Showing papers on "Keratan sulfate published in 1999"

Interactions of Alzheimer amyloid-beta peptides with glycosaminoglycans effects on fibril nucleation and growth.

Abstract: Proteoglycans and their constituent glycosaminoglycans are associated with all amyloid deposits and may be involved in the amyloidogenic pathway. In Alzheimer's disease, plaques are composed of the amyloid-beta peptide and are associated with at least four different proteoglycans. Using CD spectroscopy, fluorescence spectroscopy and electron microscopy, we examined glycosaminoglycan interaction with the amyloid-beta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to determine the effects on peptide conformation and fibril formation. Monomeric amyloid-beta peptides in trifluoroethanol, when diluted in aqueous buffer, undergo a slow random to amyloidogenic beta sheet transition. In the presence of heparin, heparan sulfate, keratan sulfate or chondroitin sulfates, this transition was accelerated with Abeta42 rapidly adopting a beta-sheet conformation. This was accompanied by the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of Abeta42. Incubation of preformed Abeta42 fibrils with glycosaminoglycans resulted in extensive lateral aggregation and precipitation of the fibrils. The glycosaminoglycans differed in their relative activities with the chondroitin sulfates producing the most pronounced effects. The less amyloidogenic Abeta40 isoform did not show an immediate structural transition that was dependent upon the shielding effect by the phosphate counter ion. Removal or substitution of phosphate resulted in similar glycosaminoglycan-induced conformational and aggregation changes. These findings clearly demonstrate that glycosaminoglycans act at the earliest stage of fibril formation, namely amyloid-beta nucleation, and are not simply involved in the lateral aggregation of preformed fibrils or nonspecific adhesion to plaques. The identification of a structure-activity relationship between amyloid-beta and the different glycosaminoglycans, as well as the condition dependence for glycosaminoglycan binding, are important for the successful development and evaluation of glycosaminoglycan-specific therapeutic interventions.

Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture.

Abstract: PURPOSE. To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes. METHODS. Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35 SO 4 . The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes. RESULTS. Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum. CONCLUSIONS. A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.

Age-related changes in fibromodulin and lumican in human intervertebral discs.

Abstract: Study design An analysis of proteoglycans of the intervertebral disc using immunoblotting of tissue extracts. Objectives To investigate the changes in structure and abundance of fibromodulin and lumican in human intervertebral discs during aging and degeneration. Summary of background data Fibromodulin and lumican are keratan sulfate proteoglycan constituents of the disc's extracellular matrix, whose interaction with collagen fibrils may contribute to the mechanical properties of the tissue. Changes in their abundance and/or structure that occur with aging and degeneration therefore may have an impact on disc function. Methods Lumbar intervertebral discs were obtained from individuals of different ages, and extracts of anulus fibrosus and nucleus pulposus were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for fibromodulin and lumican. Results The major changes in abundance observed with age were a decrease in fibromodulin in the adult nucleus pulposus and an increase in lumican in anulus fibrosus during early juvenile development. In addition, fibromodulin in the anulus fibrosus exhibited a structural change with increasing age, characterized by a shift toward the predominance of its glycoprotein form lacking keratan sulfate. Fibromodulin was more abundant in the anulus fibrosus than in nucleus pulposus at all ages, whereas lumican was much more abundant in nucleus pulposus than in anulus fibrosus in the young juvenile; in the adult, however, lumican was present in comparable levels in both tissues. With increasing degrees of degeneration, fibromodulin exhibited an increase in abundance. Conclusions Growth, aging, and degeneration of the intervertebral disc are associated with changes in the abundance and structure of fibromodulin and lumican, which presumably influence the functional properties of the tissue.

