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James W Fawcett - One of the best experts on this subject based on the ideXlab platform.
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in vitro modeling of perineuronal nets hyaluronan synthase and Link Protein are necessary for their formation and integrity
Journal of Neurochemistry, 2010Co-Authors: Jessica C F Kwok, Daniela Carulli, James W FawcettAbstract:We have previously shown that all perineuronal nets (PNNs) bearing neurons express a hyaluronan synthase (HAS), a Link Protein (usually cartilage Link Protein-1; Crtl1) and a chondroitin sulfate proteoglycan (usually aggrecan). Animal lacking Crtl1 in the CNS lacks normal PNNs. PNNs are implicated in the control of neuronal plasticity, and interventions to modulate PNN formation will be useful for manipulating plasticity. We have developed an in vitro model which demonstrates how the structural components of PNNs trigger their formation, using human embryonic kidney cells, which do not normally produce a pericellular matrix. Expression of HAS3 leads to the production of a diffuse matrix. It was converted into a compact PNN-like structure when the cells also expressed Crtl1 and aggrecan. This matrix was stained by Wisteria floribunda, contained Crtl1 and aggrecan, and like PNNs, could only be solubilized in 6 M urea. In the absence of hyaluronan produced by HAS3, aggrecan and Crtl1 dissipated into the medium, but when the cells were transfected to produce a hyaluronan matrix, Crtl1 and aggrecan were incorporated into it. Cells lacking any one of these molecules showed impaired integrity of the PNNs. Cells expressing HAS3 and Crtl1 were able to incorporate exogenous aggrecan into their pericellular matrix.
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animals lacking Link Protein have attenuated perineuronal nets and persistent plasticity
Brain, 2010Co-Authors: Daniela Carulli, Tibor T. Glant, Jessica C F Kwok, Tommaso Pizzorusso, Elena Putignano, Andrea Poli, Serhiy Forostyak, Melissa R Andrews, Sathyaseelan S Deepa, James W FawcettAbstract:Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core Protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage Link Protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.
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distribution and synthesis of extracellular matrix proteoglycans hyaluronan Link Proteins and tenascin r in the rat spinal cord
European Journal of Neuroscience, 2008Co-Authors: Clare M Galtrey, Jessica C F Kwok, Daniela Carulli, Kate E Rhodes, James W FawcettAbstract:Perineuronal nets (PNNs) are dense extracellular matrix (ECM) structures that form around many neuronal cell bodies and dendrites late in development. They contain several chondroitin sulphate proteoglycans (CSPGs), hyaluronan, Link Proteins and tenascin-R. Their time of appearance correlates with the ending of the critical period for plasticity, and they have been implicated in this process. The distribution of PNNs in the spinal cord was examined using Wisteria floribunda agglutinin lectin and staining for chondroitin sulphate stubs after chondroitinase digestion. Double labelling with the neuronal marker, NeuN, showed that PNNs were present surrounding approximately 30% of motoneurons in the ventral horn, 50% of large interneurons in the intermediate grey and 20% of neurons in the dorsal horn. These PNNs formed in the second week of postnatal development. Immunohistochemical staining demonstrated that the PNNs contain a mixture of CSPGs, hyaluronan, Link Proteins and tenascin-R. Of the CSPGs, aggrecan was present in all PNNs while neurocan, versican and phosphacan/RPTPbeta were present in some but not all PNNs. In situ hybridization showed that aggrecan and cartilage Link Protein (CRTL 1) and brain Link Protein-2 (BRAL 2) are produced by neurons. PNN-bearing neurons express hyaluronan synthase, and this enzyme and phosphacan/RPTPbeta may attach PNNs to the cell surface. During postnatal development the expression of Link Protein and aggrecan mRNA is up-regulated at the time of PNN formation, and these molecules may therefore trigger their formation.
