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Proteoglycans
Kinds of Proteoglycans Terms modified by Proteoglycans Selected AbstractsInfluence of interleukin-1, and hyaluronan on proteoglycan release from equine navicular hyaline cartilage and fibrocartilageJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 2 2000Frean Proteoglycan (PG) release, in response to recombinant human interleukin-1, (rh-IL-1,), was measured in cartilage explants obtained from the equine distal sesamoid bone (navicular bone). Fibrocartilage from the surface of the navicular bone apposing the deep digital flexor tendon and hyaline cartilage from the surface of the navicular bone articulating with the middle phalanx were labelled with 35SO4. Hyaline cartilage from the distal metacarpus was used as a control tissue. Following radiolabel incorporation, the three cartilage types were treated with rh-IL-1, (100 U/mL) in the presence of hyaluronan (0.2, 2, 20, 200 and 2000 ,g/mL). rh-IL-1,-Induced PG release was measured by scintillation assay of PG-bound radiolabel. Increases in PG release of 94% (P < 0.01), 101% (P < 0.05) and 122% (P < 0.05), in response to rh-IL-1,, were noted in fibrocartilage, navicular hyaline cartilage and metacarpal hyaline cartilage, respectively. Hyaluronan (0.2 ,g/mL) significantly reduced rh-IL-1,-induced PG release in metacarpal hyaline cartilage (P < 0.01). In fibrocartilage and navicular hyaline cartilage, hyaluronan did not reduce PG release and at some concentrations appeared to increase PG release, although this was not statistically significant. These experiments show that (i) fibrocartilage and hyaline cartilage of the navicular bone release PGs in response to rh-IL-1,, and (ii) hyaluronan does not prevent rh-IL-1,-induced breakdown of navicular bone cartilage. [source] In vivo qualitative assessments of articular cartilage in the rabbit knee with high-resolution MRI at 3 TMAGNETIC RESONANCE IN MEDICINE, Issue 3 2003Didier Laurent Abstract Proteoglycan (PG) loss and disruption of the collagen framework in cartilage are early events associated with osteoarthritis (OA). The feasibility of in vivo high-resolution MRI assessments probing both macromolecules was explored in articular cartilage of the rabbit knee. One-millimeter thick coronal images were obtained at 3 T with a 97 × 97 ,m2 pixel size. A 22% decrease in the magnetization transfer (MT) exchange rate along with an ,2-fold greater Gd(DTPA)2- -induced decrease in T1 relaxation time were measured in response to papain injection 1 day prior to the MRI session, indicative of an alteration of collagen integrity and PG depletion, respectively. A two-point method was tested as an alternative to the more time-consuming multipoint method typically used to measure T1 changes. Kinetics of Gd(DTPA)2- uptake were observed with a 10-min time resolution. The diffusive transport of Gd(DTPA)2- was characterized by a T1 decrease ,2-fold faster in papain-treated knees. These data suggest that kinetics of tracer diffusion may be used as an informative marker of PG loss, in addition to the amplitude of T1 variations. When applied to a relevant OA model, the combination of MT and Gd(DTPA)2- -MRI may help in identifying new active compounds during efficacy studies on cartilage protection. Magn Reson Med 50:541,549, 2003. © 2003 Wiley-Liss, Inc. [source] Proteoglycan-induced changes in T1, -relaxation of articular cartilage at 4TMAGNETIC RESONANCE IN MEDICINE, Issue 3 2001Sarma V.S. Akella Abstract Proteoglycan (PG) depletion-induced changes in T1, (spin-lattice relaxation in rotating frame) relaxation and dispersion in articular cartilage were studied at 4T. Using a spin-lock cluster pre-encoded fast spin echo sequence, T1, maps of healthy bovine specimens and specimens that were subjected to PG depletion were computed at varying spin-lock frequencies. Sequential PG depletion was induced by trypsinization of cartilage for varying amounts of time. Results demonstrated that over 50% depletion of PG from bovine articular cartilage resulted in average T1, increases from 110,170 ms. Regression analysis of the data showed a strong correlation (R2 = 0.987) between changes in PG and T1,. T1, values were highest at the superficial zone and decreased gradually in the middle zone and again showed an increasing trend in the region near the subchondral bone. The potentials of this method in detecting early degenerative changes of cartilage are discussed. Also, T1, -dispersion changes as a function of PG depletion are described. Magn Reson Med 46:419,423, 2001. © 2001 Wiley-Liss, Inc. [source] Repulsive guidance of axons of spinal sensory neurons in Xenopus laevis embryos: Roles of Contactin and notochord-derived chondroitin sulfate proteoglycansDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2005Naoko Fujita An immunoglobulin superfamily neuronal adhesion molecule, Contactin, has been implicated in axon guidance of spinal sensory neurons in Xenopus embryos. To identify the guidance signaling molecules that Contactin recognizes in tailbud embryos, an in situ binding assay was performed using recombinant Contactin-alkaline phosphatase fusion protein (Contactin-AP) as a probe. In the assay of whole-mount or sectioned embryos, Contactin-AP specifically bound to the notochord and its proximal regions. This binding was completely blocked by either digestion of embryo sections with chondroitinase ABC or pretreatment of Contactin-AP with chondroitin