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Biomedical subjects

E D Harris

Publications and source records attributed to E D Harris.

At least 91 records · Page 5Linked to original sources
PubMed1988

Electron microscopic studies of free and proteinase-bound duck ovostatins (ovomacroglobulins). Model of ovostatin structure and its transformation upon proteolysis.

Conformational changes of duck ovostatin (ovomacroglobulin) upon complexing with thermolysin have been studied by electron microscopy. Both free and thermolysin-bound ovostatin preparations were negatively stained with uranyl acetate, a series of three pictures were taken at 10 degrees specimen tilt intervals (+10 degrees, 0 degrees, and -10 degrees), and images of the inhibitor molecules were observed in three dimensions. Four approximately cylindrical subunits were observed in free ovostatin. Two subunits associated approximately midway from both ends to form a dimer of four arms. Two dimers associated with each other at the midpoint to form a tetramer. The proteinase susceptible "bait" regions were located near the center of the molecule. Eight arms of the tetramer take various configurations. The orthogonal extent of free tetrameric ovostatin in a two-dimensional micrograph averages 26.0 +/- 4.7 x 34.0 +/- 5.0 nm. Upon complexing with thermolysin, all eight arms curl toward the center of the molecule, having four arms upward and the other four downward. Thus, proteinase-bound ovostatin has a uniform structure with a 2-fold axis of symmetry. The overall structure of the complex is more compact with average dimensions of 16.9 +/- 0.6 x 16.9 +/- 0.6 x 19.9 +/- 0.4 nm. From these electron microscopic studies we propose that a proteinase reaches to the center of the free ovostatin molecule and attacks the bait region. Subsequent to proteolysis the subunit arms curl and entrap the enzyme within the ovostatin molecule. The results support the unique mechanism of inhibition of proteinases by alpha 2-macroglobulin and ovostatin postulated from biochemical observations (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724; Nagase, H., and Harris, E. D., Jr. (1983) J. Biol. Chem. 258, 7490-7498).

Algorithms↗
PubMed1988

Evaluation of clinical competence in rheumatology training programs.

To ensure that members of the public and the medical profession can identify physicians who have demonstrated excellence in the field of rheumatology, the American Board of Internal Medicine has called upon directors of rheumatology training programs to establish systems to evaluate, document, and substantiate those components of overall clinical competence that are considered essential for certification. Many of these abilities can be assessed only by repeated, direct observations.

Certification↗
PubMed1988

The cost effectiveness of auranofin: results of a randomized clinical trial.

In a 6-month randomized trial at 14 sites, the cost effectiveness of auranofin (AF) treatment for patients with rheumatoid arthritis was gauged in comparison with placebo. Measures of global health and of impacts on daily life suggest that the benefits of disease modification outweigh adverse effects after 4 and 6 months of treatment (p less than 0.01), with negligible differences between placebo and treated patients after 1 and 2 months. Additional medical costs directly associated with AF treatment amounted to $778/patient annually. Observed differences in less direct medical costs, help received, and earnings were not statistically significant.

Adult↗
PubMed1987

Regulation of aortic CuZn-superoxide dismutase with copper. Effects in vivo.

Cu2Zn2-superoxide dismutase (CuZn-SOD) was purified from chicken liver. The liver enzyme had a subunit Mr of 16900 and contained equimolar amounts of copper and zinc [0.26% (w/w) for each]. Aortic CuZn-SOD had the same Mr as estimated by gel filtration and cross-reacted with antibodies to the liver enzyme. Both enzymes were inhibited by 1.0 mM-NaCN. Within 24-72 h after hatching, total SOD activity in aorta rose 3-fold over the day-1 level and stayed elevated for 10 days. With low dietary copper, the total SOD activity rose as before, but then decayed progressively to non-detectable levels in 10 days. Both the cyanide-sensitive (CuZn-SOD) and insensitive (mangano-SOD) activities fell, but not at the same rate. When the 10-day-old deficient chicks were injected with 0.5 mumol of CuSO4 intraperitoneally, SOD activity in aorta was restored to control levels in about 8 h. Despite non-measurable SOD activity in aorta, extracts from the 15-day-old-deficient-chick tissue contained as much, or slightly more, immunoreactive CuZn-SOD protein as age-matched control tissue. The data show clearly that dietary copper regulates SOD activity in the aortas of young developing animals. They further suggest that a copper deficiency suppresses CuZn-SOD activity without inhibiting synthesis or accumulation of the CuZn protein in this tissue.

