Edward D Harris

Texas A&M University | TAMU · Department of Nutrition and Food Science

This book forms a technical introduction to all major and trace minerals in foods, including their chemistry, transport, absorption, bioavailability and physiological roles. The book explains the in vivo functions of food minerals and demonstrates why and how macrominerals and microminerals are necessary for proper metabolic functioning in humans....

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Fruits and vegetable have shown potential benefits to prevention of chronic diseases such as cancer and heart. Among several factors, understanding their bioactivity by isolation and purification of bioactive compounds and establishing their structure-function relationship are major challenges. Several health promoting citrus limonoids were purifie...

Limonoids, a family of triterpenoids with putative anticancer properties, occur in fruits as glucosides and aglycones. Both highly purified forms were isolated from seeds and molasses of citrus fruits and tested for toxic effects against two human cancer cell lines, SH-SY5Y neuroblastoma and Caco-2 colonic adenocarcinoma, and a noncancerous mammali...

Limonoids are triterpinoids unique to citrus and neem trees with potential cancer-preventing properties in animals and human cell lines. Antioxidant activity and apoptotic induction are thought to be the principal effects of citrus limonoids in the antiproliferative properties, but this postulate lacks firm experimental evidence. In this study four...

Citrus limonoid glucosides, a family of fruit bioactive compounds, were postulated to have free radical-scavenging and apoptosis-inducing properties against certain types of cancers. Four highly purified limonoid glucosides, limoin 17beta D-glucopypranoside (LG), obacunone 17beta D-glucopyranoside (OG), nomilinic acid 17beta D-glucopyranoside (NAG)...

Although functional only as secondary metabolites in citrus, a class of compounds called limonoids is a family of molecules that have powerful cancer prevention and possibly cancer therapeutic application in human medicine. Postulated to protect plants and fruits from marauding insects and other herbivores, limonoids have the capacity to arrest gro...

Although two decades have passed since copper was shown to stimulate blood vessel formation in the avascular cornea of rabbits, only recently have clinical trials established that Cu privation by diet or by Cu chelators diminishes a tumor's ability to mount an angiogenic response. These data have shed new light on the functional role of Cu in micro...

An oxygen-rich atmosphere obligated living organisms to cope with reactive oxygen species (O2-, H2O2, OH*) that were the unavoidable by-products of cellular metabolism. As a redox cofactor Cu was selected as a co-catalyst for numerous biological processes, many involving the utilization of oxygen. Inadequate or excessive intake of Cu can be pathoge...

This paper reviews the Second International Workshop on Iron and Copper Homeostasis, held in Pucón, Chile 10-13 November, 2001. We cover the presentations and papers published (this issue) with the intent to point out parallels, contrasts and cutting edge areas rather than to say something about every paper. Iron and copper metabolism have been int...

The gene for Menkes disease codes for a Cu-transporting ATPase that regulates Cu homeostasis in all tissues with the exception of adult liver. The basis for developmental or tissue-specific regulation at present is not understood. To learn if the regulation is associated with the promoter, we cloned and sequenced a 2.2 kb genomic DNA fragment flank...

Menkes disease is testament to a copper (Cu)-ATPase (ATP7A) playing a dominant role in the absorption and homeostasis of Cu. Significantly, the proximal promoter region of the Menkes disease gene (MNK) has cis-active elements that relate to binding sites for transcription factors which in yeast are known to be activated by a low Cu status. A respon...

Nutritionally essential metals such as zinc are moved into and out of cells by a series of transport proteins or transporters. Their tri-fold purpose is to procure zinc from the environment, to protect cells against zinc toxicity, and maintain ample supplies of zinc for metabolic purposes. Two families of zinc transporters are known: the ZIP family...

Copper transport at the cellular level is achieved by a coordinate series of interactions between passive and active membrane transport proteins, vesicles, and soluble peptides. Knowing the function of each component of this complex network has made the task of delineating the mechanism of intracellular copper homeostasis achievable.

Research in the past millennium gave us formidable advances in the fields of Cu nutrition, biochemistry, animal and human health. Insights into Cu-Fe interactions highlighted much significant advancement. The recognition that Cu was an essential mineral for Fe metabolism and hemoglobin biosynthesis traces back to a paper by Hart et al. at Wisconsin...

Extracts from three human cell lines were found to contain abridged Menkes disease gene transcripts with novel insertion sequences. The transcript variant that is the focus of the present study codes for a 103-residue protein containing the first heavy-metal-binding domain (Hmb1) of ATP7A, the Cu-ATPase associated with Menkes disease. This transcri...

