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Biomedical subjects

D E Woolley

Publications and source records attributed to D E Woolley.

At least 127 records · Page 7Linked to original sources
PubMed1979

Human collagenases: comparative and immunolocalization studies.

Four human collagenases derived from different tissues or cells were shown to have different physicochemical properties with regard to molecular size and protein charge. Such differences have been used to demonstrate that the collagenase activity of rheumatoid synovial fluids is of granulocytic origin. Immunolocalization studies have demonstrated that immunoreactive collagenase is present in a variety of human tissues. Its production by cells in normal tissues suggests a role in collagen remodelling processes, whereas its more frequent occurrence in diseased tissues suggests an important role in pathological collagen resorption. In nearly all the positive specimens examined the enzyme was restricted to a single cell or small groups of cells, or collagenous elements, suggesting microenvironmental rather than widespread collagenolytic activity.

Adenocarcinoma↗
PubMed1979

Collagenase production by rheumatoid synovial cells: morphological and immunohistochemical studies of the dendritic cell.

The dendritic cells of dissociated, adherent rheumatoid synovial cell cultures are recognised by their distinctive morphological features--compact cytoplasm around the nucleus and long, branched cytoplasmic extensions. Such cells usually composed approximately 10% of the total adherent cell population but could vary from as few as 2% to as many as 40% with different synovial specimens. Histological studies have shown the cells to contain many mitochondria and large, spherical cytoplasmic inclusions which often distort the dendritic extensions. Although lysosomes were observed, no evidence for phagocytic activity was obtained. Immunolocalisation studies by means of a monospecific antibody to human collagenase have shown that the dendritic cell attached to a collagenous substratum produces and releases this enzyme in vitro. In contrast collagenase was detected in only a few of the fibroblast- and macrophage-like cells, and it was always intracellular. It is proposed that the dendritic cell may have an important role in the pathophysiology of the rheumatoid joint, particularly with regard to collagenase-mediated cartilage destruction.

Arthritis, Rheumatoid↗
PubMed1978

Localization of the binding site for cell attachment in the alpha1(I) chain of collagen.

Certain cells such as CHO (Chinese hamster ovary) and fibroblasts attach to a substrate of type I collagen via proteins that link the cell surface to collagen. Serum contains a glycoprotein, c-CAP (collagen cell attachment protein), that can mediate this adhesion. Previously, we established that alpha1(I)-CB7 (cyanogen bromide peptide 7), which lies within residues 552 to 822 of collagen, contains a major binding site for c-CAP(alpha1(I)-CB7 has been renumbered to account for an additional triplet recently identified at residue 613 (P. P. Fietzek and R. W. Glanville, manuscript in preparation). Now we have examined the ability of various peptides derived by proteolytic digestion of alpha1(I)-CB7 to bind to c-CAP based upon their ability to inhibit cell attachment to collagen. The binding site lies within residues 757 to 791. It is likely that this is the sole binding site in the alpha1(I) chain since cleavage of the bond between residues 775 and 776 in the alpha1(I) chain destroys cell attachment activity.

Amino Acid Sequence↗
PubMed1978

Collagenase immunolocalization in cultures of rheumatoid synovial cells.

Cultures of rheumatoid synovial cells that have been enzymatically dissociated and are adherent to a culture vessel are morphologically heterogeneous. When these cells are cultured on a collagenous substrate for 2 to 6 days at 37 degrees C in serum-free medium, they produce collagenase. A monospecific antibody to human collagenase has localized the enzyme extracellularly around cytoplasmic extensions of dendritic cells and intracellularly within a few macrophage-like and fibroblast-like cells.

Arthritis, Rheumatoid↗
PubMed1978

Purification, characterization and inhibition of human skin collagenase.

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32mug of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5-8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25 degrees C, producing the two characteristic products TC(A)((3/4)) and TC(B)((1/4)). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25 degrees C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37 degrees C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the alpha-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37 degrees C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins alpha(2)-macroglobulin and beta(1)-anti-collagenase both inhibited the enzyme, but alpha(1)-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

Blood Proteins↗
PubMed1978

Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue.

1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.

Animals↗
PubMed1977

Effect of cartilage proteoglycans on human collagenase activities.

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.

Animals↗
PubMed1977

The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule.

1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.

Anaerobiosis↗
PubMed1977

Collagenase and its natural inhibitors in relation to the rheumatoid joint.

This report attempts to summarize our present knowledge of rheumatoid synovial collagenase and its natural serum inhibitors, beta1-anticollagenase and alpha2-macroglobulin, in relation to cartilage collagen resorption in the rheumatoid joint. Immunolocalization of collagenase across the cartilage/pannus junction is described, and in the light of the finding of the specific, small molecular weight beta1-anticollagenase we propose a model of cartilage erosion based on the interaction between collagenase and its natural inhibitors.

