Biomedical subjects
Publications and source records attributed to E D Harris.
Recent evidence has failed to support the claim for a pyrroloquinoline quinone (PQQ) cofactor in mammalian enzymes previously reported to have PQQ. The validity of the original analysis now has been questioned, and a second cofactor, topa quinone, has been identified in at least one enzyme.
The transport of iron from mother to fetus presents a number of challenges, none more perplexing than determining how the flow can be unidirectional, yet avoid mixing the mother's serum proteins with the fetal system. Studies with cultured cell lines suggest a possible mechanism by which this may be achieved.
This discussion was selected from the Department of Medicine Morbidity and Mortality Conference held at the Stanford University Medical Center, Stanford, California, on January 9, 1991. The editor of the conference is Philip C. Lee, MD, Chief Resident, Department of Medicine.
Multiple events give rise to rheumatoid arthritis (RA) and many different elements create an overall pathological effect. It is highly unlikely that a single cause for RA will be identified. A greater appreciation of the multiple events that lead to the development of RA is opening up a number of promising points of intervention which may serve as potential alternatives to the broader-based and often toxic drugs used in RA therapy today.
K562 cells, a human erythroleukaemic cell line blocked for differentiation, commit towards erythrocytes when exposed to haemin (20 microM). The cells synthesize fetal haemoglobins and show site-specific binding of caeruloplasmin, a plasma copper protein. These events are set into motion by haemin. On the assumption that the binding of caeruloplasmin could reflect a greater need for copper, we sought to determine whether the transfer of 67Cu from caeruloplasmin was accelerated in haemin-induced compared with non-induced K562 cells. Cu,Zn superoxide dismutase (CuZnSOD) was the recipient. Haemin induction caused the K562 cells to lose CuZnSOD activity. By 96 h, the level of SOD activity was less than 60% of that of non-induced cells. The loss was confined entirely to the CuZn form, MnSOD activity staying essentially unchanged. Although CuZnSOD activity declined with the haemin induction, the incorporation of [4,5-3H]lysine into immunoprecipitable CuZnSOD protein was unaffected. There was also no change in CuZnSOD mRNA concentration in haemin-induced cells. Thus a loss of enzyme did not correlate with a decline in the synthesis de novo of CuZnSOD protein. When 48 h-induced cells were transferred to a medium supplemented with 0.2 microM-caeruloplasmin, CuZnSOD activity was restored to control levels in 24 h. Caeruloplasmin also stimulated the incorporation of [3H]lysine into immunoprecipitable CuZnSOD protein. Caeruloplasmin addition may have affected a post-translational regulatory site for CuZnSOD biosynthesis, possibly by providing copper for the newly synthesized enzyme.
Scurvy-like symptoms have been seen in experimental copper deficiency. This forecasts a role for the vitamin in copper metabolism. Ascorbate has been known to antagonize the intestinal absorption of copper. More recent studies have characterized a postabsorption role for ascorbate in the transfer of copper ions into cells. The vitamin reacts directly or indirectly with ceruloplasmin, a serum copper protein, specifically labilizing the bound copper atoms and facilitating their cross-membrane transport. Ascorbate at physiological levels and above impedes the intracellular binding of copper to Cu,Zn superoxide dismutase. The mechanism is unclear but nonetheless suggests both positive and negative regulatory functions for ascorbate in copper metabolism.
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Stromelysin is a metalloproteinase that degrades extracellular matrix macromolecules including fibronectin, laminin, collagen IV and proteoglycans. We now report that cycloheximide, an inhibitor of protein synthesis, induces human stromelysin mRNA in fibroblast cultures in a time- and dose-dependent fashion. As determined by Northern hybridization, a 24-h treatment with cycloheximide increased stromelysin mRNA about 20-fold over the control level. In vitro translation or translation in cells after removal of cycloheximide resulted in increased levels of immunoprecipitable stromelysin suggesting that the cycloheximide-induced stromelysin mRNA was functional. Analysis of mRNA stability suggested that the cycloheximide effect is in part due to the increased activation of the stromelysin gene. In contrast to these results, cycloheximide did not induce collagenase mRNA but, rather, prevented its induction by interleukin-1 beta. These data provide evidence for discoordinate regulation of collagenase and stromelysin genes and suggest that a short-lived repressor protein may play a role in the stromelysin gene expression.
