Analysis of glycosaminoglycans in rabbit cornea after excimer laser keratectomy.
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BACKGROUND/AIMS: The biochemical basis for the development of subepithelial opacity of the cornea after excimer laser keratectomy has yet to be fully defined. The aim of this study was to evaluate the alterations of glycosaminoglycans (GAGs) after excimer laser keratectomy. METHODS: Rabbit corneas were harvested on days 5, 10, 20, and 30 after excimer laser photoablation. The amount of main disaccharide units was determined by high performance liquid chromatography (HPLC). In addition, immunohistochemical studies were performed on corneal sections 20 days after the ablation. RESULTS: The concentrations of DeltaDi-0S at 5 and 10 days were significantly lower than before the ablation. DeltaDi-6S showed a significant increase 5 days after the ablation but DeltaDi-4S did not show any significant change. There was a significant increase in DeltaDi-HA at 20 and 30 days after ablation. In immunohistochemistry, the positive staining for DeltaDi-6S and hyaluronic acid was observed in the subepithelial region. These immunohistochemical results were well correlated with the HPLC findings. CONCLUSIONS: The increase in chondroitin-6 sulphate and hyaluronic acid may be related to corneal subepithelial opacity after excimer laser keratectomy. (+info)
Chinese hamster ovary cell mutants defective in glycosaminoglycan assembly and glucuronosyltransferase I.
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The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcAbeta1, 3Galbeta1,3Galbeta1,4Xylbeta-O-, attached to specific serine residues in the core protein. Studies of Chinese hamster ovary cell mutants defective in the first or second enzymes of the pathway (xylosyltransferase and galactosyltransferase I) show that the assembly of the primer occurs by sequential transfer of single monosaccharide residues from the corresponding high energy nucleotide sugar donor to the non-reducing end of the growing chain. In order to study the other reactions involved in linkage tetrasaccharide assembly, we have devised a powerful selection method based on induced resistance to a mitotoxin composed of basic fibroblast growth factor-saporin. One class of mutants does not incorporate 35SO4 and [6-3H]GlcN into glycosaminoglycan chains. Incubation of these cells with naphthol-beta-D-xyloside (Xylbeta-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Galbeta1, 4Xylbeta-O-Np and Galbeta1, 3Galbeta1, 4Xylbeta-O-Np) and sialic acid (Siaalpha2,3Galbeta1, 3Galbeta1, 4Xylbeta-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucuronic acid:Galbeta1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Galbeta1,3Galbeta-O-naphthalenemethanol (<0.2 versus 3.6 pmol/min/mg). The mutation most likely lies in the structural gene encoding GlcAT-I since transfection of the mutant with a cDNA for GlcAT-I completely restored enzyme activity and glycosaminoglycan synthesis. These findings suggest that a single GlcAT effects the biosynthesis of common linkage region of both heparan sulfate and chondroitin sulfate in Chinese hamster ovary cells. (+info)
Apoptosis induced by granzyme B-glycosaminoglycan complexes: implications for granule-mediated apoptosis in vivo.
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Lymphocyte granule-mediated apoptosis occurs by perforin-mediated intracellular delivery of granule-associated serine proteases (granzymes). A granule-associated proteoglycan, namely serglycin, that contains chondroitin 4-sulfate (CS) glycosaminoglycans is present in the granules of cytotoxic cells. Serglycin acts as scaffold for packaging the positively charged granzymes and probably chaperones the proteases secreted extracellularly. To learn how the interaction of granzyme B (GrB) with serglycin might influence the apoptotic potential of this proteases, we have evaluated a model system where desalted CS is combined with isolated human granzyme. CS-GrB complexes were very stable, remaining undissociated in salt concentrations upwards to 500 mM (pH 7.4). On the basis of a capture enzyme immunoassay that accurately detects GrB, equivalent amounts of active free and CS-GrB, delivered by perforin or adenovirus, efficiently induced apoptosis in Jurkat cells and produced a similar time-dependent increase in caspase-3-like activity. CS-GrB processed isolated caspases-3 and -7 less efficiently than free granzyme. However, when added to cytosolic extracts, rates of processing were nearly equivalent for the two forms, suggesting cationic GrB may nonspecifically bind cytosolic proteins, leading to reduce proteolytic activity. Finally, GrB was found to be exocytosed from lymphocyte-activated killer cells as a neutral, high macromolecular weight complex, which possessed apoptotic activity. Collectively, the results indicate that neutral, high m.w. GrB has the capacity to induce cell death and will be useful to study the mechanism of cytotoxic cell-mediated apoptosis in vitro. (+info)
An IKLLI-containing peptide derived from the laminin alpha1 chain mediating heparin-binding, cell adhesion, neurite outgrowth and proliferation, represents a binding site for integrin alpha3beta1 and heparan sulphate proteoglycan.