Relationship of matrix metalloproteinases and their inhibitors to cartilage proteoglycan and collagen turnover: Analyses of synovial fluid from patients with osteoarthritis

Abstract: OBJECTIVE To determine the relationship between matrix metalloproteinases (MMPs), their inhibitors, and the turnover of matrix molecules in articular cartilage from patients with osteoarthritis (OA). METHODS Synovial fluid samples were collected from the knees of 54 patients with OA. Radiographic evaluations and magnetic resonance imaging were performed on the knees of 34 OA patients to classify the stage of the disease. Biochemical analyses and immunoassays were used to measure the concentrations of MMP-1, MMP-3, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, the disaccharide of hyaluronic acid, the proteoglycan glycosaminoglycan disaccharides of chondroitin 4-sulfate (delta di-CS4) and chondroitin 6-sulfate (delta di-CS6), the 846 epitope on chondroitin sulfate of cartilage proteoglycan aggrecan (putative biosynthetic marker), the keratan sulfate (KS) epitope of aggrecan (putative degradation marker), and the C-propeptide of cartilage type II procollagen (CPII) (biosynthetic marker). RESULTS The concentration of TIMP-1 was directly correlated with the levels of MMP-1 and MMP-3 (both were also correlated with each other), confirming earlier results. There was an inverse correlation between the delta di-CS6:delta di-CS4 ratio and the concentration of MMP-3. The level of delta di-CS6 was correlated with that of the KS epitope, and to a lesser degree, with that of the 846 epitope (the latter was also correlated with the level of delta di-CS4). The concentration of TIMP-1 correlated with that of the 846 epitope, whereas TIMP-2 levels correlated with those of CPII. There were significantly lower concentrations of delta di-CS6, delta di-CS4, the 846 epitope, and CPII in synovial fluid from patients with late-stage OA. CONCLUSION These observations suggest a link between proteolysis and inhibitor concentrations in OA cartilage. Production of TIMPs appears to be individually linked to the synthesis of specific cartilage molecules. The reduction in the amount of cartilage-matrix structural components suggests that there is a measurable loss of cartilage in the late stages of the disease, as suggested previously. The resultant composition of the cartilage suggests that the loss may primarily involve "resident" molecules originally present in healthy cartilage.

MUC-1 glycosylation in endometrium: possible roles of the apical glycocalyx at implantation.

Abstract: MUC-1 is a major epithelial apical surface glycoprotein in human endometrium. It has a large, extended and highly glycosylated ectodomain that contains keratan sulphate chains. MUC-1 is abundant at the luminal epithelial surface in the receptive phase, but keratan sulphate disappears at this time. MUC-1 has been shown experimentally to inhibit cell-cell interactions by steric hindrance of binding interactions mediated by receptors, including integrins and cadherins, so its high abundance at the time of implantation is unexpected. Here, various models for MUC-1 function in implantation are considered and its expression in different species compared. The possible evolutionary advantages of a maternal 'barrier' to implantation are discussed.

Immunocytochemical characterization of reactive optic nerve astrocytes and meningeal cells.

Abstract: Regeneration in the adult central nervous system (CNS) is thought to be hampered by the lesion-induced activation of astrocytes and meningeal cells and the consecutive formation of a glial scar. The substrate properties of reactive astrocytes differ significantly from their neonatal counterparts, which promote axon growth, but in spite of intensive studies the underlying molecular changes are still not fully understood. We have used two cell culture systems to compare the expression of certain surface molecules on neonatal astrocytes, reactive astrocytes and meningeal cells in vitro. Both neonatal and reactive adult astrocytes exhibited a very similar expression of growth promoting molecules (NCAM, L1, laminin, fibronectin, DSD-1 proteoglycan) and potential inhibitors (tenascinC, chondroitin sulfate, and NG2-proteoglycan), whereas we could not detect the inhibitory keratan sulfate on either astrocyte population. In contrast, meningeal cells expressed considerable levels of keratan sulfate, but only minimal amounts of NCAM. In addition, the much higher expression of extracellular fibronectin around meningeal cells implies an excess formation of extracellular matrix (ECM). In coculture experiments, embryonic retinal ganglion cell (RGC) axons clearly avoided meningeal cells and instead preferred even reactive adult astrocytes. Our results suggest that the expression of inhibitory keratan sulfate proteoglycans together with a lack of NCAM and an excess production of ECM may be responsible for the non-permissiveness of meningeal cells. Compared to reactive astrocytes, meningeal cells are even worse a substrate for growing axons. None of the molecules investigated, however, seems to account for the different substrate properties of neonatal and reactive adult astrocytes. GLIA 26:36–46, 1999. © 1999 Wiley-Liss, Inc.

