17 results on '"Dufner-Beattie J"'
Search Results
2. Selection for high levels of resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say) using non-transgenic foliar delivery.
- Author
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Mishra S, Dee J, Moar W, Dufner-Beattie J, Baum J, Dias NP, Alyokhin A, Buzza A, Rondon SI, Clough M, Menasha S, Groves R, Clements J, Ostlie K, Felton G, Waters T, Snyder WE, and Jurat-Fuentes JL
- Subjects
- Animals, Bacillus thuringiensis genetics, Bacillus thuringiensis Toxins genetics, Bacillus thuringiensis Toxins pharmacology, Coleoptera genetics, Coleoptera pathogenicity, Colorado, Endotoxins genetics, Endotoxins pharmacology, Hemolysin Proteins genetics, Hemolysin Proteins pharmacology, Insect Proteins genetics, Larva genetics, Larva growth & development, RNA Interference, RNA, Double-Stranded genetics, Solanum tuberosum growth & development, Solanum tuberosum parasitology, Coleoptera drug effects, Drug Resistance genetics, Insecticides pharmacology, RNA, Double-Stranded pharmacology
- Abstract
Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI β; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.
- Published
- 2021
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3. Identification of AP80978, a novel small-molecule inhibitor of hepatitis C virus replication that targets NS4B.
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Dufner-Beattie J, O'Guin A, O'Guin S, Briley A, Wang B, Balsarotti J, Roth R, Starkey G, Slomczynska U, Noueiry A, Olivo PD, and Rice CM
- Subjects
- Adenosine analogs & derivatives, Adenosine pharmacology, Biological Assay, Cell Culture Techniques, Cell Line, Cell Survival drug effects, Cyclosporine pharmacology, Genotype, Hepacivirus genetics, Hepatitis C virology, High-Throughput Screening Assays, Humans, Luciferases, Renilla, Mutagenesis, Site-Directed, Oligopeptides pharmacology, Replicon drug effects, Virus Replication drug effects, Antiviral Agents pharmacology, Furans pharmacology, Hepacivirus drug effects, Hepatitis C drug therapy, Pyridines pharmacology, Thiophenes pharmacology, Viral Nonstructural Proteins antagonists & inhibitors
- Abstract
A small-molecule inhibitor of hepatitis C virus (HCV) designated AP89652 was identified by screening a compound library with an HCV genotype 1b subgenomic replicon assay. AP89652 contains two chiral centers, and testing of two syn enantiomers revealed that activity in the replicon assay resided with only one, AP80978, whose 50% effective concentration (EC50) (the concentration at which a 50% reduction in Renilla luciferase levels was observed relative to an untreated control) was 630 nM. AP80978 was inhibitory against HCV genotypes 1a and 1b but not genotype 2a. In a replicon clearance assay, the potency and clearance rate of AP80978 were similar to those of telaprevir (VX950) and cyclosporine (CsA). AP80978 was nontoxic when tested against a panel of human cell lines, and inhibitory activity was HCV specific in that there was limited activity against negative-strand viruses, an alphavirus, and flaviviruses. By selection of resistant replicons and assessment of activity in genotype 1b/2a intergenotypic replicons, the viral protein target of this compound was identified as NS4B. NS4B F98V/L substitutions were confirmed by site-directed mutagenesis as AP80978 resistance-associated mutations. When tested against HCV produced in cell culture, the compound was significantly more potent than other HCV inhibitors, including VX950, CsA, and 2'-C-methyladenosine (2'C-meA). In addition, AP80977, the enantiomer that was inactive in the replicon assay, had activity against the virus, although it was lower than the activity of AP80978. These results suggest that AP80978 has the potential to be optimized into an effective antiviral drug and is a useful tool to further study the role of NS4B in HCV replication., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
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4. Zip3 (Slc39a3) functions in zinc reuptake from the alveolar lumen in lactating mammary gland.