sulfate A. When the spinal cord and the notochord explants were co-cultured in collagen gel, growing Contactin-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Contactin antibody, chondroitin sulfate A or chondroitinase ABC to the culture medium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gel matrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Contactin on spinal sensory axons. [source] Cloning and characterization of cDNA for syndecan core protein in sea urchin embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2000Kazuo Tomita The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription,polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae. [source] Extracellular interactome of the FGF receptor,ligand system: Complexities and the relative simplicity of the wormDEVELOPMENTAL DYNAMICS, Issue 2 2009Urszula M. Polanska Abstract Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate a multitude of biological functions in embryonic development and in adult. A major question is how does one family of growth factors and their receptors control such a variety of functions? Classically, specificity was thought to be imparted by alternative splicing of the FGFRs, resulting in isoforms that bind specifically to a subset of the FGFs, and by different saccharide sequences in the heparan sulfate proteoglycan (HSPG) co-receptor. A growing number of noncanonical co-receptors such as integrins and neural cell adhesion molecule (NCAM) are now recognized as imparting additional complexity to classic FGFR signaling. This review will discuss the noncanonical FGFR ligands and speculate on the possibility that they provide additional and alternative means to determining the functional specificity of FGFR signaling. We will also discuss how invertebrate models such as C. elegans may advance our understanding of noncanonical FGFR signaling. Developmental Dynamics 238:277,293, 2009. © 2008 Wiley-Liss, Inc. [source] NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesisDEVELOPMENTAL DYNAMICS, Issue 2 2001Ugur Ozerdem Abstract Immunofluorescence mapping demonstrates that the NG2 proteoglycan is invariably expressed by the mural cell component of mouse neovascular structures. This pattern is independent of the developmental mechanism responsible for formation of the vasculature (vasculogenesis or angiogenesis). Thus, NG2 is expressed in the embryonic heart by cardiomyocytes, in developing macrovasculature by smooth muscle cells, and in nascent microvessels by vascular pericytes. Due to the scarcity of proven markers for developing pericytes, NG2 is especially useful for identification of this cell type. The utility of NG2 as a pericyte marker is illustrated by two observations. First, pericytes are associated with endothelial tubes at an early point in microvessel development. This early interaction between pericytes and endothelial cells has important implications for the role of pericytes in the development and stabilization of microvascular tubes. Second, the pericyte to endothelial cell ratio in developing capillaries varies from tissue to tissue. Because the extent of pericyte investment is likely to affect the physical properties of the vessel in question, it is important to understand the mechanisms that control this process. Additional insight into these and other aspects of vascular morphogenesis should be possible through use of NG2 as a mural cell marker. © 2001 Wiley-Liss, Inc. [source] Retina development in zebrafish requires the heparan sulfate proteoglycan agrinDEVELOPMENTAL NEUROBIOLOGY, Issue 7 2008I-Hsuan Liu Abstract Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Tenascin-R and axon growth-promoting molecules are up-regulated in the regenerating visual pathway of the lizard (Gallotia galloti)DEVELOPMENTAL NEUROBIOLOGY, Issue 7 2008Dirk M. Lang Abstract It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down-regulation of axon growth-inhibitory proteins, and to what extent outgrowth-promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth-inhibiting extracellular matrix molecules tenascin-R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth-promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin-R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin-R and CSPG were expressed in an extracellular matrix-, oligodendrocyte/myelin- and neuron-associated pattern and up-regulated in the regenerating optic pathway. The expression pattern of tenascin-R was not indicative of a role in channeling or restriction of re-growing RGC axons. Up-regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin-R expression. Moreover, we analyzed the influence of substrates containing tenascin-R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin-R, and further improved on mixed substrates containing tenascin-R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down-regulation of tenascin-R or CSPG. Presence of tenascin-R is insufficient to prevent RGC axon growth, and concomitant up-regulation of axon growth-promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up-regulation of contactin in RGCs suggests that tenascin-R may have an instructive function during axon regeneration in the lizard optic pathway. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2008 [source] HGF induction of postsynaptic specializations at the neuromuscular junctionDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2006Raghavan Madhavan Abstract A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005 [source] Heparan sulfate proteoglycans in experimental models of diabetes: a role for perlecan in diabetes complicationsDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 6 2001Karin Conde-Knape Abstract Proteoglycans are ubiquitous extracellular proteins that serve a variety of functions throughout the organism. Unlike other glycoproteins, proteoglycans are classified based on the structure of the glycosaminoglycan carbohydrate chains, not the core proteins. Perlecan, a member of the heparan sulfate proteoglycan (HSPG) family, has been implicated in many complications of diabetes. Decreased levels of perlecan have been observed in the kidney and in other organs, both in patients with diabetes and in animal models. Perlecan has an important role in the maintenance of the glomerular filtration barrier. Decreased perlecan in the glomerular basement membrane has a central role in the development of diabetic albuminuria. The involvement of this proteoglycan in diabetic complications and the possible mechanisms underlying such a role have been addressed using a variety of models. Due to the importance of nephropathy among diabetic patients most of the studies conducted so far relate to diabetes effects on perlecan in different types of kidney cells. The various diabetic models used have provided information on some of the mechanisms underlying perlecan's role in diabetes as well as on possible factors affecting its regulation. However, many other aspects of perlecan metabolism still await full elucidation. The present review provides a description of the models that have been used to study HSPG and in particular perlecan metabolism in diabetes and some of the factors that have been found to be important in the regulation of perlecan. Copyright © 2001 John Wiley & Sons, Ltd. [source] A recombinant bispecific single-chain antibody induces targeted, supra-agonistic CD28-stimulation and tumor cell killingEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2003Ludger Grosse-Hovest Abstract Endowing tumor cells with costimulatory signals for T cell activation has emerged as a promising strategy for tumor immunotherapy. Costimulatory molecules were either transfected into tumor cells to generate vaccines or were fused, e.g. to antibodies against tumor-associated antigens, to achieve targeted T cell costimulation in vivo. Here we report the production and purification of rM28, a recombinant bispecific single-chain antibody directed to a melanoma-associated proteoglycan and to the costimulatory CD28 molecule on human T cells. We found that a dimer of the recombinant molecule, bound to tumor target cells, induced pronounced T cell activation in peripheral blood mononuclear cell preparations without additional TCR/CD3 stimulation being required. Thelytic activity generated after 3,days of stimulation effectively prevented tumor cell growth. However, it was unspecific and predominantly mediated by non T cells. Our findings demonstrate that presentation of a CD28 antibody within a suitable recombinant, bispecific format may result in a "targeted supra-agonistic stimulation" of the CD28 molecule, which leads to effective tumor cell killing after induction of unspecifically lytic cells. [source] Maintenance of the relative proportion of oligodendrocytes to axons even in the absence of BAX and BAKEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2009Kumi Kawai Abstract Highly purified oligodendroglial lineage cells from mice lacking functional bax and bak genes were resistant to apoptosis after in-vitro differentiation, indicating an essential role of the intrinsic apoptotic pathway in apoptosis of oligodendrocytes in the absence of neurons (axons) and other glial cells. These mice therefore provide a valuable tool with which to evaluate the significance of the intrinsic apoptotic pathway in regulating the population sizes of oligodendrocytes and oligodendroglial progenitor cells. Quantitative analysis of the optic nerves and the dorsal columns of the spinal cord revealed that the absolute numbers of mature oligodendrocytes immunolabeled for aspartoacylase and adult glial progenitor cells expressing NG2 chondroitin sulfate proteoglycan were increased in both white matter tracts of adult bax/bak -deficient mice and, to a lesser extent, bax -deficient mice, except that there was no increase in NG2-positive progenitor cells in the dorsal columns of these strains of mutant mice. These increases in mature oligodendrocytes and progenitor cells in bax/bak -deficient mice were unexpectedly proportional to increases in numbers of axons in these white matter tracts, thus retaining the oligodendroglial lineage to axon ratios of at most 1.3-fold of the physiological numbers. This is in contrast to the prominent expansion in numbers of neural precursor cells in the subventricular zones of these adult mutant mice. Our study indicates that homeostatic control of cell number is different for progenitors of the oligodendroglial and neuronal lineages. Furthermore, regulatory mechanism(s) operating in addition to apoptotic elimination through the intrinsic pathway, appear to prevent the overproduction of highly mitotic oligodendroglial progenitor cells. [source] Effects of agrin on the expression and distribution of the water channel protein aquaporin-4 and volume regulation in cultured astrocytesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2007Susan Noell Abstract