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upregulation of aggrecan Link Protein 1 and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum
The Journal of Comparative Neurology, 2007Co-Authors: Daniela Carulli, Kate E Rhodes, James W FawcettAbstract:Extracellular matrix molecules accumulate around central nervous system neurons during postnatal development, forming so-called perineuronal nets (PNNs). PNNs play a role in restricting plasticity at the end of critical periods. In the adult rat cerebellum, PNNs are found around large, deep cerebellar nuclei (DCN) neurons and Golgi neurons and are composed of chondroitin sulfate proteoglycans (CSPGs), tenascin-R (TN-R), hyaluronan (HA), and Link Proteins, such as cartilage Link Protein 1 (Crtll). Granule cells and Purkinje cells are surrounded by a partially organized matrix. Both glial cells and neurons surrounded by PNNs are the site of synthesis of some CSPGs and of TN-R, but only neurons produce HA synthetic enzymes (HASs), thus HA, and Link Proteins, which are scaffolding molecules for an organized matrix. To elucidate the mechanisms of formation of PNNs, we analyzed by immunohistochemistry and in situ hybridization which PNN components are upregulated during PNN formation in rat cerebellar postnatal development and what cell types express them. We observed that Wisteria floribunda agglutinin-binding PNNs develop around DCN neurons from postnatal day (P)7 and around Golgi neurons from P14. At the same time as their PNNs start to form, these neurons upregulate aggrecan, Crtll, and HASs mRNAs. However, Crtll is the only PNN component to be expressed exclusively in neurons surrounded by PNNs. The other Link Protein that shows a perineuronal net pattern in the DCN, Bral2, is upregulated later during development. These data suggest that aggrecan, HA, and, particularly, Crtll might be crucial elements for the initial assembly of PNNs.
Yoshihiko Yamada - One of the best experts on this subject based on the ideXlab platform.
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analysis of a limb specific regulatory element in the promoter of the Link Protein gene
Biochemical and Biophysical Research Communications, 2019Co-Authors: Craig Rhodes, Tomoya Matsunobu, Yoshihiko YamadaAbstract:Abstract Link Protein is encoded by the Hapln1 gene and is a prototypical Protein found in the cartilage matrix. It acts as an important component of the endochondral skeleton during early development. To study its transcriptional regulation, promoter fragments derived from the Link Protein gene were coupled to the β-galactosidase reporter and used to study in vivo transgene expression in mice. In day 15.5 mouse embryos, a Link promoter fragment spanning −1020 to +40 nucleotides demonstrated highly specific β-galactosidase staining of skeletal structures, including the appendicular and axial cartilaginous tissues. Two shorter promoter fragments, spanning −690 to +40 and −315 to +40 nucleotides, demonstrated limb- and genitalia-specific expression resembling that of homeodomain-regulated tissues. Bioinformatic analysis revealed a highly conserved, Hox-like binding site (HLBS) at approximately −220 bp of the promoter, shared by both constructs, which contained the Hox-core consensus sequence TAATTA. Electromobility shift assays demonstrated binding of Hox-B4 recombinant Protein to the HLBS, which was eliminated with nucleotide substitutions within the core-binding element. Co-transfection analysis of the HLBS demonstrated a 22-fold transcriptional activation by HoxA9 expression, which was ablated with a substitution within the core HLBS element. Together these findings establish promoter regions within the Link Protein gene that are important for in vivo expression and identify the potential role of homeodomain-containing Proteins in controlling cartilage and limb gene expression.
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Chondrodysplasia of gene knockout mice for aggrecan and Link Protein.
Glycoconjugate Journal, 2002Co-Authors: Hideto Watanabe, Yoshihiko YamadaAbstract:The proteoglycan aggregate of the cartilage is composed of aggrecan, Link Protein, and hyaluronan and forms a unique gel-like moiety that provides resistance to compression in joints and a foundational cartilage structure critical for growth plate formation. Aggrecan, a large chondroitin sulfate proteoglycan, is one of the major structural macromolecules in cartilage and binds both hyaluronan and Link Protein through its N-terminal domain G1. Link Protein, a small glycoProtein, is homologous to the G1 domain of aggrecan. Mouse cartilage matrix deficiency (cmd) is caused by a functional null mutation of the aggrecan gene and is characterized by perinatal lethal dwarfism and craniofacial abnormalities. Link Protein knockout mice show chondrodysplasia similar to but milder than cmd mice, suggesting a supporting role of Link Protein for the aggregate structure. Analysis of these mice revealed that the proteoglycan aggregate plays an important role in cartilage development and maintenance of cartilage tissue and may provide a clue to the identification of human genetic disorders caused by mutations in these genes. Published in 2003.
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Chondrodysplasia of gene knockout mice for aggrecan and Link Protein.
