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Biomedical subjects

E D Harris

Publications and source records attributed to E D Harris.

At least 55 records · Page 3Linked to original sources
PubMed1996

Functional analysis of a genetic defect of copper transport (Menkes disease) in different cell lines.

To define the function of the Cu-transporting ATPase in Menkes disease, Menkes and normal fibroblasts were incubated with 67Cu before and after brief exposure to -SH reagents, p-chloromercuribenzoate (PCMB) and dithiothreitol (DTT). Accumulation and retention were compared among these cells, BeWo cells, and rat C6 glioma cells similarly treated. The Michaelis constant for influx of 67Cu into normal and Menkes fibroblasts was practically the same (0.21 +/- 0.07 vs. 0.24 +/- 0.06 microM). The PCMB treatment stimulated 67Cu accumulation in C6 cells, inhibited accumulation in normal and Menkes fibroblasts, and did not affect BeWo cells. DTT stimulated 67Cu uptake in all cells but BeWo cells. DTT treatment after PCMB further enhanced 67Cu accumulation in normal fibroblasts and C6 cells but had no enhancing effect on Menkes fibroblasts or BeWo cells. Menkes fibroblasts and BeWo cells released 67Cu at rates considerably slower than normal fibroblasts (0.06 and 0.09 vs. 0.22%/min, respectively). The PCMB blocked 67Cu release from normal fibroblasts but did not affect Menkes fibroblasts or BeWo cells. Reverse transcription-polymerase chain reaction analysis of total RNA from BeWo cells failed to show a predicted 943-base pair fragment representing a partial transcript of the Menkes factor. The fragment was present in extracts from normal fibroblasts. We conclude that the mechanism underlying Cu homeostasis varies among different cell types. As exemplified by BeWo and Menkes cells, failure to efflux Cu ions may be linked with the failure to express a functional Cu-transporting ATPase, namely, the Menkes protein.

Animals↗
PubMed1996

In situ hybridization of stromelysin mRNA in the synovial biopsies from rheumatoid arthritis.

We examined the expression of stromelysin mRNA (SL mRNA) in synovial biopsy specimens from 12 cases of rheumatoid arthritis (RA) and 2 cases of osteoarthritis (OA) using in situ hybridization. The study demonstrated that positive cells with high levels of SL mRNA were mostly (85%) found in the synovial lining layer. The positive cells were abundant in the synovium of RA which presented well developed lymphoid follicles with massive inflammatory cells. On the other hand, the synovium of OA contained no positive cells for SL mRNA. In addition, low yet positive levels of SL mRNA were detected in the endothelial cells and vascular myocytes, and interstitial cells in the deeper layer of the synovium. Karyometric studies showed that cells positive for SL mRNA had significantly larger and more spherical nuclei than weakly positive or negative cells. The SL mRNA positive cells did not demonstrate any immunoreactivity to markers of bone marrow origin, such as Leu M1, Leukocyte Common Antigen (LCA) and lysozyme antigen. Electron microscopy of a case with many SL mRNA positive cells showed that most had well developed rough endoplasmic reticulum and numerous processes on the cell surface, and some had also well developed rough endoplasmic reticulum but without processes indicating that they may be AB and/or B synoviocytes.

Adult↗
PubMed1996

The rationale for combination therapy of rheumatoid arthritis based on pathophysiology.

Rheumatoid arthritis begins with activation of a few cells within joints. The initial process is most likely caused by presentation of an antigen or superantigen by specific HLA-DR glycoproteins to receptive lymphocytes, perhaps accompanied by retroviral activation of synoviocytes and macrophages. After this initial activation process, many different components of host immune, inflammatory, and proliferative responses are activated as well. These include: B cell proliferation, proteolytic enzyme biosynthesis, cytokine expression, activation of kinin, complement, clotting and fibrinolytic pathways, prostaglandin and leukotriene production generation of oxygen free radicals and nitric oxide, and proliferation of synoviocytes (pannocytes)/chondrocytes and their gene products. At some point after the initial stimulus, the rheumatoid synovitis may become self-sustaining, without a need for the initial inciting protein or virus. In consideration of the multiple activated pathways that become operational, it is appropriate to consider therapies that attack more than one pathway. This is the rationale for combination therapy. In practice it does not imply using 2 agents directed against the same pathway (e.g., 2 different nonsteroidal antiinflammatory drugs) but rather agents (e.g., methotrexate and cyclosporine) that have proven in vitro/in vivo to suppress different components of the immune/inflammatory/proliferative lesion. Thus, since many data suggest that irrevocable destruction of joints begins relatively early in the disease process, it is appropriate to begin substantial combination therapy early as well.