Agrin is a heparan sulfate proteoglycan of the extracellular matrix and is known for organizing the postsynaptic differentiation of the neuromuscular junction. Increasing evidence also suggests roles for agrin in the developing CNS, including the formation and maintenance of the blood,brain barrier. Here we describe effects of agrin on the expression and distribution of the water channel protein aquaporin-4 (AQP4) and on the swelling capacity of cultured astrocytes of newborn mice. If astrocytes were cultured on a substrate containing poly dl -ornithine, anti-AQP4 immunoreactivity was evenly and diffusely distributed. If, however, astrocytes were cultured in the presence of agrin-conditioned medium, we observed an increase in the intensity of AQP4-specific membrane-associated staining. Freeze-fracture studies revealed a clustering of orthogonal arrays of particles, representing a structural equivalent of AQP4, when exogenous agrin was present in the astrocyte cultures. Neuronal and non-neuronal agrin isoforms (agrin A0B0 and agrin A4B8, respectively) were able to induce membrane-associated AQP4 staining. Water transport capacity as well as the density of orthogonal arrays of intramembranous particles was increased in astrocytes cultured with the neuronal agrin isoform A4B8, but not with the endothelial and meningeal isoform A0B0. RT-PCR demonstrated that agrin A4B8 increased the level of the M23 splice variant of AQP4 and decreased the level of the M1 splice variant of AQP4. Implications for the regulation and maintenance of the blood,brain barrier including oedema formation under pathological conditions are discussed. [source] Aberrant trajectory of thalamocortical axons associated with abnormal localization of neurocan immunoreactivity in the cerebral neocortex of reeler mutant miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2005Hong-Peng Li Abstract We examined the molecular mechanisms underlying the formation of the thalamocortical pathway in the cerebral neocortex of normal and reeler mutant mice. During normal development of the mouse neocortex, thalamic axons immunoreactive for the neural cell adhesion molecule L1 rarely invaded the cortical plate and ran centered in the subplate which is immunoreactive for neurocan, a brain-specific chondroitin sulfate proteoglycan. On the other hand, in homozygous reeler mutant mice, thalamic axons took an aberrant course to run obliquely through the cortical plate. Injection of bromodeoxyuridine at embryonic day 11 specifically labeled subplate neurons in normal mice, whilst in the reeler neocortex it labeled cells scattered in the cortical plate as well as in the superficial layer (superplate). Neurocan immunoreactivity was associated with the bromodeoxyuridine-positive cells in the superplate, as well as being present in oblique bands within the cortical plate, along which L1-bearing thalamic axons preferentially ran. The present results support our previous hypothesis proposed for normal rats that a heterophilic molecular interaction between L1 and neurocan is involved in determining the thalamocortical pathway within the neocortical anlage [T. Fukuda et al. (1997)Journal of Comparative Neurology, 382, 141,152]. [source] A disaccharide derived from chondroitin sulphate proteoglycan promotes central nervous system repair in rats and mice,EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2004Asya Rolls Abstract Chondroitin sulphate proteoglycan (CSPG) inhibits axonal regeneration in the central nervous system (CNS) and its local degradation promotes repair. We postulated that the enzymatic degradation of CSPG generates reparative products. Here we show that an enzymatic degradation product of CSPG, a specific disaccharide (CSPG-DS), promoted CNS recovery by modulating both neuronal and microglial behaviour. In neurons, acting via a mechanism that involves the PKC, and PYK2 intracellular signalling pathways, CSPG-DS induced neurite outgrowth and protected against neuronal toxicity and axonal collapse in vitro. In microglia, via a mechanism that involves ERK1/2 and PYK2, CSPG-DS evoked a response that allowed these cells to manifest a neuroprotective phenotype ex vivo. In vivo, systemically or locally injected CSPG-DS protected neurons in mice subjected to glutamate or aggregated ,-amyloid intoxication. Our results suggest that treatment with CSPG-DS might provide a way to promote post-traumatic recovery, via multiple cellular targets. [source] Dynamic changes in glypican-1 expression in dorsal root ganglion neurons after peripheral and central axonal injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2004Stefan Bloechlinger Abstract Glypican-1, a glycosyl phosphatidyl inositol (GPI)-anchored heparan sulphate proteoglycan expressed in the developing and mature cells of the central nervous system, acts as a coreceptor for diverse ligands, including slit axonal guidance proteins, fibroblast growth factors and laminin. We have examined its expression in primary sensory dorsal root ganglion (DRG) neurons and spinal cord after axonal injury. In noninjured rats, glypican-1 mRNA and protein are constitutively expressed at low levels in lumbar DRGs. Sciatic nerve transection results in a two-fold increase in mRNA and protein expression. High glypican-1 expression persists until the injured axons reinnervate their peripheral targets, as in the case of a crushed nerve. Injury to the central axons of DRG neurons by either a dorsal