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Biomedical subjects

G Murphy

Publications and source records attributed to G Murphy.

At least 343 records · Page 19Linked to original sources
PubMed1990

The tissue metalloproteinase family and the inhibitor TIMP: a study using cDNAs and recombinant proteins.

Loss of connective tissue integrity occurs in many disease processes, including rheumatoid arthritis and osteoarthritis. Although there is a high incidence of these diseases in the developed world, there is no treatment that prevents the tissue damage that occurs. Several lines of evidence suggest that uncontrolled connective tissue metalloproteinase activity is responsible for the damage, and as a consequence the inhibition of these enzymes has become the target for therapeutic intervention. Several connective tissue metalloproteinases, including collagenase, stromelysin, and gelatinase, together with tissue inhibitors of metalloproteinases (TIMPs), have been described. Because of difficulties in isolating the metalloproteinases in sufficient quantity as pure separate enzymes, however, very little knowledge has accumulated about their detailed biochemistry. For similar reasons the way in which TIMPs inhibit tissue metalloproteinases is not yet fully understood. In this article it is shown how cloning metalloproteinase and TIMP cDNAs can provide information about the structure of these enzyme and inhibitor families and how the cDNAs can be used to generate recombinant cell lines from which enzymes and inhibitors can be readily purified for further studies.

Amino Acid Sequence↗
PubMed1990

Femoral neck fractures in patients receiving long-term dialysis.

The morbidity and mortality of 11 femoral neck fractures were analyzed to compare operative and conservative management of femoral neck fractures in dialysis patients. All fractures occurred in older men with severe cardiac, pulmonary, gastro-intestinal, and neurologic conditions and with advanced renal osteodystrophy. Six of the seven operated patients survived the surgery and achieved varying degrees of ambulation. Stability of the operated hip was excellent in each case. Post-operative complications included transient confusional state related to narcotics, pneumonia, decubitus ulcers, and severe hypoalbuminemia. All four patients who were managed conservatively died from complications of the fracture. Progressive deterioration was noted in each nonoperated patient, with confusion caused by narcotics and analgesics, pneumonia, hepatic coma, decubitus ulcers, severe depression, and severe hypoalbuminemia. Therefore, operative management was superior to conservative management for femoral neck fractures of patients receiving chronic dialysis with multiple medical problems and advanced renal osteodystrophy. Narcotics must be used with great caution, and efforts should be directed toward prevention of malnutrition and decubitus ulcers.

Aged↗
PubMed1989

Dissociation of tissue inhibitor of metalloproteinases (TIMP) from enzyme complexes yields fully active inhibitor.

Recombinant human tissue inhibitor of metalloproteinases (TIMP) forms complexes with high-Mr active recombinant stromelysin that are stable over long periods under physiological conditions. TIMP-stromelysin complexes could be dissociated in the presence of EDTA at pH 3, releasing free TIMP and destroying stromelysin activity. The dissociated TIMP was apparently unmodified, in contrast with other known protein inhibitors of metalloproteinases and many classes of serine-proteinase inhibitor, which are slowly cleaved.

Amino Acid Sequence↗
PubMed1989

Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members.

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.

Amino Acid Sequence↗
PubMed1989

Human and rat malignant-tumor-associated mRNAs encode stromelysin-like metalloproteinases.

Rat transin and human stromelysin 2 mRNAs, which have been associated with malignant tumors, code for potential proteins with significant sequence homology to the metalloproteinases collagenase and stromelysin. We have used an expression system that allows easy purification of these proteins after transfection of COS cells with a vector containing the corresponding cDNA. This system has allowed us to prepare transin and stromelysin 2 as active proteinases that are inhibited by inhibitors of metalloproteinases. Further analysis of these enzymes indicates that they degrade several components of the extracellular matrix including collagen types III, IV, and V and fibronectin, as well as gelatins formed from several denatured collagen types. In addition, both transin and stromelysin 2 are capable of activating procollagenase in vitro. Thus, in malignant tumors these proteinases may act, both directly and indirectly, to degrade the extracellular matrix and permit tumor invasion of neighboring tissues.

Animals↗
PubMed1989

Degradation of proteoglycan aggregate by a cartilage metalloproteinase. Evidence for the involvement of stromelysin in the generation of link protein heterogeneity in situ.