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Copper uptake from 67Cu-labeled ceruloplasmin (67CuCp) was studied in K-562 cells, a human erythroleukemic cell line. 67CuCp was prepared by an ascorbate-catalyzed exchange of recrystallized ceruloplasmin with 67CuCl2. The labeled protein was treated with Chelex-100 and gel filtration to ensure that 67Cu was tightly bound to the structure. 67CuCp bound specifically to the K-562 cells at 4 degrees C. The binding was linear with protein in the range of 200-800 nM and in the presence of 3% albumin. In this concentration range, 67CuCl2 showed no binding that could be interpreted as specific; 80-90% of the cell-bound 67Cu was removed by washing the cells with acid buffer. When binding was attempted at 37 degrees C, a significant fraction of the 67Cu resisted acid washing and with time accumulated in the cells. Fractionating the cytosolic components on Percoll gradients located the 67Cu in buoyant fractions of densities 1.030-1.05, with a peak at 1.035. Repeating the experiment with 125I-labeled ceruloplasmin failed to localize any 125I label in Percoll fractions; very little 125I was detected in the cytosol. Double-labeled 67Cu-125I-ceruloplasmin confirmed that copper and not the protein moiety of ceruloplasmin was taken up by the cells. The uptake reaction was inhibited by 1 mM bathocuproine sulfonate and by 1 mM sodium iproniazid. Ascorbate (100 microM) strongly stimulated uptake. These studies provide evidence that K-562 cells are able to extract copper atoms from ceruloplasmin and transport the copper to the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
We have determined that human stromelysin mRNA can be induced by interleukin-1 beta (IL-1 beta) and that the induced mRNA levels can be suppressed by retinoic acid and dexamethasone (Saus, J., Quinones, S., Otani, Y., Nagase, H., Harris, E.D., Jr., and Kurkinen, M. (1988) J. Biol. Chem. 263, 6742-6745). Here we show, by nuclear run-on and transient gene expression analyses, that IL-1 beta induction is a promoter function and that dexamethasone suppresses IL-1 beta-induced gene activity. For transient gene expression assays, 1.3 kilobase pairs of the stromelysin promoter region (-1303 to -11 relative to the transcription start site) and shorter fragments thereof were cloned into a human growth hormone reporter vector. In transfected human fibroblast cultures all the constructs, with the exception of the one containing the shortest promoter fragment (-53 to -11), responded to IL-1 beta induction. Interestingly, the ability of IL-1 beta to induce human growth hormone expression decreased as the length of the promoter fragment was reduced. Dexamethasone treatment suppressed the induced human growth hormone levels by approximately 50% irrespective of the promoter length. These results suggest that the 1.3-kilobase pairs stromelysin promoter fragment contains DNA elements required for IL-1 beta induction and dexamethasone suppression.
Ceruloplasmin binds to the membranes of K562 cells. The binding has been shown to result in a temperature-dependent transfer of ceruloplasmin-bound copper into the cytosol. Ascorbic acid (100 microM) stimulates the transmembrane transfer nearly 10-fold, depending on the initial concentration of 67Cu-ceruloplasmin. The protein moiety of ceruloplasmin does not enter the cells. Bathocuproine disulfonate, a chelator specific for cuprous copper, inhibits the uptake, suggesting copper atoms are reduced concomitant with their removal from ceruloplasmin. Cytosolic 67Cu from ceruloplasmin was found mainly bound to Cu, Zn superoxide dismutase, the major cytosolic copper protein in these cells. Evidence supporting the various phases in the ceruloplasmin-mediated transport mechanism are presented.
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Copper uptake from human ceruloplasmin (Cp) into cells of a human erythroleukemic cell line, K562, was investigated. The interaction between ascorbic acid and the copper atoms in ceruloplasmin was a focal point of the study. Nondenatured 67Cu-labeled ceruloplasmin (67Cu-Cp) was prepared by an ascorbate-catalyzed exchange of Cp with 67CuCl2 in vitro. The complex was stable, even in the presence of 1.0 mM ascorbate. Adding K562 cells and incubating at 37 degrees C resulted in an immediate transfer of 67Cu from ceruloplasmin to the cells. At 37 degrees C the copper accumulated by the K562 cells resisted dissociation by mild acid washing. The rate of transfer of 67Cu was proportional to the Cp concentration in the medium. Ascorbate (100 microM) enhanced the uptake of 67Cu at least fourfold. D-Isoascorbate worked as well as L-ascorbate, suggesting that the reducing potential of the vitamin (or its isomer) was important in the uptake of copper. Approximately 20% of the 67Cu absorbed into the cytosol was precipitable with antibodies to Cu-Zn superoxide dismutase (Cu-Zn SOD). Ascorbate, however, did not enhance the incorporation of radioactivity into Cu-Zn SOD, suggesting that copper may not be the only rate-limiting factor in the synthesis of this enzyme in K562 cells. The possible relevance of these observations to vitamin C deficiency is discussed.