Animals↗
PubMed1987

Regulation of aortic CuZn-superoxide dismutase with copper. Caeruloplasmin and albumin re-activate and transfer copper to the enzyme in culture.

Caeruloplasmin and albumin were compared as potential donors of copper to Cu2Zn2-superoxide dismutase (CuZn-SOD) in culture. Aortas from 15-day copper-deficient chicks were suspended in oxygenated, serum-free, Waymouth medium (752/1) for 24 h. SOD activity was restored when the medium was supplemented with CuCl2, a copper-albumin complex or caeruloplasmin, all present at a level equivalent to 5 microM-copper. Activation did not occur at 4 degrees C or with Cu-EDTA as the supplement. Mn2+ and Zn2+, alone or in combination, did not activate nor enhance the activation achieved by CuCl2. The activation with CuCl2 was not inhibited by cycloheximide or cordycepin. [67Cu]Caeruloplasmin and albumin when added to the medium transferred radioactive copper to at least three cytosolic protein fractions, one of which was determined by immunoprecipitation to be CuZn-SOD. The transfer of 67Cu from caeruloplasmin was inhibited by increasing amounts of unlabelled caeruloplasmin; disodium EDTA (1.0 mM) had no effect on the transfer of copper from caeruloplasmin. These data show that aortic SOD activity, suppressed in copper deficiency, can be restored by incubating the aortas in culture medium supplemented with copper salts. In this system, caeruloplasmin and Cu-albumin appear equally capable of activating aortic CuZn-SOD. Moreover, the transfer of copper into the enzyme structure appears to be the primary event restoring catalytic activity to the enzyme.

Animals↗
PubMed1987

Matrix metalloproteinases 1, 2, and 3 from rheumatoid synovial cells are sufficient to destroy joints.

A neutral metalloproteinase has been isolated and purified from adherent rheumatoid synovial cells in culture. This protease, named matrix metalloproteinase 3, (MMP-3) degrades gelatin, proteoglycan, fibronectin, type IV collagen, laminin, and the N propeptide of type I procollagen. It can be separated from MMP-2 (a potent gelatinase), and MMP-1, an interstitial collagenase. MMP-3 is released from cells as a proenzyme of 55 Kda. Activation by trypsin or organic mercurials produces 2 active species of 45 Kda and 28 Kda. The enzyme contains zinc as an intrinsic component and requires calcium for conformational stability. In concert, active MMP-1, -2, and -3 can destroy all significant structural proteins of joint structures.

Arthritis, Rheumatoid↗
PubMed1986

A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components. Purification and characterization.

Human rheumatoid synovial cells in culture stimulated with the conditioned culture medium of rabbit macrophages secrete three distinct latent metalloproteinases. One of them, a proteinase that digests proteoglycan and other connective tissue matrix components, was purified as two active forms after activation with 4-aminophenylmercuric acetate. The two forms were homogeneous on sodium dodecyl sulfate-gel electrophoresis with Mr = 45,000 and Mr = 28,000, whereas the latent precursor was estimated to have Mr = 51,000 by gel permeation chromatography. Both active enzymes had optimal activity at pH 7.5-7.8 and were inhibited by EDTA and 1,10-phenanthroline but not by inhibitors for cysteine, serine, or aspartic proteinases. Removal of Ca2+ from the enzyme solution resulted in a complete loss of activity that could be fully restored by the addition of 1 mM Ca2+. The activity of the apoenzyme was restored by the addition of 0.5 mM Zn2+, 5 mM Co2+, or 5 mM Mn2+ in the presence of Ca2+ but not by each metal ion alone. The identical digestion patterns of reduced, carboxymethylated protein substrates indicated that both active forms of the enzyme have the same substrate specificity. The enzyme degraded cartilage proteoglycans, type I gelatin, type IV collagen, laminin, and fibronectin, and removed the NH2-terminal propeptides from chick type I procollagen. This enzyme may play a role in the normal turnover of the connective tissue matrix as well as in the joint destruction of chronic synovitis.

