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Biomedical subjects

A R Poole

Publications and source records attributed to A R Poole.

At least 145 records · Page 8Linked to original sources
PubMed1986

Localization of a dermatan sulfate proteoglycan (DS-PGII) in cartilage and the presence of an immunologically related species in other tissues.

A monoclonal antibody to a core-protein-related epitope of a small dermatan sulfate-rich proteoglycan (DS-PGII) isolated from adult bovine articular cartilage (22) was used to localize this molecule, or molecules containing this epitope, in bovine articular cartilages, in cartilage growth plate, and in other connective tissues. Using an indirect method employing peroxidase-labeled pig anti-mouse immunoglobulin G, DS-PGII was shown to be present mainly in the superficial zone of adult articular condylar cartilage of the metacarpal-phalangeal joint. In fetal articular and epiphyseal cartilages, the molecule was uniformly distributed throughout the matrix. By approximately 10 months of age it was confined mainly to the superficial and middle zones of articular cartilage and the inter-territorial and pericellular matrix of the deep zone. DS-PGII was not detected in the primary growth plate of the fetus except in the proliferative zone, where it was sometimes present in trace amounts. In contrast, it was present throughout the adjacent matrix of developing epiphyseal cartilage. In the trabeculae of the metaphysis, strong staining for DS-PGII was seen in decalcified osteoid and bone immediately adjacent to osteoblasts. Staining was also observed on collagen fibrils in skin, tendon, and ligament and in the adventitia of the aorta and of smaller arterial vessels in the skin. These observations indicate that DS-PGII and/or molecules containing this epitope are widely distributed in collagenous tissues, where the molecule is intimately associated with collagen fibrils; in adult cartilage this association is limited mainly to the narrow parallel arrays of fibrils which are found in the superficial zone at the articular surface. From its intimate association and other studies, this molecule may play an important role in determining the sizes and tensile properties of collagen fibrils; it may also be involved in the calcification of osteoid but not of cartilage.

Animals↗
PubMed1986

Chondrocalcin and the calcification of cartilage. A review.

A protein recently identified and named chondrocalcin is associated with the calcification of hyaline cartilage matrix in health and disease. Chondrocalcin is absent from noncalcifying growth plate cartilages in disease. It is also present in noncalcifying developing epiphyseal cartilage. The molecule appears to be synthesized and secreted by chondrocytes, particularly those committed to becoming hypertrophic. It is not detectable in developing or mature bone, teeth, or other tissues, although trace amounts are present in blood. The assigned molecular weight is 69,000. The protein is basic and is constructed of two subunits of 35,500 and 34,500.

Amino Acids↗
PubMed1985

The origin of human cartilage proteoglycan link-protein heterogeneity and fragmentation during aging.

Human articular-cartilage link proteins are resolved into three components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicative of three different structures. The action of the proteinase clostripain yields a single link-protein component with electrophoretic properties analogous to those of the smallest (most mobile) native link protein, suggesting that this link protein may be derived naturally from one or both of the larger molecules by proteolytic cleavage in situ. Upon chemical deglycosylation of native link protein two components are resolved, suggesting that two of the link proteins differ only in their degree and/or type of oligosaccharide substitution. This pattern is compatible with a proteolytic origin for the smallest link protein. During aging further proteolytic fragmentation occurs, though it is only apparent on reduction of disulphide bonds. This fragmentation occurs at identical sites in all three native link proteins, indicating the existence of a large region common to all the link proteins, which appears to consist predominantly of the C-terminal half of the molecules. These observations are compatible with the variation in oligosaccharide and proteolytic heterogeneity occurring at the N-terminus of the link proteins.

Aging↗
PubMed1985

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.

Adult↗
PubMed1985

Rabbit antibodies to degraded and intact glycosaminoglycans which are naturally occurring and present in arthritic rabbits.

Using enzyme-linked immunosorbent assays and radioimmunoassays employing chondroitinase ABC-treated rabbit cartilage proteoglycan, we have shown that approximately one-third of the outbred New Zealand white rabbits we have examined possess naturally occurring antibodies which react with oligosaccharides of hyaluronic acid (independently of chain length) bearing saturated and 4,5-unsaturated glucuronosyl residues at the nonreducing ends. Such antibodies were also found in a similar proportion of rabbits with an experimental inflammatory arthritis. There was a preferential reactions in the majority of sera with unsaturated oligosaccharides of hyaluronic acid. One serum (R64) reacted only with unsaturated oligosaccharides of hyaluronic acid. Sera reacted also with unsaturated (never saturated) oligosaccharides of chondroitin 4-sulfate and with chondroitin 6-sulfate, particularly when chondroitin sulfate oligosaccharides remained bound to a proteoglycan core protein. Reactions were also observed to both unsaturated and saturated oligosaccharides of chondroitin. Some of these sera also reacted with intact hyaluronic acid and chondroitin but never with intact chondroitin sulfate. The antibodies were present in the IgG fraction of four sera studied and in the IgM fraction of one of these sera: they bound through the F(ab')2 region of the molecule. These observations suggest that, in some rabbits, humoral immunity to hyaluronic acid and/or chondroitin sulfate bound to core protein can develop after these reactive glycosaminoglycans have been degraded by eliminases or hydrolases produced by naturally occurring bacteria and rabbit cells, respectively. Immunological studies of proteoglycans and hyaluronic acid treated with eliminases and hydrolases employing rabbit antisera, and possibly those from other species, should be evaluated in the light of these observations.