Arthritis, Rheumatoid↗
PubMed1995

A quantitative test for copper using bicinchoninic acid.

We describe a direct colorimetric assay for copper in serum and biological samples using 2,2'-bicinchoninic acid (BCA), a common reagent in most laboratories. BCA offers the advantage of being highly sensitive and specific for Cu(I) which rapidly forms an intense purple complex in the presence of BCA. The complex has peak absorbances at 562 and 354.5 nm with molar absorptivities of 7.7 x 10(3) liter mol-1 cm-1 and 4.6 x 10(4) liter mol-1 cm-1, respectively. Interference by other metal ions, pH, and detergents is minimal and the results correlate strongly with atomic absorption spectrophotometry.

Animals↗
PubMed1995

The iron-copper connection: the link to ceruloplasmin grows stronger.

A rare genetic defect in ceruloplasmin biosynthesis has provided the strongest evidence to date that ceruloplasmin is essential for iron metabolism and tissue distribution in humans. A aceruloplasminemia results in massive accumulations of iron in the liver and brain and is associated with retinal degeneration and diabetes.

Amino Acid Sequence↗
PubMed1995

Copper transport and kinetics in cultured C6 rat glioma cells.

C6 rat glioma cells accumulate and efflux 67Cu. Both processes showed saturation kinetics with increasing 67Cu concentration. The Michaelis constant (Km) for uptake was 0.63 +/- 0.14 microM; maximum velocity (Vmax) was 3.29 +/- 0.57 pmol Cu.mg protein-1.min-1. The Km for efflux was 0.15 +/- 0.06 microM; Vmax was 1.08 +/- 0.71 pmol Cu.mg protein-1.min-1. p-Chloromercuribenzoate (p-CMB) totally blocked 67Cu efflux but had no effect on Km or Vmax of uptake. Total 67Cu in the cell after 50 min was partitioned equally between particulate and soluble fractions. p-CMB-treated cells accumulated more 67Cu, but < 10% was bound to the particulate (membrane) fraction. Pb also increased 67Cu accumulation without affecting Km and Vmax of 67Cu uptake. These data suggest that carriers for import and export of Cu in C6 cells are distinct or operate in two different cellular environments. Efflux is a sulfhydryl-dependent process subject to inhibition by Pb. The data are consistent with a P-type ATPase in the efflux of Cu from cells and the potential for Pb to inhibit the efflux mechanism.

Animals↗
PubMed1994

Characterization of a particulate pathway for copper in K562 cells.

More than half of the 67Cu recovered from K562 cells following a brief incubation with 67Cu-ceruloplasmin was recovered in particulate fractions of the cell. The fractions in Percoll had densities that ranged between 1.040 and 1.060 g/dl. In as early as 5 min, two fractions, densities of 1.051 and 1.056, respectively, were discernible. Components in the 1.051 fraction tested positive for clathrin and catalase. Those in the 1.056 fraction sedimented near the marker for lysosomes. The 67Cu in both fractions was stable to treatment by EDTA, nitrilotriacetate, alpha,alpha'-dipyridyl, heparinase, and ascorbate, but dissociated when treated with pronase, trypsin, or sodium dodecylsulfate. Continuous incubation with 67Cu-ceruloplasmin intensified the 67Cu activity in the 1.051 and 1.056 fractions. Cells incubated with 125I-transferrin displayed the label primarily in the 1.051 fraction. Continuous incubation intensified the label but unlike 67Cu, it did not shift to lighter or heavier fractions. Electron micrographs of the 1.051 fraction showed fields dominated by membranous structures some of which were enclosed. Micrographs of whole cells showed numerous invaginations resembling coated pits with sealed structures along and beneath the membrane surface suggesting the membrane was engaged in a rather extensive endocytosis. These data provide evidence that a large fraction of Cu from ceruloplasmin enters the K562 cell bound to membranous-like vesicles, part of which are sealed and coated with clathrin. This particulate pathway accounts for most of the copper entering the cell.

2,2'-Dipyridyl↗
PubMed1994

Iron-copper interactions: some new revelations.

Iron and copper show an uncommon but largely unexplained interdependence, the basis for which is being clarified in hemoglobin-synthesizing cells, yeast, and photosynthetic microorganisms. The interactions of the two metals with membrane transport systems and genetic regulators are more apt to be cooperative than antagonistic.