Animals↗
PubMed1986

Proactivator-dependent activation of procollagenase induced by treatment with EGTA.

A new mechanism for activation of the proactivator of procollagenase [Vater, Nagase & Harris (1983) J. Biol. Chem. 258, 9374-9382] has been found. Collagenolytic and other proteolytic enzyme activities in the medium of cultured rabbit synovial fibroblasts were found to be activated by a new mechanism: short-term incubation at 37 degrees C performed in the presence of EGTA followed by replacement of Ca2+ during enzyme assay. The crucial event in procollagenase activation is the production of a functional activator enzyme. Activation of procollagenase in the culture medium did not occur when proactivator was removed by immunoprecipitation. Proteolytic activity of proactivator was fully activated, whereas procollagenase alone could not be activated by the same sequence. EGTA treatment of the culture medium at 0 degrees C did not result in enzyme activation if Ca2+ was replaced before incubation at 37 degrees C. Certain other bivalent metal ions (e.g. Sn2+, Cd2+, Zn2+ and Mn2+) could substitute for Ca2+ to stabilize the proactivator as a zymogen and therefore prevent the appearance of proteolytic activity in culture medium. Isolation of proactivator and procollagenase from EGTA-treated radiolabelled culture medium by immunoprecipitation and subsequent analyses by fluorography revealed that a time-dependent proteolysis of both molecules occurred after replacement of Ca2+ and incubation at 37 degrees C. However, comparison of enzyme activity with fluorographic analyses showed that the maximal activation of both enzymes was achieved before any detectable decrease in Mr. The results suggest that the activation of proactivator and the subsequent activation of procollagenase may be initiated by conformational changes in structure of the proactivator molecule produced by removal of stabilizing bivalent metal ions.

Animals↗
PubMed1986

Pathogenesis of rheumatoid arthritis.

Many types of cells are activated and transformed in rheumatoid synovium, thereby contributing to amplification of the disease process. The immune response in rheumatoid arthritis is probably initiated by an antigen, although there is some evidence that anticollagen antibodies develop in response to tissue destruction, after rheumatoid arthritis has evolved clinically. Early inflammation in the synovium is characterized by a striking vascular proliferation, occurring in response to angiogenesis factors released by activated macrophages. Generalized activation of macrophages and lymphocytes typical of the immune reaction in the synovium generates antibody production, including production of rheumatoid factor. Data suggest that immune complexes deposited within cartilage attract polymorphonuclear leukocytes, which then release enzymes onto the cartilage surface. Many products of inflammation act as mediators, driving proliferation of synovial cells. Stellate cells, macrophages, and fibroblasts have been found along the pannus/cartilage junction; by various interactions, these contribute to destruction of cartilage and bone.

Animals↗
PubMed1986

Evidence for a thiol ester in duck ovostatin (ovomacroglobulin).