column injury or a dorsal root transection also up-regulates glypican-1, a feature that differs from most DRG axonal injury-induced genes, whose regulation changes only after peripheral and not central axonal injury. After axonal injury, the cellular localization of glypican-1 changes from a nuclear pattern restricted to neurons in noninjured DRGs, to the cytoplasm and membrane of injured neurons, as well as neighbouring non-neuronal cells. Sciatic nerve transection also leads to an accumulation of glypican-1 in the proximal nerve segment of injured axons. Glypican-1 is coexpressed with robo 2 and its up-regulation after axonal injury may contribute to an altered sensitivity to axonal growth or guidance cues. [source] Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cellsFEBS JOURNAL, Issue 8 2007Patryk Krzemi We characterized the expression and functional properties of the ADP-sensitive P2Y1 and P2Y12 nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y12 receptor relative to P2Y1 was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y1 receptor was low, and the P2Y12 receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y12 receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y12 receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y1 receptor, indicating the inhibitory role of P2Y1 in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y1 to P2Y12 would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation. [source] Secretion of proteases in serglycin transfected Madin,Darby canine kidney cellsFEBS JOURNAL, Issue 3 2006Lillian Zernichow Madin,Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules. [source] Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin LFEBS JOURNAL, Issue 19 2003Jeffrey P. Bocock Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine protease, cathepsin L, with a Ki of 0.7 nm, but it does not inhibit the structurally related lysosomal cysteine protease cathepsin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu. [source] Methylprednisolone inhibits the expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycans in reactivated astrocytesGLIA, Issue 13 2008Wei-Lin Liu Abstract Reactive gliosis caused by post-traumatic injury often results in marked expression of chondroitin sulfate proteoglycan (CSPG), which inhibits neurite outgrowth and regeneration. Methylprednisolone (MP), a synthetic glucocorticoid, has been shown to have neuroprotective and anti-inflammatory effects for the treatment of acute spinal cord injury (SCI). However, the effect of MP on CSPG expression in reactive glial cells remains unclear. In our study, we induced astrocyte reactivation using ,-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and cyclothiazide to mimic the excitotoxic stimuli of SCI. The expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivation, and CSPG neurocan and phosphacan were significantly elevated by AMPA treatment. The conditioned media from AMPA-treated astrocytes strongly inhibited neurite outgrowth of rat dorsal root ganglion neurons, and this effect was reversed by pretreatment with MP. Furthermore, MP downregulated GFAP and CSPG expression in adult rats with SCI. Additionally, both the glucocorticoid receptor (GR) antagonist RU486 and GR siRNA reversed the inhibitory effects of MP on GFAP and neurocan expression. Taken together, these results suggest that MP may improve neuronal repair and promote neurite outgrowth after excitotoxic insult via GR-mediated downregulation of astrocyte reactivation and inhibition of CSPG expression. © 2008 Wiley-Liss, Inc. [source] Regulation of human neutrophil-mediated cartilage proteoglycan degradation by phosphatidylinositol-3-kinaseIMMUNOLOGY, Issue 1 2001C. S. T. Hii Summary The ability of neutrophils to degrade cartilage proteoglycan suggests that the neutrophils that accumulate in the joints of rheumatoid arthritis patients are mediators of tissue damage. The regulatory mechanisms which are relevant to the proteoglycan-degrading activity of neutrophils are poorly understood. Since phosphatidylinositol 3-kinase (PI3-K), protein kinase C (PKC), the extracellular signal-regulated protein kinase (ERK)1/ERK2 and cyclic adenosine monophosphate (cAMP) have been reported to regulate neutrophil respiratory burst and/or degranulation, a role for these signalling molecules in regulating proteoglycan degradation was investigated. Preincubation of human neutrophils with GF109203X (an inhibitor of PKC), PD98059 (an inhibitor of MEK, the upstream regulator of ERK1/ERK2) or with forskolin or dibutyryl cAMP, failed to suppress proteoglycan degradation of opsonized bovine cartilage. In contrast, preincubation of neutrophils with wortmannin or LY294002, specific inhibitors of PI3-K, inhibited proteoglycan degradation. Incubation of neutrophils with cartilage resulted in the activation of PI3-K in neutrophils, consistent with a role for PI3-K in proteoglycan degradation. Activation of PI3-K and proteoglycan degradation was enhanced by tumour necrosis factor-,. Degradation caused by neutrophils from the synovial fluid of rheumatoid arthritis patients was also inhibited by wortmannin. These data demonstrate that the proteoglycan degradative activity of neutrophils required PI3-K but not PKC or the ERK1/ERK2/ERK5 cascades and was insensitive to increases in intracellular cAMP concentrations. [source] Purification and characterization of endocan (endothelial cell-specific molecule-1), a circulating