Cartilage proteoglycan aggregates were subjected to degradation by a metalloproteinase, capable of degrading proteoglycan, released from cartilage in culture. This proteinase was demonstrated to be immunologically identical with fibroblast stromelysin. An early release of hyaluronic acid-binding region and large glycosaminoglycan-attachment regions was observed. With increasing time the glycosaminoglycan-attachment regions were digested into smaller fragments and the hyaluronic acid-binding regions accumulated. The degradation of link proteins also occurred concomitantly with these events. Link proteins were converted into a component of similar size to that of the smallest native link protein component. N-Terminal sequence analysis of the three human link protein components indicated that they are all derived from the same protein core, which is closely homologous to that of the rat chondrosarcoma link protein. The two larger link proteins (Mr 48,000 and 44,000) contain the same N-terminal sequence, but they differ by the apparent presence of an N-linked oligosaccharide at residue 6 of the largest link protein component. The smallest link protein (Mr 41,000), however, has an N-terminal sequence equivalent to that commencing at residue 17 in the larger link proteins. It was found that the cartilage metalloproteinase cleaves link proteins in human neonatal cartilage proteoglycan aggregates at the His-16-Ile-17 bond, the same position at which the smallest link protein component appears to be derived naturally from the two larger link protein components. These results suggest that stromelysin secreted by chondrocytes can account for the increased accumulation of hyaluronic acid-binding regions and much of the degradation of link protein observed during aging within human articular cartilage.

Amino Acid Sequence↗
PubMed1989

Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.

The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.

Animals↗
PubMed1989

The role of plasminogen in cell-mediated collagen degradation.

The ability of VX2 tumour cells and chondrocytes to degrade radiolabelled collagen films was shown to be dependent on the presence of the serum proteinase plasminogen. Degradation of collagen films in the presence of plasminogen was inhibited by addition of exogenous TIMP indicating that such lysis was mediated by collagenase. VX2 cells required ten times less plasminogen than chondrocytes to effect comparable degradation; this result was probably related to the observation that VX2 cells did not synthesize the specific tissue inhibitor of metalloproteinases, TIMP.

Animals↗
PubMed1989

Collagenase is expressed by rabbit VX2 tumour cells in syngeneic and xenogeneic hosts.

Specific antisera for the connective tissue metalloproteinases, collagenase, gelatinase (type IV collagenase) and stromelysin were used to study their respective localizations in both rabbit primary VX2 tumours and in lung metastatic deposits (frozen immediately after excision). Collagenase was found within some cells of the primary tumour and also bound to the extracellular matrix at discrete sites. Previous studies suggest that this matrix staining represents active enzyme. Stromelysin and gelatinase had a more limited distribution, particularly the latter, but both showed cell and matrix staining. In the lung metastases collagenase and stromelysin occurred less frequently, although both cell and matrix staining were observed; gelatinase was not seen. When rabbit VX2 cells were transplanted into nude mice they grew as a discrete nodule. Cells within this nodule stained with the antiserum to collagenase, which recognizes rabbit but not mouse enzyme, and thus demonstrated that cells of tumoural origin synthesize collagenase in vivo. Stromelysin was also co-localized with collagenase in some tumour cells.

Animals↗
PubMed1989

Ultrastructural changes in red blood cells following pulsed irradiation in vitro.

The careful choice of a combination of laser parameters such as wavelength, pulse duration, and dose has provided a means for confining laser energy to specific targets within tissue such as oxyhemoglobin within the cutaneous microvasculature. In the process of achieving such vascular selectivity, certain ultrastructural changes in red blood cell (RBC) cytoplasm have been observed, such as the generation of intracytoplasmic electron-lucent spherical structures. These structures, ranging in size from 80 to 1000A, were seen in RBCs exposed to laser doses at and above threshold, and appeared to represent a morphologically novel form of highly-specific tissue injury. This in vitro study using RBC in phosphate buffered saline (PBS) was undertaken to better understand the mechanism(s) that could have been responsible for these unique morphologic changes. We conclude that the intracytoplasmic electron-lucent spherical structures seen within RBCs were heat-fixed molds formed around vaporized water bubbles and were not produced by the release of oxygen from the oxyhemoglobin moiety during 577-nm laser irradiation.