Glycoconjugate journal, 2002Co-Authors: Hideto Watanabe, Yoshihiko YamadaAbstract:The proteoglycan aggregate of the cartilage is composed of aggrecan, Link Protein, and hyaluronan and forms a unique gel-like moiety that provides resistance to compression in joints and a foundational cartilage structure critical for growth plate formation. Aggrecan, a large chondroitin sulfate proteoglycan, is one of the major structural macromolecules in cartilage and binds both hyaluronan and Link Protein through its N-terminal domain G1. Link Protein, a small glycoProtein, is homologous to the G1 domain of aggrecan. Mouse cartilage matrix deficiency (cmd) is caused by a functional null mutation of the aggrecan gene and is characterized by perinatal lethal dwarfism and craniofacial abnormalities. Link Protein knockout mice show chondrodysplasia similar to but milder than cmd mice, suggesting a supporting role of Link Protein for the aggregate structure. Analysis of these mice revealed that the proteoglycan aggregate plays an important role in cartilage development and maintenance of cartilage tissue and may provide a clue to the identification of human genetic disorders caused by mutations in these genes.
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mice lacking Link Protein develop dwarfism and craniofacial abnormalities
Nature Genetics, 1999Co-Authors: Hideto Watanabe, Yoshihiko YamadaAbstract:Link Protein (LP), an extracellular matrix Protein in cartilage, stabilizes aggregates of aggrecan and hyaluronan, giving cartilage its tensile strength and elasticity. Cartilage provides the template for endochondral ossification and is crucial for determining the length and width of the skeleton. During endochondral bone formation, hypertrophic chondrocytes die and the cartilage is replaced with bone matrix. Here, we have generated targeted mutations in mice in the gene encoding LP (Crtl1). Homozygotes showed defects in cartilage development and delayed bone formation with short limbs and craniofacial anomalies. Most Crtl1(tm1Nid/tm1Nid) mice died shortly after birth due to respiratory failure, but some survived and developed progressive dwarfism and lordosis of the cervical spine. They showed small epiphysis, slightly flared metaphysis of long bones and flattened vertebrae, characteristic of spondyloepiphyseal dysplasias. The cartilage contained significantly reduced aggrecan depositions in the hypertrophic zone, and decreased numbers of prehypertrophic and hypertrophic chondrocytes. Reduced Indian hedgehog (Ihh) expression was observed in prehypertrophic chondrocytes, and apoptosis was inhibited in hypertrophic chondrocytes. These results indicate that LP is important for the formation of proteoglycan aggregates and normal organization of hypertrophic chondrocytes, and suggest that cartilage matrix has a role in chondrocyte differentiation and maturation.
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Characterization of a glucocorticoid responsive element and identification of an AT-rich element that regulate the Link Protein gene
Nucleic Acids Research, 1995Co-Authors: Craig Rhodes, Yoshihiko YamadaAbstract:The cartilage matrix is composed of characteristic components including type II collagen, aggrecan and Link Protein. In this paper, we report two DNA elements that regulate the Link Protein gene. Using transient transfection assays with Link Protein gene constructs in chondrocytes, chloramphenicol acetyl transferase (CAT) assays were used to measure the transcriptional activity of the Link Protein gene. Previously, we identified an enhancer-like activity within the first intron of the gene. In this paper, we report an active 34 bp (+1390 to +1424) fragment within this region that contains a glucocorticoid-like response element (GRE). Both deletion of, and site-specific mutations within this sequence motif reduced the dexamethasone-inducible activity. The GRE-like sequence from the rat Link Protein gene, or the homologous sequence from the human Link Protein gene were included in vectors containing the thymidine kinase promoter Linked to the CAT gene (tkCAT). Both human and rat elements transferred the ability to respond to dexamethasone and hydrocortisone with a > 10-fold induction. Deletions through the promoter from -923 to -900 identified a second site required for both glucocorticoid and serum responsiveness. A four base substitution at this site resulted in a loss of serum responsiveness. This region contains an AT-rich element, similar to the AT-rich elements involved in homeotic Protein regulation of the growth hormone gene and the muscle creatine kinase gene. Southwestern analysis using oligonucleotides containing the AT-rich element from the Link Protein gene or the muscle creatine kinase gene, identified a 32 kDa Protein band from nuclear extracts of chick chondrocytes. Using these AT-rich oligonucleotides in band-shift analyses, nuclear extracts of chick sternal muscle, rat chondrosarcoma and chick sternal chondrocytes each showed formation of different complexes suggesting cell specificity. AT-rich elements have been identified as binding sites for homeodomain-containing Proteins and can contribute to gene regulation by serum response factors. The identification of an AT-rich element in the Link Protein gene suggests similar functions for this element.