The structure and the mechanism for proteinase inhibition of the egg white protein ovostatin (ovomacroglobulin) are similar to those of plasma alpha 2-macroglobulin, but previous studies have shown that chicken ovostatin lacks a reactive thiol ester (Nagase, H., and Harris, E. D., Jr. (1983) J. Biol. Chem. 258, 7490-7498). Here we show that duck ovostatin has conserved such a thiol ester and is capable of inhibiting both metallo- and serine proteinases stoichiometrically. Evidence for thiol esters was established by the following results with duck ovostatin: 1) autolysis into fragments of Mr = 123,000 and 60,000 occurred by heating in sodium dodecyl sulfate, but was prevented by treatment with CH3NH2; 2) covalent linkages were formed with proteinases on complex formation; 3) reaction with CH3NH2 generated 3.6 SH groups/mol, and 3.9 mol of [14C]CH3NH2 were incorporated per mol of protein; and 4) saturation with a proteinase liberated 3.8 SH groups/mol of the inhibitor. Conformational rearrangement of duck ovostatin upon reacting with CH3NH2 or proteinases was demonstrated by an increased mobility of the protein in polyacrylamide gel electrophoresis. CH3NH2-treated duck ovostatin was able to bind and inhibit proteinases without forming covalent bonds, but, unlike unmodified ovostatin, its inhibitory activity was destroyed by freezing and thawing. Complexes formed between CH3NH2-treated duck ovostatin and a proteinase were not dissociable except under denaturing conditions. These results and other evidence indicate that covalent bond formation through reaction with a thiol ester is a separate process from the trapping and inhibition of proteinases by this family of proteins.

Amino Acid Sequence↗
PubMed1986

Biochemical defect in chick lung resulting from copper deficiency.

Lung tissue from groups of chicks raised 23 d on diets deficient in copper had a higher proportion of 0.15 M NaCl-extractable collagen but lower aldehyde-reacting compounds and elastin than controls the same age. Although the lungs from deficient chicks weighed less, the ratio of lung to body weight for the two groups stayed relatively constant throughout the 23-d period. Lysyl oxidase activity in lungs of deficient chicks was strongly suppressed. Feeding the deficient chicks the diet with copper supplements restored lysyl oxidase activity in lung. This showed the close and specific control of lung lysyl oxidase by copper. The impairment in cross-linking through lysyl oxidase failure is implied from these studies. Clearly, copper is needed for the normal development of lung.

Animals↗
PubMed1986

Retrograde ileography: value of glucagon.

Many gastrointestinal diseases have a predilection for the terminal ileum. Retrograde ileography is a valuable technique for evaluating this region. However, this procedure necessitates reflux of barium (with or without air) into the terminal ileum. This does not often spontaneously occur during a barium enema study. The use of glucagon in double-contrast studies of the colon has been recommended for various reasons, one of which is to facilitate reflux of barium into the terminal ileum. This randomized, double-blind trial confirms that glucagon does promote reflux. Multiple regression analysis indicates that the independent variables of patient age, weight, and sex have little effect on the frequency of reflux. It is concluded that glucagon can facilitate examination of the distal small bowel during a double-contrast colon examination.

Female↗
PubMed1985

Purification and partial characterization of ceruloplasmin from chicken serum.

The preparation and properties of ceruloplasmin from chicken serum are described. Ethanol-CHCl3 was used to precipitate the crude protein, followed by adsorption and elution from DEAE-Sephadex. Further treatment with Sephadex G-200 and CM-Sephadex yielded an intensely blue protein judged 1572-fold purer than starting serum. epsilon-Aminocaproic acid (0.02 M) was present in all buffers and starting sera. Chicken ceruloplasmin appears to be a single polypeptide, apparent Mr 124,000, with an A610/A280 ratio of 0.07 and an absorption maximum at 602 nm. Hexose, hexosamine, and sialic acid accounted for 7.2% of the weight; copper represented 0.20%, which suggested four or five copper atoms per molecule. Chicken ceruloplasmin catalyzed the azide-sensitive oxidation of p-phenylenediamine (PPD) and N,N'-dimethyl-p-phenylenediamine (DPD), and showed ferroxidase activity similar to that of human ceruloplasmin. Its amino acid composition, although similar in many residues to human ceruloplasmin, was decidedly lower in methionine and tyrosine. The chicken protein had one-third the sialic acid content of human ceruloplasmin and showed immunochemical nonidentity with human ceruloplasmin.

Amino Acids↗