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Biomedical subjects

E D Harris

Publications and source records attributed to E D Harris.

At least 109 records · Page 6Linked to original sources
PubMed1984

Considerations of copper metabolism in osteochondrosis of suckling foals.

Of 8 Thoroughbred foals in which osteochondrosis developed before weaning, 7 had serum copper and ceruloplasmin concentrations below normal. Three foals on one farm had serum zinc content high enough to suggest zinc toxicosis, and the liver of each foal contained abnormally high content of zinc. Four foals from the second farm had extremely low serum copper content, but normal serum zinc content. Evidence of environmental exposure to excess zinc was not found on either farm. The lesions in the zones of endochondral ossification of the afflicted foals were similar in many respects to those found in other species of animals with molybdenum-induced copper deficiency and with inhibition of the function of copper-dependent lysyl oxidase by beta-aminopropionitrile, a toxic component of Lathyrus odoratus known to cause osteolathyrism.

Animals↗
PubMed1984

Specific receptor for ceruloplasmin in membrane fragments from aortic and heart tissues.

Membrane fragments from aortic and heart tissues of immature chicks were observed to bind highly purified, 125I-labeled chick ceruloplasmin. The binding reaction exhibited a linear Scatchard plot for both tissues and showed for each an apparent dissociation constant (Kd) of about 10(-8) M. On the basis of Scatchard analyses, aorta contained 1.5 pmol of receptors/mg of membrane protein, whereas receptors in the membranes from heart tissue were at least 5 times more dense. The binding of chick ceruloplasmin to aorta membranes was trypsin sensitive and neuraminidase insensitive, and showed both saturation and reversibility. Various sialoglycoproteins in 500 molar excess had very little effect on the binding. The asialo derivatives of these proteins likewise did not inhibit the binding. Human ceruloplasmin was found to bind very weakly to the chick membranes. Asialo chick ceruloplasmin bound with the same efficacy as native chick ceruloplasmin. Heat-denatured chick ceruloplasmin, however, was very ineffectual in displacing native 125I-ceruloplasmin from the membranes. These studies provide the first evidence for a homologous membrane receptor for native ceruloplasmin in the plasma membranes of animal cells.

Animals↗
PubMed1983

Evaluation of pathophysiology and drug effects on rheumatoid arthritis.

The treatment of rheumatoid arthritis has been divided into symptomatic or anti-inflammatory drug therapy, and disease-modifying or remission-inducing drug therapy. A more useful classification may be one that correlates the therapy with the component of the disease process that it affects. In general, nonsteroidal drugs do not affect cells as much as they do some of the mediators that cells produce. The effects of gold and D-penicillamine, although used later in the disease because of their toxicity, probably act early in the cascade of development of the disease by affecting the macrophages and lymphocytes. Prednisone may be effective in inhibiting synthesis by macrophages and fibroblasts of proteinases as well as arachidonic acid metabolites. Retinoids may be prototypes of drugs that selectively inhibit collagenase/proteinase production but do not affect production of normal connective tissue components.

Anti-Inflammatory Agents↗
PubMed1983

Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.

Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.

Animals↗
PubMed1983

Purification of an endogenous activator of procollagenase from rabbit synovial fibroblast culture medium.

Procollagenase and a latent activator of procollagenase were found in culture medium from rabbit synovial fibroblasts stimulated either with crystals of monosodium urate monohydrate or with phorbol myristate acetate. Procollagenase (pI 6.8) and activator (pI 5.48) could be separated by isoelectric focusing. Both procollagenase and the latent activator were purified to electrophoretic homogeneity by immunoadsorption chromatography. The activator was found to exist as a doublet of Mr = 53,400 and 51,900, with conversion to a lower Mr form upon treatment with either trypsin or 4-aminophenylmercuric acetate. In the absence of activator, purified procollagenase was not activated either by trypsin or by 4-aminophenylmercuric acetate, although both agents were capable of converting procollagenase to a lower Mr form. Activation of crude procollagenase was inhibited by immunoprecipitation of latent activator prior to addition of trypsin. Procollagenase activation increased with increased concentration of activator and with increased time of incubation with activator. The activator itself had no collagenolytic activity. The activation did not result in an apparent decrease in Mr of the procollagenase molecule as seen on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both high and low Mr procollagenase (trypsin-treated) could be activated by the activator. Purified activator, but not purified procollagenase, contained latent proteolytic activity against azocasein, gelatin, and reduced carboxymethylated bovine serum albumin substrates.

