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Biomedical subjects

A R Poole

Publications and source records attributed to A R Poole.

At least 181 records · Page 10Linked to original sources
PubMed1981

Biochemical and immunological studies of lysosomal and related proteinases in health and disease.

A review of continuing studies on the physiological and pathological roles of the lysosomal proteinases, cathepsin D and cathepsin B is presented. Intracellular release of cathepsin D from lysosomes has been demonstrated during myocardial ischemia. Both this proteinase and cathepsin B have been found, by organ culture in the presence of the appropriate antiserum, to be released into the extracellular space in rheumatoid synovium, where they may play a part in cartilage destruction. Also reviewed is the finding of a cathepsin B-like proteinase that, in organ culture, is secreted in markedly increased amounts from human malignant breast carcinomas in comparison to amounts secreted from benign tumors or normal tissue.

Arthritis, Rheumatoid↗
PubMed1981

Immunofluorescent localization of cathepsins B and D in human fibroblasts.

The proteinase cathepsin B has been localized in human fibroblasts using a monospecific antiserum that reacts in a precipitating manner with the denatured enzyme only. The distribution of staining as revealed by both direct and indirect immunofluorescence indicates a typical lysosomal localization similar to that observed for cathepsin D. Using dual localization methods employing fluorescein- and tetramethylrhodamine-labeled affinity purified antibodies both proteinases were usually observed in the same lysosome, indicating a combined role in intracellular digestion.

Cathepsin B↗
PubMed1980

Proteoglycans from bovine nasal cartilage. Immunochemical studies of link protein.

Antisera have been prepared against purified proteoglycan monomer and link protein from bovine cartilages. The immunological properties of these species have been studied by immunodiffusion, Laurell rocket immunoelectrophoresis, crossed immunoelectrophoresis, hemagglutination, and immunofluorescence. Antisera raised against either adult bovine articular or foetal bovine epiphyseal proteoglycan monomer were monospecific. They did not react with link protein, type II collagen, nor with any other molecular species isolated from bovine articular and epiphyseal cartilages. Antisera to link protein, purified from bovine nasal cartilage by dissociative density gradient centrifugation followed by chromatography under dissociative conditions on Sephacryl S-200, showed no reaction with proteoglycan monomer by immunodiffusion. However, antisera to link protein showed a weak reaction with native monomer on Laurell rocket immunoelectrophoresis (not detectable when chondroitinase ABC-treated monomer was used), and with chondroitinase ABC-treated monomer by hemagglutination assay. These traces of antibody to proteoglycan monomer were inactivated by immunoreaction with proteoglycan monomer in solution to provide a monospecific antiserum to link protein. Purified antibodies specific for proteoglycan monomer, some of which did not react with the hyaluronic acid binding region of bovine nasal monomer, were prepared from anti-link sera by affinity chromatography using chondroitinase ABC-treated bovine nasal proteoglycan monomer. Purified link protein, which contains 48,000- and 44,000-dalton subunits, contains two immunologically distinguishable species. An immunoassay for purified link protein was developed using Laurell rocket electrophoresis. A micro-method, using crossed-immunoelectrophoresis, for studying the binding of link protein to proteoglycan monomer and to hyaluronate is described. The complex of link protein bound to hyaluronate was dissociated on reaction of link protein with antibody.

Animals↗
PubMed1980

Characterization of a thiol proteinase secreted by malignant human breast tumours.

It has previously been demonstrated (Poole, A.R., Tiltman, K.J., Recklies, A.D. and Stoker, T.A.M. (1978) Nature 273, 545-547) that malignant human breast tumours maintained in organ culture secrete elevated amounts of a thiol proteinase. This enzyme has been shown to possess enzymic properties similar to those of cathepsin B (EC 3.4.22.1) with respect to specificity, affinity and pH optima for synthetic substrates. However, the tumour enzyme is much more stable than human liver cathepsin B to inactivation above neutral pH, and it also has a large molecular size and a more acidic isoenzyme pattern. The stability of this enzyme under physiological conditions may allow it to play a role in tumour invasion and metastasis.

Breast Neoplasms↗
PubMed1980

The distribution of lysosomal cathepsin D in cardiac myocytes.

Lysosomal cathepsin D has been localized with the electron microscope employing an indirect immunohistochemical method using peroxidase labeled, monospecific antibody Fab' subunits. The acid proteinase has been demonstrated within secondary lysosomes of cardiac myocytes and interstitial cells, but not in components of the Golgi apparatus or endoplasmic reticulum. Incubations with a variety of peroxidatic inhibitors suggests that the staining that is observed in secondary lysosomes is attributable to the peroxidase-labeled antibody and not to endogenous oxidation of DAB. The protocol outlined here provides a reproducible method to localize the major lysosomal acid proteinase of the heart at the subcellular level.

