scholarly journals Control of proteoglycan synthesis. Studies on the activation of synthesis observed during culture of articular cartilages

1980 ◽  
Vol 188 (1) ◽  
pp. 119-130 ◽  
Author(s):  
J D Sandy ◽  
H L Brown ◽  
D A Lowther
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Molecular Size ◽  
Fold Increase ◽  
Proteoglycan Synthesis ◽  
Adult Rabbit ◽  
Biosynthetic Activity ◽  
Dialysis Tubing ◽  
Glycosaminoglycan Synthesis ◽  
Fold Activation

When slices of adult rabbit articular cartilage were incubated in culture medium, the rate of incorporation of [35S]sulphate or [3H]acetate into glycosaminoglycans increased 4-8 fold during the first 5 days of incubation. Similar changes in biosynthetic activity were observed during culture of adult bovine cartilage. The activation of synthesis was not serum-dependent, but appeared to be a result of the depletion of tissue proteoglycan that occurs under these incubation conditions [Sandy, Brown & Lowther (1978) Biochim. Biophys. Acta 543, 536–544]. Thus, although complete activation was observed in serum-free medium, it was not observed if the cartilage was cultured inside dialysis tubing or in medium containing added proteoglycan subunit. The average molecular size of the proteoglycans synthesized by activated tissue was slightly larger than normal, as determined by chromatography on Sepharose CL-2B, and the average molecular size of the glycosaminoglycans synthesized by activated tissue was markedly increased over the normal. The increase in chain size was accompanied by an increase in the proportion of the chains degraded by chondroitinase ABC; these results are consistent with the preferential synthesis by activated chondrocytes of chondroitin sulphate-rich proteoglycans. The increase in glycosaminoglycan chain size was observed whether the chains were formed on endogenous core protein or on exogenous benzyl-beta-D-zyloside. An approximate 4-fold activation in culture of glycosaminoglycan synthesis on protein core was accompanied by a 1.54-fold increase in the rate of incorporation of [3H]serine into the chondroitin sulphate-linkage region of the proteoglycans. A 2.8-fold activation in culture of glycosaminoglycan synthesis on benzyl-beta-D-zyloside was accompanied by a 1.7-fold increase in the rate of incorporation of [3H]benzyl-beta-D-zyloside into glycosaminoglycans. The activation of glycosaminoglycan synthesis was, however, accompanied by no detectable change in the activity of xylosyltransferase (EC 2.4.2.26) in cell-free extracts. These results are discussed in relation to current ideas on the control of proteoglycan synthesis in cartilage.

scholarly journals Articular cartilage cultured with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules

1985 ◽  
Vol 227 (3) ◽  
pp. 869-878 ◽  
Author(s):  
J A Tyler
Keyword(s):  
Articular Cartilage ◽  
Enzyme Inhibitors ◽  
Core Protein ◽  
Average Length ◽  
Interleukin 1 ◽  
Fold Increase ◽  
Glycosaminoglycan Synthesis ◽  
Intracellular Degradation ◽  
Dose Dependent ◽  
Homogeneous Preparation

A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.

scholarly journals Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen

1984 ◽  
Vol 223 (2) ◽  
pp. 401-412 ◽  
Author(s):  
R M Mason ◽  
J D Lineham ◽  
M A Phillipson ◽  
C M Black
Keyword(s):  
Culture Medium ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Primary Cultures ◽  
Normal Subjects ◽  
Dermal Fibroblasts ◽  
Proteoglycan Synthesis ◽  
Dependent Manner ◽  
Glycoprotein Synthesis ◽  
Chondrocyte Cultures

Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.

scholarly journals Independent secretion of proteoglycans and collagens in chick chondrocyte cultures during acute ascorbic acid treatment

1990 ◽  
Vol 272 (1) ◽  
pp. 193-199 ◽  
Author(s):  
M Pacifici
Keyword(s):  
Acid Treatment ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Proteoglycan Synthesis ◽  
Collagen Types ◽  
Keratan Sulphate ◽  
Sulphate Proteoglycan ◽  
Chondrocyte Cultures ◽  
Chondroitin Sulphate Proteoglycan ◽  
Core Proteins

