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Biomedical subjects

G Murphy

Publications and source records attributed to G Murphy.

At least 271 records · Page 15Linked to original sources
PubMed1993

Initial validation of a test of spatial knowledge in anatomy.

The authors have developed HERCULES, a computer-based test designed to assess the spatial, non-verbal components of knowledge in anatomy. The test consists of two tasks, each requiring subjects to estimate the vertical level in the body of a set of color, cross-sectional images. In Task 1, subjects make the estimate based on a limited number of clues, where each clue is an anatomical structure that appears in the cross-section. In Task 2, subjects estimate the level based on a view of the cross-section with all structures shown. A validation study of this test using six images for each task was performed with preclinical medical students, fourth year medical students, and experienced teachers of anatomy as subjects. Results indicate that the exercise is at an appropriate level of difficulty and that a somewhat longer test than used in this study would be adequately reliable for use in actual assessment. The test appears to discriminate the expert faculty from more novice students and thus exhibits an aspect of validity that is very important in assessment exercises of this type.

Anatomy↗
PubMed1993

Rabbit models of arthritis: immunolocalization of matrix metalloproteinases and tissue inhibitor of metalloproteinase in synovium and cartilage.

The distribution of the matrix metalloproteinases, collagenase, stromelysin, gelatinases A and B, and the tissue inhibitor of metalloproteinases in cartilage and synovium removed from rabbits up to 27 days after induction of two models of arthritis was investigated by immunolocalization. Following intra-articular injection of poly-D-lysine/hyaluronic acid coacervate, collagenase and stromelysin were found bound to cartilage matrix, but there was little increase in chondrocyte synthesis of these enzymes. The synovium underwent a complex wound healing response involving invagination and encapsulation of the coacervate and inflammatory cell debris, during which all four metalloproteinases and tissue inhibitor of metalloproteinase could be immunolocalized. The second model, intra-articular injection of ovalbumin into sensitized rabbits, caused considerable chondrocyte necrosis; collagenase was found bound to cartilage matrix on day 13, although again there was little evidence of synthesis by chondrocytes. Inflammatory cell infiltration of meniscoid synovia took place initially, followed by fibrosis involving macrophagelike cells secreting gelatinase A. In both models there was rapid loss of glycosaminoglycan metachromasia from the cartilage matrix. These results are discussed in relation to current knowledge of metalloproteinase involvement in the chronic rheumatoid synovial pannus erosion of cartilage in humans. The data suggest that there are considerable differences between rheumatoid arthritis and these models, and their use must therefore be carefully defined.

Animals↗
PubMed1993

Detection and localization of mRNAs encoding matrix metalloproteinases and their tissue inhibitor in human breast pathology.

Matrix metalloproteinases (MMPs) are a group of enzymes thought to be responsible for both normal connective-tissue-matrix remodelling and the accelerated breakdown associated with tumor development. These MMPs and tissue inhibitor of MMPs (TIMP1) could be expressed by either the cancer or the stromal cells. Expression of mRNAs encoding interstitial collagenase (MMP1), 72-kD type IV collagenase (MMP2) and stromelysin (MMP3), which are probably involved in tumor invasion and metastasis, and of TIMP1 were studied in human mammary pathology by in situ hybridization and Northern blot analysis. Out of 6 benign lesions, 2 expressed MMP2 mRNAs. mRNAs encoding MMP1 and MMP3 were detectable in occasional stromal and tumor cells in 2 out of 17 carcinomas. Thirteen out of 17 cancers expressed MMP2 mRNA throughout the tumor in stromal cells close to noninvasive tumor clusters and well-differentiated invasive cancer cells. TIMP1 mRNA expression was detected in noninvasive and well-differentiated invasive tumor cells. These data suggest that there is a cooperation between tumor and stromal cells, in particular for the production of 72-kD type IV collagenase, involved in the disruption of basement membranes. A lack of TIMP1 expression from invasive cancer cells would also contribute to matrix destruction.

Blotting, Northern↗
PubMed1993

Women's satisfaction with birth control.

