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Biomedical subjects

D R Eyre

Publications and source records attributed to D R Eyre.

At least 109 records · Page 6Linked to original sources
PubMed1987

The carboxypropeptide trimer of type II collagen is a prominent component of immature cartilages and intervertebral-disc tissue.

Immature bovine cartilages and intervertebral-disc tissue all revealed a prominent protein, not present in the adult tissues, in non-denaturing extracts made with chondroitin ABC lyase (EC 4.2.2.4), Streptomyces hyaluronidase (EC 4.2.2.1) or 1 M NaCl. The protein ran on SDS-polyacrylamide electrophoresis, before disulphide reduction, as a close doublet of bands of apparent molecular weight 110,000 and 105,000. After reduction, they dissociated respectively into two protein bands at 37,000 and 35,000, indicating that the initial molecules were disulphide-bonded trimers. Amino-terminal sequence analysis established the identity of both proteins (Mr 110,000 and Mr 105,000) as forms of the carboxypropeptide of type II collagen. The larger molecule appeared to be the trimer of intact alpha 1(II) carboxypropeptides and the smaller, a version composed of chains that were ten residues shorter at their amino-terminal ends. The material appears to be identical to chondrocalcin, a protein previously found to be enriched in fetal growth plate and named on the basis that it may play a role in cartilage calcification. The present findings, however, indicate that the protein is equally abundant in all type II collagen-synthesizing young cartilages, including nucleus pulposus of the intervertebral disc and other cartilages that never calcify.

Amino Acid Sequence↗
PubMed1987

Type VI collagen of the intervertebral disc. Biochemical and electron-microscopic characterization of the native protein.

The collagen framework of the intervertebral disc contains two major fibril-forming collagens, types I and II. Smaller amounts of other types of collagen are also present. On examination of the nature and distribution of these minor collagens within bovine disc tissue, type VI collagen was found to be unusually abundant. It accounted for about 20% of the total collagen in calf nucleus pulposus, and about 5% in the annulus fibrosus. It was discovered by serially digesting disc tissue with chondroitin ABC lyase and Streptomyces hyaluronidase that native covalent polymers of type VI collagen could be extracted. Electron micrographs of this material prepared by rotary shadowing revealed the characteristic dimensions of tetramers and double tetramers of type VI molecules, with their central rods and terminal globular domains. Molecular-sieve column chromatography on agarose under non-reducing non-denaturing conditions gave a series of protein peaks with molecular sizes equivalent to the tetramer, double tetramer and higher multimers. On SDS/polyacrylamide-gel electrophoresis after disulphide cleavage, these fractions of type VI collagen all showed a main band at Mr 140,000 and four lesser bands between Mr 180,000 and 240,000. On electrophoresis without disulphide cleavage in agarose/2.4% polyacrylamide only dimeric (six chains) and tetrameric (12 chains) forms of type VI molecules were present. The ability to extract all the type VI collagen of the tissue in 4 M-guanidinium chloride, and absence of aldehyde-mediated cross-linking residues on direct analysis, showed that, in contrast with most matrix collagens, type VI collagen does not function as a covalently cross-linked structural polymer.

Animals↗
PubMed1987

Collagen type IX: evidence for covalent linkages to type II collagen in cartilage.

A major site of pyridinoline cross-linking in bovine type IX collagen was traced to a tryptic peptide derived from one of the molecule's HMW chains. This peptide gave two amino acid sequences (in 2/1 ratio) consistent with it being a three-chained structure. The major sequence matched exactly that of the C-telopeptide of type II collagen from the same tissue. A second HMW chain that contained pyridinoline cross-links also gave two amino-terminal sequences, one from its own amino terminus, the other matching exactly the N-telopeptide cross-linking sequence of type II collagen. We conclude that type IX collagen molecules are covalently cross-linked in cartilage to molecules of type II collagen, probably at fibril surfaces.

Animals↗
PubMed1987

Streaming potentials: a sensitive index of enzymatic degradation in articular cartilage.

