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Sample records for human transferrin receptor

  1. Human placental coated vesicles contain receptor-bound transferrin.

    PubMed Central

    Booth, A G; Wilson, M J

    1981-01-01

    Human placental coated vesicles have been purified by a method involving sucrose-density-gradient centrifugation and treatment with wheat-germ agglutinin. These preparations were free of contamination by placental microvillus fragments. Crossed immunoelectrophoresis demonstrated that the coated vesicles contained a single serum protein, which was identified as transferrin. This transferrin was only observed after the vesicles were treated with a non-ionic detergent, and its behaviour during crossed hydrophobic-interaction immunoelectrophoresis suggested that a large proportion of it was receptor-bound. No other serum proteins, including immunoglobulin G, could be detected in these preparations. Receptor-bound transferrin was the only antigen common to placental coated vesicles and microvilli, implying that other plasma-membrane proteins are excluded from the region of membrane involved in coated-vesicle formation. Images PLATE 2 PLATE 1 Fig. 1. Fig. 2. Fig. 3. PMID:6272755

  2. Epitope mapping of anti-human transferrin monoclonal antibodies: potential uses for transferrin-transferrin receptor interaction studies.

    PubMed

    Perera, Yasser; García, Darién; Guirola, Osmany; Huerta, Vivian; García, Yanet; Muñoz, Yasmiana

    2008-01-01

    Human transferrin (hTf) is an 80 kDa glycoprotein involved in iron transport from the absorption sites to the sites of storage and utilization. Additionally, transferrin also plays a relevant role as a bacteriostatic agent preventing uncontrolled bacterial growth in the host. In this work we describe a well-characterized Mabs panel in terms of precise epitope localization and estimate affinity for the two major hTf isoforms. We found at least four antigenic regions in the hTf molecule, narrowed down the interacting antigen residues within three of such regions, and located them on a molecular model of hTf. Two of the antigenic regions partially overlap with previously described transferrin-binding sites for both human receptor (antigenic region I: containing amino acid residues from Asp-69 to Asn-76 at the N-lobe) and bacterial receptors from two pathogenic species (antigenic region III: amino acid residues from Leu-665 to Ser-672 at the C-lobe). Hence, such monoclonal antibodies (Mabs) could be used as an additional tool for conformational studies and/or the characterization of the interaction between hTf and both types of receptor molecules.

  3. Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells.

    PubMed Central

    McGregor, S J; Naves, M L; Oria, R; Vass, J K; Brock, J H

    1990-01-01

    Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin. Images Fig. 2. PMID:2268267

  4. Serum transferrin receptor.

    PubMed

    Cook, J D; Skikne, B S; Baynes, R D

    1993-01-01

    The transferrin receptor plays a critical role in iron metabolism by precisely controlling the flow of transferrin iron into body cells. A soluble truncated form of the receptor can be detected in human serum using sensitive immunoassays, and the initial clinical experience with this new measurement indicates that it reflects the total body mass of tissue receptor. Serum receptor levels rise significantly with tissue iron deficiency and the heightened demand for iron associated with expansion of the erythroid marrow. The serum receptor provides a quantitative measure of functional iron deficiency and distinguishes the associated anemia from that of chronic disease. If iron deficiency is excluded, the serum receptor provides a quantitative measure of total erythropoiesis that is more sensitive and less invasive than bone marrow examination currently used to assess red cell precursor mass. Performed in conjunction with serum ferritin measurements, the serum receptor will be useful in establishing the true prevalence of iron deficiency anemia in population studies.

  5. Metabolic and cytoskeletal modulation of transferrin receptor mobility in mitogen-activated human lymphocytes.

    PubMed Central

    Galbraith, G M; Galbraith, R M

    1980-01-01

    The transferrin receptors which appear on mitogen-activated human peripheral blood lymphocytes were found by the use of immunofluorescence techniques to display temperature-dependent patching and capping reactions upon binding of transferrin. Lateral mobility of ligand-occupied membrane sites was accompanied by both shedding and endocytosis of receptor-transferrin complexes. In the presence of sodium azide or the microfilament inhibitor cytochalasin B, cap formation and shedding were markedly inhibited. In contrast, endocytosis of patched receptor-ligand complexes was inhibited by azide and microtubule inhibitors, including colchicine, vinblastine and vincristine. Co-capping experiments performed to elucidate further the alterations in membrane configuration involved in these reactions failed to reveal any topographical relationship between transferrin receptors and lectin-binding sites in these cells. These studied indicate that temperature-dependent mobility of transferrin receptors upon mitogen-activated peripheral blood lymphocytes is dependent upon the integrity of the cytoskeletal system and metabolic function of the cell. PMID:6258830

  6. Identification of the epitope of a monoclonal antibody that disrupts binding of human transferrin to the human transferrin receptor.

    PubMed

    Teh, Evelyn M; Hewitt, Jeff; Ung, Karen C; Griffiths, Tanya A M; Nguyen, Vinh; Briggs, Sara K; Mason, Anne B; MacGillivray, Ross T A

    2005-12-01

    The molecular basis of the transferrin (TF)-transferrin receptor (TFR) interaction is not known. The C-lobe of TF is required to facilitate binding to the TFR and both the N- and C-lobes are necessary for maximal binding. Several mAb have been raised against human transferrin (hTF). One of these, designated F11, is specific to the C-lobe of hTF and does not recognize mouse or pig TF. Furthermore, mAb F11 inhibits the binding of TF to TFR on HeLa cells. To map the epitope for mAb F11, constructs spanning various regions of hTF were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The recombinant fusion proteins were analysed in an iterative fashion by immunoblotting using mAb F11 as the probe. This process resulted in the localization of the F11 epitope to the C1 domain (residues 365-401) of hTF. Subsequent computer modelling suggested that the epitope is probably restricted to a surface patch of hTF consisting of residues 365-385. Mutagenesis of the F11 epitope of hTF to the sequence of either mouse or pig TF confirmed the identity of the epitope as immunoreactivity was diminished or lost. In agreement with other studies, these epitope mapping studies support a role for residues in the C1 domain of hTF in receptor binding.

  7. Ionic residues of human serum transferrin affect binding to the transferrin receptor and iron release.

    PubMed

    Steere, Ashley N; Miller, Brendan F; Roberts, Samantha E; Byrne, Shaina L; Chasteen, N Dennis; Smith, Valerie C; MacGillivray, Ross T A; Mason, Anne B

    2012-01-17

    Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of 11 charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A, and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF-TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367, and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and release of iron from the hTF-TFR complex.

  8. Ionic Residues of Human Serum Transferrin Affect Binding to the Transferrin Receptor and Iron Release

    PubMed Central

    Steere, Ashley N.; Miller, Brendan F.; Roberts, Samantha E.; Byrne, Shaina L.; Chasteen, N. Dennis; Smith, Valerie C.; MacGillivray, Ross T.A.; Mason, Anne B.

    2012-01-01

    Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤ 6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of eleven charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) in TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF/TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367 and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and iron release from the hTF/TFR complex. PMID:22191507

  9. Stoichiometries of Transferrin Receptors 1 and 2 in Human Liver

    PubMed Central

    Chloupková, Maja; Zhang, An-Sheng; Enns, Caroline A.

    2009-01-01

    Mutations in either the hereditary hemochromatosis protein, HFE, or transferrin receptor 2, TfR2, result in a similarly severe form of the most common type of iron overload disease called hereditary hemochromatosis. Models of the interactions between HFE, TfR1, and TfR2 imply that these proteins are present in different molar concentrations in the liver, where they control expression of the iron regulatory hormone, hepcidin, in response to body iron loading. The aim of this study was to determine in vivo levels of mRNA by quantitative RT-PCR and concentrations of these proteins by quantitative immunoblotting in human liver tissues. The level of TfR2 mRNA was 21- and 63- fold higher than that of TfR1 and HFE, respectively. Molar concentration of TfR2 protein was the highest and determined to be 1.95 nmoles/g protein in whole cell lysates and 10.89 nmoles/g protein in microsomal membranes. Molar concentration of TfR1 protein was 4.5- and 6.1-fold lower than that of TfR2 in whole cell lysates and membranes, respectively. The level of HFE protein was below 0.53 nmoles/g of total protein. HFE is thus present in substoichiometric concentrations with respect to both TfR1 and TfR2 in human liver tissue. This finding supports a model, in which availability of HFE is limiting for formation of complexes with TfR1 or TfR2. PMID:19819738

  10. The iron-chelating agent picolinic acid enhances transferrin receptors expression in human erythroleukaemic cell lines.

    PubMed

    Testa, U; Louache, F; Titeux, M; Thomopoulos, P; Rochant, H

    1985-07-01

    Picolinic acid, a metal chelating molecule, was administered to human erythroleukaemic cell lines (K 562 and HEL) that were grown in serum-containing media. Picolinic acid inhibited both iron uptake and cell growth. Furthermore, picolinic acid was shown to markedly decrease the level of ferritin in the cells. In spite of the inhibition of cell growth, picolinic acid induced a marked increase in the transferrin-binding capacity of the cells. This phenomenon was due to a two-five-fold enhancement of the rate of transferrin receptor biosynthesis. Other iron-chelating compounds, capable of reducing the level of intracellular iron, also elicited a marked enhancement of the transferrin-binding capacity of the cells. However, the addition of iron, as ferric ammonium citrate, in the culture medium elicited a marked increase in the level of ferritin and a strong decrease in the transferrin-binding capacity of the cells. On the basis of these data we propose that a feed-back mechanism is involved in the regulation of transferrin receptors: when the cells accumulate iron they decrease the number of transferrin receptors in order to prevent further accumulation of iron; when no or low iron is available to the cells, the number of transferrin receptors markedly increases as a compensatory mechanism.

  11. Mechanism for Multiple Ligand Recognition by the Human Transferrin Receptor

    PubMed Central

    Giannetti, Anthony M; Snow, Peter M; Zak, Olga

    2003-01-01

    Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf. PMID:14691533

  12. Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

    PubMed Central

    Orlandini, M.; Santucci, A.; Tramontano, A.; Neri, P.; Oliviero, S.

    1994-01-01

    In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available. PMID:7530542

  13. How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH.

    PubMed

    Eckenroth, Brian E; Steere, Ashley N; Chasteen, N Dennis; Everse, Stephen J; Mason, Anne B

    2011-08-09

    Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe(N)hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.

  14. How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH

    PubMed Central

    Eckenroth, Brian E.; Steere, Ashley N.; Chasteen, N. Dennis; Everse, Stephen J.; Mason, Anne B.

    2011-01-01

    Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of FeNhTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system. PMID:21788477

  15. How the Binding of Human Transferrin Primes the Transferrin Receptor Potentiating Iron Release at Endosomal pH

    SciTech Connect

    B Eckenroth; A Steere; N Chasteen; S Everse; A Mason

    2011-12-31

    Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-{angstrom} resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe{sub N}hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same {alpha}-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.

  16. Recombinant Human Adenovirus: Targeting to the Human Transferrin Receptor Improves Gene Transfer to Brain Microcapillary Endothelium

    PubMed Central

    Xia, Haibin; Anderson, Brian; Mao, Qinwen; Davidson, Beverly L.

    2000-01-01

    Some inborn errors of metabolism due to deficiencies of soluble lysosomal enzymes cause global neurodegenerative disease. Representative examples include the infantile and late infantile forms of the ceroid lipofuscinoses (CLN1 or CLN2 deficiency, respectively) and mucopolysaccharidoses type VII (MPS VII), a deficiency of β-glucuronidase. Treatment of the central nervous system component of these disorders will require widespread protein or enzyme replacement, either through dissemination of the protein or through dissemination of a gene encoding it. We hypothesize that transduction of brain microcapillary endothelium (BME) with recombinant viral vectors, with secretion of enzyme product basolaterally, could allow for widespread enzyme dissemination. To achieve this, viruses should be modified to target the BME. This requires (i) identification of a BME-resident target receptor, (ii) identification of motifs targeted to that molecule, (iii) the construction of modified viruses to allow for binding to the target receptor, and (iv) demonstrated transduction of receptor-expressing cells. In proof of principal experiments, we chose the human transferrin receptor (hTfR), a molecule found at high density on human BME. A nonamer phage display library was panned for motifs which could bind hTfR. Forty-three clones were sequenced, most of which contained an AKxxK/R, KxKxPK/R, or KxK motif. Ten peptides representative of the three motifs were cloned into the HI loop of adenovirus type 5 fiber. All motifs tested retained their ability to trimerize and bind transferrin receptor, and seven allowed for recombinant adenovirus production. Importantly, the fiber-modified viruses facilitated increased gene transfer (2- to 34-fold) to hTfR expressing cell lines and human brain microcapillary endothelia expressing high levels of endogenous receptor. Our data indicate that adenoviruses can be modified in the HI loop for expanded tropism to the hTfR. PMID:11070036

  17. Binding and uptake of H-ferritin are mediated by human transferrin receptor-1.

    PubMed

    Li, Li; Fang, Celia J; Ryan, James C; Niemi, Eréne C; Lebrón, José A; Björkman, Pamela J; Arase, Hisashi; Torti, Frank M; Torti, Suzy V; Nakamura, Mary C; Seaman, William E

    2010-02-23

    Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.

  18. Binding and uptake of H-ferritin are mediated by human transferrin receptor-1

    PubMed Central

    Li, Li; Fang, Celia J.; Ryan, James C.; Niemi, Eréne C.; Lebrón, José A.; Björkman, Pamela J.; Arase, Hisashi; Torti, Frank M.; Torti, Suzy V.; Nakamura, Mary C.; Seaman, William E.

    2010-01-01

    Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules. PMID:20133674

  19. Coincident expression and distribution of melanotransferrin and transferrin receptor in human brain capillary endothelium.

    PubMed

    Rothenberger, S; Food, M R; Gabathuler, R; Kennard, M L; Yamada, T; Yasuhara, O; McGeer, P L; Jefferies, W A

    1996-03-11

    One method of iron transport across the blood brain barrier (BBB) involves the transferrin receptor (TR), which is localized to the specialized brain capillary endothelium. The melanotransferrin (MTf) molecule, also called p97, has been widely described as a melanoma specific molecule, however, its expression in brain tissues has not been addressed. MTf has a high level of sequence homology to transferrin (Tf) and lactoferrin, but is unusual because it predominantly occurs as a membrane bound, glycosylphosphatidylinositol (GPI) anchored molecule, but can also occur as a soluble form. We have recently demonstrated that GPI-anchored MTf provides a novel route for cellular iron uptake which is independent of Tf and its receptor. Here we consider whether MTf may have a role in the transport of iron across the BBB. The distributions of MTf, Tf and the TR were studied immunohistochemically in human brain tissues. The distributions of MTf and TR were remarkably similar, and quite different from that of Tf. In all brain tissues examined, MTf and the TR were highly localized to capillary endothelium, while Tf itself was mainly localized to glial cells. These data suggest that MTf may play a role in iron transport within the human brain.

  20. Expression of transferrin receptors on mitogen-stimulated human peripheral blood lymphocytes: relation to cellular activation and related metabolic events.

    PubMed Central

    Galbraith, R M; Galbraith, G M

    1981-01-01

    Mitogen-activated normal human peripheral blood lymphocytes bind transferrin to specific membrane receptors. In this study, lymphocytes stimulated with phytohaemagglutinin for 0-66 hr were examined to determine the relation of this phenomenon to cellular activation and related metabolic events. Transferrin receptors were first detected at 20-24 hr. This event was consistently preceded by RNA and protein turnover which commenced during the first 6 hr of culture, whereas initiation of DNA synthesis was detected concurrently with the appearance of receptors or slightly later (24-30 hr). Exposure of cells to inhibitors of RNA and protein synthesis early during culture (at 0 or 24 hr) prevented the expression of transferrin receptors, but also caused generalized metabolic failure, and abrogated cellular activation. In contrast, later addition of these agents at 48 hr did not interfere significantly with the process of activation, but did suppress the terminal increase in receptor-bearing cells observed during the final 18 hr in control cultures lacking inhibitor. After deliberate thermal stripping of receptors from activated cells, the reappearance of membrance binding sites which normally occurred within 30 min, was also blocked by cycloheximide, puromycin and actinomycin D. However, similar inhibition of DNA which was induced by hydroxyurea had much less effect upon both the initial appearance of receptors and their reappearance after ligand-induced depletion. These results demonstrate that the appearance of transferrin receptors upon human lymphocytes is dependent upon cellular activation and requires synthesis of protein and RNA. PMID:6172372

  1. H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

    PubMed Central

    Sakamoto, Soichiro; Kawabata, Hiroshi; Masuda, Taro; Uchiyama, Tatsuki; Mizumoto, Chisaki; Ohmori, Katsuyuki; Koeffler, H. Phillip; Kadowaki, Norimitsu; Takaori-Kondo, Akifumi

    2015-01-01

    Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin. PMID:26441243

  2. Human and Host Species Transferrin Receptor 1 Use by North American Arenaviruses

    PubMed Central

    Zong, Min; Fofana, Isabel

    2014-01-01

    ABSTRACT At least five New World (NW) arenaviruses cause hemorrhagic fevers in South America. These pathogenic clade B viruses, as well as nonpathogenic arenaviruses of the same clade, use transferrin receptor 1 (TfR1) of their host species to enter cells. Pathogenic viruses are distinguished from closely related nonpathogenic ones by their additional ability to utilize human TfR1 (hTfR1). Here, we investigate the receptor usage of North American arenaviruses, whose entry proteins share greatest similarity with those of the clade B viruses. We show that all six North American arenaviruses investigated utilize host species TfR1 orthologs and present evidence consistent with arenavirus-mediated selection pressure on the TfR1 of the North American arenavirus host species. Notably, one of these viruses, AV96010151, closely related to the prototype Whitewater Arroyo virus (WWAV), entered cells using hTfR1, consistent with a role for a WWAV-like virus in three fatal human infections whose causative agent has not been identified. In addition, modest changes were sufficient to convert hTfR1 into a functional receptor for most of these viruses, suggesting that a minor alteration in virus entry protein may allow these viruses to use hTfR1. Our data establish TfR1 as a cellular receptor for North American arenaviruses, highlight an “arms race” between these viruses and their host species, support the association of North American arenavirus with fatal human infections, and suggest that these viruses have a higher potential to emerge and cause human diseases than has previously been appreciated. IMPORTANCE hTfR1 use is a key determinant for a NW arenavirus to cause hemorrhagic fevers in humans. All known pathogenic NW arenaviruses are transmitted in South America by their host rodents. North American arenaviruses are generally considered nonpathogenic, but some of these viruses have been tentatively implicated in human fatalities. We show that these North American

  3. Prospective Design of Anti‐Transferrin Receptor Bispecific Antibodies for Optimal Delivery into the Human Brain

    PubMed Central

    Kanodia, JS; Gadkar, K; Bumbaca, D; Zhang, Y; Tong, RK; Luk, W; Hoyte, K; Lu, Y; Wildsmith, KR; Couch, JA; Watts, RJ; Dennis, MS; Ernst, JA; Scearce‐Levie, K; Atwal, JK; Joseph, S

    2016-01-01

    Anti‐transferrin receptor (TfR)‐based bispecific antibodies have shown promise for boosting antibody uptake in the brain. Nevertheless, there are limited data on the molecular properties, including affinity required for successful development of TfR‐based therapeutics. A complex nonmonotonic relationship exists between affinity of the anti‐TfR arm and brain uptake at therapeutically relevant doses. However, the quantitative nature of this relationship and its translatability to humans is heretofore unexplored. Therefore, we developed a mechanistic pharmacokinetic‐pharmacodynamic (PK‐PD) model for bispecific anti‐TfR/BACE1 antibodies that accounts for antibody‐TfR interactions at the blood‐brain barrier (BBB) as well as the pharmacodynamic (PD) effect of anti‐BACE1 arm. The calibrated model correctly predicted the optimal anti‐TfR affinity required to maximize brain exposure of therapeutic antibodies in the cynomolgus monkey and was scaled to predict the optimal affinity of anti‐TfR bispecifics in humans. Thus, this model provides a framework for testing critical translational predictions for anti‐TfR bispecific antibodies, including choice of candidate molecule for clinical development. PMID:27299941

  4. Transferrin receptor function in hereditary hemochromatosis

    SciTech Connect

    Ward, J.H.; Kushner, J.P.; Ray, F.A.; Kaplan, J.

