scholarly journals Abnormal synthesis of cartilage-characteristic proteoglycan in azaserine-induced micromelial limbs

1989 ◽  
Vol 261 (2) ◽  
pp. 627-635 ◽  
Author(s):  
A Honda ◽  
I Tsuboi ◽  
K Kimata ◽  
Y Hirabayashi ◽  
K Yamada ◽  
...  
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Immunological Methods ◽  
Chick Embryos ◽  
In Ovo ◽  
Hind Limbs ◽  
Limb Morphogenesis ◽  
Indirect Immunofluorescence Technique ◽  
Chain Synthesis

Administration of azaserine (250 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs. In the present study, biosynthesis of cartilage-characteristic proteoglycan H (PG-H) as an index of limb chondrogenesis was examined in normal and micromelial hind limbs from day-7 chick embryos by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]-glucose and [35S]sulphate, followed by analysis of labelled proteoglycans by glycerol-density-gradient centrifugation under dissociative conditions, showed a marked reduction in PG-H synthesis. (2) PG-H synthesized by micromelial limbs differed from that synthesized by normal limbs in possessing a slower sedimenting velocity and much lower amounts of chondroitin sulphates. (3) The amount of PG-H core protein in micromelial limbs was significantly decreased to about 19% on a per limb basis and about 42% on a per DNA basis of that in normal limbs, as determined by e.l.i.s.a. (4) The transition from PG-M to PG-H during limb formation was retarded in micromelial limbs as judged by an indirect immunofluorescence technique using antibodies against PG-M and PG-H. (5) The deficiency of incorporation of labelled glucose into chondroitin sulphate chains of PG-H in micromelial limbs was partially restored by using [6-3H]-glucosamine as a precursor, suggesting that the synthesis of UDP-N-acetylhexosamine, required for chondroitin sulphate chain synthesis of PG-H in micromelial limbs, was decreased. These results suggest that the reduction in the synthesis of PG-H as well as the production of an abnormal form of PG-H during a critical period of limb morphogenesis may be important factors in explaining the micromelia induced by azaserine.

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.

Normal incorporation rates for precursors of collagen and mucopolysaccharide during expression of micromelia induced by 6-aminonicotinamide

Development ◽  
1974 ◽  
Vol 31 (2) ◽  
pp. 305-312
Author(s):  
Robert E. Seegmiller ◽  
Meredith N. Runner
Keyword(s):  
Core Protein ◽  
Limb Bud ◽  
Matrix Proteins ◽  
Synthesis Rate ◽  
Control Groups ◽  
In Ovo ◽  
Limb Buds ◽  
Radioactive Precursor ◽  
Hind Limbs ◽  
And Control

Further delineation of mechanisms by which 6-aminonicotinamide (6-AN) induces micromelia in the chick embryo was investigated by studies on rates of incorporation of thymidine, proline, glucosamine and sulfate as precursors to DNA, collagen and mucopolysaccharide, respectively. Twenty-four hours after in ovo administration of the vitamin antagonist, 6-AN, to day-4 chick embryos, hind limbs from experimental and control groups were excised and incubated for 1 h in medium containing 3 × 10−6m radioactive precursor. Molar incorporation of precursors into the TCA-precipitable fraction showed, in isolated limb buds, (a) that 6-AN enhanced incorporation of thymidine, (b) that 6-AN inhibited utilization of sulfate, and (c) that 6-AN did not significantly alter utilization of glucosamine and proline. Rates of incorporation of thymidine, glucosamine and proline indicate that 6-AN is not cytotoxic to the isolated limb bud. Enhanced incorporation of thymidine suggests expression of compensatory change 24 h after initial effects of 6-AN on DNA synthesis. Rate of incorporation of proline suggests that, under the influence of 6-AN, tropocollagen was synthesized in normal quantities by limb cells. Similarly, rate of incorporation of glucosamine suggests that under the influence of 6-AN normal amounts of hexosamine sugars were being attached to the nascent core-protein of chondroitin. Inhibition of sulfation and failure to complete the chondroitin sulfate molecule seem to account for 6-AN-induced micromelia. This suggests that sulfation depends upon specific NAD-dependent dehydrogenase reactions. As far as can be established by rates of incorporation of labeled precursors, 5-day limb buds, at 24 h after exposure to teratogenic levels of 6-AN, synthesize matrix proteins and hexosamine polysaccharides at normal rates.

scholarly journals Proteoglycans of the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc

1979 ◽  
Vol 179 (3) ◽  
pp. 573-578 ◽  
Author(s):  
R L Stevens ◽  
P G Dondi ◽  
H Muir
Keyword(s):  
Intervertebral Disc ◽  
Density Gradient ◽  
Core Protein ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Hydrodynamic Size ◽  
Laryngeal Cartilage ◽  
Cartilage Proteoglycans

Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.

scholarly journals β-d-xylosides and their analogues as artificial initiators of glycosaminoglycan chain synthesis. Aglycone-related variation in their effectiveness in vitro and in ovo

1987 ◽  
Vol 241 (2) ◽  
pp. 591-601 ◽  
Author(s):  
M Sobue ◽  
H Habuchi ◽  
K Ito ◽  
H Yonekura ◽  
K Oguri ◽  
...  
Keyword(s):  
Growth Rate ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Heparan Sulphate ◽  
Carbon Number ◽  
Average Chain Length ◽  
In Ovo ◽  
Ability To Act