Identification and Immunolocalization of Chondroitin Sulfate Proteoglycans in Tooth Cementum

Abstract: Proteoglycans (PGs) display a great diversity in their core proteins as well as carbohydrate structures and are thought to be involved in many biological functions. Recently we have identified and immunolocalized two keratan sulfate PGs, fibromodulin and lumican, in bovine tooth cementum (Cheng et al., Connect. Tissue Res. 34: 87-96, 1996). The objectives of this study were to identify and characterize chondroitin sulfate (CS) PGs in cementum. In order to explore their potential association with mineral, bovine cementum matrix molecules were fractionated into mineral-unbound and -bound matrices by sequential extraction. Both fractions were subjected to DEAE anion exchange column chromatography and the eluate collected was assayed for C4S and C6S isomers by dot blot immunoassay with specific monoclonal antibodies, 2-B-6 and 3-B-3, respectively. Two families of CSPGs were identified mainly in the mineral-unbound fraction. One contained only C4S glycosaminoglycan and the other both C6S and C4S. By biochemica...

Changes in serum cartilage marker levels indicate altered cartilage metabolism in families with the osteoarthritis-related type II collagen gene COL2A1 mutation.

Abstract: Objective The Arg519–Cys mutation in type II collagen results in severe, precocious familial osteoarthritis (OA) in 100% of carriers within the first 3 decades of life. The carrier population provided a well-defined patient population for the study of serum markers of familial OA with respect to pathogenesis, diagnosis, and prognosis. Methods Serum was obtained from 31 mutation-positive individuals and 16 mutation-negative individuals. OA severity was determined by clinical and radiologic assessments. Levels of serum cartilage oligomeric matrix protein (COMP), keratan sulfate (KS) epitope, the 846 epitope of aggrecan, and the C propeptide of type II collagen (CPII) were measured and were correlated with the radiologic findings. Results COMP and KS levels, both of which have been suggested to be indicative of disturbed cartilage turnover, were significantly elevated in mutation-positive individuals and in the individuals with OA regardless of mutation status. There was no statistically significant difference between mutation-positive, mutation-negative, OA-positive, and OA-negative individuals with respect to serum concentrations of epitope 846 or CPII, both of which are putative markers of cartilage repair. Conclusion Study of the macromolecular constituents of cartilage released into serum in subjects with familial OA revealed altered metabolism in OA, as demonstrated by elevated COMP and KS levels. Other constituents, the 846 epitope and CPII, were not altered, indicating dissociation of cartilage anabolism and breakdown. Future sequential studies will provide an opportunity to define biochemical changes as familial OA develops and to monitor therapeutic responses.

Immune responses to cartilage link protein and the G1 domain of proteoglycan aggrecan in patients with osteoarthritis.

Abstract: Objective To determine whether patients with osteoarthritis (OA) express cellular immunity to cartilage link protein (LP) and the G1 globular domain of proteoglycan (PG) aggrecan, and whether immunity to the G1 domain is influenced by the removal of keratan sulfate (KS). Methods LP and the G1 globular domain of PG were isolated from human and/or bovine cartilage and used in proliferation assays with peripheral blood lymphocytes (PBL) from 42 patients with OA and 40 healthy control subjects. Results Patients with OA expressed a higher prevalence of cellular immunity to human cartilage LP (42.4%) compared with the control group (13.3%). The prevalence of immune reactivity to bovine LP in patients with OA was lower (35.7%) compared with the immunity to human LP, but remained similar in the control group (13.8%). PBL from patients with OA exhibited low reactivity to the native G1 domain of bovine PG. However, removal of KS chains from the G1 globular domain resulted in increased cellular immune responses to the G1 domain in OA patients (45.8%) compared with the control group (7.7%). Conclusion These results indicate the presence of immunity to cartilage-derived LP and the G1 globular domain of PG aggrecan in patients with OA and the inhibitory effect of KS chains on the G1 domain on the expression of this immunity in OA patients. This immune reactivity is commonly observed in patients with inflammatory joint disease and can experimentally induce arthritis. Thus, it may be involved in the pathogenesis of OA.

