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Incidence of the Erythrocytic Defect associated with Drug-Sensitivity among Oriental Subjects (1959)

BEUTLER, ERNEST ; YEH, MARY K. Y. ; NECHELES, THOMAS

[s.l.] : Nature Publishing Group

Nature 183 (1959), S. 684-685

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ISSN: 1476-4687

Source: Nature Archives 1869 - 2009

Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics

Notes: [Auszug] It has been known for some time that sensitivity to drug-induced hsomolysis varies considerably in different racial groups. Application of these in vitro techniques has disclosed marked differences in the incidence of this red-cell defect in various population groups. Thus, the defect is common in ...

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1038/183684b0

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2

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Sialic acid uptake by BHK cells and subsequent incorporation into glycoproteins and glycolipids (1977)

Hirschberg, Carlos B. ; Yeh, Mary

New York, N.Y. : Wiley-Blackwell

Journal of Supramolecular Structure 6 (1977), S. 571-577

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ISSN: 0091-7419

Keywords: sialic acid uptake ; sialoglycoproteins ; sialoglycolipids ; Life Sciences ; Molecular Cell Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Chemistry and Pharmacology , Medicine

Notes: BHK cells can be grown in the presence of growth medium to which radiolabeled sialic acid has been added. After 24 h, 85% of the radioactivity in the cells is covalently bound to glycoproteins and glycolipids. No metabolism of the radiolabeled sialic acid could be detected.

Additional Material: 3 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jss.400060410

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The DMT1 Associated Protein (DAP) Regulates Iron Uptake by Erythroid Cells. (2004)

Glass, Jonathan ; Chen, Yi ; Ma, Yuxiang ; [et al.]

American Society of Hematology

In: Blood. 2004; 104(11): 53-53. Published 2004 Nov 16. doi: 10.1182/blood.v104.11.53.53.

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Publication Date: 2004-11-16

Description: The divalent metal transporter 1 (DMT1) is essential for cellular iron uptake both in the intestine and in erythroid cells. We have previously shown that with iron feeding the DMT1 expressed on the brush border membrane (BBM) of the intestine undergoes endocytosis (Am. J. Physiol. 283, G965, 2002). Using the yeast two-hybrid system we have isolated a cDNA clone encoding a protein of 526-amino acid residues with a calculated molecular mass of 60 kDa, which interacts with the C-terminus of DMT1 expressed from the IRE containing mRNA (Blood ,100, 7a, 2002). The ORF of the rat protein has been fully sequenced (Genbank #AY336075) and is now designated DMT1 associated protein (DAP). DAP is ubiquitously expressed and is especially abundant in the rat intestine and colon. In rat duodenum DAP is colocalized with DMT1 in the BBM. Both by salt and pH elution DAP was demonstrated to be a peripheral membrane protein. With iron feeding both DMT1 and DAP translocate: DAP moves from the BBM to basolateral membrane (BLM) with DMT1 and some of DMT1 undergoes endocytosis and is found in cytopasmic vesicles. Immunoprecipitation and pull-down assays confirm the interaction of DAP and DMT1 in the BBM vesicles (MMBV). We have analyzed the function of DAP by exploring the role of various consensus sequences in the DAP ORF in the cellular localization of the protein. By sequence motif analysis DAP has a nuclear localization signal, Glutamic acid-rich region, Glutamine-rich region, Arginine-rich region, PKC phosphorylation sites and GOLD domain (Golgi dynamics). The region of DAP protein interacting with the COOH-terminal cytoplasmic domain of DMT1(IRE) was found to be from 171aa to 331aa which contains Glutamic acid-rich region, Glutamine-rich region and Arginine-rich region. Immunocytochemistry confirmed that DAP is localized in the nuclei and the Golgi complex of K562, MDCK, Hela, Cos1 cells, and Caco2 (where DAP is found also in BBM). GFP-fusion constructs containing the DAP nuclear localization signal (amino acids 171–277) or GOLD domain (amino acid 278–526) were transfected into COS-1 and K562 cells and specificity of intracellular localization confirmed by fluorescence confocal microscopy. DAP expression was controlled by cellular iron content: Cells which were iron depleted had increased levels of DAP protein while cells which were iron replete had decreased DAP protein. The regulation by iron was post-transcriptional as iron levels had no affect on DAP mRNA. The levels of DAP expression was also seen to affect iron uptake. Over expression of the region of DAP which binds to DMT1 by transfection of the appropriate construct into K562 cells decreased iron uptake as measured by an increase of transferrin receptor expression and decreased levels of ferritin. Elevated DAP had no affect on endogenous DMT1 expression. Conversely, when siRNAs were used to decrease DAP mRNA in K562 cells there was increased iron uptake with decreased expression of transferrin receptor and increased ferritin expression. In these experiments siRNAs reduced DAP expression by about 60%. This is the first demonstration that a protein which interacts with DMT1 can regulate the uptake of iron into the cell and suggests that DAP may act in a regulatory pathway for iron homeostasis.

