Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage.
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ABSTRACT:
SUBMITTER: Vertel BM
PROVIDER: S-EPMC1153204 | biostudies-other | 1984 Jan
REPOSITORIES: biostudies-other
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Project description:1. Chondroitin sulphate was isolated from different regions of rat costal cartilage after extensive proteolysis of the tissues. The molecular weight, determined by gel chromatography, of the polysaccharide obtained from an actively growing region (lateral zone) near the osteochondral junction was higher than that of the polysaccharide isolated from the remaining portion of the costal cartilage (medial zone). 2. In both types of cartilage the molecular weight of chondroitin sulphate, labelled with [(35)S]sulphate, remained unchanged in vivo over a period of 10 days, approximately corresponding to the half-life of the chondroitin sulphate proteoglycan. The molecular-weight distribution of chondroitin [(35)S]sulphate, labelled in vivo or in vitro, was invariably identical with that of the bulk polysaccharide from the same tissue. It is concluded that the observed regional variations in molecular-weight distribution were established at the time of polysaccharide biosynthesis. 3. In tissue culture more than half of the (35)S-labelled polysaccharide-proteins of the two tissues was released into the medium within 10 days of incubation. The released materials were of smaller molecular size than were the corresponding native proteoglycans. In contrast, the molecular-weight distribution of the chondroitin [(35)S]sulphate (single polysaccharide chains) remained constant throughout the incubation period. 4. A portion (about 20%) of the total radioactive material released from (35)S-labelled cartilage in tissue culture was identified as inorganic [(35)S]sulphate. No corresponding decrease in the degree of sulphation of the labelled polysaccharide could be detected. These findings suggest that a limited fraction of the proteoglycan molecules had been extensively desulphated. 5. It is suggested that the initial phase of degradation involves proteolytic cleavage of the proteoglycan, but the constituent polysaccharide chains remain intact. The partially degraded proteoglycan may be eliminated from the cartilage by diffusion into the circulatory system. An additional degradative process, which may occur intracellularly, includes desulphation of the polysaccharide, probably in conjunction with a more extensive breakdown of the polymer.
| S-EPMC1174511 | biostudies-other
Project description:A biologically active, high-strength tissue adhesive is needed for numerous medical applications in tissue engineering and regenerative medicine. Integration of biomaterials or implants with surrounding native tissue is crucial for both immediate functionality and long-term performance of the tissue. Here, we use the biopolymer chondroitin sulphate (CS), one of the major components of cartilage extracellular matrix, to develop a novel bioadhesive that is readily applied and acts quickly. CS was chemically functionalized with methacrylate and aldehyde groups on the polysaccharide backbone to chemically bridge biomaterials and tissue proteins via a twofold covalent link. Three-dimensional hydrogels (with and without cells) bonded to articular cartilage defects. In in vitro and in vivo functional studies this approach led to mechanical stability of the hydrogel and tissue repair in cartilage defects.
| S-EPMC8128046 | biostudies-literature
Project description:Mounting evidence supports a key involvement of the chondroitin sulfate proteoglycans (CSPGs) NG2 and brevican (BCAN) in the regulation of axonal functions, including axon guidance, fasciculation, conductance, and myelination. Prior work suggested the possibility that these functions may, at least in part, be carried out by specialized CSPG structures surrounding axons, termed axonal coats. However, their existence remains controversial. We tested the hypothesis that NG2 and BCAN, known to be associated with oligodendrocyte precursor cells, form axonal coats enveloping myelinated axons in the human brain. In tissue blocks containing the mediodorsal thalamic nucleus (MD) from healthy donors (n = 5), we used dual immunofluorescence, confocal microscopy, and unbiased stereology to characterize BCAN and NG2 immunoreactive (IR) axonal coats and measure the percentage of myelinated axons associated with them. In a subset of donors (n = 3), we used electron microscopy to analyze the spatial relationship between axons and NG2- and BCAN-IR axonal coats within the human MD. Our results show that a substantial percentage (∼64%) of large and medium myelinated axons in the human MD are surrounded by NG2- and BCAN-IR axonal coats. Electron microscopy studies show NG2- and BCAN-IR axonal coats are interleaved with myelin sheets, with larger axons displaying greater association with axonal coats. These findings represent the first characterization of NG2 and BCAN axonal coats in the human brain. The large percentage of axons surrounded by CSPG coats, and the role of CSPGs in axonal guidance, fasciculation, conductance, and myelination suggest that these structures may contribute to several key axonal properties.
