralization. However, the staining of osteoid abruptly decreased at the mineralization front; weak staining was confined mostly to the organic material of bone nodules in mineralized bone matrix, with very weak or no staining of the rest of the bone matrix. This staining progressively decreased toward the mineralized cartilage matrix and became negative. The mineralized cartilage matrix and lamina limitans reacted strongly with all mAbs except 5-D-4. These results indicate that osteoid contains sulphated proteoglycans containing C4-S and/or DS, C6-S and KS, and subsequent bone matrix mineralization appears to require accumulation of these macromolecules within bone nodules and eventual loss of these substances for complete mineralization, whereas proteoglycans containing C0-S, C4-S and/or DS, and C6-S, still exist in mineralized cartilage matrix and lamina limitants.     

Lack of chondroitin sulphate epitope in the proliferating zone of the growth plate of chicken tibia

Summary Monoclonal antibodies specific to chondroitin sulphate (CS-56) and keratan sulphate (AH12) were used to localize proteoglycans in the proximal tibial articular cartilage and growth plate of broiler chickens. There was no CS-56 labelling in the proliferative zone of the growth plate. In contrast, intense labelling with this antibody was observed in the transitional and hypertrophic zones of the growth plate and the articular cartilage. This was confirmed by extracting chondroitin sulphate fractions from different zones of the growth plate and articular cartilage, and examining their antigenicities to CS-56 by ELISA inhibition assay. It was suggested that the maturation of chondrocytes in the growth plate is related to the production of chondroitin sulphate with CS-56 epitope, which may be a prerequisite for normal endochondral bone formation in the chicken tibia. The role of chondroitin sulphate recognized by CS-56 in the articular cartilage is unknown.     

Chondroitin sulfate proteoglycan synthesis and reutilization of beta-D-xyloside-initiated chondroitin/dermatan sulfate glycosaminoglycans in fetal kidney branching morphogenesis

Branching morphogenesis and chondroitin sulfate proteoglycan synthesis by explanted fetal mouse kidneys were previously shown to be inhibited by p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside) while glomerular development and heparan sulfate proteoglycan synthesis were unaffected. The metabolic fate of fetal kidney explant proteoglycans was investigated to determine whether or not recovery of proteoglycan synthesis and morphogenesis occur after exposure to beta-D-xyloside. Chondroitin sulfate proteoglycan synthesis resumed within 4 hr of removal of beta-D-xyloside and was enhanced once beta-D-xyloside-initiated chondroitin/dermatan-35SO4 glycosaminoglycans (GAGs) were released from the tissue. Radioactivity incorporated into beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs during labeling in the presence of beta-D-xyloside was reutilized in the synthesis of chondroitin-35SO4 proteoglycan during a 24-hr chase in nonradioactive medium without beta-D-xyloside. Further, highly purified beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs were taken up by kidneys more avidly than was free [35S]sulfate. These 35S-GAGs were degraded and reutilized in the synthesis of chondroitin-35SO4 proteoglycan. Ureteric bud branching resumed 48 hr after beta-D-xyloside was removed from the incubation medium. These findings support the idea that both chondroitin sulfate proteoglycan synthesis and proteoglycan processing may be involved in branching morphogenesis.     

The length of chondroitin sulfate chains in normal and degenerative cartilage

Content of chondroitin sulphate, the number and average length of its chains are studied in isolated glycosamineglycanes from unchanged and degeneratively changed cartilage in men of different age. The chondroitin sulphate amount in the cartilage tissue is shown to decrease without change in the number of its chains with the age and with the development of the degenerative process in the cartilage. It occurs as a result of shortening of the average length of chondroitin sulphate chains which can be induced either during atypical chondrocyte synthesis of proteoglycanes or under destruction of the latter by enzymes.     

Polyethyleneimine as a contrast agent for ultrastructural localization and characterization of proteoglycans in the matrix of cartilage and bone.

We examined the presence of proteoglycans in the extracellular matrix of cartilage and bone in fetal mouse radii at the ultrastructural level, using the cationic dye polyethyleneimine (PEI). After staining with this dye, the proteoglycans appeared as granules in the uncalcified bone matrix and as extended winding structures in the cartilage matrix. PEI-positive material was removed after treatment of the tissue with chondroitinase ABC. Inhibition of the proteoglycan synthesis by beta-D-xyloside resulted in smaller PEI-positive windings in the cartilage matrix. These observations suggest that the winding, PEI-positive structures represent proteoglycan aggregates. No loss of PEI-positive material in the calcified cartilage matrix was seen, suggesting that proteoglycans do not need to be removed to make the matrix calcifiable.     

The ratio of protein and carbohydrate components of proteoglycans from normal and degenerative articular cartilage

The protein/uronic acid ratio in monomers and aggregates of proteoglycans in the human articular cartilage is investigated. It is shown that for the first two hours of cartilage extraction by isotonic solution proteoglycans with the low concentration of chondroitin sulphate are mainly removed; in the process of the subsequent extraction proteoglycans with a large amount of chondroitin sulphate; the quantity of chondroitin sulphate in the molecule does not effect the ability of proteoglycans to aggregation. The protein/uronic acid ratio increases in cartilage proteoglycans with aging and in the process of cartilage degeneration due to a decrease in the amount of the carbohydrate part of the molecule.     

Bone matrix studies. Influences of parathyroid extract, calcitonin, and cholecalciferol and of rickets and its treatment.

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by parathyroid extract. Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamin D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.     

Proteoglycan inhibition of calcium phosphate precipitation in liposomal suspensions.

The major proteoglycan in cartilage (aggrecan) is a complex macromolecule with numerous chondroitin sulphate, keratan sulphate, and oligosaccharide substituents. It has been proposed that this macromolecule has an important role in regulating mineralization in this tissue, a process which is initiated by the deposition of apatite in matrix vesicles. We have used a liposome-centred endogenous precipitation method as a model for matrix vesicle mineralization to study the effect of the rat chondrosarcoma aggrecan and its chondroitin sulphate and core protein components on apatite formation from solution. Precipitation was initiated by encapsulating buffered (pH 7.4) 50 mmol/l KH2PO4 solutions in the aqueous centres of 7:2:1 phosphatidylcholine:dicetylphosphate:cholesterol liposomes, adding 2.25-2.65 mmol/l Ca2+ and 1.5 mmol/l total inorganic phosphate (PO4) to the suspending medium (pH 7.4, 22 degrees C), then making the intervening lipid membranes permeable to the Ca2+ ions with the calcium ionophore X-537A. Aggrecan (0.5%) in the suspending medium had no effect on intraliposomal precipitation, but severely reduced (approximately 70% reduction at 24 h) its subsequent spread into the medium. The chondroitin sulphate and core protein were similarly inhibitory. The degree to which aggrecan and its constituent parts inhibited precipitation correlated with their capacity to bind Ca2+ ions. These findings suggest that functional groups in aggrecan blocked apatite growth by linking via Ca2+ bridges to growth sites on the crystal surfaces. Similar Ca-mediated interactions may well have a critical regulatory role in cartilage mineralization.     

Replacement of proteoglycans in embryonic chick cartilage in organ culture after treatment with testicular hyaluronidase

Explants of cartilage from tibiae of 11-12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.     

Bone matrix studies Influences of parathyroid extract,calcitonin, and cholecalciferol and of rickets and its treatment

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by paratyroid extract.Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamine D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.