BACKGROUND: Contraception is a major component of preventive health care for women. There are indications that women are not satisfied with the methods of birth control currently available. Dissatisfaction with contraceptive methods may lead to unplanned pregnancies. METHODS: Adult women visiting the family health center over a 1-year period were invited to participate in a research interview. Questions were asked about demographic variables and the women's use of, and their satisfaction with, contraceptive methods. RESULTS: Many women were displeased with the present methods of birth control. This is reflected in the numerous methods used by each woman, and by frequent use of permanent sterilization as a contraceptive method. Women were as dissatisfied with oral contraceptives as they were with the less efficacious methods such as condoms, foams, gels, and rhythm. The only methods that had a greater than 70% satisfaction rate were tubal ligations and partner's vasectomies. CONCLUSIONS: There is significant dissatisfaction with the methods of contraception currently available. Increased patient-physician discussion and education may improve satisfaction with birth control methods now used. New methods of contraception may be needed to prevent unplanned pregnancies.

Adolescent↗
PubMed1992

Cell-mediated degradation of type IV collagen and gelatin films is dependent on the activation of matrix metalloproteinases.

The ability of normal rabbit dermal fibroblasts to degrade films of type IV collagen and gelatin when stimulated by phorbol ester was shown to be dependent on the induction, secretion and activation of 95 kDa gelatinase B and the secretion and activation of 72 kDa gelatinase A and stromelysin. Degradation was inhibited by exogenous human recombinant tissue inhibitor of metalloproteinases-1, specific antibodies to gelatinase and stromelysin and by the reactive-oxygen-metabolite inhibitor catalase. We discuss the various pathways for activation of matrix metalloproteinases in this model system and conclude that, although plasmin may play a key role in the activation of gelatinase B and stromelysin, gelatinase A is activated by a mechanism which has yet to be elucidated. The involvement of oxygen radicals in the direct activation of matrix metalloproteinases in this model is thought to be unlikely.

Animals↗
PubMed1992

Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: importance of the N-terminal region between cysteine 3 and cysteine 13.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.

Amino Acid Sequence↗
PubMed1992

The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.

The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.

Aggrecans↗
PubMed1992

Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.

Amino Acid Sequence↗
PubMed1992

The role of the C-terminal domain in collagenase and stromelysin specificity.

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.

Amino Acid Sequence↗
PubMed1992

The matrix metalloprotease matrilysin (PUMP) is expressed in developing human mononuclear phagocytes.

Matrilysin (PUMP, MMP-7) is a member of the metalloprotease gene family, whose constituents are responsible for the remodeling of extracellular matrix. The matrilysin protein is a 28-kDa zymogen possessing catalytic activities against a broad range of extracellular matrix substrates including proteoglycans, gelatin, fibronectin, laminin, and elastin. To gain insights into the biological expression of matrilysin in human cell types, we generated a monospecific, polyclonal antibody against a 16-amino acid sequence derived from its catalytic domain, a region which lacked significant homology with other matrix metalloenzymes. We found this antibody capable of precipitating a 28-kDa protein from the conditioned media of human bone marrow-derived promonocytes and human peripheral blood monocytes cultivated in vitro. Promonocyte matrilysin was rapidly converted to a 19-kDa form by organomercurial activation. While matrilysin was constitutively synthesized by bone marrow-derived promonocytes, its secretion was markedly up-regulated by the mononuclear phagocyte activator, lipopolysaccharide. Furthermore, despite its expression in monocyte precursors, blood monocytes, and monocyte-derived macrophages, matrilysin was not synthesized by human alveolar macrophages under any tested condition. In situ hybridization studies with matrilysin cRNA confirmed the presence of specific mRNA in both human promonocytes and monocytes. Moreover, a marked increase in hybridizable mRNA was observed with lipopolysaccharide treatment suggesting that matrilysin synthesis is pretranslationally regulated. In summary, this represents the first report documenting constitutive and regulated synthesis of matrilysin by a normal human cell type and suggests that matrilysin is expressed as a significant secreted product of mononuclear phagocytes at an intermediate stage of cellular differentiation.