- Author
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Kelleher SL, Lopez V, Lönnerdal B, Dufner-Beattie J, and Andrews GK
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- Animals, Animals, Suckling, Apoptosis, Cation Transport Proteins deficiency, Cation Transport Proteins genetics, Cell Membrane metabolism, Epithelial Cells pathology, Female, Genotype, Ion Transport, Mammary Glands, Animal pathology, Mice, Mice, Knockout, Phenotype, Zinc blood, Zinc Radioisotopes, Cation Transport Proteins metabolism, Epithelial Cells metabolism, Lactation metabolism, Mammary Glands, Animal metabolism, Milk metabolism, Zinc metabolism
- Abstract
The lactating mammary gland is composed of multiple cell types that tightly coordinate the accumulation, production, and secretion of milk components, including essential metals such as zinc (Zn). Our previous studies in animal and cell models implicated the Zn transporter Zip3 (Slc39a3) in mammary gland Zn acquisition. Herein, we investigated this hypothesis directly by utilizing Zip3-null mice. Our data verify that Zip3 is expressed in secretory mammary cells; however, Zip3 does not play a major role in Zn import from the maternal circulation. Importantly, the primary localization of Zip3 was associated with the luminal membrane of the secretory mammary cells. Consistent with this localization, Zn transfer studies using (65)Zn revealed that Zn retention in the secreted milk pool and milk Zn concentration was higher in Zip3-null compared with wild-type mice. Although total mammary gland Zn concentration was not altered, Zip3-null mice also had altered mammary tissue architecture, increased number of apoptotic cells, and reduced mammary gland weight implicating subtle changes in Zip3-mediated intracellular Zn pools in apoptosis regulation. Taken together, our data indicate that Zip3 does not participate in the acquisition of Zn from maternal circulation for secretion into milk but, in contrast, primarily plays a role in the reuptake and cellular retention of Zn in the mammary gland from the previously secreted milk pool, thus regulating cellular function.
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- 2009
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5. Novel zinc-responsive post-transcriptional mechanisms reciprocally regulate expression of the mouse Slc39a4 and Slc39a5 zinc transporters (Zip4 and Zip5).
- Author
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Weaver BP, Dufner-Beattie J, Kambe T, and Andrews GK
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- Animals, Blotting, Northern, Blotting, Western, Cell Line, Centrifugation, Density Gradient, Diet, Female, Immunohistochemistry, Immunoprecipitation, Kinetics, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Organ Culture Techniques, Polyribosomes genetics, Pregnancy, RNA, Messenger biosynthesis, RNA, Messenger genetics, RNA, Messenger isolation & purification, Reverse Transcriptase Polymerase Chain Reaction, Yolk Sac transplantation, Cation Transport Proteins biosynthesis, Cation Transport Proteins genetics, Gene Expression Regulation drug effects, Protein Processing, Post-Translational drug effects, Zinc pharmacology
- Abstract
Dietary zinc deficiency in mice is accompanied by enhanced expression of the zinc uptake transporter Slc39a4 (Zip4) and repressed expression of Slc39a5 (Zip5) in tissues which regulate zinc homeostasis (intestine, pancreas and visceral yolk sac). Herein, mechanisms controlling this differential expression were investigated. The induction of Zip4 mRNA during zinc deficiency, and its repression in response to zinc repletion were found to reflect changes in Zip4 mRNA stability and not changes in the relative rate of transcription of this gene. During zinc deficiency, ZIP4 protein levels are increased and this protein is localized on the apical membranes. Administration of an oral gavage of zinc caused ZIP4 internalization and degradation in enterocytes and visceral endoderm cells. Similarly, ZIP4 is induced by zinc deficiency in cultured mouse Hepa cells and is rapidly degraded in response to added zinc. Zip5 mRNA abundance does not change in response to zinc, but the translation of this mRNA was found to be zinc-responsive. During zinc deficiency, Zip5 mRNA remains associated with polysomes, while the protein is internalized and degraded in enterocytes, acinar cells and endoderm cells. After zinc-gavage, ZIP5 is rapidly resynthesized and targeted to the basolateral membranes of these cell types.