    1984-02-01

    The binding of /sup 125/I-diferric transferrin to cultured skin fibroblasts and phytohemagglutinin-stimulated lymphocytes was studied in cells derived from individuals homozygous for hereditary hemochromatosis and from normal individuals. Receptors with a high affinity for diferric transferrin were present on all cells. Transferrin receptor number decreased by more than 50% when fibroblasts from both normal and hemochromatotic subjects were maintained in iron-supplemented medium. The number of transferrin receptors expressed by normal and hemochromatotic lymphocytes after mitogen stimulation in iron-supplemented media was less than 50% that of lymphocytes which were mitogen stimulated in standard medium. No change in the affinity of the receptors of diferric transferrin was seen in cells maintained in iron-supplemented medium. Competition experiments in the presence of deferoxamine suggested that the transferrin receptors of fibroblasts and mitogen-stimulated lymphocytes have a 70- to 100-fold higher affinity for diferric transferrin than for apotransferrin. No differences in the properties of transferrin receptors were found between patients with hereditary hemochromatosis and normal individuals. Although transferrin binding decreases when cells are exposed to high levels of iron in the medium, the failure to totally abolish transferrin binding to the receptor suggests that the concentration of diferric transferrin to which cells are exposed may be a major determinant of cellular iron loading in hereditary hemochromatosis.

  5. The measurement of serum transferrin receptor.

    PubMed

    Cook, J D

    1999-10-01

    The concentration of the soluble fragment of transferrin receptor in serum is an important new hematological parameter. Clinical and laboratory studies have shown that this serum form of the receptor reflects the total body mass of cellular transferrin receptor, 80% of which is contained in the erythroid marrow. The two disorders that result in an elevation in the serum transferrin receptor are anemias associated with enhanced erythropoiesis and tissue iron deficiency. The concentration of soluble transferrin receptor provides a useful quantitative measure of the erythroid marrow mass and thereby assists clinically in categorizing the type of anemia. The most important clinical use of the serum transferrin receptor is in determining the cause of iron deficient erythropoiesis (that is, identifying iron deficiency anemia whether it occurs alone or in the presence of the anemia of chronic disease). Present evidence supports the routine use of the serum transferrin receptor in the clinical evaluation of anemic patients.

  6. The crystal structure of iron-free human serum transferrin provides insight into inter-lobe communication and receptor binding.

    PubMed

    Wally, Jeremy; Halbrooks, Peter J; Vonrhein, Clemens; Rould, Mark A; Everse, Stephen J; Mason, Anne B; Buchanan, Susan K

    2006-08-25

    Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependent process. The binding and release of iron result in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF), which was independently determined by two methods: 1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7-A resolution using a multiple wavelength anomalous dispersion phasing strategy, by substituting the nine methionines in hTF with selenomethionine and 2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7A by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human transferrin and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4 degrees and 49.5 degrees rotations are required to open the N- and C-lobes, respectively (compared with closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.

  7. Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced iron release from human serum transferrin

    PubMed Central

    Steere, Ashley N.; Chasteen, N. Dennis; Miller, Brendan F.; Smith, Valerie C.; MacGillivray, Ross T.A.; Mason, Anne B.

    2012-01-01

    The recent crystal structure of two monoferric human serum transferrin (FeNhTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details of this binding interaction which dictates iron delivery to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two FeNhTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca2+ binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of iron release at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of a H318A sTFR mutant allows assignment of a small pH dependent initial decrease in the fluorescent signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated iron release from the C-lobe of the Fe2hTF/sTFR Δ757–760 complex. The loss is accounted for by the inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs in order to promote receptor-stimulated iron release from the C-lobe of hTF. PMID:22356162

  8. Erythroblast transferrin receptors and transferrin kinetics in iron deficiency and various anemias

    SciTech Connect

    Muta, K.; Nishimura, J.; Ideguchi, H.; Umemura, T.; Ibayashi, H.

    1987-06-01

    To clarify the role of transferrin receptors in cases of altered iron metabolism in clinical pathological conditions, we studied: number of binding sites; affinity; and recycling kinetics of transferrin receptors on human erythroblasts. Since transferrin receptors are mainly present on erythroblasts, the number of surface transferrin receptors was determined by assay of binding of /sup 125/I-transferrin and the percentage of erythroblasts in bone marrow mononuclear cells. The number of binding sites on erythroblasts from patients with an iron deficiency anemia was significantly greater than in normal subjects. Among those with an aplastic anemia, hemolytic anemia, myelodysplastic syndrome, and polycythemia vera compared to normal subjects, there were no considerable differences in the numbers of binding sites. The dissociation constants (Kd) were measured using Scatchard analysis. The apparent Kd was unchanged (about 10 nmol/L) in patients and normal subjects. The kinetics of endocytosis and exocytosis of /sup 125/I-transferrin, examined by acid treatment, revealed no variations in recycling kinetics among the patients and normal subjects. These data suggest that iron uptake is regulated by modulation of the number of surface transferrin receptors, thereby reflecting the iron demand of the erythroblast.

  9. Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.).

    PubMed

    Zhang, Deshui; Lee, Hsin-Fang; Pettit, Steven C; Zaro, Jennica L; Huang, Ning; Shen, Wei-Chiang

    2012-11-30

    Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications. Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production. The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR

  10. Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.)

    PubMed Central

    2012-01-01

    Background Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications. Result Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production. Conclusion The functional similarity between rice derived rhTF and native hTF in their cellular iron

  11. Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced release of iron from human serum transferrin.

    PubMed

    Steere, Ashley N; Chasteen, N Dennis; Miller, Brendan F; Smith, Valerie C; MacGillivray, Ross T A; Mason, Anne B

    2012-03-13

    The recent crystal structure of two monoferric human serum transferrin (Fe(N)hTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details about this binding interaction that dictates the delivery of iron to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two Fe(N)hTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca(2+) binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of release of iron at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of an H318A sTFR mutant allows assignment of a small pH-dependent initial decrease in the magnitude of the fluorescence signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated release of iron from the C-lobe of the Fe(2)hTF/sTFR Δ757-760 complex. The inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF accounts for the loss. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs to promote receptor-stimulated release of iron from the C-lobe of hTF.

  12. Human serum transferrin: a tale of two lobes. Urea gel and steady state fluorescence analysis of recombinant transferrins as a function of pH, time, and the soluble portion of the transferrin receptor

    PubMed Central

    Byrne, Shaina L.

    2009-01-01

    Iron release from human serum transferrin (hTF) has been studied extensively; however, the molecular details of the mechanism(s) remain incomplete. This is in part due to the complexity of this process, which is influenced by lobe–lobe interactions, the transferrin receptor (TFR), the salt effect, the presence of a chelator, and acidification within the endosome, resulting in iron release. The present work brings together many of the concepts and assertions derived from previous studies in a methodical, uniform, and visual manner. Examination of earlier work reveals some uncertainty due to sample and technical limitations. We have used a combination of steady-state fluorescence and urea gels to evaluate the effect of conformation, pH, time, and the soluble portion of the TFR (sTFR) on iron release from each lobe of hTF. The use of authentic recombinant monoferric and locked species removes any possibility of cross-contamination by acquisition of iron. Elimination of detergent by use of the sTFR provides a further technical advantage. We find that iron release from the N-lobe is very sensitive to the conformation of the C-lobe, but is insensitive to the presence of the sTFR or to changes in pH (between 5.6 and 6.4). Specifically, when the cleft of the C-lobe is locked, the urea gels indicate that only about half of the iron is completely removed from the cleft of the N-lobe. Iron release from the C-lobe is most affected by the presence of the sTFR and changes in pH, but is unaffected by the conformation of the N-lobe. A model for iron release from diferric hTF is provided to delineate our findings. PMID:19290554

  13. Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis.

    PubMed

    Gironès, N; Alverez, E; Seth, A; Lin, I M; Latour, D A; Davis, R J

    1991-10-05

    It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.

  14. Structural, functional, and tissue distribution analysis of human transferrin receptor-2 by murine monoclonal antibodies and a polyclonal antiserum.

    PubMed

    Deaglio, Silvia; Capobianco, Andrea; Calì, Angelita; Bellora, Francesca; Alberti, Federica; Righi, Luisella; Sapino, Anna; Camaschella, Clara; Malavasi, Fabio

    2002-11-15

    Human transferrin receptor-2 (TFR-2) is a protein highly homologous to TFR-1/CD71 and is endowed with the ability to bind transferrin (TF) with low affinity. High levels of TFR-2 mRNA were found in the liver and in erythroid precursors. Mutations affecting the TFR-2 gene led to hemochromatosis type 3, a form of inherited iron overload. Several issues on distribution and function of the receptor were answered by raising a panel of 9 monoclonal antibodies specific for TFR-2 by immunizing mice with murine fibroblasts transfected with the human TFR-2 cDNA. A polyclonal antiserum was also produced in mice immunized with 3 peptides derived from the TFR-2 sequence, exploiting an innovative technique. The specificity of all the reagents produced was confirmed by reactivity with TFR-2(+) target cells and simultaneous negativity with TFR-1(+) cells. Western blot analyses showed a dominant chain of approximately 90 kDa in TFR-2 transfectants and HepG2 cell line. Analysis of distribution in normal tissues and in representative cell lines revealed that TFR-2 displays a restricted expression pattern--it is present at high levels in hepatocytes and in the epithelial cells of the small intestine, including the duodenal crypts. Exposure of human TFR-2(+) cells to TF-bound iron is followed by a significant up-regulation and relocalization of membrane TFR-2. The tissue distribution pattern, the behavior following exposure to iron-loaded TF, and the features of the disease resulting from TFR-2 inactivation support the hypothesis that TFR-2 contributes to body iron sensing.

  15. Meningococcal transferrin-binding proteins A and B show cooperation in their binding kinetics for human transferrin.

    PubMed

    Stokes, Russell H; Oakhill, Jonathan S; Joannou, Christopher L; Gorringe, Andrew R; Evans, Robert W

    2005-02-01

    Neisseria meningitidis, a causative agent of bacterial meningitis and septicemia, obtains transferrin-bound iron by expressing two outer membrane-located transferrin-binding proteins, TbpA and TbpB. A novel system was developed to investigate the interaction between Tbps and human transferrin. Copurified TbpA-TbpB, recombined TbpA-TbpB, and individual TbpA and TbpB were reconstituted into liposomes and fused onto an HPA chip (BIAcore). All preparations formed stable monolayers, which, with the exception of TbpB, could be regenerated by removing bound transferrin. The ligand binding properties of these monolayers were characterized with surface plasmon resonance and shown to be specific for human transferrin. Kinetic data for diferric human transferrin binding showed that recombined TbpA-TbpB had K(a) and K(d) values similar to those of copurified TbpA-TbpB. Individual TbpA and TbpB also displayed K(a) values similar to those of copurified TbpA-TbpB, but their K(d) values were one order of magnitude higher. Chemical cross-linking studies revealed that TbpA and TbpB, in the absence of human transferrin, formed large complexes with TbpA as the predominant species. Upon human transferrin binding, a complex was formed with a molecular mass corresponding to that of a TbpB-human transferrin heterodimer as well as a higher-molecular-mass complex of this heterodimer cross-linked to TbpA. This indicates that TbpA and TbpB form a functional meningococcal receptor complex in which there is cooperativity in the human transferrin binding kinetics. However, iron loss from the diferric human transferrin-TbpA-TbpB complex was not greater than that from human transferrin alone, suggesting that additional meningococcal transport components are involved in the process of iron removal.

  16. Meningococcal Transferrin-Binding Proteins A and B Show Cooperation in Their Binding Kinetics for Human Transferrin

    PubMed Central

    Stokes, Russell H.; Oakhill, Jonathan S.; Joannou, Christopher L.; Gorringe, Andrew R.; Evans, Robert W.

    2005-01-01

    Neisseria meningitidis, a causative agent of bacterial meningitis and septicemia, obtains transferrin-bound iron by expressing two outer membrane-located transferrin-binding proteins, TbpA and TbpB. A novel system was developed to investigate the interaction between Tbps and human transferrin. Copurified TbpA-TbpB, recombined TbpA-TbpB, and individual TbpA and TbpB were reconstituted into liposomes and fused onto an HPA chip (BIAcore). All preparations formed stable monolayers, which, with the exception of TbpB, could be regenerated by removing bound transferrin. The ligand binding properties of these monolayers were characterized with surface plasmon resonance and shown to be specific for human transferrin. Kinetic data for diferric human transferrin binding showed that recombined TbpA-TbpB had Ka and Kd values similar to those of copurified TbpA-TbpB. Individual TbpA and TbpB also displayed Ka values similar to those of copurified TbpA-TbpB, but their Kd values were one order of magnitude higher. Chemical cross-linking studies revealed that TbpA and TbpB, in the absence of human transferrin, formed large complexes with TbpA as the predominant species. Upon human transferrin binding, a complex was formed with a molecular mass corresponding to that of a TbpB-human transferrin heterodimer as well as a higher-molecular-mass complex of this heterodimer cross-linked to TbpA. This indicates that TbpA and TbpB form a functional meningococcal receptor complex in which there is cooperativity in the human transferrin binding kinetics. However, iron loss from the diferric human transferrin-TbpA-TbpB complex was not greater than that from human transferrin alone, suggesting that additional meningococcal transport components are involved in the process of iron removal. PMID:15664936

  17. Regulation of cell surface transferrin receptor-2 by iron-dependent cleavage and release of a soluble form

    PubMed Central

    Pagani, Alessia; Vieillevoye, Maud; Nai, Antonella; Rausa, Marco; Ladli, Meriem; Lacombe, Catherine; Mayeux, Patrick; Verdier, Frédérique; Camaschella, Clara; Silvestri, Laura

    2015-01-01

    Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency. PMID:25637053

  18. Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry

    DTIC Science & Technology

    2011-07-01

    and SABV [17,18], and a major determinant of host adaptation. However, studies on receptor use and cellular tropism suggest that the non-pathogenic...938–948. 19. Oldenburg J, Reignier T, Flanagan ML, Hamilton GA, Cannon PM (2007) Differences in tropism and pH dependence for glycoproteins from the...2010) Investigation of clade B New World arenavirus tropism by using chimeric GP1 proteins. J Virol 84: 1176–1182. 24. Bowden TA, Crispin M, Graham SC

  19. Selection of cell lines resistant to anti-transferrin receptor antibody: evidence for a mutation in transferrin receptor.

    PubMed Central

    Lesley, J F; Schulte, R J

    1984-01-01

    Some anti-murine transferrin receptor monoclonal antibodies block iron uptake in mouse cell lines and inhibit cell growth. We report here the selection and characterization of mutant murine lymphoma cell lines which escape this growth inhibition by anti-transferrin receptor antibody. Growth assays and immunoprecipitation of transferrin receptor in hybrids between independently derived mutants or between mutants and antibody-susceptible parental cell lines indicate that all of the selected lines have a similar genetic alteration that is codominantly expressed in hybrids. Anti-transferrin receptor antibodies and transferrin itself still bind to the mutant lines with saturating levels and Kd values very similar to those of the parental lines. However, reciprocal clearing experiments by immunoprecipitation and reciprocal blocking of binding to the cell surface with two anti-transferrin receptor antibodies indicate that the mutant lines have altered a fraction of their transferrin receptors such that the growth-inhibiting antibody no longer binds, whereas another portion of their transferrin receptors is similar to those of the parental lines and binds both antibodies. These results argue that the antibody-selected mutant cell lines are heterozygous in transferrin receptor expression, probably with a mutation in one of the transferrin receptor structural genes. Images PMID:6092931

  20. Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.)

    PubMed Central

    Zhang, Deshui; Nandi, Somen; Bryan, Paula; Pettit, Steve; Nguyen, Diane; Santos, Mary Ann; Huang, Ning

    2010-01-01

    Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug’s therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood-borne pathogens from the use of human- or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for native transferrin. We expressed recombinant human transferrin in rice (Oryza sativa L.) at a high level of 1% seed dry weight (10 g/kg). The recombinant human transferrin was able to be extracted with saline buffers and then purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived recombinant human transferrin was shown to be not only structurally similar to the native human transferrin, but also functionally the same as native transferrin in terms of reversible iron binding and promoting cell growth and productivity. These results indicate that rice-derived recombinant human transferrin should be a safe and low cost alternative to human or animal plasma-derived transferrin for use in cell culture-based biopharmaceutical production of protein therapeutics and vaccines. PMID:20447458

  1. Expression, purification, and characterization of recombinant human transferrin from rice (Oryza sativa L.).

    PubMed

    Zhang, Deshui; Nandi, Somen; Bryan, Paula; Pettit, Steve; Nguyen, Diane; Santos, Mary Ann; Huang, Ning

    2010-11-01

    Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug's therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood-borne pathogens from the use of human or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for native transferrin. We expressed recombinant human transferrin in rice (Oryza sativa L.) at a high level of 1% seed dry weight (10 g/kg). The recombinant human transferrin was able to be extracted with saline buffers and then purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived recombinant human transferrin was shown to be not only structurally similar to the native human transferrin, but also functionally the same as native transferrin in terms of reversible iron binding and promoting cell growth and productivity. These results indicate that rice-derived recombinant human transferrin should be a safe and low cost alternative to human or animal plasma-derived transferrin for use in cell culture-based biopharmaceutical production of protein therapeutics and vaccines. Copyright 2010 Elsevier Inc. All rights reserved.

  2. The importance of serum transferrin receptor level in the diagnosis of functional iron deficiency due to recombinant human erythropoietin treatment in haemodialysis patients.

    PubMed

    Tonbul, H Z; Kaya, H; Selçuk, N Y; Tekin, S B; San, A; Akçay, F; Akarsu, E

    1998-01-01

    In haemodialysis (HD) patients, functional iron deficiency frequently appears due to recombinant human erythropoietin (r-HuEPO) treatment. However, the diagnosis of iron deficiency is not always easy in such patients. Recent studies have shown that the serum transferrin receptor (s-TfR) level is a sensitive, quantitative measure of tissue iron deficiency. In this study, we examined the changes in s-TfR levels in patients with iron deficiency anaemia due to r-HuEPO treatment. We compared s-TfR levels of 24 patients with i.v. administered r-HuEPO (50-70 U/kg/dose) at the end of each dialysis session (three times a week) and diagnosed as having iron deficiency anaemia by routine laboratory methods (ferritin <50 microg/l and transferrin saturation <16%) with s-TfR levels of 32 patients not receiving r-HuEPO and without iron deficiency anaemia. Also, 40 healthy volunteer subjects were included in the study as a control group. Serum ferritin and transferrin receptor levels were measured with ELISAs using monoclonal reagents. There were no differences between the two groups with and without iron deficiency anaemia with respect to mean age, body weight, haemodialysis duration, haemoglobin and serum creatinine levels (p>0.05). For s-TfR levels, while no difference was present between the control and the non-iron deficiency groups (p>0.05), the iron deficiency group had higher s-TfR values than those of both the control and non-iron deficiency groups (p<0.001). Besides, there was an inverse correlation between haemoglobin and s-TfR levels in patients with iron deficiency anaemia (r = -0.85, p<0.0001). We conclude that the measurement of s-TfR levels may be useful in the diagnosis of functional iron deficiency in haemodialysis patients receiving r-HuEPO.