The mechanisms regulating the secretion of proteoglycans and collagens in chondrocytes, in particular those operating at the level of the rough endoplasmic reticulum (RER), are largely unknown. To examine these mechanisms, I studied the effects of acute ascorbate treatment on the secretion of two collagen types (types II and IX) and two proteoglycan types (PG-H and PG-Lb, the major keratan sulphate/chondroitin sulphate proteoglycan and the minor chondroitin sulphate proteoglycan respectively in cartilage) in scorbutic cultures of chick vertebral chondrocytes. I found that the scorbutic chondrocytes synthesized underhydroxylated precursors of types II and IX collagen that were secreted very slowly and accumulated in the RER. When the cultures were treated acutely with ascorbate, both macromolecules underwent hydroxylation within 1-1.5 h of treatment, and began to be secreted at normal high rates starting at about 2 h. Proteoglycan synthesis and secretion, however, remained largely unaffected by ascorbate treatment. Both the half-time of newly synthesized PG-H core protein in the RER and its conversion into completed proteoglycan were unchanged during treatment. Similarly, the overall rates of synthesis and secretion of both PG-H and PG-Lb remained at control levels during treatment. The data indicate that secretion of types II and IX collagen is regulated independently of secretion of PG-H and PG-Lb. This may be mediated by the ability of the RER of the chondrocyte to discriminate between procollagens and proteoglycan core proteins.

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

1987 ◽  
Vol 246 (3) ◽  
pp. 745-753
Author(s):  
A Honda ◽  
S Kazuno ◽  
Y Mori ◽  
K Kimata ◽  
S Suzuki
Keyword(s):  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Molecular Size ◽  
Immunological Methods ◽  
Proteoglycan Synthesis ◽  
In Ovo ◽  
Keratan Sulphate ◽  
Hind Limbs ◽  
Limb Morphogenesis ◽  
Significant Difference

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.

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

1986 ◽  
Vol 235 (2) ◽  
pp. 499-505 ◽  
Author(s):  
D J McQuillan ◽  
C J Handley ◽  
H C Robinson ◽  
K Ng ◽  
C Tzaicos
Keyword(s):  
Half Life ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Glycosaminoglycan Synthesis ◽  
First Order ◽  
The Core ◽  
First Order Kinetics ◽  
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).

scholarly journals Effects of cyclofenil diphenol, an agent which disrupts Golgi structure, on proteoglycan synthesis in chondrocytes

1992 ◽  
Vol 281 (2) ◽  
pp. 525-531 ◽  
Author(s):  
R M Mason ◽  
C A Lancaster
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Proteoglycan Synthesis ◽  
Specific Substrate ◽  
Inhibitory Mechanism ◽  
Similar Concentration ◽  
System A ◽  
Sds Page ◽  
Golgi Structure ◽  
Related Compounds

1. Cyclofenil diphenol (F6060), a weak non-steroidal oestrogen, was shown previously to inhibit [35S]proteoglycan synthesis [Mason, Lineham, Phillipson & Black (1984) Biochem. J. 223, 401-412] and to induce fragmentation of the Golgi apparatus into small vesicles [Lancaster, Fryer, Griffiths & Mason (1989) J. Cell Sci. 92, 271-280] in cultures of Swarm chondrosarcoma chondrocytes. Two structurally related compounds, F6204 and F6091, show a similar concentration-related effect, with complete inhibition of [35S]proteoglycan synthesis at 90 micrograms/ml. The apparent [3H]protein synthesis is only approx. 40% inhibited with [3H]lysine as precursor. Stilboestrol, clomiphene and tamoxiphen are also potent inhibitors of [35S]proteoglycan synthesis. 2. Syntheses of chondroitin 4-[35S]sulphate and chondroitin 6-[35S]sulphate, which are Golgi-mediated events, are inhibited 40-68% and 3-48% respectively by concentrations of cyclofenil between 50 and 70 micrograms/ml. [3H]Hyaluronan synthesis, which occurs by a different mechanism at the plasma membrane, is inhibited by 47-66%. These results suggest that cyclofenil may act via more than one inhibitory mechanism. Cyclofenil diphenol inhibits polymerization of chondroitin sulphate on to p-nitrophenyl beta-xyloside even when the chondrocytes are loaded with the initiator prior to treatment. 3. Cyclofenil diphenol interferes with the cellular uptake of amino acids via the system A carrier, as shown by inhibition of uptake of methylaminoisobutyric acid, a specific substrate for this system. The drug had no effect on the uptake of 2-deoxyglucose by the cells. 4. Cyclofenil diphenol (90 micrograms/ml) caused a decrease in the pool size of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-hexoses, but this was insufficient to account for the accompanying profound inhibition of [35S]proteoglycan synthesis. Entry of [3H]glucosamine into the cell and into the UDP-N-acetylhexosamine pool did not appear to be affected. 5. Cyclofenil diphenol inhibited the substitution of 3H-labelled proteoglycan core protein with chondroitin sulphate chains. Core protein was identified in treated cultures on the basis of immunoprecipitation with an antiserum against the hyaluronate-binding region and distinguished from precipitated proteoglycan on SDS/PAGE.