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- 2007
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6. The mouse acrodermatitis enteropathica gene Slc39a4 (Zip4) is essential for early development and heterozygosity causes hypersensitivity to zinc deficiency.
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Dufner-Beattie J, Weaver BP, Geiser J, Bilgen M, Larson M, Xu W, and Andrews GK
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- Acrodermatitis genetics, Alleles, Animals, Cation Transport Proteins metabolism, Cells, Cultured, Embryo, Mammalian metabolism, Endoderm metabolism, Female, Homozygote, Male, Mice, Mice, Inbred C57BL, Mice, Inbred Strains, Mice, Knockout, Microscopy, Fluorescence, Models, Genetic, Zinc metabolism, Cation Transport Proteins genetics, Embryonic Development, Heterozygote, Zinc deficiency
- Abstract
The human Zip4 gene (Slc39a4) is mutated in the rare recessive genetic disorder of zinc metabolism acrodermatitis enteropathica, but the physiological functions of Zip4 are not well understood. Herein we demonstrate that homozygous Zip4-knockout mouse embryos die during early morphogenesis and heterozygous offspring are significantly underrepresented. At mid-gestation, an array of developmental defects including exencephalia, anophthalmia and severe growth retardation were noted in heterozygous embryos, and at weaning, many (63/280) heterozygous offspring were hydrocephalic, growth retarded and missing one or both eyes. Maternal dietary zinc deficiency during pregnancy exacerbated these effects, whereas zinc excess ameliorated these effects and protected embryonic development of heterozygotes but failed to rescue homozygous embryos. Heterozygous Zip4 embryos were not underrepresented in litters from wild-type mothers, but were approximately 10 times more likely to develop abnormally than were their wild-type littermates during zinc deficiency. Thus, both embryonic and maternal Zip4 gene expressions are critical for proper zinc homeostasis. These studies suggest that heterozygous mutations in the acrodermatitis gene Zip4 may be associated with a wider range of developmental defects than was previously appreciated, particularly when dietary zinc is limiting.
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- 2007
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7. Targeting of the mouse Slc39a2 (Zip2) gene reveals highly cell-specific patterns of expression, and unique functions in zinc, iron, and calcium homeostasis.
- Author
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Peters JL, Dufner-Beattie J, Xu W, Geiser J, Lahner B, Salt DE, and Andrews GK
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- Animals, Calcium metabolism, Cation Transport Proteins genetics, Dendritic Cells chemistry, Dendritic Cells metabolism, Female, Gene Expression, Gene Targeting, Green Fluorescent Proteins analysis, Green Fluorescent Proteins genetics, Hepatocytes chemistry, Hepatocytes metabolism, Homeostasis, Iron metabolism, Keratinocytes chemistry, Keratinocytes metabolism, Mice, Mice, Knockout, Pregnancy, Trophoblasts chemistry, Trophoblasts metabolism, Cation Transport Proteins physiology, Gene Expression Regulation, Developmental, Zinc metabolism
- Abstract
Fourteen members of the Slc39a superfamily of metal ion uptake transporters have been identified in mice and humans, but the physiological functions of most remain obscure. Herein, we created mice with Zip2 (Slc39a2) genes in which the open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP), to study temporal and spatial patterns of Zip2 gene expression and examine the physiological roles of this transporter. Expression of this gene was remarkably cell-type specific and developmentally regulated in pericentral hepatocytes, developing keratinocytes, and a subset of immature dendritic cells in the immune system. In addition, the Zip2 gene was transiently expressed in giant trophoblast cells in the placenta. Although the Zip2 gene was not essential under conditions of normal dietary zinc, it played an important role in adapting to dietary zinc deficiency during pregnancy, and in the homeostasis of iron in the liver as well as iron and calcium in developing embryos. These studies suggest that active expression of the Zip2 gene in these few specific cell types, aforementioned, plays a particularly important role during zinc deficiency. These studies further reveal novel interactions between zinc transporter function and the homeostasis of other essential metals., (Published 2007 Wiley-Liss, Inc.)