  3. Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH.

    PubMed

    Steere, Ashley N; Byrne, Shaina L; Chasteen, N Dennis; Mason, Anne B

    2012-03-01

    Human serum transferrin (hTF) is a bilobal glycoprotein that reversibly binds Fe(3+) and delivers it to cells by the process of receptor-mediated endocytosis. Despite decades of research, the precise events resulting in iron release from each lobe of hTF within the endosome have not been fully delineated. We provide an overview of the kinetics of iron release from hTF±the transferrin receptor (TFR) at endosomal pH (5.6). A critical evaluation of the array of biophysical techniques used to determine accurate rate constants is provided. Delivery of Fe(3+)to actively dividing cells by hTF is essential; too much or too little Fe(3+) directly impacts the well-being of an individual. Because the interaction of hTF with the TFR controls iron distribution in the body, an understanding of this process at the molecular level is essential. Not only does TFR direct the delivery of iron to the cell through the binding of hTF, kinetic data demonstrate that it also modulates iron release from the N- and C-lobes of hTF. Specifically, the TFR balances the rate of iron release from each lobe, resulting in efficient Fe(3+) release within a physiologically relevant time frame. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH

    PubMed Central

    Steere, Ashley N.; Byrne, Shaina L.; Chasteen, N. Dennis; Mason, Anne B.

    2011-01-01

    Background Human serum transferrin (hTF) is a bilobal glycoprotein that reversibly binds Fe3+ and delivers it to cells by the process of receptor-mediated endocytosis. Despite decades of research, the precise events resulting in iron release from each lobe of hTF within the endosome have not been fully delineated. Scope of Review We provide an overview of the kinetics of iron release from hTF ± the transferrin receptor (TFR) at endosomal pH (5.6). A critical evaluation of the array of biophysical techniques used to determine accurate rate constants is provided. General Significance Delivery of Fe3+ by to actively dividing cells by hTF is essential; too much or too little Fe3+ directly impacts the well-being of an individual. Because the interaction of hTF with the TFR controls iron distribution in the body, an understanding of this process at the molecular level is essential. Major Conclusions Not only does TFR direct the delivery of iron to the cell through the binding of hTF, kinetic data demonstrate that it also modulates iron release from the N- and C-lobes of hTF. Specifically, the TFR balances the rate of iron release from each lobe, resulting in efficient Fe3+ release within a physiologically relevant time frame. PMID:21699959

  5. Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

    PubMed Central

    Ogunnariwo, J A; Schryvers, A B

    1990-01-01

    Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453

  6. Transferrin receptors on the surfaces of retinal pigment epithelial cells are associated with the cytoskeleton.

    PubMed

    Hunt, R C; Dewey, A; Davis, A A

    1989-04-01

    Retinal pigment epithelial cells, derived from human donor eyes, have been grown in culture as monolayers on membrane filters or plastic surfaces and shown to possess transferrin receptors with a monomeric molecular mass of 93,000. These receptors internalize 125I-labelled transferrin and recycle it to the surrounding medium in a similar manner to other cell types. Scatchard analyses show that there are about 100,000 high-affinity receptors on the surface of each cell and most of these receptors are associated with the cytoskeleton. In total cell extracts, there are additional low-affinity binding sites that do not appear to be strongly associated with the cytoskeleton. The apparent interaction of transferrin receptors with the cytoskeleton was confirmed in two ways: first, using 200 kV electron microscopy for stereo analyses, skeleton-associated transferrin receptors were detected by a monoclonal anti-receptor antibody and a colloidal gold-conjugated second antibody after Triton X-100 extraction of pigment epithelial cells grown directly on laminin-coated gold grids; and, second, when cell surface receptors were labelled with radioiodinated transferrin and then incubated for various periods of time, the labelled transferrin was observed to move from a Triton X-100-insoluble fraction (a putative cytoskeletal compartment) to a Triton-soluble compartment that was not associated with the cytoskeleton. Using either horseradish peroxidase or colloidal gold-labelled transferrin, it has been shown that basolateral and apical surface-located receptors participate in receptor-mediated endocytosis via clathrin-coated pits, endosomes and tubular structures. Initially, transferrin internalized from the apical surface is observed in small endosomes that often appear to be embedded in an apical layer of microfilaments. From these peripheral regions of the cells, the labelled receptors move to larger endosomes and multivesicular bodies deeper in the cytoplasm. These structures

  7. Synthesis and characterization of human transferrin-stabilized gold nanoclusters

    NASA Astrophysics Data System (ADS)

    Le Guével, Xavier; Daum, Nicole; Schneider, Marc

    2011-07-01

    Human transferrin has been biolabelled with gold nanoclusters (Au NCs) using a simple, fast and non-toxic method. These nanocrystals (<2 nm) are stabilized in the protein via sulfur groups and have a high fluorescence emission in the near infrared region (QY = 4.3%; λem = 695 nm). Structural investigation and photophysical measurements show a high population of clusters formed of 22-33 gold atoms covalently bound to the transferrin. In solutions with pH ranging from 5 to 10 and in buffer solutions (PBS, HEPES), those biolabelled proteins exhibit a good stability. No significant quenching effect of the fluorescent transferrin has been detected after iron loading of iron-free transferrin (apoTf) and in the presence of a specific polyclonal antibody. Additionally, antibody-induced agglomeration demonstrates no alteration in the protein activity and the receptor target ability. MTT and Vialight® Plus tests show no cytotoxicity of these labelled proteins in cells (1 µg ml - 1-1 mg ml - 1). Cell line experiments (A549) indicate also an uptake of the iron loaded fluorescent proteins inside cells. These remarkable data highlight the potential of a new type of non-toxic fluorescent transferrin for imaging and targeting.

  8. Mouse mammary tumor virus uses mouse but not human transferrin receptor 1 to reach a low pH compartment and infect cells

    SciTech Connect

    Wang Enxiu; Obeng-Adjei, Nyamekye; Ying Qihua; Davey, Robert A.; Ross, Susan R.

    2008-11-25

    Mouse mammary tumor virus (MMTV) is a pH-dependent virus that uses mouse transferrin receptor 1 (TfR1) for entry into cells. Previous studies demonstrated that MMTV could induce pH 5-dependent fusion-from-with of mouse cells. Here we show that the MMTV envelope-mediated cell-cell fusion requires both the entry receptor and low pH (pH 5). Although expression of the MMTV envelope and TfR1 was sufficient to mediate low pH-dependent syncytia formation, virus infection required trafficking to a low pH compartment; infection was independent of cathepsin-mediated proteolysis. Human TfR1 did not support virus infection, although envelope-mediated syncytia formation occurred with human cells after pH 5 treatment and this fusion depended on TfR1 expression. However, although the MMTV envelope bound human TfR1, virus was only internalized and trafficked to a low pH compartment in cells expressing mouse TfR1. Thus, while human TfR1 supported cell-cell fusion, because it was not internalized when bound to MMTV, it did not function as an entry receptor. Our data suggest that MMTV uses TfR1 for all steps of entry: cell attachment, induction of the conformational changes in Env required for membrane fusion and internalization to an appropriate acidic compartment.

  9. Uptake of 111In-labeled fully human monoclonal antibody TSP-A18 reflects transferrin receptor expression in normal organs and tissues of mice.

    PubMed

    Sugyo, Aya; Tsuji, Atsushi B; Sudo, Hitomi; Nomura, Fumiko; Satoh, Hirokazu; Koizumi, Mitsuru; Kurosawa, Gene; Kurosawa, Yoshikazu; Saga, Tsuneo

    2017-03-01

    Transferrin receptor (TfR) is an attractive molecule for targeted therapy of cancer. Various TfR-targeted therapeutic agents such as anti-TfR antibodies conjugated with anticancer agents have been developed. An antibody that recognizes both human and murine TfR is needed to predict the toxicity of antibody-based agents before clinical trials, there is no such antibody to date. In this study, a new fully human monoclonal antibody TSP-A18 that recognizes both human and murine TfR was developed and the correlation analysis of the radiolabeled antibody uptake and TfR expression in two murine strains was conducted. TSP-A18 was selected using extracellular portions of human and murine TfR from a human antibody library. The cross-reactivity of TSP-A18 with human and murine cells was confirmed by flow cytometry. Cell binding and competitive inhibition assays with [111In]TSP-A18 showed that TSP-A18 bound highly to TfR-expressing MIAPaCa-2 cells with high affinity. Biodistribution studies of [111In]TSP-A18 and [67Ga]citrate (a transferrin-mediated imaging probe) were conducted in C57BL/6J and BALB/c-nu/nu mice. [111In]TSP-A18 was accumulated highly in the spleen and bone containing marrow component of both strains, whereas high [67Ga]citrate uptake was only observed in bone containing marrow component and not in the spleen. Western blotting indicated the spleen showed the strongest TfR expression compared with other organs in both strains. There was significant correlation between [111In]TSP-A18 uptake and TfR protein expression in both strains, whereas there was significant correlation of [67Ga]citrate uptake with TfR expression only in C57BL/6J. These findings suggest that the difference in TfR expression between murine strains should be carefully considered when testing for the toxicity of anti-TfR antibody in mice and the uptake of anti-TfR antibody could reflect tissue TfR expression more accurately compared with that of transferrin-mediated imaging probe such as [67Ga]citrate.

  10. A loop in the N-lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping

    PubMed Central

    Mason, Anne B.; Byrne, Shaina L.; Everse, Stephen J.; Roberts, Samantha E.; Chasteen, N. Dennis; Smith, Valerie C.; MacGillivray, Ross T. A.; Kandemir, Banu; Bou-Abdallah, Fadi

    2015-01-01

    Transferrin (TF) is a bilobal transport protein that acquires ferric iron from the diet and holds it tightly within the cleft of each lobe (thereby preventing its hydrolysis). The iron is delivered to actively dividing cells by receptor mediated endocytosis in which diferric TF preferentially binds to TF receptors (TFRs) on the cell surface and the entire complex is taken into an acidic endosome. A combination of lower pH, a chelator, inorganic anions, and the TFR leads to the efficient release of iron from each lobe. Identification of residues/regions within both TF and TFR required for high affinity binding has been an ongoing goal in the field. In the current study, we created human TF (hTF) mutants to identify a region critical to the interaction with the TFR which also constitutes part of an overlapping epitope for two monoclonal antibodies (mAbs) to the N-lobe, one of which was previously shown to block binding of hTF to the TFR. Four single point mutants, P142A, R143A, K144A, and P145A in the N-lobe, were placed into diferric hTF. Isothermal titration calorimetry (ITC) revealed that three of the four residues (Pro142, Lys144, and Pro145) in this loop are essential to TFR binding. Additionally, Lys144 is common to the recognition of both mAbs which show different sensitivities to the three other residues. Taken together these studies prove that this loop is required for binding of the N-lobe of hTF to the TFR, provide a more precise description of the role of each residue in the loop in the interaction with the TFR, and confirm that the N-lobe is essential to high affinity binding of diferric hTF to TFR. PMID:19693784

  11. Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE.

    PubMed

    West, A P; Bennett, M J; Sellers, V M; Andrews, N C; Enns, C A; Bjorkman, P J

    2000-12-08

    The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.

  12. Hereditary Hemochromatosis and Transferrin Receptor 2

    PubMed Central

    Chen, Juxing

    2011-01-01

    Background Multicellular organisms regulate the uptake of calories, trace elements, and other nutrients by complex feedback mechanisms. In the case of iron, the body senses internal iron stores, iron requirements for hematopoiesis, and inflammatory status, and regulates iron uptake by modulating the uptake of dietary iron from the intestine. Both the liver and the intestine participate in the coordination of iron uptake and distribution in the body. The liver senses inflammatory signals and iron status of the organism and secretes a peptide hormone, hepcidin. Under high iron or inflammatory conditions hepcidin levels increase. Hepcidin binds to the iron transport protein, ferroportin (FPN), promoting FPN internalization and degradation. Decreased FPN levels reduce iron efflux out of intestinal epithelial cells and macrophages into the circulation. Derangements in iron metabolism result in either the abnormal accumulation of iron in the body, or in anemias. The identification of the mutations that cause the iron overload disease, hereditary hemochromatosis (HH), or iron-refractory iron-deficiencey anemia has revealed many of the proteins used to regulate iron uptake. Scope of the review In this review we discuss recent data concerning the regulation of iron homeostasis in the body by the liver and how transferrin receptor 2 (TfR2) affects this process. Major conclusions TfR2 plays a key role in regulating iron homeostasis in the body. General significance The regulation of iron homeostasis is important. One third of the people in the world are anemic. HH is the most common inherited disease in people of Northern European origin and can lead to severe health complications if left untreated. PMID:21864651

  13. Hemochromatosis due to mutations in transferrin receptor 2.

    PubMed

    Roetto, Antonella; Daraio, Filomena; Alberti, Federica; Porporato, Paolo; Calì, Angelita; De Gobbi, Marco; Camaschella, Clara

    2002-01-01

    A rare recessive disorder which leads to iron overload and severe clinical complications similar to those reported in HFE-related hemochromatosis has been delineated and sometimes called hemochromatosis type 3. The gene responsible is Transferrin Receptor 2 (TFR2), which maps to chromosome 7q22. The TFR2 gene presents a significative homology to transferrin receptor (TFRC) gene, encodes for a transmembrane protein with a large extracellular domain, is able to bind transferrin, even if with lower affinity than TFRC. The TFR2 function is still unclear. The transcript does not contain IRE elements and is not modified by the cellular iron status. At variance with TFRC, interactions between TFR2 and HFE do not occur, at least in their soluble forms. TFR2 is spliced in two alternative forms, alfa and beta. The alfa form is strongly expressed in the liver. The beta form, codified from a start site in exon 4 of the alpha, has a low and ubiquitous expression. Using anti-TFR2 monoclonal antibodies we have confirmed expression of the protein in the liver but also in duodenal epithelial cells, and studied the protein functional behaviour in cell lines, in response to iron addition, iron deprivation and olo-transferrin exposure. Our results suggest a regulatory role of TFR2 in iron metabolism. Five TFR2 homozygous mutations have been documented in HFE3 patients: a nonsense mutation (Y250X); a C insertion that causes a frameshift and a premature stop codon (E60X); a missense mutation (M172K); a 12 basepair deletion in exon 16, that causes 4 aminoacid loss (AVAQ 594-597del) in the extracellular domain of TFR2; a missense mutation in exon 17 (Q690P). The mutation analysis supports the hypothesis that all are private mutations. The pathogenetic role of TFR2 in hemochromatosis has been recently further demonstrated through the targeted expression of the Y250X human mutation in mice, which develop sings of iron overload identical to the human disease. Although the rarity of TFR2

  14. Serum transferrin receptors in detection of iron deficiency in pregnancy.

    PubMed

    Rusia, U; Flowers, C; Madan, N; Agarwal, N; Sood, S K; Sikka, M

    1999-08-01

    A prospective hospital-based study was conducted to evaluate the efficacy of serum transferrin receptors in the detection of iron deficiency in pregnant women. The iron status of 100 pregnant women with single uncomplicated term pregnancies in the first stage of labor was established using standard laboratory measures. These included complete hemogram, red cell indices, serum iron, percent transferrin saturation, and serum ferritin. In addition, serum transferrin receptor (STFR) was estimated. The results of 81 women with complete laboratory profiles were analyzed. Thirty-five (43.2%) women were anemic (hemoglobin <11 g/dl). Hemoglobin (Hb) showed a significant correlation with MCH, MCHC, serum iron, and percent transferrin saturation, suggesting that the anemia was likely to be due to iron deficiency. The mean STFR level was 18.05+/-9.9 mg/l in the anemic women and was significantly raised (p<0.001) compared with that of the nonanemic women. STFR correlated significantly with Hb (p<0.001), MCH (p<0.05), MCHC (p<0.01), serum iron (p<0.01), and percent transferrin saturation (p<0.01) and also showed a highly significant correlation with the degree of anemia. Serum ferritin in these women did not correlate with Hb, and only 54.4% of the women had levels <12 ng/ml, which does not reflect the true prevalence of iron deficiency. Serum transferrin receptor estimation is thus a useful measure for detecting iron deficiency in pregnancy.

  15. Nonhuman Transferrin Receptor 1 Is an Efficient Cell Entry Receptor for Ocozocoautla de Espinosa Virus

    PubMed Central

    Caì, Yíngyún; Yú, Shuĭqìng; Mazur, Steven; Dŏng, Lián; Janosko, Krisztina; Zhāng, Téngfēi; Müller, Marcel A.; Hensley, Lisa E.; Bavari, Sina; Jahrling, Peter B.

    2013-01-01

    Ocozocoautla de Espinosa virus (OCEV) is a novel, uncultured arenavirus. We found that the OCEV glycoprotein mediates entry into grivet and bat cells through transferrin receptor 1 (TfR1) binding but that OCEV glycoprotein precursor (GPC)-pseudotyped retroviruses poorly entered 53 human cancer cell lines. Interestingly, OCEV and Tacaribe virus could use bat, but not human, TfR1. Replacing three human TfR1 amino acids with their bat ortholog counterparts transformed human TfR1 into an efficient OCEV and Tacaribe virus receptor. PMID:24109228

  16. Transferrin Receptor Controls AMPA Receptor Trafficking Efficiency and Synaptic Plasticity

    PubMed Central

    Liu, Ke; Lei, Run; Li, Qiong; Wang, Xin-Xin; Wu, Qian; An, Peng; Zhang, Jianchao; Zhu, Minyan; Xu, Zhiheng; Hong, Yang; Wang, Fudi; Shen, Ying; Li, Hongchang; Li, Huashun

    2016-01-01

    Transferrin receptor (TFR) is an important iron transporter regulating iron homeostasis and has long been used as a marker for clathrin mediated endocytosis. However, little is known about its additional function other than iron transport in the development of central nervous system (CNS). Here we demonstrate that TFR functions as a regulator to control AMPA receptor trafficking efficiency and synaptic plasticity. The conditional knockout (KO) of TFR in neural progenitor cells causes mice to develop progressive epileptic seizure, and dramatically reduces basal synaptic transmission and long-term potentiation (LTP). We further demonstrate that TFR KO remarkably reduces the binding efficiency of GluR2 to AP2 and subsequently decreases AMPA receptor endocytosis and recycling. Thus, our study reveals that TFR functions as a novel regulator to control AMPA trafficking efficiency and synaptic plasticity. PMID:26880306

  17. Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes

    PubMed Central

    1991-01-01

    Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique

  18. Aluminum stimulates uptake of non-transferrin bound iron and transferrin bound iron in human glial cells.

    PubMed

    Kim, Yongbae; Olivi, Luisa; Cheong, Jae Hoon; Maertens, Alex; Bressler, Joseph P

    2007-05-01

    Aluminum and other trivalent metals were shown to stimulate uptake of transferrin bound iron and nontransferrin bound iron in erytholeukemia and hepatoma cells. Because of the association between aluminum and Alzheimer's Disease, and findings of higher levels of iron in Alzheimer's disease brains, the effects of aluminum on iron homeostasis were examined in a human glial cell line. Aluminum stimulated dose- and time-dependent uptake of nontransferrin bound iron and iron bound to transferrin. A transporter was likely involved in the uptake of nontransferrin iron because uptake reached saturation, was temperature-dependent, and attenuated by inhibitors of protein synthesis. Interestingly, the effects of aluminum were not blocked by inhibitors of RNA synthesis. Aluminum also decreased the amount of iron bound to ferritin though it did not affect levels of divalent metal transporter 1. These results suggest that aluminum disrupts iron homeostasis in the brain by several mechanisms including the transferrin receptor, a nontransferrin iron transporter, and ferritin.