A series of aryl and alkyl O-beta-D-xylosides and their analogues with S, NH or CH2 in the glycosidic linkage were prepared and examined for their ability to act as artificial chain initiators of chondroitin (dermatan) sulphate synthesis in embryonic chick cartilage, foetal rat skin and 6-week-old-rat aorta under conditions where normal protein-core synthesis was inhibited by cycloheximide. For all these tissues in culture, phenyl O-beta-D-xyloside and phenyl beta-D-thioxyloside were clearly more effective than the corresponding N-xyloside and homo-C-xyloside. Introduction of a carboxy group to the para position of their aglycone yielded derivatives with far lower initiator activity. In a concentration range lower than 0.1 mM, the effectiveness of alkyl beta-D-thioxyloside was greatly influenced by the carbon number (n) of the alkyl group and was at a maximum at n = 7 or 8 for the cartilage, at n = 5 for the skin and at n = 4 for the aorta. In the beta-xyloside-treated cartilages, the average length of newly formed chondroitin sulphate chains reflected the chain-initiator activity of added xyloside, i.e. the higher the initiator activity, the shorter the average chain length. In the skin and aorta, none of the drugs could relieve the inhibition of heparan sulphate synthesis caused by cycloheximide. Fertilized hens' eggs were each injected on day 9 with 9.2 mumol of beta-xyloside and the skeletal systems of embryos were examined a week later. The embryos treated with beta-xylosides of relatively high initiator activity showed a 30-40% decrease in the overall growth rate of skeletons, whereas those treated with beta-xylosides of low initiator activity showed little or no decrease in the growth rate. The results are consistent with the notion that the observed change in skeletal morphology results mainly, if not completely, from beta-xyloside-induced synthesis of core-protein-free chondroitin sulphate, and further suggest that a procedure employing a series of beta-xyloside homologues with various initiator activities will furnish an easily applied criterion on which to test the specificity of xyloside action on biological processes.

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

1982 ◽  
Vol 202 (2) ◽  
pp. 387-395 ◽  
Author(s):  
D Mitchell ◽  
T Hardingham
Keyword(s):  
Actinomycin D ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Chain Elongation ◽  
Chondrosarcoma Cell ◽  
Acceptor Concentration ◽  
The Core ◽  
Chain Synthesis ◽  
Cartilage Proteoglycans ◽  
Elongation Activity

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.

scholarly journals Absence of covalently linked core protein from newly synthesized hyaluronate

1982 ◽  
Vol 207 (3) ◽  
pp. 445-457 ◽  
Author(s):  
R M Mason ◽  
C d'Arville ◽  
J H Kimura ◽  
V C Hascall
Keyword(s):  
Core Protein ◽  
Cell Layer ◽  
Gradient Centrifugation ◽  
Primary Cultures ◽  
Guanidinium Chloride ◽  
Protein Pool ◽  
Cscl Density Gradient ◽  
Chain Synthesis ◽  
Covalently Linked ◽  
Zwitterionic Detergent

1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.

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

1984 ◽  
Vol 224 (3) ◽  
pp. 977-988 ◽  
Author(s):  
D J McQuillan ◽  
C J Handley ◽  
H C Robinson ◽  
K Ng ◽  
C Tzaicos ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Culture Medium ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Bovine Articular Cartilage ◽  
The Core ◽  
Elevated Rate ◽  
Chain Synthesis ◽  
Chondroitin Sulphate Chain ◽  
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.

Motor projection patterns to the hind limb of normal and paralysed chick embryos

Development ◽  
1982 ◽  
Vol 72 (1) ◽  
pp. 269-286
Author(s):  
N. G. Laing
Keyword(s):  
Spinal Cord ◽  
Cell Death ◽  
Hind Limb ◽  
Muscle Volume ◽  
Chick Embryos ◽  
Relative Contribution ◽  
Hind Limbs ◽  
Lateral Motor Column ◽  
Before And After ◽  
Major Period

Counts were made of the number of motoneurons innervating the hind limbs of 10-day normal and paralysed chick embryos whose right hind limb buds had been subjected to varying degrees of amputation prior to innervation. The number of motoneurons on the intact sides of the paralysed embryos was found to be similar to the number present in normal embryos prior to the major period of motoneuron death. Since it has previously been shown that paralysis does not increase the number of motoneurons generated, this means that normal motoneuron death was largely prevented in the paralysed embryos. There were differences in the distributions of motoneurons in the rostrocaudal axis of the spinal cord between normal and paralysed embryos. Therefore, cell death does not eliminate a uniform fraction of motoneurons throughout the rostrocaudal extent of the chick embryo lumbar lateral motor column. It is also argued that there are differences in the relative contribution of the various lumbosacral levels to different parts of the limb, e.g. the shank, before and after the period of cell death. In both normal and paralysed embryos there was a linear relationship between the volume of limb muscle which developed after amputation and the number of motoneurons surviving in the spinal cord. There was no evidence of a ‘compression’ of motoneurons into the remaining muscle either after amputation alone or after amputation combined with paralysis. Motoneurons are therefore rigidly specified for certain parts of the limb. The relationship between motoneuron number and muscle volume on the amputated side differed from that of the intact side. For a similar increase in muscle volume there was a smaller increase in motoneuron number on the intact sides. This suggested a parallel to the paradoxically small increase in motoneuron number that occurs on the addition of a supernumerary limb.

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.

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