Corneal cell proteins and ocular surface pathology.

Abstract: The cornea is a transparent and avascular tissue that functions as the major refractive structure for the eye. A wide variety of growth factors, chemokines, cytokines and their receptors are synthesized by corneal epithelial and stromal cells, and are found in tears. These molecules function in corneal wound healing and in inflammatory responses. Proteoglycans and glycoproteins are essential for normal corneal function, both at the air-epithelial interface and within the extracellular matrix. The ocular MUC mucins may play roles in forming the mucus layer of the tear film, in regulating tear film spread, and in inhibiting the adhesion of pathogens to the ocular surface. Lumican, keratocan and mimecan are the major keratan sulfate proteoglycans of the corneal stroma. They are essential, along with other proteoglycans and interfibrillar proteins, including collagens type VI and XII, for the maintenance of corneal transparency. Corneal epithelial cells interact with a specialized extracellular matrix structu...

An improved method for the structural profiling of keratan sulfates: analysis of keratan sulfates from brain and ovarian tumors.

Abstract: A previously developed method for the structural fingerprinting of keratan sulfates (Brown et al., Glycobiology, 5, 311‐317, 1995) has been adapted for use with oligosaccharides fluorescently labeled with 2-aminobenzoic acid following keratanase II digestion. The oligosaccharides are separated by high-pH anion-exchange chromatography on a Dionex AS4A-SC column. This methodology permits quantitative analysis of labeled oligosaccharides which can be detected at the sub-nanogram (~100 fmol) level. Satisfactory calibration of this method can be achieved using commercial keratan sulfate standards. Keratan sulfates from porcine brain phosphocan and human ovarian tumors have been examined using this methodology, and their structural features are discussed.

Structure and Sequence of the Gene Encoding Human Keratocan

Abstract: Keratocan is one of the three major keratan sulfate proteoglycans characteristically expressed in cornea. We have isolated cDNA and genomic clones and determined the sequence of the entire human keratocan (Kera) gene. The gene is spread over 7.65 kb of DNA and contains three exons. An open reading frame starting at the beginning of the second exon encodes a protein of 352 aa. The amino acid sequence of keratocan shows high identity among mammalian species. This evolutionary conservation between the keratocan proteins as well as the restricted expression of Kera gene in cornea suggests that this molecule might be important in developing and maintaining corneal transparency.

Sulfated glycosaminoglycans and collagen in two bovine muscles (M. Semitendinosus and M. Psoas major) differing in texture.

Abstract: M. semitendinosus (ST) and M. psoas major (PM) were used as models for tough and tender meat to study a possible role of sulfated glycosaminoglycans (GAGs) for muscle tenderness. The difference in texture was confirmed by Warner Bratzler shear force measurements. No significant difference in total amount of GAGs in the muscles was found. In contrast, a significant difference in the ratio of GAG/collagen was found between the two muscles. After separation of the GAGs by density gradient ultracentrifugation and ion-exchange chromatography, dermatan sulfate (DS), keratan sulfate (KS), chondroitin sulfate (CS), and heparan sulfate (HS) were identified by cellulose acetate electrophoresis after use of specific enzymes and chemical methods. The content of DS was higher in the tougher muscle (ST) than in PM, and the difference in DS content was statistically significant. Furthermore, a significant difference in the GAG composition pattern of the two muscles was found. The yield of GAGs extracted from the muscles was 77% for ST and 87% for PM. The residue after extraction was further analyzed and found to contain mainly HS. Immunohistochemical studies using antibodies against CS/DS showed a staining pattern of the perimysium of ST different from that of PM.