  19. Aluminum stimulates uptake of non-transferrin bound iron and transferrin bound iron in human glial cells

    SciTech Connect

    Kim, Yongbae; Olivi, Luisa; Cheong, Jae Hoon; Maertens, Alex; Bressler, Joseph P. . E-mail: Bressler@kennedykrieger.org

    2007-05-01

    Aluminum and other trivalent metals were shown to stimulate uptake of transferrin bound iron and nontransferrin bound iron in erytholeukemia and hepatoma cells. Because of the association between aluminum and Alzheimer's Disease, and findings of higher levels of iron in Alzheimer's disease brains, the effects of aluminum on iron homeostasis were examined in a human glial cell line. Aluminum stimulated dose- and time-dependent uptake of nontransferrin bound iron and iron bound to transferrin. A transporter was likely involved in the uptake of nontransferrin iron because uptake reached saturation, was temperature-dependent, and attenuated by inhibitors of protein synthesis. Interestingly, the effects of aluminum were not blocked by inhibitors of RNA synthesis. Aluminum also decreased the amount of iron bound to ferritin though it did not affect levels of divalent metal transporter 1. These results suggest that aluminum disrupts iron homeostasis in Brain by several mechanisms including the transferrin receptor, a nontransferrin iron transporter, and ferritin.

  20. Upregulation of transferrin receptor-1 induces cholangiocarcinoma progression via induction of labile iron pool.

    PubMed

    Jamnongkan, Wassana; Thanan, Raynoo; Techasen, Anchalee; Namwat, Nisana; Loilome, Watcharin; Intarawichian, Piyapharom; Titapun, Attapol; Yongvanit, Puangrat

    2017-07-01

    Labile iron pool is a cellular source of ions available for Fenton reactions resulting in oxidative stress. Living organisms avoid an excess of free irons by a tight control of iron homeostasis. We investigated the altered expression of iron regulatory proteins and iron discrimination in the development of liver fluke-associated cholangiocarcinoma. Additionally, the levels of labile iron pool and the functions of transferrin receptor-1 on cholangiocarcinoma development were also identified. Iron deposition was determined using the Prussian blue staining method in human cholangiocarcinoma tissues. We investigated the alteration of iron regulatory proteins including transferrin, transferrin receptor-1, ferritin, ferroportin, hepcidin, and divalent metal transporter-1 in cholangiocarcinoma tissues using immunohistochemistry. The clinicopathological data of cholangiocarcinoma patients and the expressions of proteins were analyzed. Moreover, the level of intracellular labile iron pool in cholangiocarcinoma cell lines was identified by the RhoNox-1 staining method. We further demonstrated transferrin receptor-1 functions on cell proliferation and migration upon small interfering RNA for human transferrin receptor 1 transfection. Results show that Iron was strongly stained in tumor tissues, whereas negative staining was observed in normal bile ducts of healthy donors. Interestingly, high iron accumulation was significantly correlated with poor prognosis of cholangiocarcinoma patients. The expressions of iron regulatory proteins in human cholangiocarcinoma tissues and normal liver from cadaveric donors revealed that transferrin receptor-1 expression was increased in the cancer cells of cholangiocarcinoma tissues when compared with the adjacent normal bile ducts and was significantly correlated with cholangiocarcinoma metastasis. Labile iron pool level and transferrin receptor-1 expression were significantly increased in KKU-214 and KKU-213 when compared with cholangiocyte

  1. Dihydroartemisinin Exerts Its Anticancer Activity through Depleting Cellular Iron via Transferrin Receptor-1

    PubMed Central

    Ba, Qian; Zhou, Naiyuan; Duan, Juan; Chen, Tao; Hao, Miao; Yang, Xinying; Li, Junyang; Yin, Jun; Chu, Ruiai; Wang, Hui

    2012-01-01

    Artemisinin and its main active metabolite dihydroartemisinin, clinically used antimalarial agents with low host toxicity, have recently shown potent anticancer activities in a variety of human cancer models. Although iron mediated oxidative damage is involved, the mechanisms underlying these activities remain unclear. In the current study, we found that dihydroartemisinin caused cellular iron depletion in time- and concentration-dependent manners. It decreased iron uptake and disturbed iron homeostasis in cancer cells, which were independent of oxidative damage. Moreover, dihydroartemisinin reduced the level of transferrin receptor-1 associated with cell membrane. The regulation of dihydroartemisinin to transferrin receptor-1 could be reversed by nystatin, a cholesterol-sequestering agent but not the inhibitor of clathrin-dependent endocytosis. Dihydroartemisinin also induced transferrin receptor-1 palmitoylation and colocalization with caveolin-1, suggesting a lipid rafts mediated internalization pathway was involved in the process. Also, nystatin reversed the influences of dihydroartemisinin on cell cycle and apoptosis related genes and the siRNA induced downregulation of transferrin receptor-1 decreased the sensitivity to dihydroartemisinin efficiently in the cells. These results indicate that dihydroartemisinin can counteract cancer through regulating cell-surface transferrin receptor-1 in a non-classical endocytic pathway, which may be a new action mechanism of DHA independently of oxidative damage. PMID:22900042

  2. Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe-lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies.

    PubMed Central

    Mason, A; He, Q Y; Tam, B; MacGillivray, R A; Woodworth, R

    1998-01-01

    Recombinant non-glycosylated human serum transferrin and mutants in which the liganding aspartic acid (D) in one or both lobes was changed to a serine residue (S) were produced in a mammalian cell system and purified from the tissue culture media. Significant downfield shifts of 20, 30, and 45 nm in the absorption maxima were found for the D63S-hTF, D392S-hTF and the double mutant, D63S/D392S-hTF when compared to wild-type hTF. A monoclonal antibody to a sequential epitope in the C-lobe of hTF reported affinity differences between the apo- and iron-forms of each mutant and the control. Cell-binding studies performed under the same buffer conditions used for the antibody work clearly showed that the mutated lobe(s) had an open cleft. It is not clear whether the receptor itself may play a role in promoting the open conformation or whether the iron remains in the cleft. PMID:9461487

  3. Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

    PubMed Central

    Steere, Ashley N.; Byrne, Shaina L.; Chasteen, N. Dennis; Smith, Valerie C.; MacGillivray, Ross T. A.

    2015-01-01

    His349 in human transferrin (hTF) is a residue critical to transferrin receptor (TFR)-stimulated iron release from the C-lobe. To evaluate the importance of His349 on the TFR interaction, it was replaced by alanine, aspartate, lysine, leucine, tryptophan, and tyrosine in a monoferric C-lobe hTF construct (FeChTF). Using a stop-ped-flow spectrofluorimeter, we determined rate processes assigned to iron release and conformational events (in the presence and in the absence of the TFR). Significantly, all mutant/TFR complexes feature dampened iron release rates. The critical contribution of His349 is most convincingly revealed by analysis of the kinetics as a function of pH (5.6–6.2). The FeChTF/TFR complex titrates with a pKa of approximately 5.9. By contrast, the H349A mutant/TFR complex releases iron at higher pH with a profile that is almost the inverse of that of the control complex. At the putative endosomal pH of 5.6 (in the presence of salt and chelator), iron is released from the H349W mutant/TFR and H349Y mutant/TFR complexes with a single rate constant similar to the iron release rate constant for the control; this suggests that these substitutions bypass the required pH-induced conformational change allowing the C-lobe to directly interact with the TFR to release iron. The H349K mutant proves that although the positive charge is crucial to complete iron release, the geometry at this position is also critical. The H349D mutant shows that a negative charge precludes complete iron release at pH 5.6 both in the presence and in the absence of the TFR. Thus, histidine uniquely drives the pH-induced conformational change in the C-lobe required for TFR interaction, which in turn promotes iron release. PMID:20711621

  4. Microfluidic Separation of Lymphoblasts for the Isolation of Acute Lymphoblastic Leukemia Using the Human Transferrin Receptor as a Capture Target.

    PubMed

    Li, Wenjie; Zhang, Ye; Reynolds, C Patrick; Pappas, Dimitri

    2017-07-18

    Acute lymphocytic leukemia (ALL) is the most prevalent pediatric cancer, and the peripheral blood lymphoblast percentage is an important index for ALL diagnosis and prognosis. We describe a microfluidic device that isolates and enumerates peripheral blood lymphoblasts using affinity separations. The innovative use of a nonspecific ligand allows a widespread "net" for cancer cells, without a priori knowledge of the cancer type. Using lymphoblasts spiked into blood, we simulated leukemia cases with lymphoblast concentrations ranging from 1 to 30% of total leukocytes. Lymphoblasts were isolated using monoclonal antibodies for the Human Transferring Receptor (CD71). Anti-CD71 antibodies were found to be more effective for capturing lymphoblasts than commonly used, ALL-specific antibodies for CD7 and CD10. CCRF-CEM lymphoblasts were isolated in the chip with 82-97% purity, with lower concentrations tested (7%) still showing >80% purity for cell capture. Patient-derived ALL cell lines COG-LL-332 and COG-LL-317 were isolated in the chip with 80%-97% and 57% -92% of purity, respectively, with the initial spike concentrations as low as 1%. The ability to capture ALL lymphoblasts present in blood at low concentrations provides a novel approach for characterization of ALL cells, including patients with low leukemic burdens during and after therapy.

  5. Soluble transferrin receptor and transferrin receptor-ferritin index in iron deficiency anemia and anemia in rheumatoid arthritis.

    PubMed

    Margetic, Sandra; Topic, Elizabeta; Ruzic, Dragica Ferenec; Kvaternik, Marina

    2005-01-01

    The aim of the study was to evaluate the clinical efficiency of soluble transferrin receptor and transferrin receptor-ferritin index (sTfR/logF) in the diagnosis of iron deficiency anemia, as well as the differential diagnosis of iron deficiency anemia and anemia in rheumatoid arthritis. The study included 96 patients with anemia and 61 healthy volunteers as a control group. In healthy subjects there were no significant sex and age differences in the parameters tested. The study results showed these parameters to be reliable in the diagnosis of iron deficiency anemia, as well as in the differential diagnosis of iron deficiency anemia and anemia of chronic disease. The results indicate that sTfR/logF could be used to help differentiate coexisting iron deficiency in patients with anemia of chronic disease. Receiver operating characteristic analysis showed a higher discriminating power of transferrin receptor-ferritin index vs. soluble transferrin receptor in the diagnosis of iron deficiency anemia, as well as in the differential diagnosis between iron deficiency anemia and anemia of chronic disease. In patients with anemia in rheumatoid arthritis, the parameters tested showed no significant differences with respect to C-reactive protein concentration. These results suggested that the parameters tested are not affected by acute or chronic inflammatory disease.

  6. Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron.

    PubMed Central

    Owen, D; Kühn, L C

    1987-01-01

    The cell-surface receptor for transferrin mediates cellular uptake of iron from serum. Transferrin receptor protein and mRNA levels are increased in cells treated with iron chelating agents, and are decreased by treatment with iron salts or hemin. Here we report that expression of human transferrin receptor cDNA constructions in stably transfected mouse fibroblasts is regulated both by the iron chelator, desferrioxamine, and by hemin. We found that sequences within the 3' noncoding region are required for the iron-dependent feed-back regulation of receptor expression, whereas the presence of the transferrin receptor promoter region is not necessary. Regulation by iron is observed when transcription is initiated at either the SV-40 early promoter or the transferrin receptor promoter, but deletion of a 2.3 kb fragment within the 2.6 kb 3' noncoding region of the cDNA abolishes regulation and increases the constitutive level of receptor expression. Furthermore, the 3' deletion does not affect the decrease in receptors which is observed in response to growth arrest, indicating that transferrin receptor expression is controlled by at least two distinct mechanisms. Images Fig. 3. Fig. 6. Fig. 8. PMID:3608980

  7. Iron uptake from plasma transferrin by a transferrin receptor 2 mutant mouse model of haemochromatosis

    PubMed Central

    Chua, Anita C.G.; Delima, Roheeth D.; Morgan, Evan H.; Herbison, Carly E.; Tirnitz-Parker, Janina E.E.; Graham, Ross M.; Fleming, Robert E.; Britton, Robert S.; Bacon, Bruce R.; Olynyk, John K.; Trinder, Debbie

    2010-01-01

    Background & Aims Hereditary haemochromatosis type 3 is caused by mutations in transferrin receptor (TFR) 2. TFR2 has been shown to mediate iron transport in vitro and regulate iron homeostasis. The aim of this study was to determine the role of Tfr2 in iron transport in vivo using a Tfr2 mutant mouse. Methods Tfr2 mutant and wild-type mice were injected intravenously with 59Fe-transferrin and tissue 59Fe uptake was measured. Tfr1, Tfr2 and ferroportin expression was measured by real-time PCR and Western blot. Cellular localisation of ferroportin was determined by immunohistochemistry. Results Transferrin-bound iron uptake by the liver and spleen in Tfr2 mutant mice was reduced by 20% and 65%, respectively, whilst duodenal and renal uptake was unchanged compared with iron-loaded wild-type mice. In Tfr2 mutant mice, liver Tfr2 protein was absent, whilst ferroportin protein was increased in non-parenchymal cells and there was a low level of expression in hepatocytes. Tfr1 expression was unchanged compared with iron-loaded wild-type mice. Splenic Tfr2 protein expression was absent whilst Tfr1 and ferroportin protein expression was increased in Tfr2 mutant mice compared with iron-loaded wild-type mice. Conclusions A small reduction in hepatic transferrin-bound iron uptake in Tfr2 mutant mice suggests that Tfr2 plays a minor role in liver iron transport and its primary role is to regulate iron metabolism. Increased ferroportin expression due to decreased hepcidin mRNA levels is likely to be responsible for impaired splenic iron uptake in Tfr2 mutant mice. PMID:20133002

  8. Transferrin receptors in rat brain: neuropeptide-like pattern and relationship to iron distribution.

    PubMed Central

    Hill, J M; Ruff, M R; Weber, R J; Pert, C B

    1985-01-01

    We have characterized and visualized the binding of 125I-labeled transferrin to sections of rat brain. This saturable, reversible, high-affinity (Kd = 1 X 10(-9) M) binding site appears indistinguishable from transferrin receptors previously characterized in other tissues. Moreover, a monoclonal antibody raised to rat lymphocyte transferrin receptors could immunoprecipitate recovered intact transferrin solubilized from labeled brain slices, indicating that labeling was to the same molecular entity previously characterized as the transferrin receptor. The pattern of transferrin receptor distribution visualized in brain with both 125I-labeled transferrin and an anti-transferrin receptor monoclonal antibody are almost indistinguishable but differ from the pattern of iron distribution. Iron-rich brain areas generally receive neuronal projections from areas with abundant transferrin receptors, suggesting that iron may be transported neuronally. However, many brain areas with a high density of transferrin receptors appear unrelated to iron uptake and neuronal transport and form a receptor distribution pattern similar to that of other known neuropeptides. This "neuropeptide-like" distribution pattern suggests that transferrin may have neuromodulatory, perhaps behavioral, function in brain. Images PMID:2989832

  9. Noncanonical interactions between serum transferrin and transferrin receptor evaluated with electrospray ionization mass spectrometry

    PubMed Central

    Leverence, Rachael; Mason, Anne B.; Kaltashov, Igor A.

    2010-01-01

    The primary route of iron acquisition in vertebrates is the transferrin receptor (TfR) mediated endocytotic pathway, which provides cellular entry to the metal transporter serum transferrin (Tf). Despite extensive research efforts, complete understanding of Tf-TfR interaction mechanism is still lacking owing to the complexity of this system. Electrospray ionization mass spectrometry (ESI MS) is used in this study to monitor the protein/receptor interaction and demonstrate the ability of metal-free Tf to associate with TfR at neutral pH. A set of Tf variants is used in a series of competition and displacement experiments to bracket TfR affinity of apo-Tf at neutral pH (0.2–0.6 μM). Consistent with current models of endosomal iron release from Tf, acidification of the protein solution results in a dramatic change of binding preferences, with apo-Tf becoming a preferred receptor binder. Contrary to the current models implying that the apo-Tf/TfR complex dissociates almost immediately upon exposure to the neutral environment at the cell surface, our data indicate that this complex remains intact. Iron-loaded Tf displaces apo-Tf from TfR, making it available for the next cycle of iron binding, transport and delivery to tissues. However, apo-Tf may still interfere with the cellular uptake of engineered Tf molecules whose TfR affinity is affected by various modifications (e.g., conjugation to cytotoxic molecules). This work also highlights the great potential of ESI MS as a tool capable of providing precise details of complex protein-receptor interactions under conditions that closely mimic the environment in which these encounters occur in physiological systems. PMID:20404192

  10. Serum transferrin receptor: a quantitative measure of tissue iron deficiency.

    PubMed

    Skikne, B S; Flowers, C H; Cook, J D

    1990-05-01

    This study was undertaken to evaluate the role of serum transferrin receptor measurements in the assessment of iron status. Repeated phlebotomies were performed in 14 normal volunteer subjects to obtain varying degrees of iron deficiency. Serial measurements of serum iron, total iron-binding capacity, mean cell volume (MCV), free erythrocyte protoporphyrin (FEP), red cell mean index, serum ferritin, and serum transferrin receptor were performed throughout the phlebotomy program. There was no change in receptor levels during the phase of storage iron depletion. When the serum ferritin level reached subnormal values there was an increase in serum receptor levels, which continued throughout the phlebotomy program. Functional iron deficiency was defined as a reduction in body iron beyond the point of depleted iron stores. The serum receptor level was a more sensitive and reliable guide to the degree of functional iron deficiency than either the FEP or MCV. Our studies indicate that the serum receptor measurement is of particular value in identifying mild iron deficiency of recent onset. The iron status of a population can be fully assessed by using serum ferritin as a measure of iron stores, serum receptor as a measure of mild tissue iron deficiency, and hemoglobin concentration as a measure of advanced iron deficiency.

  11. Transferrin Binding to Peripheral Blood Lymphocytes Activated by Phytohemagglutinin Involves a Specific Receptor

    PubMed Central

    Galbraith, Robert M.; Werner, Phillip; Arnaud, Philippe; Galbraith, Gillian M. P.

    1980-01-01

    Immunohistological studies have indicated that membrane sites binding transferrin are present upon activated human peripheral blood lymphocytes. In this study, we have investigated transferrin uptake in human lymphocytes exposed to phytohemagglutinin (PHA), by quantitative radiobinding and immunofluorescence in parallel. In stimulated lymphocytes, binding was maximal after a 30-min incubation, being greatest at 37°C, and greater at 22°C than at 4°C. Although some shedding and endocytosis of transferrin occurred at 22° and 37°C, these factors, and resulting synthesis of new sites, did not affect measurement of binding which was found to be saturable, reversible, and specific for transferrin (Ka 0.5-2.5 × 108 M−1). Binding was greater after a 48-h exposure to PHA than after 24 h, and was maximal at 66 h. Sequential Scatchard analysis revealed no significant elevation in affinity of interaction. However, although the total number of receptors increased, the proportion of cells in which binding of ligand was detected immunohistologically increased in parallel, and after appropriate correction, the cellular density of receptors remained relatively constant throughout (60,000-80,000 sites/cell). Increments in binding during the culture period were thus due predominantly to expansion of a population of cells bearing receptors. Similar differences in binding were apparent upon comparison of cells cultured in different doses of PHA, and in unstimulated cells binding was negligible. Transferrin receptors appear, therefore, to be readily detectable only upon lymphocytes that have been activated. Images PMID:6253523

  12. ALUMINUM STIMULATES UPTAKE OF NON-TRANSFERRIN BOUND IRON AND TRANSFERRIN BOUND IRON IN HUMAN GLIAL CELLS

    PubMed Central

    Kim, Yongbae; Olivi, Luisa; Cheong, Jae Hoon; Maertens, Alex; Bressler, Joseph P.

