Thesis (M.S.)--University of Delaware, 2008. / Principal faculty advisor: Mary C. Farach-Carson, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (doctoral)--Umeä Universitet, 1982. / Extra t.p. with thesis statement inserted. Includes bibliographies.

Thesis (doctoral)--Umeä Universitet, 1982. / Extra t.p. with thesis statement inserted. Includes bibliographies.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

This research examined the role of the cartilaginous end-plate (CEP) in ageing and degeneration of the human intervertebral disc (IVD). The matrix component affected primarily during degeneration of the IVD is proteoglycan (PG) (Pearce et al., 1987). The CEP, a hyaline cartilage found between the nucleus pulposus (NP) and the anulus fibrosus (AF) and the vertebral body, has been proposed as the source of the PG of the AF and NP. This study was undertaken to: 1) assess the similarity of CEP PG to PG from articular cartilage and IVD, (2) compare the CEP PG from healthy young discs with those from older degenerate discs (3) distinguish the changes in CEP PG due to ageing from those due to degeneration. The combined effects of ageing and degeneration were studied using end-plates from three healthy young spines and three post-mature spines; those to degeneration alone by comparison of two healthy with one degenerate disc in each of three spines. Altogether 86 CEP from 10 lumbar spines were examined. The CEP PG were prepared from 4M guanidinium chloride tissue extracts by density gradient ultracentrifugation under associative conditions. PG were separated into high and low molecular weight (M) components by Sepharose CL-2B chromatography. The PG and the high M and low M fractions were analysed for hexose (hex) and hexuronate (hexA) as measures of keratan sulphate and chondroitin sulphate, respectively. Also, the two fractions were analysed by composite agarose-polyacrylamide gel electrophoresis. The CEP PG resembled the IVD PG more closely than those of articular cartilage PG: the fraction excluded from Sepharose CL-2B was low, the hex/hexA ratio was high, and five electrophoretically distinct subspecies were seen. With degeneration, several properties of the CEP PG altered irrespective of age: the extractable .total proteoglycan fell, the ratio hex/hexA rose and number of electrophoretically distinct PG subspecies declined. With age, the sizes of the high M and low M fractions fell and the electrophoretic mobilities of the subspecies changed. These results suggested that degeneration involves both a conversion of aggregating to non-aggregating PG and the preferential biosynthesis of a keratan sulphate-rich over a chondroitin sulphate-rich PG. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

A predominant species of heparan sulfate proteoglycan that consisted of at least two subunits linked by disulfide bonding was isolated from cell layers of normal ("cobblestone") bovine vascular endothelial cells in culture. Treatment of the parent molecules with dithiothreitol caused their complete cleavage and permitted the subsequent separation of the larger and smaller subunits on Sepharose CL4B columns. Removal of dithiothreitol by dialysis resulted in the reformation of large disulfide-bonded molecules but such recombination of the subunits was prevented by prior reductive alkylation using iodoacetamide. Buoyant density gradient analysis as well as gel chromatography on Sepharose CL6B columns, following alkaline borohydride and nitrous ac i d treatment of individual carbohydrate-rich subunits, showed that the latter consisted of core proteins associated solely with heparan sulfate glycosaminoglycans. The sizes of the latter were estimated by chromatographic techniques to be approximately 50 000 and 14 000 daltons in the case of the larger and smaller subunits, respectively. This is the first description of disulfide-bonded proteoheparan sulfates in bovine aortic endothelial cells. Studies of the effects of various extracellular matrices on the proliferative behaviour of bovine aortic endothelial cells in culture revealed that extracellular matrix material from rat smooth muscle cells stimulated proliferation more than did other matrices. Bovine aortic endothelial cells also changed their morphology and cell-surface proteoglycan profiles in response to particular extracellular matrices. Enzymic modifications of matrices did not, however, cause noticeable changes in the cell surface proteoglycans synthesized by bovine aortic endothelial cells. This discrepancy suggested that the observed differences in cell-surface proteoglycan profiles cannot be ascribed to any specific single constituent of the extracellular matrix but that its overall architecture may be the sole determinant of such differences. When the turnover of endothelial cell proteoglycans was assessed, degradation of both intracellular and pericellular proteoglycans was inhibited by lysosomotropic agents. This indicated that these macromolecules may be degraded within the lysosomes; the cell layer proteoglycans are apparently internalized prior to their degradation in this location. Failure by both NH₄Cl and chloroquine completely to block the degradation of intracellular as well as pericellular proteoglycans suggested that other mechanisms of degradation also exist. The results extend biochemical data on endothelial cell surface proteoglycans.