Recurrent Macular Corneal Dystrophy Type II 49 Years After Penetrating Keratoplasty

Abstract: Recurrence of macular corneal dystrophy after keratoplasty is rare. We report light microscopic, immunohistochemical, electron microscopic, and serologic findings in a 78-year-old woman who underwent regrafting 49 years following the first penetrating keratoplasty. Examination of the corneal button revealed deposits of glycosaminoglycans in the graft beneath the Bowman layer, throughout the stroma, and in the endothelium with positive staining for antigenic keratan sulfate. By transmission electron microscopy, intracellular and extracellular deposits of a fibrillogranular material were detected in the stroma, Descemet membrane, and endothelium. The serum level of antigenic keratan sulfate was normal. Our findings indicate that macular corneal dystrophy type II may show late recurrence after penetrating keratoplasty with intense deposition of antigenic keratan sulfate in all corneal layers.

Measurement of activities of human serum sulfotransferases which transfer sulfate to the galactose residues of keratan sulfate and to the nonreducing end N-acetylglucosamine residues of N-acetyllactosamine trisaccharide: comparison between normal controls and patients with macular corneal dystrophy.

Abstract: Human serum sulfotransferase activities were measured in normal controls and patients with macular corneal dystrophy (MCD), an inherited disorder characterized by the decreased sulfation of keratan sulfate in the corneal stroma and serum, using two kinds of acceptor: partially desulfated keratan sulfate and a trisaccharide with a GlcNAc residue at the nonreducing terminal, GlcNAcbeta1-3Galbeta1-4GlcNAc. When partially desulfated keratan sulfate was used as the acceptor, only sulfotransferase activity which transfers sulfate to position 6 of the Gal residues was detected. In contrast, when GlcNAcbeta1-3Galbeta1-4GlcNAc was used as the acceptor, sulfotransferase activity which transfers sulfate to position 6 of the nonreducing terminal GlcNAc residue could be detected. Although keratan sulfate levels in the sera of MCD patients determined by ELISA were much lower than those in normal controls, there were no detectable differences in either the sulfotransferase activity responsible for the sulfation of position 6 of Gal residues or that responsible for the sulfation of position 6 of nonreducing end GlcNAc residues between normal controls and MCD patients. These results suggest that the sulfotransferase involved in the sulfation of keratan sulfate, which is assumed to be deficient in MCD patients, may not be secreted into the serum, and that direct measurement of the sulfotransferase activity present in affected tissues such as the cornea instead of serum may be necessary to confirm the postulated deficiency in the biosynthesis of keratan sulfate in MCD.

Immunohistochemical analysis of proteoglycan biosynthesis during early development of the chicken cornea.

Abstract: Antibodies to core proteins of chicken corneal keratan sulfate proteoglycan and chondroitin sulfate proteoglycan were prepared and purified by use of an affinity column. Using these antibodies and monoclonal antibody 5-D-4 to keratan sulfate (commercial), the localization of proteoglycans in developing corneas (Days 5 to 17 of embryonic age and 2 days after hatching) was determined immunohistochemically. Keratan sulfate proteoglycan antigen was not detected in cornea on Day 5, but it was detected uniformly over the whole stroma on Day 6, ca. 12 h after invasion of the primary stroma by mesenchymal cells. The absence of the antigen in cornea of Day 5 was confirmed by Western blotting of the corneal extract. Immunohistochemistry with 5-D-4 antibody revealed that the keratan sulfate chain was undersulfated in corneas of Days 6 to 7, because the staining was much weaker than that in cornea of Day 8. In addition, keratan sulfate proteoglycan antigen was detected uniformly over the whole stroma on Days 7 to 17 and 2 days after hatching, but not in the epithelial layer on Day 13 and after: because the epithelial layer was clearly not observed on photomicrographs until Day 13, it is not known whether keratan sulfate proteoglycan was synthesized by the epithelium during Days 6 to 12. In contrast, chondroitin sulfate proteoglycan antigen was detected in cornea on Day 5 and also, like keratan sulfate proteoglycan, uniformly over the whole stroma on Day 6 through 2 days after hatching. Furthermore, the chondroitin sulfate proteoglycan was not detected in the epithelial layer on Day 13 and after. These results show that keratan sulfate proteoglycan is synthesized by the stromal cells which invade the primary stroma between Day 5.5 and 6, while chondroitin sulfate proteoglycan is synthesized by epithelial and/or endothelial cells before the invasion, and also by the stromal cells after the invasion.