    2011-01-01

    Aluminum and other trivalent metals were shown to stimulate uptake of transferrin bound iron and nontransferrin bound iron in erytholeukemia and hepatoma cells. Because of the association between aluminum and Alzheimer’s Disease, and findings of higher levels of iron in Alzheimer’s disease brains, the effects of aluminum on iron homeostasis were examined in a human glial cell line. Aluminum stimulated dose- and time-dependent uptake of nontransferrin bound iron and iron bound to transferrin. A transporter was likely involved in the uptake of nontransferrin iron because uptake reached saturation, was temperature-dependent, and attenuated by inhibitors of protein synthesis. Interestingly, the effects of aluminum were not blocked by inhibitors of RNA synthesis. Aluminum also decreased the amount of iron bound to ferritin though it did not affect levels of divalent metal transporter 1. These results suggest that aluminum disrupts iron homeostasis in the brain by several mechanisms including the transferrin receptor, a nontransferrin iron transporter, and ferritin. PMID:17376497

  13. Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro.

    PubMed Central

    Young, S P; Garner, C

    1990-01-01

    Following suggestions that transferrin present in fetal-bovine serum, a common supplement used in tissue-culture media, may not bind well to human cells, we have isolated the protein and investigated its interaction with both human and bovine cells. Bovine transferrin bound to a human cell line, K562, at 4 degrees C with a kd of 590 nM, whereas human transferrin bound with a kd of 3.57 nM, a 165-fold difference. With a bovine cell line, NBL4, bovine transferrin bound with the higher affinity, kd 9.09 nM, whereas human transferrin bound with a kd of 41.7 nM, only a 5-fold difference. These values were reflected in an 8.6-fold difference in the rate of iron delivery by the two proteins to human cells, whereas delivery to bovine cells was the same. Nevertheless, the bovine transferrin was taken up by the human cells by a specific receptor-mediated process. Human cells cultured in bovine diferric transferrin at 40 micrograms/ml, the concentration expected in the presence of 10% fetal-bovine serum, failed to thrive, whereas cells cultured in the presence of human transferrin proliferated normally. These results suggest that growth of human cells in bovine serum could give rise to a cellular iron deficiency, which may in turn lead to the selection of clones of cells adapted for survival with less iron. This has important consequences for the use of such cells as models, since they may have aberrant iron-dependent pathways and perhaps other unknown alterations in cell function. PMID:2302189

  14. Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites.

    PubMed

    West, A P; Giannetti, A M; Herr, A B; Bennett, M J; Nangiana, J S; Pierce, J R; Weiner, L P; Snow, P M; Bjorkman, P J

    2001-10-19

    The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 A resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR. Copyright 2001 Academic Press.

  15. Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms

    SciTech Connect

    Alcantara, O.; Phillips, J.L.; Boldt, D.H.

    1986-12-01

    Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. The authors studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester-induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf-induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf-induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf-induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. Phorbol diesters cause TfR downregulation by a cytoskeleton-independent mechanism. These data indicate that TfR expression is regulated by two independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.

  16. Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15.

    PubMed

    Fertrin, Kleber Yotsumoto; Lanaro, Carolina; Franco-Penteado, Carla Fernanda; de Albuquerque, Dulcinéia Martins; de Mello, Mariana Rezende Bandeira; Pallis, Flávia Rubia; Bezerra, Marcos André Cavalcanti; Hatzlhofer, Betania Lucena Domingues; Olbina, Gordana; Saad, Sara Terezinha Olalla; da Silva Araújo, Aderson; Westerman, Mark; Costa, Fernando Ferreira

    2014-04-01

    Growth differentiation factor 15 (GDF-15) is a bone marrow-derived cytokine whose ability to suppress iron regulator hepcidin in vitro and increased concentrations found in patients with ineffective erythropoiesis (IE)suggest that hepcidin deficiency mediated by GDF-15 may be the pathophysiological explanation for nontransfusional iron overload. We aimed to compare GDF-15 production in anemic states with different types of erythropoietic dysfunction. Complete blood counts, biochemical markers of iron status, plasma hepcidin, GDF-15, and known hepcidin regulators [interleukin-6 and erythropoietin (EPO)] were measured in 87 patients with red cell disorders comprising IE and hemolytic states: thalassemia, sickle cell anemia, and cobalamin deficiency. Healthy volunteers were also evaluated for comparison. Neither overall increased EPO,nor variable GDF-15 concentrations correlated with circulating hepcidin concentrations (P = 0.265 and P = 0.872). Relative hepcidin deficiency was found in disorders presenting with concurrent elevation of GDF-15 and soluble transferrin receptor (sTfR), a biomarker of erythropoiesis, and sTfR had the strongest correlation with hepcidin (r(s) = 0.584, P < 0.0001). Our data show that high concentrations of GDF-15 in vivo are not necessarily associated with pathological hepcidin reduction, and hepcidin deficiency was only found when associated with sTfR overproduction. sTfR elevation may be a necessary common denominator of erythropoiesis-driven mechanisms to favor iron absorption in anemic states and appears a suitable target for investigative approaches to iron disorders.

  17. Transferrin receptor facilitates TGF-β and BMP signaling activation to control craniofacial morphogenesis

    PubMed Central

    Lei, R; Zhang, K; Liu, K; Shao, X; Ding, Z; Wang, F; Hong, Y; Zhu, M; Li, H; Li, H

    2016-01-01

    The Pierre Robin Sequence (PRS), consisting of cleft palate, glossoptosis and micrognathia, is a common human birth defect. However, how this abnormality occurs remains largely unknown. Here we report that neural crest cell (NCC)-specific knockout of transferrin receptor (Tfrc), a well known transferrin transporter protein, caused micrognathia, cleft palate, severe respiratory distress and inability to suckle in mice, which highly resemble human PRS. Histological and anatomical analysis revealed that the cleft palate is due to the failure of palatal shelves elevation that resulted from a retarded extension of Meckel's cartilage. Interestingly, Tfrc deletion dramatically suppressed both transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling in cranial NCCs-derived mandibular tissues, suggesting that Tfrc may act as a facilitator of these two signaling pathways during craniofacial morphogenesis. Together, our study uncovers an unknown function of Tfrc in craniofacial development and provides novel insight into the etiology of PRS. PMID:27362800

  18. The Complex Role of Multivalency in Nanoparticles Targeting the Transferrin Receptor for Cancer Therapies

    PubMed Central

    Wang, Jin; Tian, Shaomin; Petros, Robby A.; Napier, Mary E.; DeSimone, Joseph M.

    2010-01-01

    Transferrin receptor (TfR, CD71) has long been therapeutic target due to its over-expression on many malignant tissues. In this study, PRINT® nanoparticles were conjugated with TfR ligands for targeted drug delivery. Cylindrical poly(ethylene glycol)-based PRINT nanoparticles (diameter [d] = 200 nm, height [h] = 200 nm) labeled with transferrin receptor antibody (NP-OKT9) or human transferrin (NP-hTf), showed highly specific TfR-mediated uptake by all human tumor cell lines tested, relative to negative controls (IgG1 for OKT9 or bovine transferrin (bTf) for hTf). The targeting efficiency was dependent on particle concentration, ligand density, dosing time and cell surface receptor expression level. Interestingly, NP-OKT9 or NP-hTf showed little cytotoxicity on all solid tumor cell lines tested but were very toxic to Ramos B-cell lymphoma, whereas free OKT9 or hTf was not toxic. There was a strong correlation between TfR ligand density on particle surface and cell viability and particle uptake. NP-OKT9 and NP-hTf were internalized into acidic intracellular compartments but were not localized in EEA1 enriched early endosomes or lysosomes. Elevated caspase 3/7 activity indicates activation of apoptosis pathways upon particle treatment. Supplementation of iron suppressed the toxicity of NP-OKT9 but not NP-hTf, suggesting different mechanisms by which NP-hTf and NP-OKT9 exerts cytotoxicity on Ramos cells. Based on such an observation, the complex role of multivalency in nanoparticles is discussed. In addition, our data clearly reveal that one must be careful in making claims of “lack of toxicity” when a targeting molecule is used on nanoparticles and also raise concerns for unanticipated off-target effects when one is designing targeted chemotherapy nano-delivery agents. PMID:20698697

  19. The complex role of multivalency in nanoparticles targeting the transferrin receptor for cancer therapies.

    PubMed

    Wang, Jin; Tian, Shaomin; Petros, Robby A; Napier, Mary E; Desimone, Joseph M

    2010-08-18

    Transferrin receptor (TfR, CD71) has long been a therapeutic target due to its overexpression in many malignant tissues. In this study, PRINT() nanoparticles were conjugated with TfR ligands for targeted drug delivery. Cylindrical poly(ethylene glycol)-based PRINT nanoparticles (diameter (d) = 200 nm, height (h) = 200 nm) labeled with transferrin receptor antibody (NP-OKT9) or human transferrin (NP-hTf) showed highly specific TfR-mediated uptake by all human tumor cell lines tested, relative to negative controls (IgG1 for OKT9 or bovine transferrin (bTf) for hTf). The targeting efficiency was dependent on particle concentration, ligand density, dosing time, and cell surface receptor expression level. Interestingly, NP-OKT9 or NP-hTf showed little cytotoxicity on all solid tumor cell lines tested but were very toxic to Ramos B-cell lymphoma, whereas free OKT9 or hTf was not toxic. There was a strong correlation between TfR ligand density on the particle surface and cell viability and particle uptake. NP-OKT9 and NP-hTf were internalized into acidic intracellular compartments but were not localized in EEA1-enriched early endosomes or lysosomes. Elevated caspase 3/7 activity indicates activation of apoptosis pathways upon particle treatment. Supplementation of iron suppressed the toxicity of NP-OKT9 but not NP-hTf, suggesting different mechanisms by which NP-hTf and NP-OKT9 exerts cytotoxicity on Ramos cells. On the basis of such an observation, the complex role of multivalency in nanoparticles is discussed. In addition, our data clearly reveal that one must be careful in making claims of "lack of toxicity" when a targeting molecule is used on nanoparticles and also raise concerns for unanticipated off-target effects when one is designing targeted chemotherapy nanodelivery agents.

  20. Transferrin receptor-targeted theranostic gold nanoparticles for photosensitizer delivery in brain tumors

    NASA Astrophysics Data System (ADS)

    Dixit, Suraj; Novak, Thomas; Miller, Kayla; Zhu, Yun; Kenney, Malcolm E.; Broome, Ann-Marie

    2015-01-01

    Therapeutic drug delivery across the blood-brain barrier (BBB) is not only inefficient, but also nonspecific to brain stroma. These are major limitations in the effective treatment of brain cancer. Transferrin peptide (Tfpep) targeted gold nanoparticles (Tfpep-Au NPs) loaded with the photodynamic pro-drug, Pc 4, have been designed and compared with untargeted Au NPs for delivery of the photosensitizer to brain cancer cell lines. In vitro studies of human glioma cancer lines (LN229 and U87) overexpressing the transferrin receptor (TfR) show a significant increase in cellular uptake for targeted conjugates as compared to untargeted particles. Pc 4 delivered from Tfpep-Au NPs clusters within vesicles after targeting with the Tfpep. Pc 4 continues to accumulate over a 4 hour period. Our work suggests that TfR-targeted Au NPs may have important therapeutic implications for delivering brain tumor therapies and/or providing a platform for noninvasive imaging.

  1. Cord blood transferrin receptors to assess fetal iron status

    PubMed Central

    Sweet, D.; Savage, G.; Tubman, R.; Lappin, T.; Halliday, H.

    2001-01-01

    OBJECTIVE—To study iron status at different gestational ages using cord blood serum transferrin receptors (STfRs).
METHODS—STfRs, iron, ferritin, total iron binding capacity, haemoglobin, and reticulocytes were measured in 144 cord blood samples. The babies were divided into three groups according to gestation (26 very preterm (24-29 weeks); 50 preterm (30-36 weeks); 68 term (37-41 weeks)).
RESULTS—Serum iron, ferritin, and total iron binding capacity were highest at term, whereas reticulocytes were highest in the very preterm. STfR levels were not influenced by gestation. Haemoglobin (r = 0.46; p < 0.0001) and reticulocytes (r = 0.42; p < 0.0001) were the only indices that independently correlated with STfR levels.
CONCLUSIONS—STfR levels in cord blood are not directly influenced by gestation and probably reflect the iron requirements of the fetus for erythropoiesis.

 PMID:11420322

  2. Transferrin receptor expression by stimulated cells in mixed lymphocyte culture.

    PubMed Central

    Salmon, M; Bacon, P A; Symmons, D P; Walton, K W

    1985-01-01

    Transferrin receptor (TRFr) expression by cells in mixed lymphocyte culture increases steadily for the first 5 days, but then reaches a plateau. By the sixth day in culture, about 20% of viable cells express TRFr in two-way mixed lymphocyte reactions. This subpopulation of TRFr-positive cells represents the proliferating population; it is heterogeneous, containing T-cell blasts and smaller cells which are a mixture of T and non-T cells. A small group of non-T cells have phenotypic similarity to natural killer (NK) cells. T cells appear to divide earlier in the course of the response than non-T cells. The biphasic nature of this response and the slower non-T reactivity may be due to a secondary stimulation of non-T cells by factors released from activated T cells (such as interleukin-2). PMID:2982734

  3. Metabolic Catastrophe in Mice Lacking Transferrin Receptor in Muscle

    PubMed Central

    Barrientos, Tomasa; Laothamatas, Indira; Koves, Timothy R.; Soderblom, Erik J.; Bryan, Miles; Moseley, M. Arthur; Muoio, Deborah M.; Andrews, Nancy C.

    2015-01-01

    Transferrin receptor (Tfr1) is ubiquitously expressed, but its roles in non-hematopoietic cells are incompletely understood. We used a tissue-specific conditional knockout strategy to ask whether skeletal muscle required Tfr1 for iron uptake. We found that iron assimilation via Tfr1 was critical for skeletal muscle metabolism, and that iron deficiency in muscle led to dramatic changes, not only in muscle, but also in adipose tissue and liver. Inactivation of Tfr1 incapacitated normal energy production in muscle, leading to growth arrest and a muted attempt to switch to fatty acid β oxidation, using up fat stores. Starvation signals stimulated gluconeogenesis in the liver, but amino acid substrates became limiting and hypoglycemia ensued. Surprisingly, the liver was also iron deficient, and production of the iron regulatory hormone hepcidin was depressed. Our observations reveal a complex interaction between iron homeostasis and metabolism that has implications for metabolic and iron disorders. PMID:26870796

  4. Lethal Cardiomyopathy in Mice Lacking Transferrin Receptor in the Heart.

    PubMed

    Xu, Wenjing; Barrientos, Tomasa; Mao, Lan; Rockman, Howard A; Sauve, Anthony A; Andrews, Nancy C

    2015-10-20

    Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure.

  5. Transferrin receptor targeted PLA-TPGS micelles improved efficacy and safety in docetaxel delivery.

    PubMed

    Singh, Rahul Pratap; Sharma, Gunjan; Sonali; Agrawal, Poornima; Pandey, Bajarangprasad L; Koch, Biplob; Muthu, Madaswamy S

    2016-02-01

    The aim of this work was to develop targeted polymeric micelles of poly-lactic acid-D-α-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS), which are assembled along with D-alpha-tocopheryl polyethylene glycol 1000 succinate-transferrin conjugate (TPGS-Tf), and loaded docetaxel (DTX) as a model drug for enhanced treatment of lung cancer in comparison to non-targeted polymeric micelles and DTX injection (Docel™). A549 human lung cancer cells were employed as an in vitro model to access cytotoxicity study of the DTX loaded polymeric micelles. The safety of DTX formulations were studied by the measurement of alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and total protein levels in bronchoalveolar lavage (BAL) fluid of rats after the treatments. The IC50 values demonstrated that the non-targeted and transferrin receptor targeted polymeric micelles could be 7 and 70 folds more effective than Docel™ after 24 h treatment with the A549 cells. Results suggested that transferrin receptor targeted polymeric micelles have showed better efficacy and safety than the non-targeted polymeric micelles and Docel™.

  6. Transferrin receptor ligand-targeted toxin conjugate (Tf-CRM107) for therapy of malignant gliomas.

    PubMed

    Weaver, Michael; Laske, Douglas W

    2003-10-01

    The authors review the preclinical and clinical results of the ligand-targeted toxin conjugate Transferrin-CRM107 (Tf-CRM107), for the treatment of malignant gliomas. Tf-CRM107 is a conjugate protein of diphtheria toxin with a point mutation (CRM107) linked by a thioester bond to human transferrin (Tf). This conjugate exhibits potent cytotoxicity in vitro against mammalian cells expressing the transferrin receptor with activity at picomolar concentrations. Phase I clinical trial results demonstrated that Tf-CRM107, delivered via a high-flow convection method utilizing stereotactically placed catheters, produced tumor response in patients with malignant brain tumors refractory to conventional therapy without severe neurologic or systemic toxicity. The results of a Phase II study are also summarized. Tf-CRM107 treatment results in complete and partial tumor response without severe toxicity in 35% of the evaluable patients. These data warrant a Phase III study as well as continued research in the field of targeted toxin therapy. Future directions of research include optimizing Tf-CRM107 delivery to targeted brain regions, and improving the treatment efficacy by combining with other toxin conjugates targeted to different receptors.

  7. Characterization of the interaction between diferric transferrin and transferrin receptor 2 by functional assays and atomic force microscopy*

    PubMed Central

    Ikuta, Katsuya; Yersin, Alexandre; Ikai, Atsushi; Aisen, Philip; Kohgo, Yutaka

    2010-01-01

    Transferrin receptor (TfR2), a homologue of classical transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein, but the β form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2α we expressed the protein with FLAG-tagging in transferrin receptor-deficient Chinese hamster ovary cells. The association constant for binding of diferric transferrin (Tf) to TfR2α is 5.6 × 106 M−1, which is about 50 times lower than that of TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2α without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2α with Tf was further investigated using atomic force microscopy (AFM), a powerful tool for investigation of the interaction between ligand and receptor at the single molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2α and TfR1, with Tf-TfR1 unbinding characterized by 2 energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2α by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2α may help account for the different physiological roles of the two receptors. PMID:20096706

  8. Bacterial receptors for host transferrin and lactoferrin: molecular mechanisms and role in host-microbe interactions.

    PubMed

    Morgenthau, Ari; Pogoutse, Anastassia; Adamiak, Paul; Moraes, Trevor F; Schryvers, Anthony B

    2013-12-01

    Iron homeostasis in the mammalian host limits the availability of iron to invading pathogens and is thought to restrict iron availability for microbes inhabiting mucosal surfaces. The presence of surface receptors for the host iron-binding glycoproteins transferrin (Tf) and lactoferrin (Lf) in globally important Gram-negative bacterial pathogens of humans and food production animals suggests that Tf and Lf are important sources of iron in the upper respiratory or genitourinary tracts, where they exclusively reside. Lf receptors have the additional function of protecting against host cationic antimicrobial peptides, suggesting that the bacteria expressing these receptors reside in a niche where exposure is likely. In this review we compare Tf and Lf receptors with respect to their structural and functional features, their role in colonization and infection, and their distribution among pathogenic and commensal bacteria.

  9. Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice.

    PubMed

    Yan, Jingbin; Gong, Xiuli; Pan, Shubiao; Guo, Xinbing; Ren, Zhaorui; Zeng, Yitao

    2014-06-01

    Human transferrin (hTF) belongs to the iron-binding glycoprotein family. It plays an important role in iron transport throughout the body. Transgenic mice are a good model to study how to produce functional hTF on a large-scale. We have improved the expression of hTF and investigated its regulatory mechanism in transgenic mice. Three expression constructs were prepared in which hTF expression was controlled by different regulatory cassettes of rabbit transferrin (rTF). hTF was secreted into serum of transgenic mice when its expression was controlled by the rTF promoter and enhancer, whereas the rTF enhancer in tandem with the rTF promoter repressed hTF secretion into milk. A significant inverse relationship between methylation of the rTF promoter and hTF expression was observed in liver, heart, mammary gland, and muscle of transgenic mice. The highest concentration of hTF was 700 μg/ml in milk.