Animals↗
PubMed1985

Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages.

Two species of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from mature bovine articular cartilages. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis at low ionic strength in 0.01 M phosphate the dermatan sulfate proteoglycans appeared as a single polydisperse species whose molecular weight ranged from 80,000 to 140,000. The dermatan sulfate proteoglycans eluted as a single peak on Sepharose CL-4B chromatography in 4 M guanidine hydrochloride and showed no tendency to separate into two components. Following chondroitinase AC and ABC digestion, a core protein was obtained whose molecular weight was 45,000. However, what appeared to be a single dermatan sulfate proteoglycan was consistently separated into two species of distinctly different mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at high ionic strength in 0.375 M Tris. The molecular weight of the smaller species (DS-PGII) ranged from 87,000 to 120,000. The molecular weight of the larger species (DS-PGI) ranged from 165,000 to 285,000. DS-PGI self-associates in 0.375 M Tris, while DS-PGII does not. This phenomenon was exploited to separate DS-PGI and DS-PGII by preparative electrophoresis on 5 to 20% gradient slab gels. The immunological identities of the individual species, DS-PGI and DS-PGII, were examined by enzyme-linked immunosorbent assay using polyclonal antiserum to cartilage-specific proteoglycan monomer from bovine articular cartilage and polyclonal and monoclonal antibodies to DS-PGII. The polyclonal antiserum to cartilage-specific proteoglycan monomer did not react with DS-PGI or DS-PGII, indicating that DS-PGI and DS-PGII possess different core proteins from cartilage-specific proteoglycan monomer. Polyclonal and monoclonal antibodies raised against the mixture of DS-PGI and DS-PGII reacted strongly with DS-PGII, but weakly or not at all with DS-PGI. These results suggest that DS-PGI and DS-PGII possess different core proteins and may represent two different species of dermatan sulfate proteoglycans.

Animals↗
PubMed1985

Secretion of a cysteine proteinase from a hormone-independent cell population of cultured explants of murine mammary gland.

Cultured explants of mouse mammary gland were investigated for their capacity to secrete a cathepsin B-like cysteine proteinase. This enzyme had been shown previously to be secreted excessively from cultured explants of human breast tumors and spontaneous mouse mammary carcinomas. We now show that secretion is also observed from cultured explants of mammary gland. Lactating tissue and tissues obtained from mid- and late-pregnant mice were found to secrete the cysteine proteinase at very high rates, but secretion was also detectable from explants of virgin mammary glands and from tissues obtained from retired breeders. However, in all cases, it was found that secretion was greatest from explants maintained in hormone-free medium and did not depend on the maintenance of normal mammary gland function. Secretion was greatly reduced in the presence of the lactogenic hormone combination of insulin, prolactin, and hydrocortisone, and this suppression was found to be due to hydrocortisone. Insulin and prolactin, while resulting in better tissue maintenance in culture, had no effect on the secretion. Enzyme release was reversibly inhibited by cycloheximide and required the presence of viable tissue, ruling out the possibility that the accumulation of enzyme activity in the culture medium is due to dying cells. The presence of metabolically active cells in explants cultured in the absence of hormones was also demonstrated by the incorporation of radiolabeled precursors into protein and DNA. DNA synthesis in cultured explants of lactating tissue was not stimulated by the addition of hormones. Histological studies revealed that, while large areas of the explants showed severe degeneration after culture without added hormones, clusters of cells persisted which displayed a relatively high mitotic activity and which showed a lack of normal epithelial organization. These observations suggest the presence of a hormone-independent cell population in the mammary gland, which secretes the stable cysteine proteinase. The secretion process itself, however, is inhibited by corticosteroids.

Animals↗
PubMed1984

Cartilage link proteins. Biochemical and immunochemical studies of isolation and heterogeneity.