Erythrocytes↗
PubMed1989

Gingival fibroblasts degrade type I collagen films when stimulated with tumor necrosis factor and interleukin 1: evidence that breakdown is mediated by metalloproteinases.

We previously suggested that periodontal pathogens might mediate connective tissue degradation in periodontal diseases through the ability of antigens from their cell walls to stimulate cytokine production by circulating mononuclear cells. Such cytokines would then induce metalloproteinase (MP) synthesis by resident gingival cells and thus initiate matrix degradation. In the present investigation human gingival fibroblasts (HGFs) were grown on [14C]-labelled type I collagen films and stimulated with either tumor necrosis factor (TNF) or interleukin-1 (IL-1) for 48 h. Collagenolysis occurred in a dose-dependent manner; the optimal dose for human rTNF alpha was 100 ng/ml and for rIL-1 alpha and rIL-1 beta, 1 ng/ml. Collagen degradation was accompanied by increased synthesis and release of the MPs collagenase, gelatinase and stromelysin, and there was a reduction in free TIMP (tissue inhibitor of metalloproteinases): collagenase and stromelysin were detected in both active and latent forms. Cytokine-stimulated collagenolysis was abolished by the addition of exogenous human rTIMP (5 units/ml). We also measured collagenase and TIMP by ELISAs which recognize all forms of collagenase (latent, active or complexed) and TIMP (free or complexed). These showed that while collagenase activity (0.6-1.2 microgram/ml) correlated with lysis, total TIMP levels remained unchanged at approximately 0.2 microgram/ml. These results demonstrate important roles for MPs and TIMP in regulating type I collagen degradation by HGFs, and support the hypothesis that connective tissue destruction during inflammatory diseases may be initiated, at least in part, by TNF and IL-1.

Cells, Cultured↗
PubMed1989

Gelatinase (type IV collagenase) immunolocalization in cells and tissues: use of an antiserum to rabbit bone gelatinase that identifies high and low Mr forms.

An antiserum was raised to rabbit bone gelatinase (type IV collagenase). It was shown by immunoblotting to detect both the low Mr (72,000) enzyme produced by connective tissue cells from rabbit, pig, human and mouse, as well as the high Mr (94,000-97,000) enzymes secreted by macrophages and polymorphonuclear leucocytes from these species, and by rabbit chondrocytes and endothelial cells. Crossed immunoblotting, antibody inhibition and deglycosylation studies indicated that the high and low Mr forms of gelatinase are immunologically distinct gene products, although their substrate specificity profiles are identical. The anti-gelatinase antiserum was used to immunolocalize the enzyme. Gelatinase was most efficiently detected in rabbit monocytes and connective tissue cells, but cells derived from the human and pig gave poor immunostaining, although mouse gelatinase stained well. The anti-gelatinase antiserum stained cells of the synovial tissue of rabbits at 14 days after induction of an antigen-induced arthritis, demonstrating its usefulness as a tool to assess the role of this enzyme in degradative events.

Animals↗
PubMed1989

Clinical significance of hemodialysis performed during the course of continuous ambulatory peritoneal dialysis.

We analyzed causes and clinical associations of hemodialysis performed during the course of CAPD in 71 patients (34 diabetic) on CAPD between 1978 and 1988. Thirty seven patients had at least one hemodialysis during CAPD. Causes (multiple for some patients) included poor compliance with CAPD (20 patients), peritoneal catheter loss from infection (16 patients), and miscellaneous other conditions (18 patients). Hemodialysis rates were similar between diabetic and non-diabetic CAPD patients. Compared to non-hemodialyzed patients, CAPD patients hemodialyzed for poor compliance had higher rates of peritonitis (one episode per 8.3 patient months vs one episode per 23.4 patient months, p less than 0.001) and catheter loss (one loss per 27.7 patient months vs one loss per 112.0 patient months, p less than 0.001), and exhibited a trend towards more deaths (annual mortality 30.9% vs 14.3%, p = 0.067). Hemodialysis performed in CAPD patients because of poor compliance is associated with higher rates of peritonitis and catheter loss, and, probably, with increased mortality.

Adult↗