proteoglycan involved in tumour progression and inflammatory diseasesINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Stéphane Sarrazin Introduction By virtue of the multiplicity of their protein-binding partners (e.g. growth factors, cytokines/chemokines), proteoglycans have been shown to be involved in the regulation of a large number of pathophysiological processes including cancer and inflammatory diseases. We have studied and characterized endocan, also called endothelial cell-specific molecule-1 (ESM-1), which represents a new group of circulating proteoglycans. Endocan is mainly expressed by endothelial cells but also by epithelial cells from lung, gut and kidney. Structurally, endocan is constituted of a mature polypeptide of 165 amino acids with a single glycosaminoglycan chain covalently linked to the serine at position 137 (Béchard et al. 2001). Methods and results We showed that human umbilical vein endothelial cells expressed endocan specifically with a single chain of dermatan sulfate (DS) as glycosaminoglycan moiety. As shown by surface plasmon resonance, the DS chain directly interacts with cytokines and growth factors including hepatocyte growth factor/scatter factor and could be responsible for endocan's biological activities. Human embryonic kidney 293 cells, which have been genetically engineered to overexpress endocan, induce tumour growth when injected subcutaneously in SCID mice. Moreover, inflammatory cytokines such as TNF-a and IL-1 have been shown to increase the synthesis and the secretion of endocan from human umbilical vein endothelial cells. Conclusion These results suggest that circulating levels of endocan may represent a novel marker for cancer and inflammatory diseases. Further studies on its GAG structure could help us to better understand the biological activities of endocan and to design future glycomic-based therapies. [source] Soluble syndecan-1 (sCD138) as a prognostic factor independent of mutation status in patients with chronic lymphocytic leukemiaINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 1 2009I. JILANI Summary Syndecan-1 (sCD138) is a transmembrane heparan sulfate-bearing proteoglycan expressed in epithelial cells as well as hematopoietic cells that demonstrate plasmacytoid differentiation. Higher levels of sCD138 correlate with poor outcome in myeloma. We examined the association of circulating sCD138 levels in plasma with clinical behavior in 104 patients with chronic lymphocytic leukemia. sCD138 levels were significantly higher in patients (median, 52.8 ng/ml; range, 13.4,252.7 ng/ml) than in healthy control subjects (median, 19.86; range, 14.49,33.14 ng/ml) (P < 0.01). Elevated sCD138 (>median, 52.8 ng/ml) was associated with significantly shorter survival (P = 0.0004); this association was independent of IgVH mutation status, ,2-microglobulin (,2-M) level, and treatment history. Patients with mutated IgVH but high sCD138 levels (>52.8 ng/ml) had significantly shorter survival than those with mutated IgVH and lower levels of sCD138. Similarly, patients with unmutated IgVH but high sCD138 levels had significantly shorter survival than those with lower sCD138 levels and unmutated IgVH (P = 0.007). In a multivariate Cox regression model, only Rai stage, ,2-M, and sCD138 remained predictors of survival. These data suggest that sCD138 when combined with ,2-M and Rai stage, may replace the need for testing IgVH mutation status. [source] Disc structure function and its potential for repairINTERNATIONAL JOURNAL OF RHEUMATIC DISEASES, Issue 1 2002J. Melrose The intervertebral disc (IVD) is the largest predominantly avascular, aneural, alymphatic structure of the human body. It provides articulation between adjoining vertebral bodies and also acts as a weight-bearing cushion dissipating axially applied spinal loads. The IVD is composed of an outer collagen-rich annulus fibrosus (AF) and a central proteoglycan (PG)-rich nucleus pulposus (NP). Superior and inferior cartilaginous endplates (CEPs), thin layers of hyaline-like cartilage, cover the ends of the vertebral bodies. The AF is composed of concentric layers (lamellae) which contain variable proportions of type I and II collagen, this tissue has high tensile strength. The NP in contrast is a gelatinous PG-rich tissue which provides weight-bearing properties to the composite disc structure. With the onset of age, cells in the NP progressively die as this tissue becomes depleted of PGs, less hydrated and more fibrous as the disc undergoes an age-dependent fibrocartilaginous transformation. Such age-dependent cellular and matrix changes can decrease the discs' biomechanical competence and trauma can further lead to failure of structural components of the disc. Annular defects are fairly common and include vertebral rim-lesions, concentric (circumferential) annular tears (separation of adjacent annular lamellae) and radial annular tears (clefts which initiate within the NP). While vascular in-growth around annular tears has been noted, evidence from human post-mortem studies indicate they have a limited ability to undergo repair. Several experimental approaches are currently under evaluation for their ability to promote the repair of such annular lesions. These include growth of AF fibrochondrocytes on a resorbable polycaprolactone (PCL) bio-membrane.1 Sheets of fibrochondrocytes lay down type-I collagen and actin stress fibres on PCL. These matrix components are important