Animals↗
PubMed1993

Morning report.

Explore the source record for details and available documents.

Diagnosis↗
PubMed1993

Menkes' disease: perspective and update on a fatal copper disorder.

Although the causes of the abnormal copper utilization seen in Menkes' disease remain unknown, a candidate gene reported by three laboratories has narrowed the search for the defective or missing factor. These genetic studies also suggest that a copper ATPase may be important in normal copper metabolism.

Copper↗
PubMed1993

The transport of copper.

In summary, one concern of copper transport is to identify the serum component(s) that deliver copper to cells. Another, to learn how copper ions penetrate cells and engage enzymes. Ceruloplasmin and albumin have received major attention as transport agents but there could be others. Copper transport operates within a system that is sensitive to copper concentrations and poised to engage copper ions should they build up to toxic levels. Uptake studies with cultured cells suggest the presence of specific membrane channels for passing copper ions and specific binding proteins that work against concentration barriers to deliver the copper internally. Diffusion, aqueous-phase pinocytosis, may also play roles in transporting copper into cells. Most work to date supports the existence of specific transporters in the membrane, yet no such carriers have been isolated or characterized outside the environment of the cell. Model system work has opened up new and intriguing ideas that link membrane potential to transmembrane transport. Studies with lower life forms have confirmed that there may be other copper transport mechanisms functional in cells. Finally, we must recognize that nearly all the copper ions in fluids and cells exists in a protein-bound form and, to a much lesser extent, as complexes with amino acids. The metal must dissociate from its protein environment to be functional. Amino acids may perform this function with albumin-bound copper. Cells, however, appear also to have membrane receptors for binding ceruloplasmin as well as mechanisms for reducing and dissociating the copper atoms bound. In this way cells are able to tap the richest excellular source of copper for metabolic purposes. We have yet to understand the finer details of that mechanism.

Amino Acids↗
PubMed1992

Copper as a cofactor and regulator of copper,zinc superoxide dismutase.

Copper,zinc superoxide dismutase (CuZnSOD), an antioxidant enzyme, is unique in requiring two essential metals for catalytic function. Yet, only one, copper, seems to regulate the expression of functional activity. Restricting dietary copper quickly impairs catalytic functioning of CuZnSOD in numerous tissues. Diets supplemented with copper or small amounts of CuCl2 administered intraperitoneally restore the enzyme activity in animals deprived of copper. Thus, CuZnSOD has been considered a good marker of copper status. A metal-free (apo) form of CuZnSOD could exist in tissues at all times, but especially when an animal is deprived of copper. Restoring CuZnSOD activity with copper permits elucidation of the pathway of copper incorporation into the enzyme. Ceruloplasmin and albumin transport copper to the enzyme in vitro. K562 cells, a human erythroleukemic cell line, can extract copper from ceruloplasmin and incorporate it into CuZnSOD. Ascorbic acid stimulates the transfer of 67Cu transfer from ceruloplasmin to the cells, and somewhat unexpectedly, appears to restrict the amount of transferred copper that becomes bound to the enzyme. Reactivation of CuZnSOD in healthy individuals has the potential of being a useful tool for assessing copper status. This approach has merit, but one must consider that the levels of apo-enzyme that prevail in tissue could be influenced by other metals.

Animals↗
PubMed1992

Regulation of antioxidant enzymes.

Free radicals generated by a partial reduction of O2 pose a serious hazard to tissues and vital organs, especially membrane lipids, connective tissues, and the nucleic acids of cells. For protection, enzymes have evolved that specifically attack O2-, hydrogen, and organic peroxides, and repair any damage incurred to DNA. With few exceptions, antioxidant enzymes are found in all aerobic and aerotolerant anaerobic organisms. Logic assumes that a basal level of antioxidant enzyme activity is maintained at all times. This may be true. Yet cells must have ways to amplify antioxidant enzyme activity to counter sudden increases in oxygen metabolites. The full details of that regulation are slowly coming to light. Bacteria possess a series of elaborate and interacting genes that can sense specific increases in intracellular H2O2 and O2-. In higher organisms, hormones and metal ion cofactors impose pre- and posttranslational control over the genetic expression of antioxidant enzymes. Furthermore, aging, cellular differentiation, and organ specificity must also be factored into the final equation in higher organisms. This review will discuss some of the more recent findings relevant to antioxidant enzyme regulation in bacteria and higher organisms.

Aging↗