Daniela Carulli - One of the best experts on this subject based on the ideXlab platform.
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in vitro modeling of perineuronal nets hyaluronan synthase and Link Protein are necessary for their formation and integrity
Journal of Neurochemistry, 2010Co-Authors: Jessica C F Kwok, Daniela Carulli, James W FawcettAbstract:We have previously shown that all perineuronal nets (PNNs) bearing neurons express a hyaluronan synthase (HAS), a Link Protein (usually cartilage Link Protein-1; Crtl1) and a chondroitin sulfate proteoglycan (usually aggrecan). Animal lacking Crtl1 in the CNS lacks normal PNNs. PNNs are implicated in the control of neuronal plasticity, and interventions to modulate PNN formation will be useful for manipulating plasticity. We have developed an in vitro model which demonstrates how the structural components of PNNs trigger their formation, using human embryonic kidney cells, which do not normally produce a pericellular matrix. Expression of HAS3 leads to the production of a diffuse matrix. It was converted into a compact PNN-like structure when the cells also expressed Crtl1 and aggrecan. This matrix was stained by Wisteria floribunda, contained Crtl1 and aggrecan, and like PNNs, could only be solubilized in 6 M urea. In the absence of hyaluronan produced by HAS3, aggrecan and Crtl1 dissipated into the medium, but when the cells were transfected to produce a hyaluronan matrix, Crtl1 and aggrecan were incorporated into it. Cells lacking any one of these molecules showed impaired integrity of the PNNs. Cells expressing HAS3 and Crtl1 were able to incorporate exogenous aggrecan into their pericellular matrix.
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animals lacking Link Protein have attenuated perineuronal nets and persistent plasticity
Brain, 2010Co-Authors: Daniela Carulli, Tibor T. Glant, Jessica C F Kwok, Tommaso Pizzorusso, Elena Putignano, Andrea Poli, Serhiy Forostyak, Melissa R Andrews, Sathyaseelan S Deepa, James W FawcettAbstract:Chondroitin sulphate proteoglycans in the extracellular matrix restrict plasticity in the adult central nervous system and their digestion with chondroitinase reactivates plasticity. However the structures in the extracellular matrix that restrict plasticity are unknown. There are many changes in the extracellular matrix as critical periods for plasticity close, including changes in chondroitin sulphate proteoglycan core Protein levels, changes in glycosaminoglycan sulphation and the appearance of dense chondroitin sulphate proteoglycan-containing perineuronal nets around many neurons. We show that formation of perineuronal nets is triggered by neuronal production of cartilage Link Protein Crtl1 (Hapln1), which is up-regulated in the visual cortex as perineuronal nets form during development and after dark rearing. Mice lacking Crtl1 have attenuated perineuronal nets, but the overall levels of chondroitin sulphate proteoglycans and their pattern of glycan sulphation are unchanged. Crtl1 knockout animals retain juvenile levels of ocular dominance plasticity and their visual acuity remains sensitive to visual deprivation. In the sensory pathway, axons in knockout animals but not controls sprout into the party denervated cuneate nucleus. The organization of chondroitin sulphate proteoglycan into perineuronal nets is therefore the key event in the control of central nervous system plasticity by the extracellular matrix.