for the spatial assembly of the collagenous lamella during annular development and correct phenotypic expression of cells in biomatrices.1 An alternative approach employs preparation of tissue engineered IVDs where AF and NP cells are separately cultured in polyglycolic acid and sodium alginate biomatrices, either separately or within a manifold designed to reproduce the required IVD dimensions for its use as a prospective implant device.2 AF and NP cells have also been grown on tissue culture inserts after their recovery from alginate bead culture to form plugs of tissue engineered cartilage.3 A key component in this latter strategy was the stimulation of the high density disc cell cultures with osteogenic protein-1 (OP-1) 200 ng/mL.3 This resulted in the production of tissue engineered AF and NP plugs with compositions, histochemical characteristics and biomechanical properties approaching those of the native disc tissues.2,3 Such materials hold reat promise in future applications as disc or annular implants. The introduction of appropriate genes into disc cells by gene transduction methodology using adenoviral vectors or ,gene-gun' delivery systems also holds considerable promise for the promotion of disc repair processes.4 Such an approach with the OP-1 gene is particularly appealing.5 The anchoring of discal implants to vertebral bodies has also been evaluated by several approaches. A 3D fabric based polyethylene biocomposite holds much promise as one such anchorage device6 while biological glues used to seal fibrocartilaginous structures such as the AF and meniscus8 following surgical intervention, also hold promise in this area. Several very promising new experimental approaches and strategies are therefore currently under evaluation for the improvement of discal repair. The aforementioned IVD defects are a common cause of disc failure and sites of increased nerve in-growth in symptomatic IVDs in man and are thus often sources of sciatic-type pain. Annular defects such as those described above have formerly been considered incapable of undergoing spontaneous repair thus a clear need exists for interventions which might improve on their repair. Based on the rapid rate of progress and the examples outlined above one may optimistically suggest that a successful remedy to this troublesome clinical entity will be developed in the not so distant future. References 1JohnsonWEBet al. (2001) Directed cytoskeletal orientation and intervertebral disc cell growth: towards the development of annular repair techniques. Trans Orthop Res Soc26, 894. 2MizunoHet al. (2001) Tissue engineering of a composite intervertebral disc. Trans Orthop Res Soc26, 78. 3MatsumotoTet al. (2001) Formation of transplantable disc shaped tissues by nucleus pulposus and annulus fibrosus cells: biochemical and biomechanical properties. Trans Orthop Res Soc26, 897. 4NishidaKet al. (2000) Potential applications of gene therapy to the treatment of intervertebral disc disorders. Clin Orthop Rel Res379 (Suppl), S234,S241. 5MatsumotoTet al. (2001) Transfer of osteogenic protein-1 gene by gene gun system promotes matrix synthesis in bovine intervertebral disc and articular cartilage cells. Trans Orthop Res Soc26, 30. 6ShikinamiY , Kawarada (1998) Potential application of a triaxial three-dimensional fabric (3-DF) as an implant. Biomaterials19, 617,35. [source] Molecular regulation of postsynaptic differentiation at the neuromuscular junctionIUBMB LIFE, Issue 11 2005Raghavan Madhavan Abstract The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs. IUBMB Life, 57: 719-730, 2005 [source] In vitro degradation of articular cartilage: does trypsin treatment produce consistent results?JOURNAL OF ANATOMY, Issue 2 2006H. R. Moody Abstract It is common practice in laboratories to create models of degraded articular cartilage in vitro and use these to study the effects of degeneration on cartilage responses to external stimuli such as mechanical loading. However, there are inconsistencies in the reported action of trypsin, and there is no guide on the concentration of trypsin or the time to which a given sample can be treated so that a specific level of proteoglycan depletion is achieved. This paper argues that before any level of confidence can be established in comparative analysis it is necessary to first obtain samples with similar properties. Consequently, we examine the consistency of the outcome of the artificial modification of cartilage relative to the effects of the common enzyme, trypsin, used in the process of in vitro proteoglycan depletion. The results demonstrate that for a given time and enzyme concentration, the action of trypsin on proteoglycans is highly variable and is dependent on the initial distribution and concentration of proteoglycans at different depths, the intrinsic sample depth, the location in the joint space and the medium type, thereby sounding a note of caution to researchers attempting to model a proteoglycan-based degeneration of articular cartilage in their experimental studies. [source] Studies on bioemulsifier production by Acinetobacter strains isolated from healthy human skinJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2001J.R. Patil Aims: In recent years, interest has been growing in the search for novel bioemulsifiers. Many bacterial genera including Acinetobacter have been reported to produce bioemulsifiers. The present study aims to screen Acinetobacter isolates from