- Published
- 2007
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8. Expression and regulation of SLC39A family zinc transporters in the developing mouse intestine.
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Huang ZL, Dufner-Beattie J, and Andrews GK
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- Adaptation, Physiological genetics, Animals, Animals, Newborn, Embryo, Mammalian, Gene Expression Regulation, Developmental physiology, Homeostasis, Mice, Tissue Distribution, Zinc physiology, Cation Transport Proteins genetics, Gene Expression Regulation, Intestines growth & development, Zinc metabolism
- Abstract
Several ZIP genes (SLC39A family of metal transporters) play roles in zinc homeostasis. Herein, the temporal and spatial patterns of expression of the mouse ZIP1, 3, 4, and 5 genes in the developing intestine and the effects of maternal dietary zinc deficiency on these patterns of expression were examined. ZIP1 and ZIP3 genes, conserved members of the ZIP subfamily II, were found to be coexpressed during development. Expression of these genes was detected on day 14 of gestation in smooth muscle and the pseudostratified endoderm. By 5 days post-partum, prominent expression became restricted to muscle and connective stroma. In contrast, expression of ZIP4 and ZIP5 genes, members of the ZIP subfamily called LIV-1, coincided with epithelial morphogenesis. ZIP5 expression was detected on d16 of gestation and localized to the basolateral membranes of the single-layered epithelium. ZIP4 expression was detected on d18 of gestation and localized to the apical membrane of villus epithelial cells. When dams were fed a zinc-deficient diet beginning at parturition, ZIP4 expression in the nursing neonate was greatly induced. In contrast, neonatal ZIP5 expression remained unchanged, but this protein was removed from the basolateral membrane of the enterocyte. These responses to dietary zinc deficiency mimic those found in the adult intestine. These studies reveal cell-type-specific expression of ZIP genes during development of the intestine, and suggest that the mouse intestine can elicit an adaptive response to dietary zinc availability at birth.
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- 2006
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9. Mouse ZIP1 and ZIP3 genes together are essential for adaptation to dietary zinc deficiency during pregnancy.
- Author
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Dufner-Beattie J, Huang ZL, Geiser J, Xu W, and Andrews GK
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- Adaptation, Physiological, Animals, Cation Transport Proteins genetics, Congenital Abnormalities etiology, Diet, Embryo Loss etiology, Embryo, Mammalian metabolism, Female, Gene Expression, Mice, Mice, Inbred C57BL, Mice, Knockout, Mutation, Pregnancy, Zinc metabolism, Cation Transport Proteins metabolism, Embryonic Development, Pregnancy Complications metabolism, Zinc deficiency
- Abstract
Subfamily II of the solute-linked carrier 39A superfamily contains three well-conserved zinc transporters (ZIPs1, 2, 3) whose physiological functions are unknown. We generated mice homozygous for knockout alleles of ZIP1 and both ZIP1 and ZIP 3 (double-knockout). These mice were apparently normal when dietary zinc was replete, but when dietary zinc was limited during pregnancy embryos from ZIP1 or ZIP3 knockout mice were two to three times more likely to develop abnormally than those in wildtype mice, and 91% (71/78) of embryos developed abnormally in ZIP1, ZIP3 double-knockout mice. Analysis of the patterns of expression of these genes in mice revealed predominate expression in intestinal stromal cells, nephric-tubular epithelial cells, pancreatic ductal epithelial cells, and hepatocytes surrounding the central vein. This suggests that these zinc transporters function, at least in part, in the redistribution and/or retention of zinc rather than its acquisition from the diet. In conclusion, mutations in the ZIP1 and ZIP3 zinc transporter genes are silent when dietary intake of zinc is normal, but can dramatically compromise the success of pregnancy when dietary intake of zinc is limiting.
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- 2006
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10. Generation and characterization of mice lacking the zinc uptake transporter ZIP3.