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We synthesized and characterized several peptides containing the IKLLI sequence in the alpha1 chain of laminin-1. The IKLLI-containing peptides, such as LA4 (CSRNLSEIKLLISRARK), LA5 (EIKLLIS) and LA5L (SEIKLLIS), were found to mediate heparin binding and cell adhesion, while also promoting neurite outgrowth in PC12 cells. Furthermore, peptides LA4 and LA5 also mediated proliferation. However, a scrambled peptide, LA5S (ILEKSLI), did not show any of these activities. Anti-LA4 antibodies inhibited laminin- and LA5-mediated cell adhesion and neurite outgrowth, and anti-(integrin alpha3) and anti-(integrin beta1) antibodies inhibited LA5-mediated cell adhesion and neurite outgrowth. Heparin and heparan sulphate inhibited LA5-mediated heparin binding and PC12 cell adhesion in a dose- dependent manner. The IC50 for inhibition of heparin binding and cell adhesion was observed with 9 microM and 8 microM heparin/heparan sulphate respectively. Furthermore, heparan sulphate proteoglycan also inhibited LA5-mediated PC12 cell adhesion with an IC50 of 100 micrograms/ml. However, chondroitin sulphate (dermatan sulphate) did not inhibit cell adhesion. These data suggest that an IKLLI-containing peptide derived from the laminin alpha1 chain may be an active site of laminin and that its cell adhesion may thus interact with both integrin alpha3beta1 and cell- surface heparan sulphate proteoglycan. (+info)
Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan--protein linkage region. Use of a sensitive analytical method developed by chromophore-labeling of linkage glycoserines using dimethylaminoazobenzenesulfonyl chloride.
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Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate. Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan-protein linkage region -HexA-HexNAc-GlcA-Gal-Gal-Xyl-Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N-acetylgalactosamine or N-acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide-serines derived from the linkage region. In the case of oligosaccharide-serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis. Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C-4 or C-6 of the GalNAc residue and C-6 of the Gal residues, as well as 2-O-phosphorylation of the Xyl residue. In contrast, it did not act on the GalNAc-GlcA linkage when attached to a 4-O-sulfated Gal residue. Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide-serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage. These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides. (+info)
Characterization of a Chinese hamster ovary cell line developed by retroviral insertional mutagenesis that is resistant to Sindbis virus infection.
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The alphavirus Sindbis virus (SV) has a wide host range and infects many types of cultured cells in vitro. The outcome of infection is dependent on the strain of virus used for infection and the properties of the cells infected. To identify cellular determinants of susceptibility to SV infection we mutagenized Chinese hamster ovary (CHO) cells by retroviral insertion with a vector containing the neomycin resistance gene that allowed selection for integration into transcriptionally active genes. Cells were then selected for survival after infection with SV. The most resistant cell line (CHO-18.4m) exhibited delayed virus replication and virus-induced cell death, had a single retroviral insertion, and was defective in SV binding to the cell surface. Further analysis revealed that CHO-18.4m cells were deficient in the expression of the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate. This further confirms the importance of heparan sulfate as an attachment molecule for SV in vitro and demonstrates the usefulness of this technique for identifying cellular genes that are important for virus replication. (+info)
TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages.
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ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation. (+info)
Alcohol promotes in vitro chondrogenesis in embryonic facial mesenchyme.
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Ethanol is a well-recognized teratogen in vertebrates that can perturb the development of the facial primordia and various other embryonic structures. However,the mechanisms underlying alcohol's effects on embryogenesis are currently unclear. Recent evidence suggests that the cranial neural crest, which forms the entire facial skeleton, may be a particularly sensitive target of ethanol teratogenicity. In the present study we have examined the influence of in vitro ethanol exposure on cartilage differentiation in micromass cultures of mesenchymal cells isolated from the various facial primordia (maxillary, mandibular, frontonasal, and hyoid processes) of the stage 24 chick embryo. In all four populations of facial mesenchyme, exposure to 1-1.5% ethanol promoted marked increases in Alcian blue-positive cartilage matrix formation, a rise in 35SO4 accumulation into matrix glycosaminoglycans, and enhanced expression of cartilage-characteristic type II collagen and aggrecan gene transcripts. In frontonasal and mandibular mesenchyme cultures, which undergo extensive spontaneous cartilage formation, ethanol treatment quantitatively elevated both matrix production and cartilage-specific gene transcript expression. In cultures of maxillary process and hyoid arch mesenchyme, which form little or no cartilage spontaneously, ethanol exposure induced the formation of chondrogenic cell aggregates and the appearance of aggrecan and type II collagen mRNAs. These actions were not restricted to ethanol, since tertiary butanol treatment also enhanced cartilage differentiation in facial mesenchyme cultures. Our findings demonstrate a potent stimulatory effect of alcohol on the differentiation of prechondrogenic mesenchyme of the facial primordia. Further analysis of this phenomenon might yield insight into the developmental mechanisms underlying the facial dysmorphologies associated with embryonic ethanol exposure. (+info)