Exposure to potentially neurotoxic levels of lead (Pb) occurs in about 9% of American children under 6 years of age. Astroglia in the brain serve as a Pb depot, sequestering Pb and preventing its contact with the more sensitive neurons. Astroglia have the capacity to adapt to Pb exposure, and as such are able to tolerate relatively high intracellul...

The transport and cellular metabolism of Cu depends on a series of membrane proteins and smaller soluble peptides that comprise a functionally integrated system for maintaining cellular Cu homeostasis. Inward transport across the plasma membrane appears to be a function of integral membrane proteins that form the channels that select Cu ions for pa...

C6 rat glioma cells resemble rat astroglia in culture in that both cell types accumulate lead (Pb) intracellularly from the medium. As such, C6 cells are a model for Pb accumulation by the brain. In this study, an increase in intracellular Pb accumulation induced by p-chloromercuribenzoate (PCMB) after exposure to 10 microM Pb acetate suggests a ro...

The 5' region of MNK cDNAs has a 45 bp insert terminating at the 5'end with an AGATG sequence. The ATG in the sequence is in-frame with the ATG downstream identified by Vulpe et al (1993) as a translation start site for MNK mRNA. Inserts of 192 bp and 45 bp have been found in the 5' region of MNK mRNAs from BeWo cells, Caco-2 cells and normal human...

We have been concerned with the functional significance of small poly (A)+ RNAs with partial Menkes mRNA sequence that are found in non-Menkes human cells. One of these RNAs appears to have formed through a splicing event that excised exons 3–15 and formed a union between exons 2 and 16. This variant is common to nearly all the human cells lines th...

The metabolism of Cu is intimately linked with its nutrition. From gut to enzymes, Cu bioavailability to key enzymes and other components operates through a complex mechanism that uses transport proteins as well as small molecular weight ligands. Steps in Cu transport through the blood, absorption by cells, and incorporation into enzymes are slowly...

We isolated cDNA fragments from four human cell lines that had sequences for the Menkes Cu-transporting ATPase (ATP7A). Primers designed to generate a 4.8 kb cDNA with the complete open reading frame generated a 1.9 kb cDNA in addition to the expected 4.8 kb product. Sequence analysis revealed that the 1.9 kb cDNA encoded one of the six Cu-binding...

Previously, we showed that the transport of Cu by PC12 pheochromocytoma cells and C6 glioma cells correlated with the expression of a Cu-transporting ATPase (Atp7a) that has been linked to Menkes disease. Here, we show that cerebrovascular endothelial (CVE) cells that comprise the blood-brain barrier (BBB) also express the gene for the Cu-ATPase. B...

The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism. Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and pro...

Rat Atp7a occupied a single open reading frame (274502) which coded for a protein of 1492 residues. Rat Atp7a was 98% and 95% identical to published sequences for the mouse and Chinese hamster, respectively, and 94% homologous to human ATP7A. Compared to ATP7A, the rat transcript coded for an additional alanine (A446) in the heavy metal binding (Hm...

Amyotrophic lateral sclerosis (ALS) is an age-dependent degeneration of motor neurons in the central nervous system. ALS is not caused by faulty nutrition. Recent data suggest that ALS could be an oxidative neurotoxicity induced by a mutation in the SOD1 protein. This finding extends beyond the simple loss of an antioxidant enzyme.

The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism. Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and pro...

Molecular understandings of copper transport and metabolism have advanced significantly in the past 10 years. In this review we examine two areas where the new information has been particularly rapid and the insights more profound: membrane copper transport and the ferroxidase function of ceruloplasmin. The first is highlighted by discoveries of th...

We previously reported that copper efflux from C6 rat glioma cells was blocked by a brief exposure to sulfhydryl reagents p-chloromercuribenzoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, suggesting the possible involvement of a Cu-transporting ATPase in the efflux mechanism. In this report, we show that copper efflux from PC12...

This study evaluated the use of IEC-6 cells as a model for studying lead (Pb) transport by intestinal epithelial cells (IECs) and examined potential transport mechanisms for Pb uptake and extrusion. Pb accumulation in IEC-6 cells exposed to 5 and 10 microM Pb for up to 60 min was time- and dose-dependent. Reduction of incubation temperature signifi...

A unique construct of low-density lipoprotein (LDL), lipoprotein[a] has been shown to be a valuable independent risk factor for coronary artery disease. While not subject to dietary modulation, lipoprotein[a] nonetheless has provided one of the more promising leads to understanding genetic predisposition to arterial disease.

We have reported Cu efflux from rat C6 cells was blocked by sulfhydryl reagents (e.g. PCMB), as well as ATPase inhibitor, suggesting Cu-ATPase involvement in efflux mechanism. This report shows Cu efflux from PC-12 cells, a neuron-like cell line from rat adrenal pheochromocytoma, is also inhibited by PCMB exposure. RT-PCR detected both C6 and PC-12...