  10. Transport and expression in human melanomas of a transferrin-like glycosylphosphatidylinositol-anchored protein.

    PubMed

    Food, M R; Rothenberger, S; Gabathuler, R; Haidl, I D; Reid, G; Jefferies, W A

    1994-01-28

    Melanotransferrin, also called p97, is a cell surface glycoprotein which was first described as a marker antigen for human melanoma cells. Although p97 has a striking structural similarity to human serum transferrin and lactoferrin, its function has not yet been determined. One feature that distinguishes p97 from the other members of the transferrin family is the presence of a stretch of 24 hydrophobic amino acids at the C terminus, previously assumed to form a proteinacious transmembrane domain. In this study, sensitivity to bacterial phosphatidylinositol-specific phospholipase C, biosynthetic labeling with [3H]ethanolamine, and partitioning in Triton X-114 are used to establish that p97 is expressed at the cell surface as a glycosylphosphatidylinositol-anchored protein. In addition, to gain insight into the intracellular transport of p97, biosynthetic transport studies were performed on a melanoma cell line. These studies resulted in the identification of an additional form of p97 which is found in the medium and which likely does not originate from an alternatively spliced form of the p97 mRNA. These findings, together with our recent observation of the co-localization of p97 and the transferrin receptor in brain capillary endothelium (W. A. Jefferies, M. R. Food, R. Gabathuler, S. Rothenberger, T. Yamada, and P. L. McGeer, manuscript submitted) raise important questions about the function of the two forms of p97 detected and the possible involvement of this protein in a cellular iron uptake mechanism that is independent from the transferrin/transferrin receptor system.

  11. Evaluation of Efficacy of Radioimmunotherapy with 90Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models.

    PubMed

    Sugyo, Aya; Tsuji, Atsushi B; Sudo, Hitomi; Okada, Maki; Koizumi, Mitsuru; Satoh, Hirokazu; Kurosawa, Gene; Kurosawa, Yoshikazu; Saga, Tsuneo

    2015-01-01

    Pancreatic cancer is an aggressive tumor and the prognosis remains poor. Therefore, development of more effective therapy is needed. We previously reported that 89Zr-labeled TSP-A01, an antibody against transferrin receptor (TfR), is highly accumulated in a pancreatic cancer xenograft, but not in major normal organs. In the present study, we evaluated the efficacy of radioimmunotherapy (RIT) with 90Y-TSP-A01 in pancreatic cancer mouse models. TfR expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2) was evaluated by immunofluorescence staining. 111In-labeled anti-TfR antibodies (TSP-A01, TSP-A02) were evaluated in vitro by cell binding assay with the three cell lines and by competitive inhibition assay with MIAPaCa-2. In vivo biodistribution was evaluated in mice bearing BxPC-3 and MIAPaCa-2 xenografts. Tumor volumes of BxPC-3 and MIAPaCa-2 were sequentially measured after 90Y-TSP-A01 injection and histological analysis of tumors was conducted. MIAPaCa-2 cells showed the highest TfR expression, followed by AsPC-1 and BxPC-3 cells. 111In-TSP-A01 and 111In-TSP-A02 bound specifically to the three cell lines according to TfR expression. The dissociation constants for TSP-A01, DOTA-TSP-A01, TSP-A02, and DOTA-TSP-A02 were 0.22, 0.28, 0.17, and 0.22 nM, respectively. 111In-TSP-A01 was highly accumulated in tumors, especially in MIAPaCa-2, but this was not true of 111In-TSP-A02. The absorbed dose for 90Y-TSP-A01 was estimated to be 8.3 Gy/MBq to BxPC-3 and 12.4 Gy/MBq to MIAPaCa-2. MIAPaCa-2 tumors treated with 3.7 MBq of 90Y-TSP-A01 had almost completely disappeared around 3 weeks after injection and regrowth was not observed. Growth of BxPC-3 tumors was inhibited by 3.7 MBq of 90Y-TSP-A01, but the tumor size was not reduced. 90Y-TSP-A01 treatment achieved an almost complete response in MIAPaCa-2 tumors, whereas it merely inhibited the growth of BxPC-3 tumors. 90Y-TSP-A01 is a promising RIT agent for pancreatic cancer, although further investigation is

  12. Transferrin Receptor 1 Facilitates Poliovirus Permeation of Mouse Brain Capillary Endothelial Cells*

    PubMed Central

    Mizutani, Taketoshi; Ishizaka, Aya; Nihei, Coh-ichi

    2016-01-01

    As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS. PMID:26637351

  13. Variability of the transferrin receptor 2 gene in AMD.

    PubMed

    Wysokinski, Daniel; Blasiak, Janusz; Dorecka, Mariola; Kowalska, Marta; Robaszkiewicz, Jacek; Pawlowska, Elzbieta; Szaflik, Jerzy; Szaflik, Jacek Pawel

    2014-01-01

    Oxidative stress is a major factor in the pathogenesis of age-related macular degeneration (AMD). Iron may catalyze the Fenton reaction resulting in overproduction of reactive oxygen species. Transferrin receptor 2 plays a critical role in iron homeostasis and variability in its gene may influence oxidative stress and AMD occurrence. To verify this hypothesis we assessed the association between polymorphisms of the TFR2 gene and AMD. A total of 493 AMD patients and 171 matched controls were genotyped for the two polymorphisms of the TFR2 gene: c.1892C>T (rs2075674) and c.-258+123T>C (rs4434553). We also assessed the modulation of some AMD risk factors by these polymorphisms. The CC and TT genotypes of the c.1892C>T were associated with AMD occurrence but the latter only in obese patients. The other polymorphism was not associated with AMD occurrence, but the CC genotype was correlated with an increasing AMD frequency in subjects with BMI < 26. The TT genotype and the T allele of this polymorphism decreased AMD occurrence in subjects above 72 years, whereas the TC genotype and the C allele increased occurrence of AMD in this group. The c.1892C>T and c.-258+123T>C polymorphisms of the TRF2 gene may be associated with AMD occurrence, either directly or by modulation of risk factors.

  14. Serum transferrin receptor status of healthy adult Arabs.

    PubMed

    Knox-Macaulay, Huxley; Gravell, David; Elender, Frances

    2007-01-01

    Several studies have provided reference ranges for the concentration of serum transferrin receptor (sTfR) in various white populations, but there is a dearth of relevant reference sTfR data in non-whites. The aim of this investigation was to establish sTfR reference ranges and mean values for a healthy non-white Arab population that could be used also for Arabs worldwide. sTfR and serum ferritin concentrations were estimated by immunoassays and blood counts were determined by conventional methods. Analysis of the data of 114 volunteer Arab blood donors (91 male, 23 female) revealed a higher mean sTfR concentration in males of 22.6+/-8.1 nmol/L (range 10.9-38.7 nmol/L) compared to that in females of 18.7+/-4.4 nmol/L (range 10.7-25.8 nmol/L, p=0.001). There was no significant correlation of sTfR concentration with age, serum ferritin level, or blood haemoglobin level, but a strong inverse correlation was demonstrated with mean cell volume and mean cell haemoglobin of red cells. Iron-replete volunteer subjects with alpha-thalassaemia trait appear to have relatively high mean sTfR concentration. We recommend the use of gender-dependent sTfR reference values for Arabs.

  15. Downregulation of transferrin receptor surface expression by intracellular antibody

    SciTech Connect

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin . E-mail: guanxin_shen@yahoo.com.cn

    2007-03-23

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 {+-} 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.

  16. Variability of the Transferrin Receptor 2 Gene in AMD

    PubMed Central

    Blasiak, Janusz; Dorecka, Mariola; Kowalska, Marta; Pawlowska, Elzbieta; Szaflik, Jerzy; Szaflik, Jacek Pawel

    2014-01-01

    Oxidative stress is a major factor in the pathogenesis of age-related macular degeneration (AMD). Iron may catalyze the Fenton reaction resulting in overproduction of reactive oxygen species. Transferrin receptor 2 plays a critical role in iron homeostasis and variability in its gene may influence oxidative stress and AMD occurrence. To verify this hypothesis we assessed the association between polymorphisms of the TFR2 gene and AMD. A total of 493 AMD patients and 171 matched controls were genotyped for the two polymorphisms of the TFR2 gene: c.1892C>T (rs2075674) and c.−258+123T>C (rs4434553). We also assessed the modulation of some AMD risk factors by these polymorphisms. The CC and TT genotypes of the c.1892C>T were associated with AMD occurrence but the latter only in obese patients. The other polymorphism was not associated with AMD occurrence, but the CC genotype was correlated with an increasing AMD frequency in subjects with BMI < 26. The TT genotype and the T allele of this polymorphism decreased AMD occurrence in subjects above 72 years, whereas the TC genotype and the C allele increased occurrence of AMD in this group. The c.1892C>T and c.−258+123T>C polymorphisms of the TRF2 gene may be associated with AMD occurrence, either directly or by modulation of risk factors. PMID:24648608

  17. Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor.

    PubMed

    Lebrón, J A; Bennett, M J; Vaughn, D E; Chirino, A J; Snow, P M; Mintier, G A; Feder, J N; Bjorkman, P J

    1998-04-03

    HFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 A crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex.

  18. The structural basis of transferrin sequestration by transferrin-binding protein B

    SciTech Connect

    Calmettes, Charles; Alcantara, Joenel; Yu, Rong-Hua; Schryvers, Anthony B.; Moraes, Trevor F.

    2012-03-28

    Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin during infection through a surface transferrin receptor system composed of proteins TbpA and TbpB. Here we present the crystal structures of TbpB from N. meningitidis in its apo form and in complex with human transferrin. The structure reveals how TbpB sequesters and initiates iron release from human transferrin.

  19. Interaction of the Hereditary Hemochromatosis Protein, HFE, with Transferrin Receptor 2 Is Required for Transferrin-Induced Hepcidin Expression

    PubMed Central

    Gao, Junwei; Chen, Juxing; Kramer, Maxwell; Tsukamoto, Hidekazu; Zhang, An-Sheng; Enns, Caroline A.

    2009-01-01

    SUMMARY The mechanisms that allow the body to sense iron levels in order to maintain iron homeostasis are unknown. Patients with the most common form of hereditary iron overload have mutations in the hereditary hemochromatosis protein, HFE. They have lower levels of hepcidin, than unaffected individuals. Hepcidin, a hepatic peptide hormone, negatively regulates iron efflux from the intestines into the blood. We report two hepatic cell lines, WIF-B cells and HepG2 cells transfected with HFE, where hepcidin expression responded to iron-loaded transferrin. The response was abolished when endogenous transferrin receptor 2 (TfR2) was suppressed or in primary hepatocytes lacking either functional TfR2 or HFE. Furthermore, transferrin-treated HepG2 cells transfected with HFE chimeras containing only the α3 and cytoplasmic domains could upregulate hepcidin expression. Since the HFE α3 domain interacts with TfR2, these results supported our finding that TfR2/HFE complex is required for transcriptional regulation of hepcidin by holo-Tf. PMID:19254567

  20. The influence of transferrin stabilised magnetic nanoparticles on human dermal fibroblasts in culture.

    PubMed

    Berry, Catherine C; Charles, Stuart; Wells, Stephen; Dalby, Matthew J; Curtis, Adam S G

    2004-01-09

    Magnetic nanoparticles have been used for bio-medical purposes including drug delivery, cell destruction and as MRI contrast agents for several years. A more recent biological application has focused on targeted drug delivery. To this end, a wide variety of iron oxide nanoparticles have been synthesised. This study involves the use of magnetic nanoparticles synthesised and derivatised with human transferrin, compared to identical underivatised particles. Human fibroblasts were used, representative of a tissue cell-type. The influence in vitro was determined using light and fluorescence microscopy, scanning and transmission electron microscopy, and 1718 gene microarray. The results indicate that the transferrin derivatised particles appear to localise to the cell membrane without instigating receptor-mediated endocytosis, and also induce up-regulation in the cells for many genes, particularly in the area of cytoskeleton and cell signalling. The microscopy results further support these findings.

  1. Transferrin receptor regulates pancreatic cancer growth by modulating mitochondrial respiration and ROS generation

    SciTech Connect

    Jeong, Seung Min; Hwang, Sunsook; Seong, Rho Hyun

    2016-03-11

    The transferrin receptor (TfR1) is upregulated in malignant cells and its expression is associated with cancer progression. Because of its pre-eminent role in cell proliferation, TfR1 has been an important target for the development of cancer therapy. Although TfR1 is highly expressed in pancreatic cancers, what it carries out in these refractory cancers remains poorly understood. Here we report that TfR1 supports mitochondrial respiration and ROS production in human pancreatic ductal adenocarcinoma (PDAC) cells, which is required for their tumorigenic growth. Elevated TfR1 expression in PDAC cells contributes to oxidative phosphorylation, which allows for the generation of ROS. Importantly, mitochondrial-derived ROS are essential for PDAC growth. However, exogenous iron supplement cannot rescue the defects caused by TfR1 knockdown. Moreover, we found that TfR1 expression determines PDAC cells sensitivity to oxidative stress. Together, our findings reveal that TfR1 can contribute to the mitochondrial respiration and ROS production, which have essential roles in growth and survival of pancreatic cancer. - Highlights: • Pancreatic ductal adenocarcinoma (PDAC) exhibits an elevated transferrin receptor (TfR1) expression in comparison with non-transformed pancreatic cells. • TfR1 is required for PDAC growth by regulating mitochondrial respiration and ROS production. • TfR1 functions as a determinant of cell viability to oxidative stress in PDAC cells.

  2. Serum transferrin receptor concentration indicates increased erythropoiesis in Kenyan children with asymptomatic malaria.

    PubMed

    Verhoef, H; West, C E; Ndeto, P; Burema, J; Beguin, Y; Kok, F J

    2001-12-01

    Serum transferrin receptor concentrations indicate both erythropoietic activity and the deficit of functional iron in the erythron. In contrast with serum ferritin concentrations, serum transferrin receptor concentrations are not or are only marginally influenced by the inflammatory response to infection. We assessed iron status and examined the relation between serum transferrin receptor concentrations and malaria in children aged 2-36 mo who were asymptomatic for malaria. This was a community-based cluster survey (n = 318). Prevalences of malaria, anemia (hemoglobin concentration <110 g/L), iron deficiency (serum ferritin concentration <12 microg/L), and iron deficiency anemia were 18%, 69%, 53%, and 46%, respectively. Malaria was associated with lower mean hemoglobin concentrations (92.7 compared with 104.1 g/L; P = 0.0001) and higher geometric mean serum concentrations of transferrin receptor (11.4 compared with 7.8 mg/L; P = 0.005), ferritin (21.6 compared with 11.9 microg/L; P = 0.05), and C-reactive protein (12.5 compared with 6.8 mg/L; P = 0.004). There was no evidence for an association between serum concentrations of C-reactive protein and transferrin receptor. Children with malaria had higher serum transferrin receptor concentrations than expected for the degree of anemia, even after adjustment for inflammation indicated by serum C-reactive protein concentration quartiles (P = 0.02). Our findings are consistent with the notion that malaria-induced hemolysis is accompanied by increased erythropoiesis. Serum transferrin receptor concentration is not useful for detecting iron deficiency in individuals with malaria. Individuals with high concentrations of serum C-reactive protein or similar acute phase reactants should be excluded from analysis if serum ferritin concentrations <12 microg/L are to be used to measure iron deficiency in malaria-endemic areas.

  3. Structure, function and clinical significance of transferrin receptors.

    PubMed

    Feelders, R A; Kuiper-Kramer, E P; van Eijk, H G

    1999-01-01

    Iron plays an essential role in a spectrum of metabolic processes. Cellular iron uptake is facilitated by transferrin receptor (TfR)-mediated endocytosis. In recent years more insight has been obtained in TfR physiology and the regulation of cellular iron homeostasis. The synthesis of TfR and the iron storage protein ferritin is regulated reciprocally at the post-transcriptional level according to the cellular iron status. As a result of externalization of TfR during the endocytic cycle, a soluble form of TfR can be detected in serum. The serum TfR (sTfR) level is closely related to erythroid TfR turnover and the prime determinants of the sTfR concentration are cellular iron demands and erythroid proliferation rate. In the absence of a hyperplastic erythropoiesis the sTfR level is a sensitive parameter of early tissue iron deficiency. The entire spectrum of body iron status can be assessed by measurement of serum ferritin and sTfR levels, with ferritin as marker of tissue iron stores and sTfR as index of tissue iron needs. The sTfR may be a promising tool to detect iron deficiency in inflammatory states and in the anaemia of chronic disease as its concentration is, in contrast to ferritin levels, not influenced by the acute phase response. Determination of sTfR levels may also improve assessment of body iron stores during pregnancy and in neonates. Finally, the sTfR may be a useful parameter to monitor erythropoiesis in various clinical settings, for instance in the prediction of the haematological response to erythropoietin treatment. However, standardization of the sTfR assay, with definition of reference and pathological ranges, is necessary for the definitive introduction of the sTfR as major parameter of iron metabolism.

  4. Soluble transferrin receptor, ferritin and soluble transferrin receptor--Ferritin index in assessment of anaemia in rhaeumatoid arthritis.

    PubMed

    Pavai, S; Jayaranee, S; Sargunan, S

    2007-10-01

    Anaemia of chronic disease (ACD) is a frequent complication of rheumatoid arthritis (RA). A diagnostic difficulty in RA is the distinction between iron deficiency anaemia (IDA) and ACD. The aim of our study was to evaluate the usefulness of serum soluble transferrin receptor (sTfR) and sTfR/log ferritin (TfR-F) index to diagnose iron deficiency in RA patients with anaemia. Routine laboratory indices of anaemia and sTfR were measured in 20 healthy persons to form the control group, 30 patients with iron deficiency anaemia and 28 RA patients with anaemia. Serum sTfR levels were significantly elevated above the cut-off value in patients with IDA and those in the iron depleted RA subgroup (ferritin < 60 microg/L) compared with those in the control and iron repleted RA subgroup (ferritin > 60 microg/L). The same was observed for TfR-F index. However, five patients in the iron repleted RA sub group had an elevated sTfR level, of which two had increased TfR-F index. Serum sTfR correlated well with the markers of anaemia and not with ESR. Ferritin had no correlation with markers of anaemia but correlated well with ESR. Measurement of sTfR and TfR-F index are good indicators of iron deficiency in RA patients with anaemia. To be cost effective, sTfR can be estimated in RA patients with anaemia when the ferritin level is more than 60 microg/L.

  5. Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin.

    PubMed

    Boradia, Vishant Mahendra; Malhotra, Himanshu; Thakkar, Janak Shrikant; Tillu, Vikas Ajit; Vuppala, Bhavana; Patil, Pravinkumar; Sheokand, Navdeep; Sharma, Prerna; Chauhan, Anoop Singh; Raje, Manoj; Raje, Chaaya Iyengar

    2014-08-28

    Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages.