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Dufner-Beattie J, Huang ZL, Geiser J, Xu W, and Andrews GK
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- Animals, Animals, Newborn, Blastocyst metabolism, Cation Transport Proteins genetics, Cells, Cultured, Crosses, Genetic, Electroporation, Embryonic Development, Female, Genes, Reporter, Genetic Vectors, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Pregnancy, Stem Cells cytology, Tissue Distribution, Zinc analysis, Zinc deficiency, Carrier Proteins genetics, Carrier Proteins metabolism, Cation Transport Proteins metabolism, Gene Expression Regulation, Developmental, Zinc metabolism
- Abstract
The mouse ZIP3 (SLC39A3) gene encodes an eight-transmembrane-domain protein that has been conserved in mammals and can function to transport zinc. To analyze the expression of ZIP3 in the early embryo and neonate and to determine its in vivo function, we generated ZIP3 null mice in which the ZIP3 open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP) reporter. EGFP fluorescence revealed that ZIP3 was expressed in the inner cell mass of the blastocyst and later during embryonic development in many tissues. Elevated expression was apparent in the embryonic brain and neurotube and neonatal gonads. Homozygous knockout mice were viable and fertile and under normal growth conditions exhibited no obvious phenotypic abnormalities. Deletion of ZIP3 did not alter zinc homeostasis at the molecular level as assessed by essential metal levels and the expression of zinc-responsive genes. In knockout mice stressed with a zinc-deficient diet during pregnancy or at weaning, a subtle increase in the sensitivity to abnormal morphogenesis of the embryo and to depletion of thymic pre-T cells, respectively, was noted. These results suggest that this protein plays an ancillary role in zinc homeostasis in mice.
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- 2005
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11. The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5.
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Dufner-Beattie J, Kuo YM, Gitschier J, and Andrews GK
- Subjects
- Amino Acid Sequence, Animals, Biological Transport, Blotting, Northern, Blotting, Western, Cation Transport Proteins chemistry, Cell Membrane metabolism, Computational Biology, Conserved Sequence, DNA, Complementary metabolism, Dietary Supplements, Evolution, Molecular, Exons, Female, Immunohistochemistry, Insulin metabolism, Mice, Microscopy, Fluorescence, Models, Genetic, Molecular Sequence Data, Pancreas cytology, Pancreas embryology, Pancreas metabolism, RNA metabolism, Sequence Homology, Amino Acid, Time Factors, Tissue Distribution, Yolk Sac metabolism, Zinc chemistry, Zinc metabolism, Cation Transport Proteins metabolism, Endoderm metabolism, Gene Expression Regulation, Developmental, Zinc pharmacology
- Abstract
The ZIP5 gene encodes a protein closely related to ZIP4, a zinc transporter mutated in the human genetic disorder acrodermatitis enteropathica. Herein, we demonstrate that mouse ZIP5 and ZIP4 genes are co-expressed in several tissues involved in zinc homeostasis (intestine, pancreas, embryonic yolk sac). However, unlike expression of the ZIP4 gene, which is induced during periods of zinc deficiency, ZIP5 gene expression is unaltered by dietary zinc. Immunohistochemistry localizes ZIP5 to the basolateral surfaces of enterocytes, acinar cells, and visceral endoderm cells in mice fed a zinc-adequate diet. However, this protein is removed from these cell surfaces and internalized during dietary zinc deficiency. In contrast, ZIP4 is induced and recruited to the apical surface of enterocytes and endoderm cells during zinc deficiency. In the pancreas, ZIP4 is expressed in beta-cells, whereas ZIP5 is expressed in acinar cells. These results suggest that the function of ZIP5 is antagonistic to that of ZIP4 in the control of zinc homeostasis; rather than functioning in the acquisition of dietary zinc, as does ZIP4, ZIP5 may function in the removal of zinc from the body. Thus, during periods when dietary zinc is replete, ZIP5 may function to remove zinc from the blood via the pancreas and intestine, the major sites of zinc excretion in mammals, whereas the acquisition of dietary zinc by intestinal ZIP4 would be minimal. In contrast, during periods of dietary zinc deficiency when secretion of zinc by the pancreas and intestine is minimized, ZIP5 is removed from the cell surface, and the intestinal uptake of zinc is augmented by induction of ZIP4.