Animals↗
PubMed1983

Ovostatin: a novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin.

The inhibition mechanism of ovostatin was studied using rabbit synovial collagenase and thermolysin. When enzymes were complexed with ovostatin, only the proteolytic activity towards high molecular weight substrates was inhibited. Activity towards low molecular weight substrates was partially modified: the catalytic activity of collagenase bound to ovostatin was inhibited by only 40% towards 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and that of thermolysin bound to ovostatin was activated about 2.6-fold towards benzyloxycarbonyl-Gly-Leu-NH2 and benzyloxycarbonyl-Gly-Phe-NH2. Collagenase-ovostatin complexes failed to react with anti-(collagenase) antibody. Saturation of ovostatin with thermolysin prevented the subsequent binding of collagenase. Ovostatin-proteinase complexes ran faster than free ovostatin on 5% polyacrylamide gel electrophoresis. Complexing ovostatin with either collagenase or thermolysin resulted in the cleavage of the quarter-subunit of ovostatin (Mr = 165,000) into two fragments with Mr = 88,000 and 78,000. On the other hand, when the inhibitory capacity of ovostatin was tested with trypsin, chymotrypsin, and papain, only partial inhibition of their proteolytic activities was observed towards azocasein. Stronger inhibition was noted when Azocoll was a substrate, however. Analyses of ovostatin-enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the quarter-subunit of ovostatin was cleaved into several fragments by those enzymes. These results led us to propose that ovostatin inhibits metalloproteinases in preference to proteinases of other classes in a manner similar to alpha 2-macroglobulin; hydrolysis of a peptide bond by a proteinase in the susceptible region of the ovostatin polypeptide chain triggers a conformational change in the ovostatin molecule and the enzyme becomes bound to ovostatin in such a way that the proteinase is sterically hindered from access to large protein substrates and yet is accessible to small synthetic substrates. A kinetic study of collagenase binding to ovostatin gave the value of k2/Ki = 6.3 X 10(5) M-1 min-1. The results indicate that ovostatin is equally as good a substrate for collagenase as type I collagens.

Animals↗
PubMed1983

Ovostatin: a novel proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties, and tissue distribution of ovostatin.

A proteinase inhibitor which has strong anti-collagenase activity was found in chicken egg white. The inhibitor (pI = 4.9) was purified by poly(ethylene glycol) (5.5-10%) precipitation and chromatography on Ultrogel AcA 34, DEAE-cellulose, and Sephacryl S-300. The final product was homogeneous on 5% polyacrylamide gel electrophoresis. Stoichiometric inhibition was observed with the inhibitor and rabbit synovial collagenase and thermolysin (1:1 molar ratio with thermolysin). The inhibitor ran on sodium dodecyl sulfate-gel electrophoresis with reduction as a single protein band of Mr = 165,000. The molecular weight of the native inhibitor was estimated to be 780,000 by sedimentation equilibrium centrifugation. Centrifugation analysis in 6 M guanidine hydrochloride and of the reduced sample gave M omega = 380,000 and M omega = 195,000, respectively, where M omega is the weight-average molecular weight determined by equilibrium ultra-centrifugation. The results indicated that the inhibitor molecule is a tetramer of identical subunits linked in pairs by disulfide bonds. Since the molecular weight and the quaternary structure of the inhibitor were similar to those of alpha 2-macroglobulin (alpha 2M) in plasma, chicken alpha 2M was isolated and compared with the inhibitor. The inhibitor was not sensitive to methylamine, whereas chicken alpha 2M was. No immunocross-reactivity was observed between the inhibitor and chicken alpha 2M. The NH2-terminal sequence of the egg white inhibitor is Lys-Glu-Pro-Glu-Pro-Gln-Tyr-Val-Leu-Met-Val-Pro-Ala. The sequence of chicken alpha 2M is Ser-Thr-Val-Thr-Glu-Pro-Gln-Tyr-Met-Val-Leu-Leu-Pro-Phe. Considerable homology was found between the two sequences and to the NH2-terminal sequence of human alpha 2M. Monospecific antibody raised against the egg white inhibitor was employed to examine the tissue distribution of the inhibitor. The inhibitor was found only in oviduct and egg white, but not in other tissues or serum of chickens.

Amino Acid Sequence↗
PubMed1983

Evaluation of (+)-catechin action on lysyl oxidase activity in aortic tissue.