Native and clostripain-derived link proteins from proteoglycan aggregates were separated by gel chromatography in 4 M guanidine HCl from low-buoyant-density proteoglycan and proteoglycan hyaluronic acid-binding region after extraction from adult bovine nasal cartilage and the Swarm rat chondrosarcoma. Separations were monitored with Laurell immunoelectrophoresis using precipitating antibodies to link protein, hyaluronic acid-binding region, and low-buoyant-density proteoglycan. These immunoanalyses, with sodium dodecyl sulfate-polyacrylamide gel analyses, revealed the usefulness of this combined approach when assessing the purities and identities of these molecules. Using these isolated link proteins we provide data suggesting the presence of two immunologically detectable forms of link proteins of the same molecular weight. On immunoelectrophoresis both rat and bovine link proteins of a single molecular weight each produced two precipitin reactions with antibody to link protein. They were of similar intensity for native molecules but of different intensity for clostripain-isolated link protein. Isoelectric focusing in 6 M urea revealed that the 48,000 and 44,000 molecular weight link proteins of bovine nasal cartilage together produce a complex pattern of many bands. Link proteins of a single molecular size produced a much simpler, predominantly five-banded focusing pattern. Immunoelectrophoresis of the electrofocused clostripain rat link protein (42,000) revealed that the three major central bands each produced double precipitin reactions. Mixing of native and clostripain-derived link protein in 4 M guanidine HCl followed by dialysis to 6 M urea prior to isoelectric focusing did not change the focusing position of the individual bands. This suggests that the heterogeneity of focusing forms was due to the existence of different isoforms. The double precipitin reactions may be due to the existence of two different conformations expressing different epitopes of the kind reported previously by Thonar et al. (Thonar, E. J.-M. A., Kimura, J. H., Hascall, V. C., and Poole, A. R. (1982) J. Biol. Chem. 257, 14173-14180).

Animals↗
PubMed1984

The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage.

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.

Adult↗
PubMed1984

Extracellular presence of the lysosomal proteinase cathepsin B in rheumatoid synovium and its activity at neutral pH.

The presence of the lysosomal proteinases cathepsin B and cathepsin D at extracellular sites in rheumatoid synovium was demonstrated using the antibody capture technique. Unlike cathepsin D, the cysteine proteinase cathepsin B was commonly detected only at the edges of the synovial explants. Radioimmunoassay and enzyme activity assay of these proteinases demonstrated that both were released from rheumatoid synovial cells in comparable amounts. Since lysosomal cathepsin B is unstable and denatured at physiologic pH and the antibody used only recognizes inactivated enzyme, we believe the selective detection of cathepsin B at the edge of the synovium may be due to the proteinase maintaining a native conformation within the explant, where the pH may be low enough to permit this. By use of a fluorescent substrate in a sensitive, continuous enzyme assay, cathepsin B was shown to express significant activity at neutral and alkaline pH before being inactivated. This and earlier work from this laboratory indicate that cathepsin B secreted by rheumatoid synovial cells may possess extracellular activity in vivo and be involved in the degradation of connective tissue macromolecules.

Antigen-Antibody Complex↗
PubMed1984

Activation of latent collagenase by serum proteinases that interact with immobilized immunoglobulin G.

It has been previously observed that collagen destruction occurs in the vicinity of immune complexes present in articular cartilages of patients with rheumatoid arthritis. When IgG is covalently linked to Sepharose it behaves as if it has reacted with an antigen to form an immune complex, in that it binds the complement component C1 from human serum. Other serum components also interact with this matrix, though their interaction may not be specific for IgG. Two of these components were shown to possess proteolytic activity, one being kallikrein and the other having the properties of plasmin. Both of the activities could activate latent human collagenase. Whilst the binding of the plasmin activity is probably nonspecific, the binding of the kallikrein activity may be selective for IgG (although it is not certain whether this binding is direct or indirect via another molecule). These results therefore suggest that active proteinases such as plasma kallikrein may be selectively concentrated on immune complexes in vivo, where they may locally activate latent proteinases such as collagenase thereby initiating tissue destruction.

Antigen-Antibody Complex↗
PubMed1984

Connective tissue antigens stimulate collagenase production in arthritic diseases.