Smooth muscle cells isolated from neonatal rat hearts synthesize and secrete radioactively labelled proteoglycans into two distinct extracellular compartments, the pericellular (cell surface/matrix layer) and the culture medium (extracellular). Cultures grown in the presence of ascorbic acid synthesize proteoglycans that are more highly sulphated than those produced in the absence of ascorbate. The glycosaminoglycan chains associated with the proteoglycans synthesized by rat smooth muscle cells were heparan sulphate, chondroitin sulphate and dermatan sulphate. There was no evidence for the synthesis of hyaluronic acid by these cells. Most of the heparan sulphate was found to be associated with the pericellular and intracellular compartments, whereas the extracellular compartment contained the bulk of the chondroitin sulphate. In the presence of ascorbate there was an increase in dermatan sulphate content of the pericellular compartment at the expense of heparan sulphate, whilst in the absence of ascorbate the heparan sulphate content of this compartment was significantly increased. Hyaluronic acid and the antibiotic Tunicamycin had no effect on the biosynthesis of sulphated macromolecules produced by the rat smooth muscle cells. However, p-nitrophenyl-β-D-xyloside increased by 10-fold the amount of radioactive sulphate incorporation into macromolecules in the extracellular compartment. This increase was due to increased sulphation of glycosaminoglycan chains synthesized in the presence of the exogenous acceptor, as evidenced by the sulphate/ uronate ratio of these sulphated macromolecules. Furthermore, heparan sulphate secretion into the extracellular compartment was decreased whilst dermatan sulphate increased in the presence of xyloside. Pulse-chase experiments with radioactive sulphate were used to study the pathways and kinetics of secretion in the rat smooth muscle cell system. The data from these studies are consistent with a very rapid intracellular sulphation mechanism followed by rapid secretion to the pericellular compartment of macromolecular sulphated proteoglycans. Subsequently some of these molecules then travel to the extracellular compartment. The time that different proteoglycan species remain associated with the pericellular compartment is influenced by the different matrix connective tissue proteins found in this compartment as a result of ascorbate supplementation or deprivation. During the course of these investigations, it was observed that the pericellular compartment contributed to catabolism of sulphated macromolecules. The sulphated proteoglycans associated with this compartment are acted upon by a sulphatase or sulphatases to give rise to free radioactive inorganic sulphate and macromolecules which have been desulphated. That this process occurs in the pericellular compartment only was proven by the use of intracellular lysomotrophic inhibitors and by the continuous exposure of sulphate labelled macromolecules to the extracellular extract. Neither resulted in the release of radiolabelled inorganic sulphate from sulphated macromolecules.

published_or_final_version / Physiology / Master / Master of Philosophy

No description available.

Glycoproteins, and their subset proteoglycans, are an important group of molecules in joint tissues, providing crucial functions such as cartilage structural integrity and lubrication at cartilage surfaces. The functionality of these glycoproteins is attributable to their oligosaccharide components, however surprisingly little is known about their fine structural details. With the use of glycoproteomic methods, this thesis presents the development and incorporation of mass spectrometric, biochemical and immunological methods to elucidate glycoprotein structures in synovial fluids, chondrocytes and synoviocytes in order to provide insight into how their structures may contribute to their functions. Initially, anion exchange chromatography was used to extract the acidic fraction containing glycoproteins and proteoglycans in arthritic synovial fluid (SF) samples, followed by proteomic analysis to identify the main glycoproteins in 1D-SDS-PAGE gels. To complement these findings, an in-gel enzymatic digest method for glycosaminoglycan (GAG) and oligosaccharide analysis was developed for analysis of glycoproteins by graphitised carbon liquid chromatography mass spectrometry (LC-MS). Further characterization of the major glycoprotein, lubricin, was pursued by investigating its interactions with the surrounding extracellular matrix (ECM) from its cellular sources and characterising the secreted lubricin with Western blot and proteomic analysis. Finally, the graphitised carbon LC-MS method was applied to analyse the overall glycosylation profiles of lubricin. The major glycoprotein found in arthritic synovial fluid was lubricin, as identified by peptide LC-MS and Western blot. Graphitised carbon LC-MS identified the major chondroitin sulfate (CS) repeat region disaccharides and linkage region oligosaccharides of aggrecan with confirmation through tandem mass spectra and Western blots using CS linkage region stub antibodies. Application of this method to lubricin led to the discovery of O-linked oligosaccharide structures which were previously undescribed for lubricin. A higher proportion of sialylated oligosaccharide structures were detected in the rheumatoid arthritis (RA) samples compared to the osteoarthritic (OA) samples, which signifies a diagnostic difference between these diseases. Sulfated oligosaccharide structures were also detected on synovial fluid lubricin, correlated with Western blot reactivity with the MECA-79 antibody, thus suggesting a role for lubricin in inflammation. Overall the results demonstrated that glycosylation structure indicates additional functional properties for the glycoproteins such as lubricin.