Nancy C. Andrews

Bio: Nancy C. Andrews is an academic researcher from Duke University. The author has contributed to research in topics: Hepcidin & Hemochromatosis. The author has an hindex of 79, co-authored 185 publications receiving 30811 citations. Previous affiliations of Nancy C. Andrews include Howard Hughes Medical Institute & Harvard University.

A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells.

Abstract: We have developed a simple and rapid method for preparing DNA-binding protein extracts from mammalian cells. The protocol is derived from the large scale procedure of Dignam et al. (1) and utilizes hypotonic lysis followed by high salt extraction of nuclei. The technique described by Dignam has several drawbacks, including the need for large numbers of cells and lengthy incubation and dialysis steps. It is labor-intensive and precludes preparation of multiple samples simultaneously. Our aim in developing this micropreparation procedure was to easily and rapidly extract DNA-binding proteins from small numbers of cells. Frequently, the quantity of cells available for extraction of DNA-binding proteins is limiting, as in analysis of clinical samples, of multiple clones of transfected cells, or of COS cell pools transiently transfected with a cDNA expression library. Ideally, such a technique would allow processing of many samples simultaneously and quickly on the benchtop. The method described in this report accomplishes these goals. In addition, it gives an excellent yield of DNA-binding proteins, comparable to that of the large scale Dignam protocol with minimal proteolysis. We typically start with between 5X10 and 10 cells. All centrifugations of less than 30 seconds are carried out in a room temperature microfuge; between steps, the samples are placed on ice. Adherent cells are scraped into 1.5 ml of cold phosphatebuffered saline (PBS); non-adherent cells are pelleted and resuspended in 1.5 ml cold PBS. The cell suspension is then transferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 yX cold Buffer A (10 mM HEPES-KOH pH 7.9 at 4°C, 1.5 mm MgCl2, 10 mM KC1, 0.5 mM dithiothreitol, 0.2 mM PMSF) by flicking the tube. The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 20—100 y\ (according to starting number of cells) of cold Buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 minutes at 4C and the supernatant fraction (containing DNA binding proteins) is stored at -70°C. The yield is 50 to 75 /tg protein per 10 cells.

Disorders of iron metabolism.

Abstract: Iron has the capacity to accept and donate electrons readily, interconverting between ferric (Fe2+) and ferrous (Fe3+) forms. This capability makes it a useful component of cytochromes, oxygen-bind...

Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter

Abstract: Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMT1 (refs 1,2,3). A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology

Abstract: For a long time, superoxide generation by an NADPH oxidase was considered as an oddity only found in professional phagocytes. Over the last years, six homologs of the cytochrome subunit of the phag...

Global quantification of mammalian gene expression control

Abstract: Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.

Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

Abstract: Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.

Hepcidin Regulates Cellular Iron Efflux by Binding to Ferroportin and Inducing Its Internalization

Abstract: Hepcidin is a peptide hormone secreted by the liver in response to iron loading and inflammation. Decreased hepcidin leads to tissue iron overload, whereas hepcidin overproduction leads to hypoferremia and the anemia of inflammation. Ferroportin is an iron exporter present on the surface of absorptive enterocytes, macrophages, hepatocytes, and placental cells. Here we report that hepcidin bound to ferroportin in tissue culture cells. After binding, ferroportin was internalized and degraded, leading to decreased export of cellular iron. The posttranslational regulation of ferroportin by hepcidin may thus complete a homeostatic loop: Iron regulates the secretion of hepcidin, which in turn controls the concentration of ferroportin on the cell surface.