| S-EPMC9298528 | biostudies-literature
Project description:Chondroitin sulphate proteoglycan 4 (CSPG4) is a cell surface proteoglycan highly expressed by tumour, perivascular and oligodendrocyte cells and known to be involved cell adhesion and migration. This study showed that CSPG4 was present as a proteoglycan on the cell surface of two melanoma cell lines, MM200 and Me1007, as well as shed into the conditioned medium. CSPG4 from the two melanoma cell lines differed in the amount of chondroitin sulphate (CS) decoration, as well as the way the protein core was fragmented. In contrast, the CSPG4 expressed by a colon carcinoma cell line, WiDr, was predominantly as a protein core on the cell surface lacking glycosaminoglycan (GAG) chains. This study demonstrated that CSPG4 immunopurified from the melanoma cell lines formed a complex with perlecan synthesized by the same cultured cells. Mechanistic studies showed that CSPG4 bound to perlecan via hydrophobic protein-protein interactions involving multiple sites on perlecan including the C-terminal region. Furthermore, this study revealed that CSPG4 interacted with perlecan to support cell adhesion and actin polymerization. Together these data suggest a novel mechanism by which CSPG4 expressing cells might attach to perlecan-rich matrices so as those found in connective tissues and basement membranes.
| S-EPMC5905647 | biostudies-literature
Project description:Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.
| S-EPMC1163134 | biostudies-other
Project description:Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.
| S-EPMC7349878 | biostudies-literature
Project description:The excretion and degradation was studied of (35)S-labelled 4-chondroitin sulphate injected into guinea pigs in the form of proteoglycan isolated from cartilage and in the form of free chondroitin 4-sulphate prepared from the same proteoglycan by proteolysis. When the proteoglycan was injected there was a delay of about 15-20min before significant amounts or radioactivity were excreted, whereas after injection of chondroitin 4-sulphate a considerable amount of radioactivity was excreted within 10min and a much higher proportion of the radioactive dose was excreted in 1h or 24h compared with the proteoglycan. In both cases, however, a major part of the radioactivity was not excreted even in 24h. Sterile conditions were used to collect the radioactive material directly from the bladder. When chondroitin 4-sulphate was injected, the molecular sizes of injected and excreted materials were similar, as assessed by gel chromatography on Sephadex G-200, whereas when proteoglycan was injected the molecular size of the excreted labelled material was similar to that of the chondroitin 4-sulphate chains in the original proteoglycan. In neither case did the size of the excreted labelled material change with time over 1h, and low-molecular-weight labelled material was virtually absent. In contrast, when urine was collected for 24h without preservative the labelled material in it was extensively degraded after either the proteoglycan or chondroitin 4-sulphate had been given. Chondroitin 4-sulphate became similarly degraded when incubated with non-sterile urine, but not when the urine was passed through a bacterial filter, suggesting that degradation was caused by contaminating micro-organisms in the experiments in which urine was collected for 24 h. It is concluded that chondroitin 4-sulphate chains of about 18000 molecular weight can be excreted readily as such, whereas intact proteoglycans must be degraded to free glycosaminoglycans first, although both are taken up by the tissues more rapidly than they are excreted.
| S-EPMC1174440 | biostudies-other
Project description:Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.
| S-EPMC1135148 | biostudies-other
Project description:Nanostructured three-dimensional constructs combining layer-by-layer technology (LbL) and template leaching were processed and evaluated as possible support structures for cartilage tissue engineering. Multilayered constructs were formed by depositing the polyelectrolytes chitosan (CHT) and chondroitin sulphate (CS) on either bidimensional glass surfaces or 3D packet of paraffin spheres. 2D CHT/CS multi-layered constructs proved to support the attachment and proliferation of bovine chondrocytes (BCH). The technology was transposed to 3D level and CHT/CS multi-layered hierarchical scaffolds were retrieved after paraffin leaching. The obtained nanostructured 3D constructs had a high porosity and water uptake capacity of about 300%. Dynamical mechanical analysis (DMA) showed the viscoelastic nature of the scaffolds. Cellular tests were performed with the culture of BCH and multipotent bone marrow derived stromal cells (hMSCs) up to 21 days in chondrogenic differentiation media. Together with scanning electronic microscopy analysis, viability tests and DNA quantification, our results clearly showed that cells attached, proliferated and were metabolically active over the entire scaffold. Cartilaginous extracellular matrix (ECM) formation was further assessed and results showed that GAG secretion occurred indicating the maintenance of the chondrogenic phenotype and the chondrogenic differentiation of hMSCs.
| S-EPMC3577876 | biostudies-literature
Project description:The electrostatic interaction of chondroitin sulphate and the chondroitin sulphate-proteoglycan with collagen was studied by chromatography of the glycosaminoglycan and the proteoglycan on a collagen gel. The observed binding between the macromolecules increased with decreasing pH and ionic strength, and was significant under physiological conditions. A study of the interaction between chondroitin sulphate and a preparation of soluble collagen, with a partition-equilibrium technique, afforded similar results.
| S-EPMC1176559 | biostudies-other