Two active forms (Mr 45,000 and 28,000) of a metalloendopeptidase that digest proteoglycans and other extracellular matrix components of connective tissues have previously been purified from rheumatoid synovial cells and characterized [Okada, Nagase & Harris (1986) J. Biol. Chem. 261, 14245-14255]. To study the mechanisms of activation the precursor of this metalloendopeptidase has now been purified. The final products are homogeneous on SDS/polyacrylamide-gel electrophoresis and identified as a set of zymogens of Mr 57,000 and 59,000, in which the latter form is probably the product of post-translational glycosylation of the Mr 57,000 zymogen, as it binds to concanavalin A. The zymogen can be activated by trypsin, chymotrypsin, plasma kallikrein, plasmin and thermolysin, but not by thrombin. Although the activated metalloendopeptidase is further degraded by trypsin, plasma kallikrein and thermolysin during a prolonged incubation, it is relatively stable against plasmin and chymotrypsin. Activation with 4-aminophenylmercuric acetate is dependent on its concentration. It requires the reaction with the zymogen, possibly through thiol groups, and the continued presence of the agent. During this treatment the zymogen undergoes a sequential processing; first it becomes active without changing its apparent molecular mass, and then it is processed to low-Mr species of Mr 46,000, 45,000 (HMM) and 28,000 (LMM). The rate of conversion of the precursor into an initial intermediate of Mr 46,000 follows first-order kinetics (t1/2 2.0 h with 1.5 mM-4-amino-phenylmercuric acetate at 37 degrees C) and is independent of the initial concentration of the zymogen or the presence of up to a 676-fold molar excess of substrate, whereas the generation of HMM and LMM species is affected by these parameters. These results indicate that activation of the prometalloendopeptidase by an organomercurial compound is initiated by the molecular perturbation of the zymogen that results in conversion into the 46,000-Mr intermediate by an intramolecular action; the subsequent processing of this intermediate in HMM and LMM species is a bimolecular reaction. In vivo it is probable that the precursor of this metalloendopeptidase is activated either by direct limited proteolysis by tissue or plasma endopeptidases, or, alternatively, by factors that cause certain conformational changes in the zymogen molecule.
Four Thoroughbred foals were weaned from their dams when they were 1 day old and were fed a liquid milk-replacer diet containing approximately 1.7 micrograms of copper/g from plastic buckets for 4 to 7 months. They were kept in stalls with fiberglass walls and asphalt floors covered with rubber pads. Serum copper and zinc concentrations were determined 3 times/week by atomic absorption spectrophotometry, and liver copper and zinc concentrations were determined similarly after acid digestion of tissues taken at necropsy. The amount of soluble collagen in articular cartilage and aortic tissue was determined after necropsy. Clinical signs of illness, particularly evidence of lameness, were monitored daily. The foals were weighed weekly, and growth rate was monitored by measurement of height at the withers. Packed cell volumes and total and differential WBC counts were measured each time blood was drawn for copper and zinc concentration determinations. The foals were examined by necropsy at the end of the experiment, and the tissues were examined histologically. The foals developed intermittent, but nondebilitating, diarrhea with the onset of low serum copper concentrations. Considering the totally liquid diet, the foals grew well. Serum copper concentrations decreased to less than 0.1 micrograms/ml in 13 to 16 weeks. Lameness was evident 2 to 6 weeks after serum copper concentrations decreased to their lowest value (less than 0.1 micrograms/ml). All foals developed stilted gaits and ultimately walked on the front of their hooves. Major hematologic changes and alterations of hair color were not evident. Soluble collagen of articular cartilage and aortic tissue increased from 340 to 600% greater than that of control foals.(ABSTRACT TRUNCATED AT 250 WORDS)
We have determined the complete primary structure for human matrix metalloproteinase-3 (MMP-3), which has 477 residues including a 17-residue signal peptide. The result indicates that MMP-3 is identical with stromelysin (Whitham, S. E., Murphy, G., Angel, P., Rahmsdorf, H.-J., Smith, B. J., Lyons, A., Harris, T. J. R., Reynolds, J. J., Herrlich, P., and Docherty, A. J. P. (1986) Biochem. J. 240, 913-916). A striking result is that MMP-3 and collagenase are 54% identical in sequence, suggesting a common origin for the evolution of the two proteinases. We also show that in human synovial fibroblast cultures human recombinant interleukin-1 beta rapidly induces high levels of MMP-3 mRNA and, conversely, that retinoic acid or dexamethasone can suppress the MMP-3 mRNA levels. Similar results were obtained for human synovial collagenase mRNA. The data suggest that MMP-3 and collagenase expression are coordinately modulated in synovial fibroblast cultures.