  6. DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin.

    PubMed

    Werner, P A; Galbraith, R M; Arnaud, P

    1983-10-01

    Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

  7. Determination of human transferrin concentrations in mouse models of neisserial infection.

    PubMed

    Perera, Yasser; Cobas, Karen; Garrido, Yainelis; Nazabal, Consuelo; Brown, Enma; Pajon, Rolando

    2006-04-20

    Transferrin constitutes the major protein involved in the transport of iron from the sites of absorption to the sites of storage and utilization. Despite the high affinity of transferrin for iron, most bacterial pathogens, such as the human restricted Neisseria meningitidis, have developed iron acquisition mechanisms. Several animal models of bacterial infection that include the exogenous supply of human transferrin have been implemented, and tests using transgenic mouse models are underway. Here we describe an ELISA sandwich procedure based on two monoclonal antibodies with negligible cross-reactivity to murine transferrin, to estimate human transferrin concentrations in mouse sera. The assay can detect as little as 10 ng/ml of human transferrin with coefficients of variation ranging from 1.6% to 4.4% (intra-assay) and 3.8% to 5% (inter-assay). The recovery values range from 90% to 110% in the assay working range (25-400 ng/ml). Human transferrin concentrations estimated in sera from 41 human transferrin transgenic mice ranged from 2 to 14 microg/ml. Further estimations of human transferrin levels in mouse sera of a previously described mouse model of N. meningitidis were also carried out. The intraperitoneal injection of 8 mg of human transferrin achieved a sustained value of human transferrin in mouse sera in the range of 1-2mg/ml over the first 24h, indicating that bacteria reaching the blood stream during this time would be exposed to levels of hTf found in normal human serum.

  8. Patterns of structural and sequence variation within isotype lineages of the Neisseria meningitidis transferrin receptor system

    PubMed Central

    Adamiak, Paul; Calmettes, Charles; Moraes, Trevor F; Schryvers, Anthony B

    2015-01-01

    Neisseria meningitidis inhabits the human upper respiratory tract and is an important cause of sepsis and meningitis. A surface receptor comprised of transferrin-binding proteins A and B (TbpA and TbpB), is responsible for acquiring iron from host transferrin. Sequence and immunological diversity divides TbpBs into two distinct lineages; isotype I and isotype II. Two representative isotype I and II strains, B16B6 and M982, differ in their dependence on TbpB for in vitro growth on exogenous transferrin. The crystal structure of TbpB and a structural model for TbpA from the representative isotype I N. meningitidis strain B16B6 were obtained. The structures were integrated with a comprehensive analysis of the sequence diversity of these proteins to probe for potential functional differences. A distinct isotype I TbpA was identified that co-varied with TbpB and lacked sequence in the region for the loop 3 α-helix that is proposed to be involved in iron removal from transferrin. The tightly associated isotype I TbpBs had a distinct anchor peptide region, a distinct, smaller linker region between the lobes and lacked the large loops in the isotype II C-lobe. Sequences of the intact TbpB, the TbpB N-lobe, the TbpB C-lobe, and TbpA were subjected to phylogenetic analyses. The phylogenetic clustering of TbpA and the TbpB C-lobe were similar with two main branches comprising the isotype 1 and isotype 2 TbpBs, possibly suggesting an association between TbpA and the TbpB C-lobe. The intact TbpB and TbpB N-lobe had 4 main branches, one consisting of the isotype 1 TbpBs. One isotype 2 TbpB cluster appeared to consist of isotype 1 N-lobe sequences and isotype 2 C-lobe sequences, indicating the swapping of N-lobes and C-lobes. Our findings should inform future studies on the interaction between TbpB and TbpA and the process of iron acquisition. PMID:25800619

  9. Serum transferrin receptor distinguishes the anemia of chronic disease from iron deficiency anemia.

    PubMed

    Ferguson, B J; Skikne, B S; Simpson, K M; Baynes, R D; Cook, J D

    1992-04-01

    Recent studies have shown that the serum transferrin receptor is a sensitive, quantitative measure of tissue iron deficiency. This study was undertaken to determine the serum transferrin receptor's ability to distinguish iron-deficiency anemia from the anemia of chronic inflammation and to identify iron deficiency in patients with liver disease. The mean transferrin receptor level in 17 normal controls was 5.36 +/- 0.82 mg/L compared with 13.91 +/- 4.63 mg/L in 17 patients with iron-deficiency anemia (p less than 0.001). The mean serum receptor level was normal in all 20 patients with acute infection, including five with acute hepatitis, and was also normal in 8 of 10 anemic patients with chronic liver disease. Receptor levels were in the normal range in all but 4 of 41 patients with anemia of chronic disease. We conclude that unlike serum ferritin levels, which are disproportionately elevated in relation to iron stores in patients with inflammation or liver disease, the serum transferrin receptor level is not affected by these disorders and is therefore a reliable laboratory index of iron deficiency anemia.

  10. An unusual case of iron deficiency anemia is associated with extremely low level of transferrin receptor.

    PubMed

    Hao, Shuangying; Li, Huihui; Sun, Xiaoyan; Li, Juan; Li, Kuanyu

    2015-01-01

    A case study of a female patient, diagnosed with iron deficiency anemia, was unresponsive to oral iron treatment and only partially responsive to parenteral iron therapy, a clinical profile resembling the iron-refractory iron deficiency anemia (IRIDA) disorder. However, the patient failed to exhibit microcytic phenotype, one of the IRIDA hallmarks. Biochemical assays revealed that serum iron, hepcidin, interluekin 6, and transferrin saturation were within the normal range of references or were comparable to her non-anemic offspring. Iron contents in serum and red blood cells and hemoglobin levels were measured, which confirmed the partial improvement of anemia after parenteral iron therapy. Strikingly, serum transferrin receptor in patient was almost undetectable, reflecting the very low activity of bone-marrow erythropoiesis. Our data demonstrate that this is not a case of systemic iron deficiency, but rather cellular iron deficit due to the low level of transferrin receptor, particularly in erythroid tissue.

  11. An unusual case of iron deficiency anemia is associated with extremely low level of transferrin receptor

    PubMed Central

    Hao, Shuangying; Li, Huihui; Sun, Xiaoyan; Li, Juan; Li, Kuanyu

    2015-01-01

    A case study of a female patient, diagnosed with iron deficiency anemia, was unresponsive to oral iron treatment and only partially responsive to parenteral iron therapy, a clinical profile resembling the iron-refractory iron deficiency anemia (IRIDA) disorder. However, the patient failed to exhibit microcytic phenotype, one of the IRIDA hallmarks. Biochemical assays revealed that serum iron, hepcidin, interluekin 6, and transferrin saturation were within the normal range of references or were comparable to her non-anemic offspring. Iron contents in serum and red blood cells and hemoglobin levels were measured, which confirmed the partial improvement of anemia after parenteral iron therapy. Strikingly, serum transferrin receptor in patient was almost undetectable, reflecting the very low activity of bone-marrow erythropoiesis. Our data demonstrate that this is not a case of systemic iron deficiency, but rather cellular iron deficit due to the low level of transferrin receptor, particularly in erythroid tissue. PMID:26339443

  12. Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor.

    PubMed

    Byrne, Shaina L; Leverence, Rachael; Klein, Joshua S; Giannetti, Anthony M; Smith, Valerie C; MacGillivray, Ross T A; Kaltashov, Igor A; Mason, Anne B

    2006-05-30

    Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.

  13. MicroRNA-152-mediated dysregulation of hepatic transferrin receptor 1 in liver carcinogenesis.

    PubMed

    Kindrat, Iryna; Tryndyak, Volodymyr; de Conti, Aline; Shpyleva, Svitlana; Mudalige, Thilak K; Kobets, Tetyana; Erstenyuk, Anna M; Beland, Frederick A; Pogribny, Igor P

    2016-01-12

    Over-expression of transferrin receptor 1 (TFRC) is observed in hepatocellular carcinoma (HCC); however, there is a lack of conclusive information regarding the mechanisms of this dysregulation. In the present study, we demonstrated a significant increase in the levels of TFRC mRNA and protein in preneoplastic livers from relevant experimental models of human hepatocarcinogenesis and in human HCC cells. Additionally, using the TCGA database, we demonstrated an over-expression of TFRC in human HCC tissue samples and a markedly decreased level of microRNA-152 (miR-152) when compared to non-tumor liver tissue. The results indicated that the increase in levels of TFRC in human HCC cells and human HCC tissue samples may be attributed, in part, to a post-transcriptional mechanism mediated by a down-regulation of miR-152. This was evidenced by a strong inverse correlation between the level of TFRC and the expression of miR-152 in human HCC cells (r = -0.99, p = 4. 7 × 10-9), and was confirmed by in vitro experiments showing that transfection of human HCC cell lines with miR-152 effectively suppressed TFRC expression. This suggests that miR-152-specific targeting of TFRC may provide a selective anticancer therapeutic approach for the treatment of HCC.

  14. The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information.

    PubMed Central

    Dargemont, C; Le Bivic, A; Rothenberger, S; Iacopetta, B; Kühn, L C

    1993-01-01

    Wild-type human transferrin receptor (hTfR), like endogenous canine receptor, is expressed almost exclusively (97%) at the basolateral membrane of transfected Madin-Darbey canine kidney (MDCK) cells. We investigated the role of two distinct features of the hTfR cytoplasmic domain, namely the endocytic signal and the unique phosphorylation site, in polarized cell surface delivery. Basolateral location was not altered by point mutation of Ser24-->Ala24, indicating that phosphorylation is not involved in vectorial sorting of hTfR. The steady state distribution of hTfR was partially affected by a deletion of 36 cytoplasmic residues encompassing the internalization sequence. However, 80% of the receptors were still basolateral. As assessed by pulse-chase experiments in combination with biotinylation, newly synthesized wild-type and deletion mutant receptors were directly sorted to the domain of their steady state residency. Although both receptors could bind human transferrin, endocytosis of the deletion mutant was strongly impaired at either surface. These data indicate that the predominant basolateral targeting signal of hTfR is independent of the internalization sequence. Images PMID:8467813

  15. Mutation analysis of the transferrin receptor-2 gene in patients with iron overload.

    PubMed

    Lee, P L; Halloran, C; West, C; Beutler, E

    2001-01-01

    Three mutations in the transferrin receptor-2 gene have recently been identified in four Sicilian families with iron overload who had a normal hemochromatosis gene, HFE (C. Camaschella, personal communication). To determine the extent to which mutations in the transferrin receptor-2 gene occur in other populations with iron overload, we have completely sequenced this gene in 17 whites, 10 Asians, and 8 African Americans with iron overload and a C282C/C282C HFE genotype, as well as 4 subjects without iron overload and homozygous for the mutant HFE C282Y genotype, 5 patients with iron overload and homozygous for the mutant HFE C282Y genotype, and 5 normal individuals. None of the individuals exhibited the Sicilian mutations, Y250X in exon 6, M172K in exon 4, and E60X in exon 2. One iron-overloaded individual of Asian descent exhibited a I238M mutation which was subsequently found to be a polymorphism present in the Asian population at a frequency of 0.0192. The presence of the I238M mutation was not associated with an increase in ferritin or transferrin saturation levels. Three silent polymorphisms were also identified, nt 1770 (D590D) and nt 1851 (A617A) and a polymorphism at nt 2255 in the 3' UTR. Thus, mutations in the transferrin receptor-2 gene were not responsible for the iron overload seen in our subjects.

  16. Transferrin-conjugated boron nitride nanotubes: protein grafting, characterization, and interaction with human endothelial cells.

    PubMed

    Ciofani, Gianni; Del Turco, Serena; Genchi, Giada Graziana; D'Alessandro, Delfo; Basta, Giuseppina; Mattoli, Virgilio

    2012-10-15

    In this paper we report on a covalent grafting of boron nitride nanotubes with human transferrin. After silanization of the nanotube wall, transferrin was linked to the nanotubes through carbamide binding. The obtained transferrin-conjugated boron nitride nanotubes (tf-BNNTs) resulted stable in aqueous environments and were characterized in terms of scanning electron microscopy, transmission electron microscopy, size distribution analysis and Z-potential measurement. Effective covalent grafting of transferrin was demonstrated by Fourier transform infrared spectroscopy and UV-Vis spectrophotometry. The obtained tf-BNNTs were thereafter tested on human umbilical vein endothelial cells (HUVECs); in particular cellular up-take was investigated by confocal, scanning and transmission electron microscopy, demonstrating the key role of transferrin during the internalization process. Here reported for the first time in the literature, the covalent BNNT functionalization with a targeting ligand represents a fundamental step towards BNNT exploitation as smart and selective nanocarriers in a number of nanomedicine applications.

  17. A Novel Rat Model of Hereditary Hemochromatosis Due to a Mutation in Transferrin Receptor 2

    PubMed Central

    Bartnikas, Thomas B; Wildt, Sheryl J; Wineinger, Amy E; Schmitz-Abe, Klaus; Markianos, Kyriacos; Cooper, Dale M; Fleming, Mark D

    2013-01-01

    Sporadic iron overload in rats has been reported, but whether it is due to genetic or environmental causes is unknown. In the current study, phenotypic analysis of Hsd:HHCL Wistar rats revealed a low incidence of histologically detected liver iron overload. Here we characterized the pathophysiology of the iron overload and showed that the phenotype is heritable and due to a mutation in a single gene. We identified a single male rat among the 132 screened animals that exhibited predominantly periportal, hepatocellular iron accumulation. This rat expressed low RNA levels of the iron regulatory hormone hepcidin and low protein levels of transferrin receptor 2 (Tfr2), a membrane protein essential for hepcidin expression in humans and mice and mutated in forms of hereditary hemochromatosis. Sequencing of Tfr2 in the iron-overloaded rat revealed a novel Ala679Gly polymorphism in a highly conserved residue. Quantitative trait locus mapping indicated that this polymorphism correlated strongly with serum iron and transferrin saturations in male rats. Expression of the Gly679 variant in tissue culture cell lines revealed decreased steady-state levels of Tfr2. Characterization of iron metabolism in the progeny of polymorphic rats suggested that homozygosity for the Ala679Gly allele leads to a hemochromatosis phenotype. However, we currently cannot exclude the possibility that a polymorphism or mutation in the noncoding region of Tfr2 contributes to the iron-overload phenotype. Hsd:HHCL rats are the first genetic rat model of hereditary hemochromatosis and may prove useful for understanding the molecular mechanisms underlying the regulation of iron metabolism. PMID:23582421

  18. Lethal iron deprivation induced by non-neutralizing antibodies targeting transferrin receptor 1 in malignant B cells.

    PubMed

    Rodríguez, José A; Luria-Pérez, Rosendo; López-Valdés, Héctor E; Casero, David; Daniels, Tracy R; Patel, Shabnum; Avila, David; Leuchter, Richard; So, Sokuntheavy; Ortiz-Sánchez, Elizabeth; Bonavida, Benjamin; Martínez-Maza, Otoniel; Charles, Andrew C; Pellegrini, Matteo; Helguera, Gustavo; Penichet, Manuel L

    2011-11-01

    A number of antibodies have been developed that induce lethal iron deprivation (LID) by targeting the transferrin receptor 1 (TfR1/CD71) and either neutralizing transferrin (Tf) binding, blocking internalization of the receptor and/or inducing its degradation. We have developed recombinant antibodies targeting human TfR1 (ch128.1 and ch128.1Av), which induce receptor degradation and are cytotoxic to certain malignant B-cells. We now show that internalization of TfR1 bound to these antibodies can lead to its sequestration and degradation, as well as reduced Tf uptake, and the induction of a transcriptional response consistent with iron deprivation, which is mediated in part by downstream targets of p53. Cells resistant to these antibodies do not sequester and degrade TfR1 after internalization of the antibody/receptor complex, and accordingly maintain their ability to internalize Tf. These findings are expected to facilitate the rational design and clinical use of therapeutic agents targeting iron import via TfR1 in hematopoietic malignancies.

  19. Lethal iron deprivation induced by non-neutralizing antibodies targeting transferrin receptor 1 in malignant B cells

    PubMed Central

    Rodríguez, JoséA.; Luria-Pérez, Rosendo; López-Valdés, Héctor E.; Casero, David; Daniels, Tracy R.; Patel, Shabnum; Avila, David; Leuchter, Richard; So, Sokuntheavy; ánchez, Elizabeth Ortiz-S; Bonavida, Benjamin; Martínez-Maza, Otoniel; Charles, Andrew .C; Pellegrini, Matteo; Helguera, Gustavo; Penichet, Manuel L.

    2013-01-01

    A number of antibodies have been developed that induce lethal iron deprivation (LID) by targeting the transferrin receptor 1 (TfR1/CD71) and either neutralizing transferrin (Tf) binding, blocking internalization of the receptor and/or inducing its degradation. We have developed recombinant antibodies targeting human TfR1 (ch128.1 and ch128.1Av), which induce receptor degradation and are cytotoxic to certain malignant B-cells. We now show that internalization of TfR1 bound to these antibodies can lead to its sequestration and degradation, as well as reduced Tf uptake, and the induction of a transcriptional response consistent with iron deprivation, which is mediated in part by downstream targets of p53. Cells resistant to these antibodies do not sequester and degrade TfR1 after internalization of the antibody/receptor complex, and accordingly maintain their ability to internalize Tf. These findings are expected to facilitate the rational design and clinical use of therapeutic agents targeting iron import via TfR1 in hematopoietic malignancies. PMID:21870996

  20. Transferrin receptor-1 gene polymorphisms are associated with type 2 diabetes.

    PubMed

    Fernández-Real, José Manuel; Mercader, Josep Maria; Ortega, Francisco José; Moreno-Navarrete, Jose Maria; López-Romero, Pedro; Ricart, Wifredo

    2010-07-01

    Iron is involved in oxidative stress and type 2 diabetes (T2D). Transferrin receptor (TFRC) constitutes the major receptor by which most cells take up iron. The aim of this study was to evaluate whether TFRC gene polymorphisms are associated with T2D. We evaluated TFRC gene polymorphism (rs3817672, 210AG, S142G) in a sample of T2D patients and nondiabetic controls (n = 722), and 39 SNPs within the TFRC genomic region analysed by the Welcome Trust Case Control Consortium (WTCCC) (1921 T2D subjects and 3000 controls). In a subset of subjects, glucose tolerance and insulin sensitivity were also studied. The frequency of the G allele at the position 210 of the TFRC gene was significantly higher in T2D patients. Both GG and GA genotypes had a 69% (P < 0.01) greater risk of developing T2D estimated under a dominant model. The increased prevalence of the G allele run in parallel to increased sex-adjusted log-serum ferritin and slightly increased soluble transferrin receptor among patients with T2D. Furthermore, post-load glucose and insulin sensitivity were significantly associated with circulating soluble transferrin receptor, and insulin sensitivity was significantly associated with serum ferritin among G allele carriers, (r = -0.33, P = 0.001) but not in AA homozygotes. Sixteen other TFRC SNPs were also associated to T2D according to the Welcome Trust Case Control Consortium data. TFRC gene variants are associated with T2D.

  1. HIF-1-mediated activation of transferrin receptor gene transcription by iron chelation.

    PubMed

    Bianchi, L; Tacchini, L; Cairo, G

    1999-11-01

    Treatment with iron chelators mimics hypoxic induction of the hypoxia inducible factor (HIF-1) which activates transcription by binding to hypoxia responsive elements (HRE). We investigated whether HIF-1 is involved in transcriptional activation of the transferrin receptor (TfR), a membrane protein which mediates cellular iron uptake, in response to iron deprivation. The transcription rate of the TfR gene in isolated nuclei was up-regulated by treatment of Hep3B human hepatoma cells with the iron chelator desferrioxamine (DFO). The role of HIF-1 in the activation of TfR was indicated by the following observations: (i) DFO-dependent activation of a luciferase reporter gene in transfected Hep3B cells was mediated by a fragment of the human TfR promoter containing a putative HRE sequence; (ii) mutation of this sequence prevented stimulation of luciferase activity; (iii) binding to this sequence of HIF-1alpha, identified by competition experiments and supershift assays, was induced by DFO. Furthermore, in mouse hepatoma cells unable to assemble functional HIF-1, inducibility of TfR transcription by DFO was lost and TfR mRNA up-regulation was reduced. These results, which show the role of HIF-1 in the control of TfR gene expression in conditions of iron depletion, give insights into the mechanisms of transcriptional regulation which concur with the well-characterized post-transcriptional control of TfR expression to expand the extent of response to iron deficiency.