600 MHz NMR studies of human articular cartilage keratan sulfates.

Abstract: The use of high-field two-dimensional 1H-correlation data is described for the detailed comparison of intact keratan sulfate polymer chains derived from human articular cartilage sources as a function of age. For fetal material the nonreducing chain termini are shown to be sparsely capped by sialyl groups which, if present, are exclusively (α2–3)-linked to an unsulfated galactose residue. The asialo capping segment has the structure: Gal-GlcNAc6S-Gal-GlcNAc6S-. Examination of keratan sulfate from 10-year-old cartilage shows that capping by sialyl groups is complete, with (α2–3)-linkages predominant; for both this and the 38-year-old cartilage the three capping structures: NeuAc(α2–3)-Gal-GlcNAc6S-Gal-GlcNAc6S-, NeuAc(α2–3)-Gal-GlcNAc6S-Gal6S-GlcNAc6S-, and NeuAc(α2–3)-Gal6S-GlcNAc6S-Gal6S-GlcNAc6S- are clearly recognizable. The level of (α2–6)-linked chain capping sialyl groups is significant for 38-year-old cartilage keratan sulfate. Structural information concerning the linkage region to protein and the distribution of galactose environments is readily obtained from the spectra. Signal complexities severely limit the usefulness of two-dimensional correlation spectroscopy at 600 MHz for the examination of N-acetylglucosamine residues within the poly(N-acetyllactosamine) repeat sequence and signals representing fucose placements remain undifferentiated. This nondestructive approach complements current degradative methods for the structural examination of keratan sulfates.

Bone induction promoter

Abstract: PROBLEM TO BE SOLVED: To obtain a bone induction promoter which can effectively promote bone induction even when it is administered in a small dosage or in a low concentration by using as the active ingredient a lipidbonded glycosaminoglycan composed of a glycosaminoglycan and a lipid bonded thereto or a pharmacologically acceptable salt thereof. SOLUTION: This promoter is desirably selected from among a bone induction promoter in which the glycosaminoglycan is hyaluronic acid, chondroitin, chondroitin sulfate, chondroitin polysulfate, dermatan sulfate, heparin, keratan sulfate, or keratan polysulfate, a bond induction promoter in which the lipid is a glycerolipid, a bone induction promoter in which the glycerolipid is a phospholipid, a bone induction promoter in which the phospholipid is phosphatidylethanolamine, a bone induction promoter in which the glycosaminoglycan is hyaluronic acid and the lipid is phosphatidylethanolamine, and a bone induction promoter in which the lipid is covalently bonded to the reducing terminal of the glycosaminoglycan.

Keratan sulfate glycosaminoglycans in murine eosinophil-specific granules.

Abstract: SUMMARY We examined the presence of sialyl glycoconjugates in specific granules from murine bone marrow eosinophils. Lectin cytochemistry using Maackia amurensis lectin II (MAL II) specific for sialyl � -2,3 galactose residues demonstrated positive labeling in both immature and mature specific granules. Pretreatment with Clostridium neuraminidase or keratanase II eliminated the positive labeling of MAL II in the specific granules. High iron diamine–thiocarbohydrazide–silver proteinate physical development (HID-TCH-SP-PD) staining, which is specific for sulfated glycoconjugates, also positively labeled immature specific granules lacking crystalloids but not mature granules with crystalloids. Pretreatment with a combination of chondroitinase ABC and keratanase, or a combination of chondroitinase ABC and keratanase II, eliminated the positive labeling obtained with HID-TCH-SP-PD. These results indicate that the sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan, which appears to be of the corneal type in view of its sensitivity to keratanase and keratanase II.