Print ISSN: 0006-4971

Electronic ISSN: 1528-0020

Topics: Biology , Medicine

Published by American Society of Hematology

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DMT1 Expression Is Regulated by DMT1 Associated Protein (DAP) in K562 Cells. (2007)

Okazaki, Yasumasa ; Yin, Hong ; Ma, Yuxiang ; [et al.]

American Society of Hematology

In: Blood. 2007; 110(11): 2661-2661. Published 2007 Nov 16. doi: 10.1182/blood.v110.11.2661.2661.

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Publication Date: 2007-11-16

Description: While iron is essential for cell growth and survival excess iron through oxidative stress may produce hepatitic cirrhosis and hepatocellular carcinoma, diabetes mellitus, and cardiomyopathy. Iron is absorbed across the duodenum with transport across the brush border mediated by DMT1 and across the basolateral surface by ferroportin with mechanisms that are inversely regulated by body iron concentrations. We have identified in rat intestine DAP, a novel protein that binds to the C-terminus of DMT1 (IRE) but not to the C-terminus of the non-IRE isoform (Blood, Nov 2004; 104: 53). DAP is a 526 amino acid protein that has been previously described as binding to the peripheral benzodiazepine receptor, an intrinsic mitochondrial protein involved in steriodogenesis and possibly in protoporphyrin IX transport into the mitochrondria. To investigate if DAP may have a role in regulation of intracellular iron transport DAP expression was down regulated using a vector containing a siRNA for DAP transfected into K562 cells by electroporation. Expression levels of DAP, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1) and ferritin were examined by western blot and quantitative quantitivative PCR assays from days 1 to 6 after transfection. Following transfection with the DAP siRNA DAP mRNA levels were decreased 50% by day 1 with DAP protein levels decreasing by 50% at day 3. The DAP siRNA also decreased DMT1 protein expression by about 50% for the DMT1 (IRE) protein but had no effect on the protein derived from the non-IRE isoform. The leels of DMT1 mRNA were not affected by DAP siRNA. The decrease of DAP expression was not associated with any change in TfR1 or ferritin expression, suggesting that altered levels of DAP did not affect intracellular iron pools. Transfection with the DAP siRNA resulted also in more protean effects decreasing cell proliferation, the transition from S-phase to G2 in cell cycle, and protein synthesis. These data are consistent with DAP regulating DMT1 expression in K562 cells by modulating turnover of DMT1 (IRE) protein and also having more global effects on cellular metabolism.

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Protoporphyrin IX Down-Regulates DMT1 Associated Protein (DAP) in K562 Cells. (2006)

Okazaki, Yasumasa ; Yin, Hong ; Ma, Yuxiang ; [et al.]

American Society of Hematology

In: Blood. 2006; 108(11): 1548-1548. Published 2006 Nov 16. doi: 10.1182/blood.v108.11.1548.1548.

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Publication Date: 2006-11-16

Description: The final steps of heme biosynthesis include the transport of coproporphyrin with the transport step probably mediated by the peripheral benzodiazepine receptor (PBR). Within the mitochondria copropoprhyrin is then converted to protoporphyrin IX (PPIX) which in turn is converted to hemin with insertion of iron by ferrochelatase. PBR is ubiquitously expressed and has been implicated in steriodogenesis, apoptosis, erythroid differentiation, and inflammation. Interestingly, PPIX is among several high affinity ligands for PBR. Various cytosolic proteins that interact with PBR have also been defined including PBR associated protein 7 (PAP7). The various PBR ligands including PPIX may affect the binding of these proteins to PBR. We have demonstrated (Blood, Nov 2004; 104: 53) that DAP, a protein highly homologous to PAP7, binds to the C-terminus of DMT1 and may have a role in regulation of intracellular iron transport. We, therefore, examined the effects of PPIX on the functions of DAP and other proteins that affect cellular iron metabolism. DAP is 526 amino acid protein with a nuclear localization signal domain (aa 212–229) and a Golgi localization domain (aa 380–524), and is distributed in the cytoplasm, Golgi apparatus, and nuclei of K562 cells. K562 cells were grown in the presence of 5 μM PPIX for 24 hours and then the expression of DAP, transferrin receptor 1 (TfR1), and ferritin examined by western blot analysis. In addition, cells were grown in medium of either normal iron content (3.5 μM from ferri-transferrin), high iron content (217 μM from the addition of ferric ammonium citrate), or low iron content (by the addition of 50 mM desferroxamine). Under all three iron conditions PPIX induced differentiation but down-regulated ferritin expression and up-regulated TfR1 expression. Additionally, PPIX had a striking effect on DAP expression markedly decreasing DAP levels but only in cells grown either in normal or low iron medium. In addition, PPIX affected the expression of the iron transporter DMT1in parallel with DAP. As PPIX induced erythroid differentiation of K562 cells we examined the effects of hemin which can also induce differentiation of K562 cells. In contrast to PPIX, hemin caused strong down-regulation of TfR and up-regulation of ferritin and DAP. The down-regulation of DAP induced by PPIX was restored by the addition of hemin. These results indicate that PPIX affects DAP expression and other important elements involved in cellular iron metabolism and that these effects are partially modified by the iron status of the cell.