scholarly journals Low-Dose Fractionated Irradiation Selectivly Regulate CSPGs and PNNs, Promote Serotonergic Axonal Regeneration

2021 ◽  
Author(s):  
Qiang Zhang ◽  
Jiao Xu ◽  
Xiaoxiao Xiong ◽  
Bifeng Zhu ◽  
Bo Zhu ◽  
...  
Keyword(s):  
Axonal Regeneration ◽  
Low Dose ◽  
Axon Regeneration ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Canine Model ◽  
Inhibitory Effect ◽  
Fractionated Irradiation ◽  
Chondroitin Sulphate Proteoglycans ◽  
Cord Injury

Abstract Chondroitin sulphate proteoglycans (CSPGs) are major components to impeding axonal regeneration, condense in the extracellular-matrix to form perineuronal nets (PNNs) which interdigitate with axonal contacts. Each CSPG comprises a core protein with covalently attached chondroitin-sulfate glycosaminoglycan side chains (CS moieties). In the past, the representative treatment for CSPGs were chondroitinase-ABC which destroys all CS moieties. However, recent rodents researches found some CS moieties promote axon regeneration rather than inhibit axon regeneration. Using a canine model of spinal cord injury (SCI), which is a superior translational model for progressing rodent data into clinical practice, we showed that specific sulfation patterns of CS moieties play different role in modulation of axon re-growth. Upregulated CS-A expression occurred at 1-day post-SCI, earlier than CS-C expression which was increased at 14-days post-SCI. CS-A was mainly colocalized with astrocytes but CS-C was upregulated in both astrocytes and neurons/axons. Treatment with low-dose fractionated irradiation (LDI) significantly inhibited the expressions of astrocyte-associated CS-A and CS-A-enriched PNNs, but no inhibitory effect on CS-C and CS-C-enriched PNNs. There was a positive correlation between a reduction of CS-A-enriched PNNs and an increase of serotonergic (5-hydroxytryptamine, 5-HT) axonal sprouting. Increased serotonergic axon sprouting proximal to the lesion accompanied 5HT receptor up regulation following LDI treatment. Furthermore, LDI treatment promoted hindlimb motor function recovery following SCI. Taken together, our findings show that specific sulfation patterns of CS moieties and CSPG-enriched PNNs involved in carrying instructions for regulating axonal regeneration and that LDI treatment may be an efficacious strategy for treating SCI.

scholarly journals Proteoglycan and glycosaminoglycan synthesis in embryonic mouse salivary glands: effects of beta-D-xyloside, an inhibitor of branching morphogenesis.