Under physiological conditions, the extracellular matrix of articular cartilage contains a high fixed-charge density, associated with its ionized proteoglycan (PG) molecules. Compression of the highly charged cartilage matrix within the physiologic range leads to the production of electrical streaming potentials. We observed significant changes in the potential response due to chemical modifications of the matrix, such as extraction of PG and glycosaminoglycan (GAG) moieties using chondroitinase-ABC adn trypsin. The streaming potential was a sensitive index of the degradative loss of these matrix constituents and of the kinetics of the enzymatic degradative process.

Animals↗
PubMed1987

A growing family of collagens in articular cartilage: identification of 5 genetically distinct types.

Cartilage displays an extensive polymorphism of collagen types. All hyaline cartilages are known to contain type II collagen (90-95% of the collagen) and 2 other cartilage-specific minor collagens called types IX (or M) and XI (or 1 alpha 2 alpha 3 alpha). In the present work, the molecular heterogeneity of articular cartilage collagen was investigated in more detail. Two additional genetic types of collagen were found in the mature bovine tissue, types V and VI, each accounting for about 1-2% of the tissue dry weight. Type V, a close relative of type XI, apparently had accumulated after birth. Type VI is a short-helix, microfibrillar collagen of wide tissue distribution but unknown function. In understanding the physical changes in the collagen network of cartilage that are believed to herald the onset of cartilage degeneration in osteoarthrosis, all five of these collagen types deserve attention.

Animals↗
PubMed1986

Nonexpression of cartilage type II collagen in a case of Langer-Saldino achondrogenesis.

A lethal short-limbed dwarfism was diagnosed at autopsy as the Langer-Saldino variant of achondrogenesis by radiological, histological, and gross pathological criteria. Cartilage was obtained for biochemical and ultrastructural analyses from the ends of long bones, from ribs and from a scapula of the newborn infant. At all sites, it had an abnormal gelatinous texture and translucent appearance. Biochemical analyses of the cartilages to identify pepsin-solubilized collagen alpha-chains and collagen-specific CNBr-peptides failed to detect type II collagen at any site where it would normally be the main constituent. Instead, type I was the predominant collagen present. However, three cartilage-specific minor collagen chains identified as 1 alpha, 2 alpha, and 3 alpha chains by their electrophoretic mobility were present at about 10% of the total collagen. Cartilage-specific proteoglycans also appeared to be abundant in the tissue judging by its high hexosamine content and high ratio of galactosamine to glucosamine. The findings indicate that a chondrocyte phenotype had differentiated but without the expression of type II collagen. In addition to the skeletal abnormalities, the severe pulmonary hypoplasia was also felt to be directly related to the underlying pathology in collagen expression. The term chondrogenesis imperfecta rather than achondrogenesis should be considered a more accurate description of this and related conditions.

Bone and Bones↗
PubMed1985

A heterozygous collagen defect in a variant of the Ehlers-Danlos syndrome type VII. Evidence for a deleted amino-telopeptide domain in the pro-alpha 2(I) chain.

A structural defect in the alpha 2(I) chain of type I collagen was characterized in a new case of the Ehlers-Danlos syndrome type VII. The patient's skin, fascia, and bone collagens all showed an abnormal additional chain, pN-alpha 2(I)s, running slower than the alpha 2(I) chain on electrophoresis. The extension was shown to be on the amino-terminal fragment of pN-alpha (I)s by cleavage with human collagenase, but pepsin was unable to convert pN-alpha 2(I)s to alpha 2(I). Skin collagen was 4-fold more extractable and contained fewer beta-dimers and a lower concentration of cross-linking amino acids than control skin collagen. Electron micrographs of both dermis and bone showed markedly irregular ragged outlines of the collagen fibrils in cross-section, although the patient had no clinical signs of bone disease. Procollagen secreted by her skin fibroblasts in culture showed equal amounts of the normal and abnormal alpha 2(I) chains on pepsin digestion. Before pepsin, the pN-alpha 2(I) component ran as a doublet on electrophoresis; pepsin removed only the normal slower chain. The suspected deletion in pN-alpha 2(I)s was traced by CNBr peptide analysis to the N-propeptide fragment, which behaved on electrophoresis about 15-20 residues smaller than that from the normal pN-alpha 2(I) chain. The simplest genetic explanation is a spontaneous heterozygote in which one normal and one abnormal allele for the pro-alpha 2(I) gene are expressed, the protein defect being a deletion of the junction domain that spans the N-propeptidase cleavage site and the N-telopeptide cross-linking sequence.