- Published
- 2004
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12. Zinc-stimulated endocytosis controls activity of the mouse ZIP1 and ZIP3 zinc uptake transporters.
- Author
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Wang F, Dufner-Beattie J, Kim BE, Petris MJ, Andrews G, and Eide DJ
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- Animals, Cation Transport Proteins, Cell Line, Cell Membrane metabolism, Humans, Immunoblotting, Mice, Microscopy, Fluorescence, Plasmids metabolism, Protein Processing, Post-Translational, RNA, Messenger metabolism, Subcellular Fractions metabolism, Time Factors, Transfection, Zinc chemistry, Carrier Proteins metabolism, Endocytosis, Zinc metabolism
- Abstract
The mouse mZip1 and mZip3 zinc transporters have been implicated in zinc acquisition by the cells of many tissues. This hypothesis raised the question of whether activity of these proteins is regulated to maintain zinc homeostasis. Neither mZIP1 nor mZIP3 mRNA levels are highly regulated by zinc status. Therefore, we investigated whether zinc controls the activity of these proteins post-translationally by altering their subcellular distribution. When expressed in transfected cells grown in zinc-replete medium, both mZip1 and mZip3 were largely present in intracellular organelles. However, these proteins were found to rapidly transit between the plasma membrane and intracellular compartments in zinc-replete cells. Zinc deficiency increased plasma membrane levels of mZip1 and mZip3 by decreasing their rates of endocytosis. Greater zinc deficiency was required to alter mZip3 distribution than was needed to affect mZip1. Increased surface levels correlated with increased zinc uptake activity. Taken together, these results suggest that post-translational control of mZip1 and mZip3 localization plays a role in zinc homeostasis. Moreover, our results indicate that zinc-responsive endocytosis is a conserved mechanism controlling activity of many mammalian zinc uptake transporters.
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- 2004
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13. Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter.
- Author
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Wang F, Kim BE, Dufner-Beattie J, Petris MJ, Andrews G, and Eide DJ
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- Animals, Cation Transport Proteins genetics, Cells, Cultured, Disease Models, Animal, Glycosylation, Immunoblotting, Mice, Microscopy, Fluorescence, Mutation, Missense genetics, Plasmids genetics, Protein Structure, Tertiary, Transfection, Acrodermatitis genetics, Alleles, Cation Transport Proteins metabolism, Endocytosis genetics, Zinc metabolism
- Abstract
The Zip4 protein is involved in dietary zinc uptake from the intestinal lumen. The human ZIP4 gene (SLC39A4) was identified because of its association with acrodermatitis enteropathica (AE), a genetic disorder of zinc absorption. To date, several SLC39A4 mutations have been identified in AE patients. To investigate the effects of these mutations on function of the Zip4 transporter, we introduced six AE-associated missense mutations into the orthologous mouse ZIP4 gene for functional expression in cultured cells. All mutations decreased 65Zn uptake activity of mZip4, thereby providing a causal link to AE. The mutants fell into two groups based on their phenotypic effects. Several alleles (G340D, L382P, G384R, G643R) failed to localize on the cell surface at high levels. These defects were attributable to misfolding and/or mislocalization in the secretory pathway. Two other alleles (P200L and G539R) accumulated to high levels in the plasma membrane and had wild-type apparent Km values for 65Zn uptake. However, these mutations decreased the Vmax of uptake to approximately 30% of wild-type. We showed previously that wild-type mZip4 is regulated post-translationally in response to zinc status. In zinc-replete cells, mZip4 is found largely in intracellular compartments. In zinc-limited cells, surface levels increase markedly because the rate of endocytosis decreases. Surprisingly, endocytosis of both P200L and G539R is no longer zinc responsive; these proteins are endocytosed at a slow rate regardless of zinc status. These effects suggest a zinc sensing mechanism for regulating Zip4 trafficking in response to zinc.