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distribution and synthesis of extracellular matrix proteoglycans hyaluronan Link Proteins and tenascin r in the rat spinal cord
European Journal of Neuroscience, 2008Co-Authors: Clare M Galtrey, Jessica C F Kwok, Daniela Carulli, Kate E Rhodes, James W FawcettAbstract:Perineuronal nets (PNNs) are dense extracellular matrix (ECM) structures that form around many neuronal cell bodies and dendrites late in development. They contain several chondroitin sulphate proteoglycans (CSPGs), hyaluronan, Link Proteins and tenascin-R. Their time of appearance correlates with the ending of the critical period for plasticity, and they have been implicated in this process. The distribution of PNNs in the spinal cord was examined using Wisteria floribunda agglutinin lectin and staining for chondroitin sulphate stubs after chondroitinase digestion. Double labelling with the neuronal marker, NeuN, showed that PNNs were present surrounding approximately 30% of motoneurons in the ventral horn, 50% of large interneurons in the intermediate grey and 20% of neurons in the dorsal horn. These PNNs formed in the second week of postnatal development. Immunohistochemical staining demonstrated that the PNNs contain a mixture of CSPGs, hyaluronan, Link Proteins and tenascin-R. Of the CSPGs, aggrecan was present in all PNNs while neurocan, versican and phosphacan/RPTPbeta were present in some but not all PNNs. In situ hybridization showed that aggrecan and cartilage Link Protein (CRTL 1) and brain Link Protein-2 (BRAL 2) are produced by neurons. PNN-bearing neurons express hyaluronan synthase, and this enzyme and phosphacan/RPTPbeta may attach PNNs to the cell surface. During postnatal development the expression of Link Protein and aggrecan mRNA is up-regulated at the time of PNN formation, and these molecules may therefore trigger their formation.
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upregulation of aggrecan Link Protein 1 and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum
The Journal of Comparative Neurology, 2007Co-Authors: Daniela Carulli, Kate E Rhodes, James W FawcettAbstract:Extracellular matrix molecules accumulate around central nervous system neurons during postnatal development, forming so-called perineuronal nets (PNNs). PNNs play a role in restricting plasticity at the end of critical periods. In the adult rat cerebellum, PNNs are found around large, deep cerebellar nuclei (DCN) neurons and Golgi neurons and are composed of chondroitin sulfate proteoglycans (CSPGs), tenascin-R (TN-R), hyaluronan (HA), and Link Proteins, such as cartilage Link Protein 1 (Crtll). Granule cells and Purkinje cells are surrounded by a partially organized matrix. Both glial cells and neurons surrounded by PNNs are the site of synthesis of some CSPGs and of TN-R, but only neurons produce HA synthetic enzymes (HASs), thus HA, and Link Proteins, which are scaffolding molecules for an organized matrix. To elucidate the mechanisms of formation of PNNs, we analyzed by immunohistochemistry and in situ hybridization which PNN components are upregulated during PNN formation in rat cerebellar postnatal development and what cell types express them. We observed that Wisteria floribunda agglutinin-binding PNNs develop around DCN neurons from postnatal day (P)7 and around Golgi neurons from P14. At the same time as their PNNs start to form, these neurons upregulate aggrecan, Crtll, and HASs mRNAs. However, Crtll is the only PNN component to be expressed exclusively in neurons surrounded by PNNs. The other Link Protein that shows a perineuronal net pattern in the DCN, Bral2, is upregulated later during development. These data suggest that aggrecan, HA, and, particularly, Crtll might be crucial elements for the initial assembly of PNNs.
Christopher J Handley - One of the best experts on this subject based on the ideXlab platform.
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metabolic processing of newly synthesized Link Protein in bovine articular cartilage explant cultures
Matrix Biology, 1999Co-Authors: Angus M Tester, Clem H Robinson, Mirna Z Ilic, Christopher J HandleyAbstract:Abstract In explant cultures of articular cartilage from cattle of different ages radiolabeled leucine was shown to be incorporated into Link Proteins 1, 2 and 3. The newly synthesized Link Proteins were incorporated into and lost from the cartilage extracellular matrix with time. The levels of radiolabeled Link Proteins 1 and 2 remaining in the matrix declined over the culture period, but there was an initial increase in the amount of radiolabeled Link Protein 3, before its level declined. The turnover time of the radiolabeled Link Proteins 1 and 2 were similar, indicating that neither Link Protein was preferentially processed to generate Link Protein 3, nor lost from the extracellular matrix. The majority of the radiolabeled Link Protein lost from the cartilage matrix could not be recovered from the culture medium, suggesting that turnover of the radiolabeled aggrecan complexes involves the newly synthesized Link Protein being internalized by the chondrocytes. Inclusion of cytotoxic Proteinase inhibitors to the culture medium resulted in a marked decrease in the rate of loss of Link Protein from the cartilage, suggesting that the catabolism of Link Protein is cell-mediated and dependent on metabolically active cells.