Arthritis, Rheumatoid↗
PubMed1976

A neutral collagenase from human gastric mucosa.

Biopsy specimens of human gastric mucosa, maintained in culture for 7 days in the absence of serum, released a collagen-degrading enzyme into the medium. The yield of active enzyme reached a maximum after 2-3 days, and viable tissue, capable of protein synthesis, was essential for its production. 2. At 25 degrees C the enzyme attacked undenatured collagen in solution, resulting in a 55% loss of specific viscosity and producing the two products TCA and TCB characteristic of neutral-collagenase action. 3. Electron microscopy of segment-long-spacing crystallites of these reaction products showed the exact cleavage locus of the collagen molecules to be between bands 43 and 44 (I-43). The larger TCA and smaller TCB products were fragments representing 77 and 23% respectively of the length of the collagen molecule. 4. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a mol.wt. of approx. 38000 was derived from gel-filtration studies. 5. The enzyme was shown to be inhibited by the human serum proteins alpha2-macroglobulin and a smaller component of mol.wt. approx. 40000; alpha1-anti-trypsin was not inhibitory. 6. EDTA, 1, 10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. 7. The gastric enzyme has properties similar to other well characterized collagenases, but differences exist with respect to its molecular size and the site of attack on the collagen molecule.

Blood Proteins↗
PubMed1976

Some aspects of neurophysiological basis of insecticide action.

When the insecticide parathion was administered to awake, unrestrained rats with chronically implanted brain electrodes, it was observed that the latency of the averaged flash-evoked potential in the visual cortex and superior colliculus was increased and the amplitude was decreased 2 to 4 hours later with responses returning to pretreatment levels about 8 hours after administration. Similarly, after administration of several dose levels of parathion in the rat, durations of phases of the maximal electroshock seizure (MES) pattern were altered to the greatest extent 4 hours later, but effects disappeared at 24 hours. These effects of parathion on the MES and evoked potentials coincided with a fall in blood and brain acetylcholinesterase (AChe) activities but disappeared after AChe inhibition had reached its peak and stabilized. Brain AChe activities required 2 to 4 weeks for recovery whereas blood AChe activity recovered in 1 week following inhibition by parathion (at least 2 mg/kg body weight). Studies in the monkey demonstrated similar results. Because these measurements of central nervous system function returned to normal despite continued inhibition of AChe activity, the results are interpreted to mean either that adaptation of evoked potentials or MES responses to prolonged AChe inhibition can occur in the rat and monkey after parathion administration or that some of the effects of parathion do not depend on AChe inhibition. Administration of DDT (100 mg/kg by mouth) to awake, unrestrained rats markedly increased the amplitude of spontaneous electrical activity in the cerebellum, whereas there was much less effect on electrical activity recorded simultaneously in the occipital cortex, reticular formation, and medial geniculate body. Similarly, DDT administration had marked effects on the averaged, sound evoked potential recorded in the cerebellum; DDT caused the appearance and increased the amplitude of an early component of this response not usually present during control recordings. Sound-evoked potentials recorded simultaneously from the frontal and occipital cortex and reticular formation were affected less or were decreased in amplitude by administration of DDT.

Acetylcholinesterase↗
PubMed1975

Action of rheumatoid synovial collagenase on cartilage collagen. Different susceptibilities of cartilage and tendon collagen to collagenase attack.

The action of purified rheumatoid synovial collagenase on purified cartilage collagen, alpha-1(II)-3, in solution at 25 degrees C has been characterised. The enzyme attacked cartilage collagen in solution producing a 58% reduction in specific viscosity and resulting in the appearance of two reaction products which represented approximately three-quarter and one-quarter fragments of the intact molecule as shown by disc electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The alpha-chain fragments which comprised each of these components corresponded to molecular weights of approximately 74000 and 21000. Electron microscopy of segment-long-spacing crystallites of the reaction products revealed three-quarter (TC-a) and one-quarter (TC-b) length fragments, and permitted accurate localization of the cleavage locus between bands 41 and 42 (I-41). This cleavage site and the formation of TC-a and TC-b reaction products are very similar to those found for type-I collagen substrates. Cartilage collagen in solution was found to be more resistant to collagenase attack than tendon collagen, the rate of cartilage collagen degradation being six times slower than that for tendon collagen, as judged by viscometry. The mid-point melting temperatures (T-m) for lathyritic cartilage and tendon collagen were 40.5 and 41.5 degrees C, and for the collagenase-produced reaction products 38.5 and 37.5 degrees C, respectively. The significance of these findings is discussed in relation to the structure of type I and II collagens.

Animals↗