Animals↗
PubMed1980

Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study.

Using monospecific antisera and immunofluorescence microscopy, proteoglycan monomer (PG), and link proteins were demonstrated throughout the extracellular matrix of bovine articular cartilage. A narrow band of strong pericellular staining was usually observed for both molecules, indicating a pericellular concentration of proteoglycan monomer: this conclusion was supported by dye-binding studies. Whereas PG was evenly distributed throughout the remaining matrix, more link protein was detectable in interterritorial sites in middle and deep zones. Well-defined zones of weaker territorial staining for link protein stained strongest for chondroitin sulfate. Trypsin treatment of cartilage resulted in a loss of most of the PG staining, but some selective retention of link protein, particularly around chondrocytes in the superficial zone at and near the articular surface. This residual staining was largely removed if sections were fixed after chondroitinase treatment. After extraction of cartilage with 4M guanidine hydrochloride, only PG remained and this was concentrated in the superficial zone. These observations are shown to support the concept of aggregation of PG and link protein with hyaluronic acid (HA) in cartilage matrix, and the binding of PG and link protein to HA, which is attached to the chondrocyte surface. Culture of cartilage depleted of PG and link protein by trypsin demonstrated that individual chondrocytes can secrete both PG and link proteins and that the organization of cartilage matrix can be regenerated in part over a period of 4 days.

Animals↗
PubMed1980

Secretion of proteinases from malignant and nonmalignant human breast tissue.

Both malignant (adenocarcinomas) and nonmalignant (fibroadenomas and normal tissue) human breast tissues were maintained in organ culture for up to 10 days to study the secretion of lysosomal and neutral proteinases. Little difference was observed between the different tissue groups in the release of the lysosomal proteinase cathepsin D into the culture medium. Similar results were obtained when media were tested for plasminogen activator activity. The secretion of collagenolytic activity was investigated with fibroadenoma and adenocarcinoma explants and found to be very low for both tissue groups. The average accumulation of collagenase activity during a 2-day period was 0.002 units/microgram DNA for adenocarcinomas and 0.008 units/microgram DNA for fibroadenomas. The only proteinase that was secreted in substantially higher amounts from explants of malignant tissue was a cathepsin B-like thiol proteinase. Media from adenocarcinoma explants (n = 38) contained on the average 11 times more activity than did media from fibroadenoma (n = 20) and normal tissue explants (n = 8). Metastases of mammary adenocarcinomas (n = 7) secreted the thiol proteinase at about one third of the rate of primary tumors. The secretion of this enzyme is dependent upon protein synthesis as its release was completely inhibited 24 hr after the addition of cycloheximide. In some cases, it was also observed that the presence of sheep serum in the tissue culture medium reduced the accumulation of activity.

Adenofibroma↗
PubMed1980

Lysosomal alterations in hypoxic and reoxygenated hearts. II. Immunohistochemical and biochemical changes in cathepsin D.

Sublethal hypoxic injury in rat and rabbit hearts was accompanied by a biochemical redistribution of cathepsin D activity from the particulate to the supernatant fraction of the tissue homogenate, which was partially reversible on reoxygenation. Immunofluorescent staining for cathepsin D failed to reveal major anatomic release of the acid hydrolase until necrosis was present, suggesting that the earlier biochemical redistribution was primarily a result of increased lysosomal fragility during homogenization, with significant intracellular diffusion of the enzyme occurring only as irreversible damage took place. Hypoxia produced enlargement of both cathepsin-D-staining lysosomes and nonstaining vacuoles, as well as their aggregation. These changes were intensified during reoxygenation and recovery of reversibly damaged hearts, suggesting a possible role for the lysosomal-vacuolar apparatus in myocytic repair following hypoxic injury.

Animals↗
PubMed1980

Resistance to ischemic damage in hearts of starved rabbits: Correlation with lysosomal alterations and delayed release of cathepsin D.

Prolonged starvation produces dramatic changes both in the lysosomal properties of the heart and in its energy stores and, therefore, might be expected to alter some of the characteristic cardiac responses to ischemia. To test this possibility we ligated the circumflex coronary artery of rabbits that had been fed normally or starved for 6 days. Ultrastructural evidence of myocytic damage following 30 to 120 minutes of ischemia was much less severe in the starved animals than in the normally fed group. The development of signs of irreversible injury (e.g., osmiophilic densities in mitochondria) was delayed for 1 hour or more by starvation. A similar delay occurred in the biochemical redistribution of cathepsin D activity and in the cytoplasmic release of acid hydrolases from lysosomes and sarcoplasmic reticulum. These results indicate a marked protective effect of starvation against myocardial ischemia. In addition, both in starved and in fed animals, ischemically induced release of lysosomal enzymes was closely linked temporally to the development of subcellular damage.

Acid Phosphatase↗