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effect of insulin like growth factor i on the synthesis and distribution of Link Protein and hyaluronan in explant cultures of articular cartilage
Biochimica et Biophysica Acta, 1992Co-Authors: Andrea J Curtis, Chee Keng Ng, Christopher J Handley, Clem H RobinsonAbstract:Abstract Addition of 20% (v/v) fetal calf serum or insulin-like growth factor-I (IGF-I; 20 ng/ml) to the medium of explant cultures of adult articular cartilage resulted in an increased rate of synthesis of the three components of the proteoglycan aggregate — namely Link Protein, hyaluronan and aggrecan. Fetal calf serum also stimulated the synthesis of other matrix Proteins by articular cartilage compared with tissue mantained in medium alone or medium containing IGF-I. Although addition of fetal calf serum or IGF-I to the culture medium of cartilage explant cultures stimulated both hyaluronan and aggrecan synthesis, no change in the distribution of these two macromolecules between tissue and medium was observed. Approx. 50% of the newly synthesized hyaluronan was retained by the tissue compared to 93% of the labelled aggrecan. Culture conditions had some influence on the distribution of Link Protein, in cultures maintained in medium alone or in medium containing IGF-I, less than 12% of the newly synthesized Link Protein was lost to the medium of the cultures. However, in cultures maintained with fetal calf serum between 25% and 19% of the radiolabelled Link Protein was lost from the matrix of the explants. This work suggests that the chondrocyte synthesizes the macromolecules that make up the proteoglycan aggregate in a co-ordinated manner, thereby retaining the relative amounts of each component of this functionally important complex.
Clem H Robinson - One of the best experts on this subject based on the ideXlab platform.
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metabolic processing of newly synthesized Link Protein in bovine articular cartilage explant cultures
Matrix Biology, 1999Co-Authors: Angus M Tester, Clem H Robinson, Mirna Z Ilic, Christopher J HandleyAbstract:Abstract In explant cultures of articular cartilage from cattle of different ages radiolabeled leucine was shown to be incorporated into Link Proteins 1, 2 and 3. The newly synthesized Link Proteins were incorporated into and lost from the cartilage extracellular matrix with time. The levels of radiolabeled Link Proteins 1 and 2 remaining in the matrix declined over the culture period, but there was an initial increase in the amount of radiolabeled Link Protein 3, before its level declined. The turnover time of the radiolabeled Link Proteins 1 and 2 were similar, indicating that neither Link Protein was preferentially processed to generate Link Protein 3, nor lost from the extracellular matrix. The majority of the radiolabeled Link Protein lost from the cartilage matrix could not be recovered from the culture medium, suggesting that turnover of the radiolabeled aggrecan complexes involves the newly synthesized Link Protein being internalized by the chondrocytes. Inclusion of cytotoxic Proteinase inhibitors to the culture medium resulted in a marked decrease in the rate of loss of Link Protein from the cartilage, suggesting that the catabolism of Link Protein is cell-mediated and dependent on metabolically active cells.
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effect of insulin like growth factor i on the synthesis and distribution of Link Protein and hyaluronan in explant cultures of articular cartilage
Biochimica et Biophysica Acta, 1992Co-Authors: Andrea J Curtis, Chee Keng Ng, Christopher J Handley, Clem H RobinsonAbstract:Abstract Addition of 20% (v/v) fetal calf serum or insulin-like growth factor-I (IGF-I; 20 ng/ml) to the medium of explant cultures of adult articular cartilage resulted in an increased rate of synthesis of the three components of the proteoglycan aggregate — namely Link Protein, hyaluronan and aggrecan. Fetal calf serum also stimulated the synthesis of other matrix Proteins by articular cartilage compared with tissue mantained in medium alone or medium containing IGF-I. Although addition of fetal calf serum or IGF-I to the culture medium of cartilage explant cultures stimulated both hyaluronan and aggrecan synthesis, no change in the distribution of these two macromolecules between tissue and medium was observed. Approx. 50% of the newly synthesized hyaluronan was retained by the tissue compared to 93% of the labelled aggrecan. Culture conditions had some influence on the distribution of Link Protein, in cultures maintained in medium alone or in medium containing IGF-I, less than 12% of the newly synthesized Link Protein was lost to the medium of the cultures. However, in cultures maintained with fetal calf serum between 25% and 19% of the radiolabelled Link Protein was lost from the matrix of the explants. This work suggests that the chondrocyte synthesizes the macromolecules that make up the proteoglycan aggregate in a co-ordinated manner, thereby retaining the relative amounts of each component of this functionally important complex.