- Published
- 2004
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14. Zn2+-stimulated endocytosis of the mZIP4 zinc transporter regulates its location at the plasma membrane.
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Kim BE, Wang F, Dufner-Beattie J, Andrews GK, Eide DJ, and Petris MJ
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- Animals, Cation Transport Proteins chemistry, Cation Transport Proteins genetics, Cell Line, Cell Membrane metabolism, Endocytosis, Humans, Mice, Models, Molecular, Protein Processing, Post-Translational, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Transfection, Zinc deficiency, Zinc pharmacology, Cation Transport Proteins metabolism, Zinc metabolism
- Abstract
Zinc is an essential nutrient for all organisms. Its requirement in humans is illustrated dramatically by the genetic disorder acrodermatitis enteropathica (AE). AE is caused by the reduced uptake of dietary zinc by enterocytes, and the ensuing systemic zinc deficiency leads to dermatological lesions and immune and reproductive dysfunction. The gene responsible for AE, SLC39A4, encodes a member of the ZIP family of metal transporters, hZIP4. The mouse ZIP4 protein, mZIP4, stimulates zinc uptake in cultured cells, and studies in mice have demonstrated that zinc treatment decreases mZIP4 mRNA levels in the gut. In this study, we demonstrated using transfected cultured cells that the mZIP4 protein is also regulated at a post-translational level in response to zinc availability. Zinc deficiency increased mZIP4 protein levels at the plasma membrane, and this was associated with increased zinc uptake. Significantly, treating cells with low micromolar zinc concentrations stimulated the rapid endocytosis of the transporter. Zinc-regulated localization of the human ZIP4 protein was also demonstrated in cultured cells. These findings suggest that zinc-regulated trafficking of human and mouse ZIP4 is a key mechanism controlling dietary zinc absorption and cellular zinc homeostasis.
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- 2004
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15. Structure, function, and regulation of a subfamily of mouse zinc transporter genes.
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Dufner-Beattie J, Langmade SJ, Wang F, Eide D, and Andrews GK
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- Amino Acid Sequence, Animals, Biological Transport, Blotting, Northern, Cation Transport Proteins, Cations, Cell Line, Cells, Cultured, DNA, Complementary metabolism, Dose-Response Relationship, Drug, Female, Gene Expression Regulation, Humans, Intestinal Mucosa metabolism, Male, Mice, Models, Genetic, Molecular Sequence Data, Plasmids metabolism, Protein Structure, Tertiary, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Structure-Activity Relationship, Time Factors, Tissue Distribution, Transfection, Zinc chemistry, Zinc deficiency, Zinc metabolism, Carrier Proteins chemistry, Carrier Proteins genetics, Carrier Proteins physiology
- Abstract
Zinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eight-transmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of zinc in transfected cultured cells, similar to the previously demonstrated functions of human ZIP1 and ZIP2 (Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564; Gaither, L. A., and Eide, D. J. (2001) J. Biol. Chem. 276, 22258-22264). No ZIP3 orthologs have been previously studied. Furthermore, this first systematic comparative study of the in vivo expression and dietary zinc regulation of this subfamily of zip genes revealed that 1) zip1 mRNA is abundant in many mouse tissues, whereas zip2 and zip3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted in their accumulation; and 2) unlike mouse metallothionein I and zip4 mRNAs (Dufner-Beattie, J., Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D., and Andrews, G. K. (2003) J. Biol. Chem. 278, 33474-33481), the abundance of zip1, zip2, and zip3 mRNAs is not regulated by dietary zinc in the intestine and visceral endoderm, tissues involved in nutrient absorption. These studies suggest that all three of these ZIP proteins may play cell-specific roles in zinc homeostasis rather than primary roles in the acquisition of dietary zinc.
- Published
- 2003
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16. The acrodermatitis enteropathica gene ZIP4 encodes a tissue-specific, zinc-regulated zinc transporter in mice.