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Topics: Biology , Medicine

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Iron Transport in Intestinal Epithelial Cells Occurs by Transcytosis. A Fluorescent Metal-Sensor Study. (2005)

Ma, Yuxiang ; Yeh, Mary ; Yeh, Kwo-yih ; [et al.]

American Society of Hematology

In: Blood. 2005; 106(11): 3583-3583. Published 2005 Nov 16. doi: 10.1182/blood.v106.11.3583.3583.

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Publication Date: 2005-11-16

Description: The molecular mechanisms ensuring directionality of iron transport across the intestinal epithelium are still poorly understood. Iron is transported across the brush-border membrane (BBM) by the divalent metal transporter 1 (DMT1) and then must be targeted to specific organelles and other transporters for transport across the basolateral membrane (BLM). We have previously shown that in Caco2 cells grown in bicameral chambers with the addition of iron to the apical surface, DMT1 on the BBM undergoes endocytosis and fuses with endocytic vesicles derived from the basal surface. However, which cytosolic proteins mediate the endocytosis of DMT1 and the interaction of apical derived vesicles with basal derived vesicles are still unknown. The present study focuses on trying to confirm our hypothesis that iron absorption in the intestinal epithelium is through endocytic processes and involves a pathway in which apical derived vesicles fuse with basolateral-derived vesicles. Calcein is a fluorescent metal sensor whose fluorescence is quenched with chelation of iron. Calcein, which is hydrophilic and membrane-impermeable, is taken up by cells by endocytosis (Glickstein H et al. Blood First Edition Paper, prepublished online July 14, 2005). To monitor the flux of iron via transcytosis we took advantage of the properties of calcein and used Caco2 cells grown as a polarized cell layer in bicameral chambers as a model system to study iron flux. In the Caco2 model the apical chamber baths the brush border surface of the cells and the basal chamber baths the basolateral surface. When calcein was offered in the basal chamber along with apo-transferrerin (apo-Tf), calcein was found to undergo endocytosis and co-localize with apo-Tf in the sub-apical cytoplasm with a coefficient of co-localization of 36.8 ± 3.6 %. Under these conditions offering ferrous iron in the apical chamber caused the calcein to be quenched and the coefficient of co-localization decreased to 22.5 ± 5.7 %. The addition of a permeable iron chelator restored calcein fluorescence and the co-localization increased to 58.6 ± 20.5 %. Iron chelated to calcein, which markedly quenches calcein fluorescence, was then offered in the apical chamber as a source of iron. Apo-Tf was offered simultaneously in the basal chamber. After internalization calcein fluorescence was subsequently restored and calcein was observed to co-localize in vesicles with apo-Tf with a co-localization coefficient was 26.4 ± 5.8 %. These studies strongly suggest that iron is transported across the intestinal epithelium by transcytosis with apical derived vesicles fusing with basolateral-derived vesicles. Early endosome antigen 1 (EEA1) and the small GTPase, Rab5, are required for endosomal transport and fusion in mammalian cells and might provide directionality to vesicular transport from the membrane to the early endosomes. We examined if both EEA1 and Rab5 are involved in the transcytosis of DMT1 and iron and found that after iron feeding that there was increased co-localization of DMT1 with both EEA1 and Rab5 in the sub-apical compartment. Taken together these studies support our hypothesis that iron absorption in the intestinal epithelium is through endocytic processes and involves a transcytosis pathway.

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Erythrocyte Glutathione Reductase (1963)

BEUTLER, ERNEST ; YEH, MARY K. Y.

American Society of Hematology

In: Blood. 1963; 21(5): 573-585. Published 1963 May 01. doi: 10.1182/blood.v21.5.573.573.

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Publication Date: 1963-05-01

Description: 1. The physiologic roles of glutathione in the red cell and of glutathione reductase are reviewed briefly. 2. The partial purification of glutathione reductase by a combination of salting out, heating and chromatographic procedures is described. 3. Throughout purification, no appreciable change in the ratio of activity of the enzyme with DPN and TPN as a coenzyme has been found. 4. Potassium chloride and sodium chloride increased markedly the enzymatic activity when TPNH served as hydrogen donor. However, when DPNH was the hydrogen donor these salts caused a decrease in enzyme activity. Potassium phosphate buffer increased enzymatic activity with TPNH but produced little change with DPNH. 5. Michaelis-Menten constants were computed for the purified enzyme preparations. 6. The DPN-linked system did not result in GSSG reduction when lactate served as a substrate in intact erythrocytes.

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