1983 ◽  
Vol 96 (5) ◽  
pp. 1443-1450 ◽  
Author(s):  
H A Thompson ◽  
B S Spooner
Keyword(s):  
Salivary Glands ◽  
Dermatan Sulfate ◽  
Branching Morphogenesis ◽  
Proteoglycan Synthesis ◽  
Embryonic Mouse ◽  
Glycosaminoglycan Synthesis ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent ◽  
Stimulation Of

The proteoglycans and glycosaminoglycans synthesized by embryonic mouse salivary glands during normal morphogenesis and in the presence of beta-xyloside, an inhibitor of branching morphogenesis, have been partially characterized. Control and rho-nitrophenyl-beta-D-xyloside-treated salivary rudiments synthesize proteoglycans that are qualitatively similar, based on mobility on Sepharose CL-4B under dissociative conditions and glycosaminoglycan composition. However, beta-xyloside inhibits total proteoglycan-associated glycosaminoglycan synthesis by 50%, and also stimulates synthesis of large amounts of free chondroitin (dermatan) sulfate. This free glycosaminoglycan accounts for the threefold stimulation of total glycosaminoglycan synthesis in beta-xyloside-treated cultures. Several observations suggest that the disruption of proteoglycan synthesis rather than the presence of large amounts of free glycosaminoglycan is responsible for the inhibition of branching morphogenesis. (a) We have been unable to inhibit branching activity by adding large amounts of chondroitin (dermatan) sulfate, extracted from beta-xyloside-treated cultures, to the medium of salivary rudiments undergoing morphogenesis. (b) In the range of 0.1-0.4 mM beta-xyloside, the dose-dependent inhibition of branching morphogenesis is directly correlated with the inhibition of proteoglycan synthesis. The stimulation of free glycosaminoglycan synthesis is independent of dose in this range, since stimulation is maximal even at the lowest concentration used, 0.1 mM. The data strongly suggest that the inhibition of branching morphogenesis is caused by the disruption of proteoglycan synthesis in beta-xyloside-treated salivary glands.

scholarly journals Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase

2020 ◽  
Vol 69 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Brenna M. Zimmer ◽  
Joseph J. Barycki ◽  
Melanie A. Simpson
Keyword(s):  
Glucose Dehydrogenase ◽  
Sugar Metabolism ◽  
Structural Features ◽  
Proteoglycan Synthesis ◽  
Allosteric Modulators ◽  
Uridine Diphosphate ◽  
Cellular Mechanisms ◽  
Glycosaminoglycan Synthesis ◽  
Post Translational Modifications ◽  
Multiple Levels

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways:

scholarly journals Modulation of polyamine-biosynthetic activity by S-adenosylmethionine depletion

1988 ◽  
Vol 249 (2) ◽  
pp. 581-586 ◽  
Author(s):  
D L Kramer ◽  
J R Sufrin ◽  
C W Porter
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Enzyme Inhibitors ◽  
Fold Increase ◽  
Negative Control ◽  
Biosynthetic Activity ◽  
Drug Induced ◽  
Polyamine Biosynthesis ◽  
Sequence Characterization ◽  
Adomet Synthetase

The methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), was used to study the early effects of AdoMet depletion on polyamine biosynthesis. In the presence of decreased methionine (30 microM) in the medium, treatment of cultured L1210 cells with 1 mM-L-cisAMB resulted in a near-total (95%) depletion of cellular AdoMet pools by 4 h. This was accompanied by a 3-fold increase in ornithine decarboxylase (ODC) activity, a 2.5-fold increase in AdoMet decarboxylase (AdoMetDC) activity and a 20% decrease in spermidine and spermine pools. The increase in enzyme activities seemed to be partially due to prolongation of enzyme activity half-life, since that of ODC was extended from 30 to 50 min and that of AdoMetDC from 65 to 310 min. By temporal sequence characterization (0-4 h), the onset of elevations of enzyme activity (0.5-1 h) seemed to be causally related to an earlier (0-0.5 h) decline in AdoMet pools, as opposed to the 20% decrease in spermidine and spermine pools, which occurred much later (2-4 h); the latter are known to regulate decarboxylase activities negatively. Drug-induced elevations in ODC and, to a lesser extent, AdoMetDC activities were reversed by later treatment with exogenous AdoMet. However, because the latter also increased spermine pools (which could not be prevented with various enzyme inhibitors), the reversal of elevations in enzyme activities could not be directly linked to AdoMet. Although not definitive, the data raise the interesting possibility that, in addition to being negatively regulated by polyamines, ODC and AdoMetDC activities may also be subject to negative control by cellular AdoMet (or an AdoMet metabolite). The net effect of either or both of these influences would be to conserve polyamine-biosynthetic activity in the face of declining AdoMet supplies.

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