Peripheral blood mononuclear cells from patients with rheumatoid arthritis (n = 27), systemic lupus erythematosus (n = 24), juvenile rheumatoid arthritis (n = 30), osteoarthritis (n = 20), apparently healthy adults (n = 12), and nonarthritic children (n = 8) were exposed to several putative connective tissue antigens to determine if the monokine, mononuclear cell factor, was released. Release of this factor was detected by bioassay in which enhancement of collagenase production from human synovial cells or dermal fibroblasts was measured. The antigens, all of homologous tissue origin, included cyanogen bromide-derived peptides of type I, II, and III collagens, type I and II helical collagens, and cartilage proteoglycan. Of the subjects examined, 44% of the rheumatoid group, 42% of the systemic lupus group, 33% of the juvenile rheumatoid group but only 10% of the osteoarthritic group and 5% of the control group released monokine after exposure of peripheral blood mononuclear cells to at least one of these connective tissue antigens. Patients with rheumatoid arthritis most frequently responded to type II peptides (but not to type II helical collagen) although the frequencies of responses to type I peptides, type I helical collagen and proteoglycan were also elevated over levels observed in the control population. Positive responses in these patients typically occurred to only one antigen, were transient, often occurred close to the onset of arthritis, and appeared to be unrelated to disease activity. The profiles of responses in patients with juvenile rheumatoid arthritis and systemic lupus shared many features in common and were distinct from those of adult rheumatoid arthritis. Patients with systemic lupus or juvenile rheumatoid arthritis responded to all of the antigens tested. Positive responses often occurred simultaneously to several antigens. Responses to type II helical collagen were most common while sensitization to type II peptides was infrequently detected. Positive responses were transient, unrelated to overall disease activity, type of juvenile arthritis, or duration of disease in lupus patients. Stimulation of mononuclear cell factor release by connective tissue molecules and their degradation products may make an important contribution to the chronic inflammation commonly seen in these diseases.

Adult↗
PubMed1984

Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation.

We examined bovine fetal epiphyseal and growth plate cartilages by immunofluorescence microscopy and immunoelectron microscopy using monospecific antibodies to a newly discovered cartilage-matrix calcium-binding protein that we now call chondrocalcin. Chondrocalcin was evenly distributed at relatively low concentration in resting fetal epiphyseal cartilage. In growth plate cartilage, it was absent from the extracellular matrix in the zone of proliferating chondrocytes but was present in intracellular vacuoles in proliferating, maturing and upper hypertrophic chondrocytes. The protein then disappeared from the lower hypertrophic chondrocytes and appeared in the adjoining extracellular matrix, where it was selectively concentrated in the longitudinal septa in precisely the same location where amorphous mineral was deposited in large amounts as demonstrated by von Kossa staining and electron microscopy. Mineral then spread out from these "nucleation sites" to occupy much of the surrounding matrix. Matrix vesicles were identified in this calcifying matrix but they bore no observable morphological relationship to these major sites of calcification where chondrocalcin was concentrated. Since chondrocalcin is a calcium-binding protein and has a strong affinity for hydroxyapatite, these observations suggest that chondrocalcin may play a fundamental role in the creation of nucleation sites for the calcification of cartilage matrix in endochondral bone formation.

Animals↗
PubMed1984

The association of a newly discovered protein, called chondrocalcin, with cartilage calcification.

A newly identified calcium binding protein called chondrocalcin with two subunits of molecular weight approximately 35 000 has been studied in bovine, rat and human cartilage matrix using a monospecific polyclonal antibody. Although it is present in small amounts in non-calcifying cartilage, it occurs in local high concentrations wherever cartilage calcification is observed, namely in the calcifying part of the growth plate and in calcified articular cartilage. Immunoelectron microscopy revealed that it is present in exactly the same discrete sites where mineral is first detected. Thus it may act as a nucleating agent for apatite formation. It is deposited in the same sites where unusual local high concentrations of proteoglycan and link protein are detected by immunoelectron microscopy. Chondrocalcin may bind either directly or indirectly to these molecules. Its occurrence within hypertrophic chondrocytes immediately prior to its extracellular appearance suggests that it is synthesised and released by these cells. Its absence from osteoid during intramembranous calcification indicates a selective involvement in endochondral calcification.

Animals↗
PubMed1983

Age-related changes in the structure of proteoglycan link proteins present in normal human articular cartilage.

Link proteins were identified immunologically in human articular-cartilage protein preparations from various individuals. Irrespective of age, all cartilages contained three link proteins of mol.wts. 48000, 44000 and 41000. However, with increasing age, multiple additional components of mol.wts. 26000-30000 were commonly observed under conditions where disulphide bonds were reduced.

Aging↗
PubMed1983

Biochemical and morphologic studies of cartilage from the adult human sacroiliac joint.

The cartilage of the adult sacroiliac joint contains type II collagen and a high concentration of glycosaminoglycan. Furthermore, large aggregating proteoglycans and link proteins were extracted from the sacral cartilage. In these respects the sacroiliac cartilage is similar to that in the peripheral joints. However, while the organization of the collagen in the sacral cartilage is typical of an articular cartilage, the organization of the ilial cartilage is very different, and throughout its depth it possesses narrow collagen fibrils arranged parallel to the articular surface.

Adult↗