- Author
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Dufner-Beattie J, Wang F, Kuo YM, Gitschier J, Eide D, and Andrews GK
- Subjects
- Amino Acid Sequence, Animals, Biological Transport, Blotting, Northern, Cation Transport Proteins chemistry, Cations, Cell Membrane metabolism, Dose-Response Relationship, Drug, Female, Immunohistochemistry, Intestinal Mucosa metabolism, Intestine, Small embryology, Kinetics, Male, Metals chemistry, Mice, Models, Genetic, Molecular Sequence Data, Peptides chemistry, Plasmids metabolism, Protein Structure, Tertiary, RNA metabolism, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Tissue Distribution, Transfection, Zinc metabolism, Acrodermatitis metabolism, Carrier Proteins metabolism, Cation Transport Proteins metabolism, Cation Transport Proteins physiology
- Abstract
The human ZIP4 gene (SLC39A4) is a candidate for the genetic disorder of zinc metabolism acrodermatitis enteropathica. To understand its role in zinc homeostasis, we examined the function and expression of mouse ZIP4. This gene encodes a well conserved eight-transmembrane protein that can specifically increase the influx of zinc into transfected cells. Expression of this gene is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by zinc. Dietary zinc deficiency causes a marked increase in the accumulation of ZIP4 mRNA in these tissues, whereas injection of zinc or increasing zinc content of the diet rapidly reduces its abundance. Zinc can also regulate the accumulation of ZIP4 protein at the apical surface of enterocytes and visceral endoderm cells. These results provide compelling evidence that ZIP4 is a zinc transporter that plays an important role in zinc homeostasis, a process that is defective in acrodermatitis enteropathica in humans.
- Published
- 2003
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17. Pancreatic metallothionein-I may play a role in zinc homeostasis during maternal dietary zinc deficiency in mice.
- Author
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Lee DK, Geiser J, Dufner-Beattie J, and Andrews GK
- Subjects
- Animals, Diet, Female, Genotype, Homeostasis, Metallothionein genetics, Mice, Mice, Transgenic, Pancrelipase metabolism, Pregnancy, Zinc administration & dosage, Metallothionein physiology, Pancrelipase physiology, Zinc deficiency
- Abstract
Herein, the function of pancreatic metallothionein (MT)-I during zinc deficiency in pregnancy was examined using transgenic mice, which constitutively express the mouse MT-I gene driven by the rat elastase I promoter. Pancreatic MT protein levels and zinc levels were elevated significantly in the transgenic mice compared with those in control mice. Pregnant transgenic and control mice were fed zinc-deficient (1 micro g/g beginning at d 8) or zinc-adequate (50 micro g/g) diets during pregnancy, and the effects on the morphology of embryos were determined at d 14 of pregnancy (d 1 = vaginal plug). As other indicators of zinc deficiency, maternal pancreatic MT levels, as well as the expression of zinc-regulated genes in the embryonic visceral yolk sac were examined. Under these experimental conditions of moderate dietary zinc deficiency, 21.3% of the embryos in control mice exhibited morphological defects, whereas only 5.8% of the embryos in the elastase-MT-I transgenic females had developed abnormally by d 14. Surprisingly, dietary zinc deficiency caused a >95% decrease in pancreatic MT protein concentration in these transgenic mice. This suggests the post-transcriptional control of MT protein levels during zinc deficiency because the rat elastase I promoter is not metal-regulated. The decrease in pancreatic MT protein levels was paralleled by a dramatic decrease in the relative abundance of MT-I mRNA and a dramatic increase in the relative abundance of the zinc/iron regulated transporter-related zinc transporter-4 (ZIP4) mRNA in the embryonic visceral yolk sac. Thus, the constitutive overexpression of pancreatic MT-I in these mice attenuated, but did not prevent the effects of maternal or embryonic zinc deficiency under these conditions. Overall, these findings are consistent with the hypothesis that mouse pancreatic MT-I may participate in providing a labile pool of maternal zinc for the developing embryo during periods of zinc deficiency.
- Published
- 2003
- Full Text
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