Showing papers on "Keratan sulfate published in 2009"

On-line separations combined with MS for analysis of glycosaminoglycans

Abstract: The glycosaminoglycan (GAG) family of polysaccharides includes the unsulfated hyaluronan and the sulfated heparin, heparan sulfate, keratan sulfate, and chondroitin/dermatan sulfate. GAGs are biosynthesized by a series of enzymes, the activities of which are controlled by complex factors. Animal cells alter their responses to different growth conditions by changing the structures of GAGs expressed on their cell surfaces and in extracellular matrices. Because this variation is a means whereby the functions of the limited number of protein gene products in animal genomes is elaborated, the phenotypic and functional assessment of GAG structures expressed spatially and temporally is an important goal in glycomics. On-line mass spectrometric separations are essential for successful determination of expression patterns for the GAG compound classes due to their inherent complexity and heterogeneity. Options include size exclusion, anion exchange, reversed phase, reversed phase ion pairing, hydrophilic interaction, and graphitized carbon chromatographic modes and capillary electrophoresis. This review summarizes the application of these approaches to on-line MS analysis of the GAG classes.

The effect of growth factor signaling on keratocytes in vitro and its relationship to the phases of stromal wound repair.

Abstract: Purpose To determine the relationship between signaling by different growth factors and the phases of corneal stromal wound repair. The authors hypothesize that the process involves sequential signaling, resulting first in proliferation and then in extracellular matrix (ECM) synthesis. Methods The effects of IGF-I, TGF-beta1, FGF-2, and PDGF on proliferation and ECM production by primary cultured bovine keratocytes were evaluated. DNA synthesis was determined by (3)H-thymidine incorporation, and maximal cell density was determined by measurement of DNA content. Relative levels of ECM components synthesized by keratocytes and secreted into the media were evaluated by (3)H-glycine incorporation into total ECM protein and collagen, by (3)H-glucosamine incorporation into chondroitin sulfate, keratan sulfate, and hyaluronan, and by Western blotting with antibodies specific to procollagen types Iota and IotaIotaIota. Results FGF-2 stimulated the highest level of proliferation and the lowest level of glycosaminoglycan synthesis and inhibited the synthesis of collagen types Iota and IotaIotaIota. IGF-I, in contrast, stimulated the lowest level of proliferation and the highest levels of collagen synthesis. PDGF and TGF-beta1 had intermediate effects on proliferation and collagen synthesis. Although FGF-2 inhibited collagen production, it could be restored by subsequent treatment with IGF-I, TGF-beta1, and PDGF. Conclusions The results of this study showed that the level of proliferation induced by the growth factors was inversely related to the levels of collagen production. The authors suggest that FGF-2 initiates the hypercellular phase of corneal wound healing and that IGF-I and PDGF are involved in the restoration of a normal ECM.

Structural characterization of glycosaminoglycans from zebrafish in different ages

Abstract: The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5 days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using 1H-NMR.

Macular corneal dystrophy in a Chinese family related with novel mutations of CHST6.

Abstract: PURPOSE To identify mutations in the carbohydrate sulfotransferase gene (CHST6) for a Chinese family with macular corneal dystrophy (MCD) and to investigate the histopathological changes in the affected cornea. METHODS A corneal button of the proband was obtained by penetrating keratoplasty. The half button and ultrathin sections from the other half button were examined with special stains under a light microscope (LM) and an electron microscope (EM) separately. Genomic DNA was extracted from peripheral blood of 11 family members, and the coding region of CHST6 was amplified by the polymerase chain reaction (PCR) method. The PCR products were analyzed by direct sequencing and restriction enzyme digestion. RESULTS The positive reaction to colloidal iron stain (extracellular blue accumulations in the stroma) was detected under light microscopy. Transmission electron microscopy revealed the enlargement of smooth endoplasmic reticulum and the presence of intracytoplasmic vacuoles. The compound heterozygous mutations, c.892C>T and c.1072T>C, were identified in exon 3 of CHST6 in three patients. The two transversions resulted in the substitution of a stop codon for glutamine at codon 298 (p.Q298X) and a missense mutation at codon 358, tyrosine to histidine (p.Y358H). The six unaffected family individuals carried alternative heterozygous mutations. These two mutations were not detected in any of the 100 control subjects. CONCLUSIONS Those novel compound heterozygous mutations were thought to contribute to the loss of CHST6 function, which induced the abnormal metabolism of keratan sulfate (KS) that deposited in the corneal stroma. It could be proved by the observation of a positive stain reaction and the enlarged collagen fibers as well as hyperplastic fibroblasts under microscopes.