Small amounts of (+)-catechin (5 mg/kg body wt) administered intramuscularly to 8-day-old chicks raised the lysyl oxidase activity in aorta about 20%. (+)-Catechin had no effect on chicks that were copper-deficient. In the deficient chicks, (+)-catechin treatment prompted a substantially stronger increase in lysyl oxidase activity in response to CuSO4. The observed increments in lysyl oxidase activity in vivo were sensitive to inhibition by beta-aminopropionitrile (BAPN), suggesting that (+)-catechin was affecting the enzyme. (+)-Catechin in the assay medium partially inhibited lysyl oxidase activity. With excess copper ions present, (+)-catechin catalyzed a very strong release of volatile tritium from the substrate proteins. The release of tritium, indicative of lysyl oxidase activity, was not blocked by BAPN, suggesting that the activity in vitro was not enzyme catalyzed.

Amino Acid Oxidoreductases↗
PubMed1983

Glucocorticoid use in rheumatoid arthritis.

Although the early hopes and enthusiasm held for glucocorticoids in rheumatoid arthritis therapy have been greatly modified, there is still a secondary therapeutic role for these drugs, one which has been refined by clinical experience.

Adult↗
PubMed1983

Low dose prednisone therapy in rheumatoid arthritis: a double blind study.

Prednisone, 5 mg taken each morning, was added to other drugs in 18 patients with rheumatoid arthritis. Sixteen patients were given a placebo in this double blind study. After 24 weeks, all patients were given the placebo. Slight functional improvement was noted in the prednisone group during the 24-week period, but deterioration after switching to placebo was sustained for at least 8 weeks. Progression of hand erosions occurred in 1 prednisone-treated patient, and in 4 controls. An asymptomatic vertebral spine compression fracture developed in 2 patients given prednisone; this was the only toxicity noted possibly due to this therapy. Minimal dose prednisone may be useful as "bridge" therapy between nonsteroidal antiinflammatory therapy and use of disease-modifying drugs.

Adolescent↗
PubMed1982

Increased level of translatable collagenase messenger ribonucleic acid in rabbit synovial fibroblasts treated with phorbol myristate acetate or crystals of monosodium urate monohydrate.

We studied mechanisms governing production of the neutral proteinase collagenase by synovial cells. We used a model system of monolayer cultures of rabbit synovial fibroblasts stimulated to produce collagenase by treatment with phorbol myristate acetate or crystals of monosodium urate monohydrate. mRNAs from these and untreated cells were translated in a wheat germ cell-free system. Collagenase was not present in the culture medium or in the in vitro translation products of mRNA from untreated cells but was present in both the medium and translation products of stimulated cells, as analyzed by gel electrophoresis and immunoprecipitation with monospecific antibody. Induction of collagenase was prevented by treatment of the cells with alpha-amanitin (2 mug/ mL), an inhibitor of mRNA synthesis. We have concluded that the induction of collagenase synthesis by either phorbol myristate acetate or urate crystals is due to an increased level of translatable mRNA.

Amanitins↗
PubMed1982

Comparison of pathways of copper metabolism in aorta and liver. A functional test of metallothionein.

Soluble fractions from chick liver and aorta were examined for copper-binding proteins. In liver a zinc-binding thionein appeared to be the major binding protein for copper. Aortic tissue contained only traces of this thionein protein. Unlike liver, moderate amounts of soluble copper in aorta showed no association with macromolecules. Chicks fed on copper-deficient diets for 8 days had one-third the liver copper concentrations of controls. Aortic copper concentration was decreased only slightly, but the activity of lysyl oxidase, a copper-dependent enzyme in aorta, was decreased significantly. Treating the deficient chicks with CuSO4 (1 mg/kg) restored liver copper rapidly. The increase correlated with the binding of copper to a 10 000-mol.wt. component in the soluble fraction. Aortic copper concentrations responded much less to the CuSO4 treatment, but lysyl oxidase activity was again measurable in the tissue. Radioactive isotopes of copper bound almost exclusively to the 10 000-mol.wt. component in liver and to components of mol.wt. 30 000 or above in aorta. Hardly any of the administered radioactivity appeared with the 10 000-mol.wt. components in aorta, and none was found with unbound copper. The 30 000-mol.wt. components in aorta showed superoxide dismutase activity that was sensitive to NaCN. They also showed the highest specific activity of copper of any other aorta component. A clear distinction was seen between the metabolism of copper in liver and aortic tissues. Whereas a copper thionein, metallothionein, was a major component in the liver pathway, it is doubtful that this protein plays a major role in the intracellular metabolism of copper in aortic tissue.

Animals↗