Impact of various glycosaminoglycans upon cAMP-dependent protein kinase.

Abstract: The physiological effects of the second messenger cAMP are displayed by cAMP-dependent protein kinase-medicated phosphorylation of specific target proteins which in turn control diverse cellular functions. We have determined this enzyme substrate phosphorylation in the presence of various glycosaminoglycans using a cAMP-dependent protein kinase isolated from rat liver. The results indicate that sulfated and unsulfated polysaccharides are able to inhibit phosphorylation of histone type IIa catalysed by cAMP-dependent protein kinase. Based on their impact upon substrate phosphorylation, glycosaminoglycans can be divided into three groups: group I with the highest inhibitory effect: dermatan sulfate and heparan sulfate; group II: chondroitin 4-sulfate and group III with the lowest inhibitory effect: chondroitin 6-sulfate, keratan sulfate and hyaluronic acid. Copyright © 1999 John Wiley & Sons, Ltd.

Sulfate group transferase preparation

Abstract: PROBLEM TO BE SOLVED: To obtain the subject new enzyme preparation capable of specifically transferring a sulfate group from a sulfate group donor to an OH group at the 6-position of N-acetylgalactosamine 4-sulfate residue of glycosaminoglycan, suitable for production of a sulfated polysaccharide useful for a medicine, etc., function analysis, etc. SOLUTION: This new sulfate group transferase preparation specifically transfers a sulfate group from a sulfate group donor to an OH group at the 6-position of N-acetylgalactosamine 4-sulfate residue of glycosaminoglycan, transfers the sulfate group to chondroitinsulfuric acid A derived from a whale cartilage, chondroitinsulfuric acid C derived from a shark cartilage and dermatan sulfate derived from-swine skin but not to chondroitinsulfuric acid E derived from a squid cartilage, keratan sulfate derived from bovine cornea, heparan sulfate derived from bovine kidney and CDSNS-heparin, increases activity in the presence of Mn2+, Mg2+, Ca2+, NaCl, KCl, protamine, etc., inhibits the sulfate group from transferring to chondroitinsulfuric acid A by chondroitinsulfuric acid E. The preparation has uniformity and is obtained by extraction from an organism body such as squid cartilage, etc. COPYRIGHT: (C)2001,JPO

Endothelial cell surface-associated keratan sulfate after excimer laser photoablation of the anterior rabbit cornea.

Abstract: PURPOSE The expression of keratan sulfate on the surfaces of corneal endothelial cells is altered when the cells are responding to injury. The purpose of this study was to investigate whether excimer laser surgery affected corneal endothelial cells and the levels of keratan sulfate associated with them. METHODS We performed 14 bilateral, transepithelial phototherapeutic keratectomies in rabbits using a Nidek EC-5000 excimer laser. Ablations were 6 mm in diameter and 50 microm, 150 microm, or 240 microm deep. At various times following surgery the endothelium was immunolabeled for keratan sulfate and examined by scanning electron microscopy. Four untreated corneas were also examined. RESULTS Three days after surgery, endothelial cells were not flat but were rounded or domed, a finding that was more pronounced after deeper ablations. No rounded cells, however, were seen at post-operative day 12. Keratan sulfate immunolabel was elevated on endothelial cells 3 days after surgery. By postoperative day 36, its expression was normal under the 50-microm ablations, but remained elevated under one of two 240-microm ablations. CONCLUSIONS Corneal endothelial cells take on a rounded appearance in the early stages after excimer laser photoablations in rabbits, especially after deeper ablations. The apical surface of the endothelium also transiently expresses elevated levels of cell surface-associated keratan sulfate following surgery. These changes appear to be responses to some aspect of the surgery, and may have physiological implications.