healthy human skin for bioemulsifier production. Methods and Results: Acinetobacter junii SC14 produced maximum bioemulsifier in the presence of almond oil during stationary growth phase at 37°C and pH 7·2. Partially purified, nondialysable bioemulsifier from SC14 was a proteoglycan. The protein and polysaccharide fractions resulted in 95·2% reconstitution of the emulsification activity. The role of esterase in the release of cell-bound emulsifier and the contribution of capsular polysaccharide to the emulsification activity were observed. Conclusion:Acinetobacter strains from human skin exhibited better emulsification activity than that by burn wound or soil isolates, owing to the inherent differences in chemical microenvironment of their habitats. Significance and Impact of the Study: Investigation of skin commensals, especially acinetobacters, would lead to the discovery of novel bioemulsifiers with interesting properties. Attempts of screening and strain improvement directed towards skin commensals will open up new avenues for strains producing bioemulsifier on a commercial scale. [source] Characterization of cartilagenous tissue formed on calcium polyphosphate substrates in vitroJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 3 2002Stephen D. Waldman Abstract Successful joint resurfacing by tissue-engineered cartilage has been limited, in part, by an inability to secure the implant to bone. To overcome this, we have developed the methodology to form a cartilage implant in vitro consisting of a layer of cartilagenous tissue overlying a porous, biodegradable calcium polyphosphate (CPP) substrate. As bone will grow into the CPP after implantation, it will result in anchorage of the cartilage. In this study, the cartilagenous tissue formed in vitro after 8 weeks in culture was characterized and compared to native articular cartilage. Light microscopic examination of histological sections showed that there was a continuous layer of cartilagenous tissue on, and integrated with the subsurface of, the CPP substrate. The in vitro -formed tissue achieved a similar thickness to native articular cartilage (mean ± SEM: in vitro = 0.94 ± 0.03 mm; ex vivo = 1.03 ± 0.01 mm). The cells in the in vitro -formed tissue synthesized large proteoglycans (Kav ± SEM: in vitro = 0.27 ± 0.01; ex vivo = 0.27 ± 0.01) and type II collagen similar to the chondrocytes in the ex-vivo cartilage. The in vitro -formed tissue had a similar amount of proteoglycan (GAG ,g/mg dry wt.: in vitro = 198 ± 10; ex vivo = 201 ± 13) but less collagen than the native cartilage (hydroxyproline ,g/mg dry wt.: in vitro = 21 ± 1; ex vivo = 70 ± 8). The in vitro -formed tissue had only about 3% of the load-bearing capacity and stiffness of the native articular cartilage, determined from unconfined mechanical compression testing. Although low, this was within the range of properties reported by others for tissue-engineered cartilage. It is possible that the limited load-bearing capacity is the result of the low collagen content and further studies are required to identify the conditions that will increase collagen synthesis. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 62:323,330, 2002 [source] The Assembly and Remodeling of the Extracellular Matrix in the Growth Plate in Relationship to Mineral Deposition and Cellular Hypertrophy: An In Situ Study of Collagens II and IX and Proteoglycan,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2002Fackson Mwale Abstract The recent development of new specific immunoassays has provided an opportunity to study the assembly and resorption of type II and IX collagens of the extracellular matrix in relationship to endochondral calcification in situ. Here, we describe how in the bovine fetal physis prehypertrophic chondrocytes deposit an extensive extracellular matrix that, initially, is rich in both type II and type IX collagens and proteoglycan (PG; principally, aggrecan). The majority of the ,1(IX)-chains lack the NC4 domain consistent with our previous studies with cultured chondrocytes. During assembly, the molar ratio of type II/COL2 domain of the ,1(IX)-chain varied from 8:1 to 25:1. An increase in the content of Ca2+ and inorganic phosphate (Pi) was initiated in the prehypertrophic zone when the NC4 domain was removed selectively from the ,1(IX)-chain. This was followed by the progressive loss of the ,1(IX) COL2 domain and type II collagen. In the hypertrophic zone, the Ca2+/Pi molar ratio ranged from 1.56 to a maximum of 1.74, closely corresponding to that of mature hydroxyapatite (1.67). The prehypertrophic zone had an average ratio Ca2+/Pi ranging from 0.25 to 1, suggesting a phase transformation. At hypertrophy, when mineral content was maximal, type II collagen was reduced maximally in content coincident with a peak of cleavage of this molecule by collagenase when matrix metalloproteinase 13 (MMP-13) expression was maximal. In contrast, PG (principally aggrecan) was retained when hydroxyapatite was formed consistent with the view that this PG does not inhibit and might promote calcification in vivo. Taken together with earlier studies, these findings show that matrix remodeling after assembly is linked closely to initial changes in Ca2+ and Pi to subsequent cellular hypertrophy and mineralization. These changes involve a progressive and selective removal of types II and IX collagens with the retention of the PG aggrecan. [source] | ||||||||||||||||||||||||||||||||||