Bone and Bones↗
PubMed1984

Cartilage type IX collagen is cross-linked by hydroxypyridinium residues.

Type IX collagen, a recently discovered, unusual protein of cartilage, has a segmented triple-helical structure containing interchain disulfides. Its polymeric form and function are unknown. When prepared by pepsin from bovine articular cartilage, type IX collagen was found to contain a high concentration of hydroxypyridinium cross-links, similar to that in type II collagen. Fluorescence spectroscopy located the hydroxylysyl pyridinoline and lysyl pyridinoline cross-linking residues exclusively in the high-molecular-weight collagen fraction, from which they were recovered predominantly in a single CNBr-derived peptide. The results point to a structural role for type IX collagen in cartilage matrix, possibly as an adhesion material to type II collagen fibrils.

Animals↗
PubMed1984

Identification of hydroxypyridinium cross-linking sites in type II collagen of bovine articular cartilage.

In mature cartilage, collagen fibrils are strengthened by covalent intermolecular bonds provided by 3- hydroxypyridinium cross-linking residues. To determine the location of these trifunctional cross-links within the type II collagen molecule, CNBr peptides were analyzed from pepsin-soluble collagen and from guanidine hydrochloride insoluble collagen of bovine articular cartilage. The presence of hydroxypyridinium residues in collagen alpha chains and CNBr-derived peptides was detected by their characteristic natural fluorescence. Quantitatively, about one in three alpha chains from pepsin-soluble collagen was found to contain a hydroxypyridinium residue. Its distribution in the chains was limited to two CNBr peptides, which were purified by column chromatography on CM-cellulose and Bio-Gel P-30 followed by slab-gel electrophoresis in sodium dodecyl sulfate-polyacrylamide. The composition and properties of the two peptides indicated that the main component of one was alpha 1(II)- CB9 ,7 and of the other alpha 1(II) CB12 . It was suspected that two amino-terminal telopeptides were cross-linked by hydroxylysylpyridinoline to alpha 1(II) CB9 ,7 and two carboxy-terminal telopeptides to alpha 1(II) CB12 . The properties of fluorescent CNBr peptides isolated from digests of insoluble cartilage collagen supported this conclusion. Cleavage of the 3- hydroxypyridinium ring by UV light was exploited to confirm the identity of the cross-linked peptides. On UV irradiation, one cross-linked peptide released alpha 1(II) CB9 ,7, and the other, alpha 1(II) CB12 . The findings indicate there are only two hydroxypyridinium cross-linking sites within the triple-helical region of the type II collagen molecule, probably placed symmetrically at opposite ends at residues 87 and 930, where telopeptide aldehydes are known to react to form the initial "head to tail" intermolecular bonds.

Amino Acids↗
PubMed1984

All three chains of 1 alpha 2 alpha 3 alpha collagen from hyaline cartilage resist human collagenase.

A previous report that the 3 alpha collagen chain of hyaline cartilage was cleaved by human collagenase could not be confirmed when the 1 alpha 2 alpha 3 alpha collagen fraction was freed of all contaminating type II collagen. All three minor collagen chains, 1 alpha, 2 alpha and 3 alpha, were totally resistant to highly purified collagenases from both rheumatoid synovial and gastric mucosal tissues. This finding and CNBr-peptide patterns suggest that, despite the close homology with alpha 1 (II), the 3 alpha chain is a unique collagen component, possibly combined with 1 alpha and 2 alpha in heterotrimeric molecules. In contrast, a 3 alpha-like component from fibrocartilage was cleaved by collagenase and gave a CNBr-peptide pattern more typical of alpha 1 (II) than of the collagenase-resistant 3 alpha of hyaline cartilage.

Animals↗
PubMed1984

Quantitation of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography.