Menkes disease is an X-linked disorder of Cu metabolism. The Menkes gene codes for a P-type ATPase (ATP7A) believed to force Cu membrane transfer. In BeWo cells, a human choriocarcinoma cell line, Cu efflux is coincident with ATP7A gene expression. ATP7A has a 45 kb ORF. We have now prepared a 5.8 kb PCR fragment with a complete ORF and cloned the...

Molecular biology has provided nutrition science with a powerful experimental tool for exploring the molecular basis of essential nutrient deficiencies. Differential display polymerase chain reaction has emerged as an instrument of unlimited potential for assessing the manner by which nutrients regulate cell functions.

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. Th...

BeWo cells, a human choriocarcinoma cell line, have a high-affinity system for transporting copper ions into the cell (Km = 0.21 microM) but are sluggish in releasing copper back into the medium from preloaded cells. The slow efflux rate has recently been shown to correlate with a failure of BeWo cells to express the Menkes transcript [Y. Qian, E....

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.m...

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.

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...

We describe a 21-year-old man who developed copper deficiency manifested as a demyelinating neuropathy, chronic intestinal pseudo-obstruction, osteoporosis, testicular failure, retinal degeneration, and cardiomyopathy with a tortuous aorta. His serum copper was low and did not increase despite administration of large doses of intravenous copper sul...

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

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 i...

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.

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 co...

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.

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.

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, antiox...

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 smal...

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 direct...

Copper transport continues to show unexpected and, in some cases, surprising departures from traditional mechanisms. Although both iron and copper bind to plasma proteins, the recent data suggest that each works through separate and distinct mechanisms for gaining access to cells. Ceruloplasmin and albumin as postulated transport carriers have with...

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 caeruloplasmi...

Copper from ceruloplasmin (Cp) is taken up by K562 cells independently of Cp and incorporated into superoxide dismutase. The process of release from Cp, transport across the plasma membrane and into the enzyme target is unclear. The authors now have evidence that copper from Cp may be converted into a vesicular form during the uptake process. After...

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 b...

Broadly interpreted, copper transport encompasses events and intermediates that move copper progressively from gut to cells and to copper requiring enzymes within. Ceruloplasmin, the major copper-binding protein in plasma, may be an important, but not exclusive source of copper for extrahepatic cells. We have attempted to learn the mechanism by whi...

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...

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 ceruloplasmi...

Human erythroleukemic cells (K562) show enhanced binding of ceruloplasmin and transport more copper internally when induced to differentiate with hemin (20 μM). Hemin makes these leukemic cells synthesize fetal and embryonic hemoglobins. We asked if induction causes a greater need for copper possibly as an antioxidant or to synthesize copper-depen...

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 determ...

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...

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 caerulo...

Transport of trace metals to cells and enzymes may be thought to occur in three phases: (1) an extracellular phase dominated by a centralized protein or binding factor that conveys the metal to the cell, (2) a membrane phase characterized by a membrane receptor or carrier protein that mediates the transfer of the metal-bound complex or the free met...

Understanding how cuproenzymes are regulated in connective tissues is vital to understanding the role copper and copper complexes play in ameliorating chronic inflammations. The rapidly growing chick aorta is an excellent model system to test the effects of copper availability on cuproenzyme production because chicks are readily made deficient, cop...

Copper in a biological setting is primarily an essential metal cofactor for numerous biocatalysts. Transport systems that specifically handle copper are known to exist but have not been fully characterized. We are left, therefore, with an incomplete understanding as to how copper becomes incorporated into cells and into the cellular enzymes. In thi...

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 thr...

The efficacy of copper and its complexes to control or reverse inflammation hinges on a unique ability to interact with specific target enzymes in the afflicted tissues. One such enzyme is lysyl oxidase. This copper-metalloenzyme plays a key role in the maturation of connective tissue proteins. The enzyme catalyzes formation of the crosslinking com...

Lysyl oxidase activity was found in the isthmus (the membrane-forming region) of the hen's oviduct in a copper-rich region proximal to the shell gland. Desmosine and isodesmosine, cross-linking compounds associated with mature elastin, were found in hydrolysates of the shell membrane, confirming the necessity for lysyl oxidase in its biosynthesis....

An amine oxidase, lysyl oxidase, which catalyzes the oxidation of peptidyl lysine has been isolated from immature chicken aortas. Although insoluble in salt buffers, the enzyme was extracted from the tissue with 4 M urea. A combination of DEAE-cellulose and affinity chromatography was used to purify the enzyme to a level 2400 times the concentratio...