Heterogeneity, polydispersity, and physiologic role of corneal proteoglycans.

Abstract: Gel chromatography, affinity chromatography, ultracentrifugation, enzymic fragmentation, and analysis of amino acids, hexosamines and neutral sugars were used to characterize a heterogeneous fraction of proteoglycans from bovine corneal stroma. The results indicate that the fraction largely consists of a mixture of the 2 main types of corneal proteoglycans described earlier, namely keratan sulfate proteoglycans and chondroitin sulfate-rich proteoglycans with covalently bound oligosaccharides. Models for the structure of proteoglycans are suggested, an it is concluded that the molecular size of corneal proteoglycans makes them appropriate as ‘spacers’ between the collagen fibrils, a property important for corneal transparency. Cornea is softer than cartilage because corneal proteoglycans are less underhydrated than cartilage proteoglycans.

Keratan sulfate‐containing proteoglycans in sheep brain with particular reference to phosphacan and synaptic vesicle proteoglycan isoforms

Abstract: Proteoglycans (PGs) are widely expressed in all areas of the brain. In this study, the keratan sulfate-containing PGs (KS-PGs) from cerebrum (CB), cerebellum (CL) and brainstem (BS) of young sheep brain were isolated, purified and characterized. The amount of KS-PGs in CL was significantly lower than that in CB and BS. KS-PGs were characterized by increased extent of glycosylation and heterogeneity of KS chains in CL. Western blot analyses demonstrated the presence of the KS-PGs phosphacan, SV2A and SV2B isoforms of synaptic vesicle proteoglycan in all three areas of the young sheep brain. Phosphacan predominated in BS and CB, showing significant molecular heterogeneity. SV2A and SV2B were found in two forms of high and low molecular sizes according to their extent of glycosylation in sheep brain. SV2A predominated in CL, where forms with very high molecular sizes were detected. Immunohistochemical examination revealed that SV2A was localized in the extracellular matrix of both gray and white matter. In contrast, phosphacan and SV2B were mainly localized in the white matter in all brain regions. The results of the present study demonstrated that KS-PGs are present in the three areas of the sheep brain, showing significant variations in their content, structure and localization among the distinct areas. These differences may be important for the physiology of the brain. Copyright © 2008 John Wiley & Sons, Ltd.

Method for assaying keratan sulfate, assay kit therefor and method for detecting joint disease by using same.

Abstract: The inventions provides a method for immunologically determining a keratan sulfate level which method includes bringing an anti-keratan sulfate monoclonal antibody into contact with a biological sample, the anti-keratan sulfate monoclonal antibody exhibiting a relative reaction specificity between keratan sulfate-I and keratan sulfate-II represented by IC50 KS-I/KS-II of 0.4 to 5, to thereby provide a signal; and detecting keratan sulfate contained in the biological sample from the signal. On the basis of the method, the invention also provides a joint disease detection method and a method for assessing the effect of a remedy for a joint disease and a candidate substance therefor. Through these methods, a very small amount of keratan sulfate contained in a sample, can be determined. Particularly, these methods can determine, at high-sensitivity and high-specificity, the total keratan sulfate including keratan sulfate-I, which have been difficult to determine through a conventional technique. The methods also enables detect a joint disease and assess the effect of a remedy for a joint disease or a candidate substance therefor.

Method for producing keratan sulfate oligosaccharide fraction

Abstract: PROBLEM TO BE SOLVED: To provide a method for producing a keratan sulfate oligosaccharide having substantially no contamination of impurities. SOLUTION: There is provided the method for producing the keratan sulfate oligosaccharide fraction represented by formulas (I), (II) and (III), and having properties (A) to (C). COPYRIGHT: (C)2010,JPO&INPIT

Agent for applying to mucosa and its production method

Abstract: PROBLEM TO BE SOLVED: To provide an agent for applying to the mucosa, which can persistently exert therapeutic effects on disorders such as inflammation and lesions in the mucosa even by low-frequency administration because the agent can stay at a diseased site for a long period of time by exhibiting high retentivity in a mucosal epithelial layer. SOLUTION: The agent contains, as an active component, a glycosaminoglycan (GAG) introduced with a hydrophobic group through a bonding chain. The glycosaminoglycan is selected from hyaluronic acid, chondroitin, chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate and keratan sulfate including their salts. COPYRIGHT: (C)2010,JPO&INPIT