An HPLC method for quantifying the 3-hydroxypyridinium crosslinks of collagen is described. It can be applied to crude hydrolysates of all types of connective tissue. Mineralized tissues can be hydrolyzed directly and analyzed without interference from the mineral ions. The hydroxylysyl (HP) and lysyl (LP) forms of hydroxypyridinium residue were resolved on a reverse-phase C18 column using a gradient of acetonitrile in water and 0.01 M n-heptafluorobutyric acid as an ion-pairing agent. The crosslinking amino acids were accurately quantified down to 2 PM (1 ng) injected, by detecting their natural fluorescence with a spectrofluorometer. Tissues in which hydroxypyridinium crosslinks were plentiful included all forms of cartilage, bone, dentin, ligament, tendon, fascia, intervertebral disc, lung, gut, cervix, aorta, and vitreous humor. Among normal tissues, LP, the minor form of the crosslink, was present in significant amounts relative to HP only in bone and dentin. Both crosslinks were essentially absent from skin, cornea, rat tail tendon, and basement membranes.

Amino Acids↗
PubMed1983

Collagen of fibrocartilage: a distinctive molecular phenotype in bovine meniscus.

Fibrocartilage of bovine knee meniscus was analyzed for major and minor collagen constituents. The main fraction (approximately 98%) of pepsin-solubilized collagen consisted of type I with a small proportion (less than 10%) of type III molecules. The minor fraction (1--2%) isolated by salt precipitation could be further resolved into type V collagen that consisted of alpha 1(V) and alpha 2(V) chains and a type II-like molecule with chains that had all the characteristics of the 3 alpha variant of alpha 1(II) found in hyaline cartilage. The articular surface zone of the meniscus appeared 2--3-fold enriched in these minor collagens compared with deeper tissue, though qualitatively the same distinctive collagen phenotype was evident throughout.

Amino Acids↗
PubMed1983

Early changes in material properties of rabbit articular cartilage after meniscectomy.

We have correlated early material and biochemical changes in articular cartilage in a surgical model for cartilage degeneration. Medial meniscectomy was performed on the left knee of 17 adult, female New Zealand white rabbits. The equilibrium Young's modulus of cartilage was assessed by an indentation test in situ at defined sites on the medial and lateral tibial plateaus of the operated and control knees; the cartilage was then excised and analyzed biochemically. Focal changes were consistently observed in the medial surface of the operated knee. The equilibrium modulus and the glycosaminoglycan content fell rapidly, reaching a minimum by 2 weeks after surgery; the lateral tibial surface was essentially unaffected. Six months after surgery, the glycosaminoglycan content had returned to normal and the modulus to near normal. Independent measurements on cored plugs from the medial surface 2 weeks after surgery revealed a significant decrease in both the dynamic stiffness and the streaming potential in the operated knee compared with the control. The findings suggest that normal ambulatory loads in vivo will deform the affected medial cartilage much more than normal. It remains to be seen if altered mechanical stresses are solely responsible for initiating and sustaining matrix remodeling by the chondrocytes.

Animals↗
PubMed1983

Collagen crosslinks in human dentin: increasing content of hydroxypyridinium residues with age.

The hydroxypyridinium crosslinks of collagen are believed to derive from reducible, divalent crosslinks. To study this concept further, both types of crosslink were quantified as a function of age in dentin, a tissue thought to have minimal collagen turnover. Human (5, 15, 28 and 56 years) and bovine (fetal and adult) root dentin was analyzed by a procedure that measures both hydroxypyridinium and reducible crosslinks on the amino acid analyzer. In human dentin, hydroxypyridinium crosslinks increased with age and became the predominant crosslinks as the two reducible residues, dehydrodihydroxylysinonorleucine and dehydrohydroxylysinonorleucine, diminished. Similarly in adult bovine dentin, hydroxypyridinium residues were sixfold more concentrated than in fetal bovine dentin. Borohydride treatment of tissue did not influence the measured content of hydroxypyridinium residues. The analyses also ruled out natural reduction as a stabilizing reaction for the divalent, reducible crosslinks. Though hydroxypyridinium residues became the major aldehyde-mediated crosslinks of adult dentin collagen, significant levels of reducible crosslinks remain throughout the tooth's adult life.

Adolescent↗