Amino Acid Sequence↗
PubMed1992

The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases.

Recombinant 72 kDa gelatinase A and a truncated form lacking the C-terminal domain were shown to be activated by organomercurials and to possess similar activities towards a number of substrates. The truncated proenzyme differed from the full-length gelatinase in that it could not be activated by a membrane activator and did not bind tissue inhibitor of metalloproteinase (TIMP)-2. Kinetic studies also showed that the inhibition of the activated truncated enzyme, by both TIMP-1 and TIMP-2, was considerably decreased compared with the full-length enzyme. We conclude that the C-terminal domain plays an important role in the regulation of gelatinase A by a potential physiological activator and inhibitors.

Base Sequence↗
PubMed1992

A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases.

(7-methoxycoumarin-4-yl)Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L- 2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly fluorescent 7-methoxycoumarin group is efficiently quenched by energy transfer to the 2,4-dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3.4.24.23) cleaves the substrate at the Gly-Leu bond with a 190-fold increase in fluorescence (lambda cx 328 nm, lambda cm 393 nm). In assays of the human matrix metalloproteinases. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 is about 50 to 100 times more sensitive than dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (kcat/Km) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.

Amino Acid Sequence↗
PubMed1992

The matrix metalloproteinases and their natural inhibitors: prospects for treating degenerative tissue diseases.

Uncontrolled matrix metalloproteinase activity is thought to be a cause of the tissue damage observed in many disease processes. None of the drugs currently in use can prevent tissue destruction, and strategies for the development of synthetic inhibitors have been hampered by a poor understanding of the biochemistry of matrix metalloproteinases. Recent cDNA cloning efforts and characterization of recombinant human matrix metalloproteinases have permitted structure-function analysis of the enzymes and their inhibitors. Progress in this area should help indicate a route to rational strategies for designing lead therapeutic compounds.

Amino Acid Sequence↗
PubMed1992

Inverted repeats in the long-terminal repeats of the wheat retrotransposon Wis 2-1A.

The wheat insertion sequence Wis 2-1A possesses all the structural features characteristic of retrotransposons. Its long-terminal repeats (LTRs) are unusually long (1,755 bp) compared with those of other retrotransposons. Sequence analysis revealed that they differ from each other by only six point mutations. They contain a few tandem direct repeats, which could be explained by slippage mechanisms during replication. Almost half (44%) of the length of the LTRs is occupied by hairpin structures, which may relate to their large size. Possible origins of these inverted repeats are proposed, including the insertion and imprecise excision of transposable elements and errors when the DNA replication intermediate switches RNA template during retrotransposon replication.

Base Sequence↗
PubMed1992

The ambulatory medical record project at Group Health Cooperative: what did a decade of experience teach us?

Computer-based record systems are documented to improve patient care (Barnett, 1984; McDonald & Tierney, 1988) and their importance in the future is widely accepted. The report of the Institute of Medicine on patient computer-based record systems (Detmer, 1991) will help guide the development of future computer-based record systems and will likely stimulate renewed interest in them. We believe computing systems have great value to an HMO but understand that the benefits do not come without the risk of setbacks. We plan to build on what we have learned from our decade of experience.

Ambulatory Care Information Systems↗
PubMed1992

The transoral approach to the cervical spine.

The transoral surgical approach is useful for operating on structures at the base of the brain and the upper cervical spinal cord. For example, this route has been used for resecting spinal tumors and clipping vertebrobasilar aneurysms. In the past, this surgical approach was not advocated due to concerns about exposure and infection. However, the availability of the microscope, computed tomography, computed myelotomography, magnetic resonance imaging and intraoperative radiography as well as more effective techniques have improved the diagnosis of pathology of the craniovertebral junction and surgical performance. An understanding of the operative procedures involved with this approach assists the neuro-science nurse in preoperative teaching and anticipating potential postoperative complications.

Arthritis, Rheumatoid↗