  2. Snx3 regulates recycling of the transferrin receptor and iron assimilation.

    PubMed

    Chen, Caiyong; Garcia-Santos, Daniel; Ishikawa, Yuichi; Seguin, Alexandra; Li, Liangtao; Fegan, Katherine H; Hildick-Smith, Gordon J; Shah, Dhvanit I; Cooney, Jeffrey D; Chen, Wen; King, Matthew J; Yien, Yvette Y; Schultz, Iman J; Anderson, Heidi; Dalton, Arthur J; Freedman, Matthew L; Kingsley, Paul D; Palis, James; Hattangadi, Shilpa M; Lodish, Harvey F; Ward, Diane M; Kaplan, Jerry; Maeda, Takahiro; Ponka, Prem; Paw, Barry H

    2013-03-05

    Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc) and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.

  3. Snx3 regulates recycling of the transferrin receptor and iron assimilation

    PubMed Central

    Chen, Caiyong; Garcia-Santos, Daniel; Ishikawa, Yuichi; Seguin, Alexandra; Li, Liangtao; Fegan, Katherine H.; Hildick-Smith, Gordon J.; Shah, Dhvanit I.; Cooney, Jeffrey D.; Chen, Wen; King, Matthew J.; Yien, Yvette Y.; Schultz, Iman J.; Anderson, Heidi; Dalton, Arthur J.; Freedman, Matthew L.; Kingsley, Paul D.; Palis, James; Hattangadi, Shilpa M.; Lodish, Harvey F.; Ward, Diane M.; Kaplan, Jerry; Maeda, Takahiro; Ponka, Prem; Paw, Barry H.

    2013-01-01

    SUMMARY Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc), and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism. PMID:23416069

  4. Plant-derived recombinant human serum transferrin demonstrates multiple functions.

    PubMed

    Brandsma, Martin E; Diao, Hong; Wang, Xiaofeng; Kohalmi, Susanne E; Jevnikar, Anthony M; Ma, Shengwu

    2010-05-01

    Human serum transferrin (hTf) is the major iron-binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these applications is critically dependent on the availability of high-quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 microg/g fresh leaf weight). Furthermore, plant-derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum-free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of particular interest is the use of plant rhTf as a novel carrier for cell-specific or oral delivery of protein/peptide drugs for the treatment of human diseases such as diabetes.To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon-like peptide 1 (GLP-1) or its derivative in plants. Here, we show that plant-derived hTf-GLP-1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro.

  5. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes

    PubMed Central

    1985-01-01

    Using ferritin-labeled protein A and colloidal gold-labeled anti-rabbit IgG, the fate of the sheep transferrin receptor has been followed microscopically during reticulocyte maturation in vitro. After a few minutes of incubation at 37 degrees C, the receptor is found on the cell surface or in simple vesicles of 100-200 nm, in which the receptor appears to line the limiting membrane of the vesicles. With time (60 min or longer), large multivesicular elements (MVEs) appear whose diameter may reach 1-1.5 micron. Inside these large MVEs are round bodies of approximately 50-nm diam that bear the receptor at their external surfaces. The limiting membrane of the large MVEs is relatively free from receptor. When the large MVEs fuse with the plasma membrane, their contents, the 50-nm bodies, are released into the medium. The 50-nm bodies appear to arise by budding from the limiting membrane of the intracellular vesicles. Removal of surface receptor with pronase does not prevent exocytosis of internalized receptor. It is proposed that the exocytosis of the approximately 50-nm bodies represents the mechanism by which the transferrin receptor is shed during reticulocyte maturation. PMID:2993317

  6. Ovine uterine space restriction alters placental transferrin receptor and fetal iron status during late pregnancy

    PubMed Central

    Sun, Mary Y.; Habeck, Jason M.; Meyer, Katie M.; Koch, Jill M.; Ramadoss, Jayanth; Blohowiak, Sharon E.; Magness, Ronald R.; Kling, Pamela J.

    2013-01-01

    Background Fetal growth restriction is reported to be associated with impaired placental iron transport. Transferrin receptor (TfR) is a major placental iron transporter in humans, but is unstudied in sheep. TfR is regulated by both iron and nitric oxide (NO), the molecule produced by endothelial NOS (eNOS). We hypothesized that limited placental development downregulates both placental TfR and eNOS expression, thereby lowering fetal tissue iron. Methods An ovine surgical uterine space restriction (USR) model, combined with multifetal gestation, tested the extremes of uterine and placental adaptation. Blood, tissues, and placentomes from non-space restricted (NSR) singletons were compared to USR fetuses at 120 or 130 days of gestation (GD). Results When expressed proportionate to fetal weight, liver iron content did not differ while renal iron was higher in USR vs. NSR fetuses. Renal TfR protein expression did not differ, but placental TfR expression was lower in USR fetuses at GD130. Placental levels of TfR correlated to eNOS. TfR was localized throughout the placentome, including the hemophagous zone, implicating a role for TfR in ovine placental iron transport. Conclusion In conclusion, fetal iron was regulated in an organ-specific fashion. In USR fetuses, NO-mediated placental adaptations may prevent the normal upregulation of placental TfR at GD130. PMID:23202722

  7. Receptor recognition of transferrin bound to lanthanides and actinides: a discriminating step in cellular acquisition of f-block metals

    PubMed Central

    Deblonde, Gauthier J.-P.; Sturzbecher-Hoehne, Manuel; Mason, Anne B.; Abergel, Rebecca J.

    2013-01-01

    Following an internal contamination event, the transport of actinide and lanthanide metal ions through the body is facilitated by endogenous ligands such as the human iron-transport protein transferrin (Tf). The recognition of resulting metallo-transferrin complexes (M2Tf) by the cognate transferrin receptor (TfR) is therefore a critical step for cellular uptake of these metal ions. A high performance liquid chromatography-based method has been used to probe the binding of M2Tf with TfR, yielding a direct measurement of the successive thermodynamic constants that correspond to the dissociation of TfR(M2Tf)2 and TfR(M2Tf) complexes for Fe3+, Ga3+, La3+, Nd3+, Gd3+, Yb3+, Lu3+, 232Th4+, 238UO22+, and 242Pu4+. Important features of this method are (i) its ability to distinguish both 1:1 and 1:2 complexes formed between the receptor and the metal-bound transferrin, and (ii) the requirement for very small amounts of each binding partner (<1 nmol of protein per assay). Consistent with previous reports, the strongest receptor affinity is found for Fe2Tf (Kd1 = 5 nM and Kd2 = 20 nM), while the lowest affinity was measured for Pu2Tf (Kd1 = 0.28 µM and Kd2 = 1.8 µM) binding to the TfR. Other toxic metal ions such as ThIV and UVI, when bound to Tf, are well recognized by the TfR. Under the described experimental conditions, the relative stabilities of TfR:(MxTf)y adducts follow the order Fe3+ >> Th4+ □ UO22+ □ Cm3+ > Ln3+ □ Ga3+ >>> Yb3+ □ Pu4+. This study substantiates a role for Tf in binding lanthanide fission products and actinides, and transporting them into cells by receptor mediated endocytosis. PMID:23446908

  8. Receptor recognition of transferrin bound to lanthanides and actinides: a discriminating step in cellular acquisition of f-block metals.

    PubMed

    Deblonde, Gauthier J-P; Sturzbecher-Hoehne, Manuel; Mason, Anne B; Abergel, Rebecca J

    2013-06-01

    Following an internal contamination event, the transport of actinide (An) and lanthanide (Ln) metal ions through the body is facilitated by endogenous ligands such as the human iron-transport protein transferrin (Tf). The recognition of resulting metallo-transferrin complexes (M2Tf) by the cognate transferrin receptor (TfR) is therefore a critical step for cellular uptake of these metal ions. A high performance liquid chromatography-based method has been used to probe the binding of M2Tf with TfR, yielding a direct measurement of the successive thermodynamic constants that correspond to the dissociation of TfR(M2Tf)2 and TfR(M2Tf) complexes for Fe(3+), Ga(3+), La(3+), Nd(3+), Gd(3+), Yb(3+), Lu(3+), (232)Th(4+), (238)UO2(2+), and (242)Pu(4+). Important features of this method are (i) its ability to distinguish both 1 : 1 and 1 : 2 complexes formed between the receptor and the metal-bound transferrin, and (ii) the requirement for very small amounts of each binding partner (<1 nmol of protein per assay). Consistent with previous reports, the strongest receptor affinity is found for Fe2Tf (Kd1 = 5 nM and Kd2 = 20 nM), while the lowest affinity was measured for Pu2Tf (Kd1 = 0.28 μM and Kd2 = 1.8 μM) binding to the TfR. Other toxic metal ions such as Th(IV) and U(VI), when bound to Tf, are well recognized by the TfR. Under the described experimental conditions, the relative stabilities of TfR:(MxTf)y adducts follow the order Fe(3+) > Th(4+) ~ UO2(2+) ~ Cm(3+) > Ln(3+) ~ Ga(3+) > Yb(3+) ~ Pu(4+). This study substantiates a role for Tf in binding lanthanide fission products and actinides, and transporting them into cells by receptor-mediated endocytosis.

  9. A missense mutation in TFRC, encoding transferrin receptor 1, causes combined immunodeficiency.

    PubMed

    Jabara, Haifa H; Boyden, Steven E; Chou, Janet; Ramesh, Narayanaswamy; Massaad, Michel J; Benson, Halli; Bainter, Wayne; Fraulino, David; Rahimov, Fedik; Sieff, Colin; Liu, Zhi-Jian; Alshemmari, Salem H; Al-Ramadi, Basel K; Al-Dhekri, Hasan; Arnaout, Rand; Abu-Shukair, Mohammad; Vatsayan, Anant; Silver, Eli; Ahuja, Sanjay; Davies, E Graham; Sola-Visner, Martha; Ohsumi, Toshiro K; Andrews, Nancy C; Notarangelo, Luigi D; Fleming, Mark D; Al-Herz, Waleed; Kunkel, Louis M; Geha, Raif S

    2016-01-01

    Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.

  10. Serum transferrin receptor levels in the evaluation of iron deficiency in the neonate.

    PubMed

    Rusia, U; Flowers, C; Madan, N; Agarwal, N; Sood, S K; Sikka, M

    1996-10-01

    Iron deficiency anemia (IDA) is a major global problem. Early onset of iron deficiency in developing countries makes it imperative to identify iron deficiency in neonates. Most conventional laboratory parameters of iron status fail to distinguish neonates with iron deficient erythropoiesis. Serum transferrin receptor (STFR) levels are a recent sensitive measure of iron deficiency and the present study was carried out to evaluate the usefulness of cord serum transferrin receptors in identifying iron deficient erythropoiesis in neonates. A complete hemogram, red cell indices, iron profile: serum iron (SI), percent transferrin saturation (TS%) and serum ferritin (SF) was carried out in 100 full-term neonates and their mothers at parturition. Cord and maternal STFR levels were estimated using a sensitive enzyme-linked immunosorbent assay (ELISA) technique. Anemic women had a significantly lower SI, their TS% and high STFR levels suggesting that iron deficiency was responsible for the anemia. In the neonates of iron deficient mothers, cord SI, TS% and cord ferritin were not significantly different from those of neonates born to non-anemic mothers. Cord STFR level correlated well with hemoglobin (Hb) and laboratory parameters of iron status, and its level was significantly higher in neonates born to anemic mothers than in those born to non-anemic mothers. It was the only laboratory parameter to differentiate between neonates born to anemic and non-anemic mothers. Therefore, STFR is a sensitive index of iron status in neonates and identifies neonates with iron deficient erythropoiesis.

  11. Separation of Albumin, Ceruloplasmin, and Transferrin from Human Plasma.

    ERIC Educational Resources Information Center

    Barnes, Grady; Frieden, Earl

    1982-01-01

    Procedures are provided for separating the principal metalloproteins (albumin, ceruloplasmin, and transferrin) from plasma using column chromatographic techniques. The experiment can be completed in two separate three-hour laboratory periods during which column chromatography is illustrated and the effect of pH on charge and affinity of a protein…

  12. Separation of Albumin, Ceruloplasmin, and Transferrin from Human Plasma.

    ERIC Educational Resources Information Center

    Barnes, Grady; Frieden, Earl

    1982-01-01

    Procedures are provided for separating the principal metalloproteins (albumin, ceruloplasmin, and transferrin) from plasma using column chromatographic techniques. The experiment can be completed in two separate three-hour laboratory periods during which column chromatography is illustrated and the effect of pH on charge and affinity of a protein…

  13. Influence of endurance exercise (triathlon) on circulating transferrin receptors and other indicators of iron status in female athletes.

    PubMed

    Röcker, Lothar; Hinz, Katrin; Holland, Karsten; Gunga, Hanns-Christian; Vogelgesang, Jens; Kiesewetter, Holger

    2002-01-01

    Numerous reports have described a poor iron status in female endurance athletes. However, the traditionally applied indicators of iron status (hemoglobin, ferritin, transferrin) may not truly reflect the iron status. Therefore we studied the newly developed soluble transferrin receptor and other indicators of iron status in twelve female endurance athletes before and after a triathlon race. Resting values showed a poor iron status in the participants of the race. Serum TfR concentration increased slightly after the race. However, if the values are corrected for hemoconcentration no change could be found. Hemoglobin, serum ferritin and transferrin values were increased after the race.

  14. Analysis of the Immunological Responses to Transferrin and Lactoferrin Receptor Proteins from Moraxella catarrhalis

    PubMed Central

    Yu, Rong-hua; Bonnah, Robert A.; Ainsworth, Samuel; Schryvers, Anthony B.

    1999-01-01

    Moraxella catarrhalis expresses surface receptor proteins that specifically bind host transferrin (Tf) and lactoferrin (Lf) in the first step of the iron acquisition pathway. Acute- and convalescent-phase antisera from a series of patients with M. catarrhalis pulmonary infections were tested against Tf and Lf receptor proteins purified from the corresponding isolates. After the purified proteins had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, we observed strong reactivity against Tf-binding protein B (TbpB; also called OMP1) and Lf-binding protein B (LbpB) but little or no reactivity against Tf-binding protein A (TbpA) or Lf-binding protein A (LbpA), using the convalescent-phase antisera. Considerable antigenic heterogeneity was observed when TbpBs and LbpBs isolated from different strains were tested with the convalescent-phase antisera. Comparison to the reactivity against electroblotted total cellular proteins revealed that the immune response against LbpB and TbpB constitutes a significant portion of the total detectable immune response to M. catarrhalis proteins. Preparations of affinity-isolated TbpA and LbpA reacted with convalescent-phase antisera in a solid-phase binding assay, but blocking with soluble TbpB, soluble LbpB, or extracts from an LbpA− mutant demonstrated that this reactivity was attributed to contaminants in the TbpA and LbpA preparations. These studies demonstrate the immunogenicity of M. catarrhalis TbpB and LbpB in humans and support their potential as vaccine candidates. PMID:10417140

  15. Common pathway for tumor cell uptake of gallium-67 and iron-59 via a transferrin receptor

    SciTech Connect

    Larson, S.M.; Rasey, J.S.; Allen, D.R.; Nelson, N.J.; Harp, G.D.; Williams, D.L.

    1980-01-01

    We studied the tumor uptake of (/sup 67/Ga)citrate, (/sup 59/Fe)citrate, and /sup 125/I-labeled transferrin (TF) by the in vitro growth form of EMT-6, a sarcoma-like mammary tumor of BALB/c mice. In analyzing the binindg experiments, we developed a new mathematical model based on a formulation originally used to express the interaction of hormones with specific tissue receptors. The uptake of both carrier-free /sup 67/Ga and /sup 59/Fe by tumor cells was mediated by kinetically identical TF receptors. We also studied teric acid extracts of the stroma of EMT-6 tumors grown both in vivo and in vitro. Chromatography of these extracts on Sephacryl S-200 SF demonstrated that the cellular stroma contained specific TF-binding macromolecules. On the basis of these findings, we proposed the transferrin receptor hypothesis for the mechanism of /sup 67/Ga uptake by tumors. According to this view, a tumor-assisted TF receptor is the functional unit responsible for the affinity of gallum for certain neoplasms. This receptor was also active in the uptake of iron by tumors.

  16. Sequence and structural diversity of transferrin receptors in Gram-negative porcine pathogens.

    PubMed

    Curran, David M; Adamiak, Paul J; Fegan, Jamie E; Qian, Chenzhe; Yu, Rong-Hua; Schryvers, Anthony B

    2015-10-13

    Actinobacillus pleuropneumoniae, Actinobacillus suis, and Haemophilus parasuis are bacterial pathogens from the upper respiratory tract that are responsible for a substantial burden of porcine disease. Although reduction of disease has been accomplished by intensive management practices, immunization remains an important strategy for disease prevention, particularly when intensive management practices are not feasible or suitable. An attractive target for vaccine development is the surface receptor involved in acquiring iron from host transferrin, since it is common to all three pathogenic species and has been shown to be essential for survival and disease causation. It has also recently been demonstrated that an engineered antigen derived from the lipoprotein component of the receptor, transferrin-binding protein B (TbpB), was more effective at preventing infection by H. parasuis than a commercial vaccine product. This study was initiated to explore the genetic and immunogenic diversity of the transferrin receptor system from these species. Nucleic acid sequences were obtained from a geographically and temporally diverse collection of isolates, consisting of 41 A. pleuropneumoniae strains, 30 H. parasuis strains, and 2 A. suis strains. Phylogenetic analyses demonstrated that the receptor protein sequences cluster independently of species, suggesting that there is genetic exchange between these species such that receptor-based vaccines should logically target all three species. To evaluate the cross-reactive response of TbpB-derived antigens, pigs were immunized with the intact TbpB, the TbpB N-lobe and the TbpB C-lobe from A. pleuropneumoniae strain H49 and the resulting sera were tested against a representative panel of TbpBs; demonstrating that the C-lobe induces a broadly cross-reactive response. Overall our results indicate that there is a common reservoir for transferrin receptor antigenic variation amongst these pathogens. While this could present a

  17. Involvement of multiple distinct Bordetella receptor proteins in the utilization of iron liberated from transferrin by host catecholamine stress hormones

    PubMed Central

    Armstrong, Sandra K.; Brickman, Timothy J.; Suhadolc, Ryan J.

    2012-01-01

    Summary Bordetella bronchiseptica is a pathogen that can acquire iron using its native alcaligin siderophore system, but can also use the catechol xenosiderophore enterobactin via the BfeA outer membrane receptor. Transcription of bfeA is positively controlled by a regulator that requires induction by enterobactin. Catecholamine hormones also induce bfeA transcription and B. bronchiseptica can use the catecholamine norepinephrine for growth on transferrin. In this study, B. bronchiseptica was shown to use catecholamines to obtain iron from both transferrin and lactoferrin in the absence of siderophore. In the presence of siderophore, norepinephrine augmented transferrin utilization by B. bronchiseptica, as well as siderophore function in vitro. Genetic analysis identified BfrA, BfrD and BfrE as TonB dependent outer membrane catecholamine receptors. The BfeA enterobactin receptor was found to not be involved directly in catecholamine utilization; however, the BfrA, BfrD and BfrE catecholamine receptors could serve as receptors for enterobactin and its degradation product 2,3-dihydroxybenzoic acid. Thus, there is a functional link between enterobactin-dependent and catecholamine-dependent transferrin utilization. This investigation characterizes a new B. bronchiseptica mechanism for iron uptake from transferrin that uses host stress hormones that not only deliver iron directly to catecholamine receptors, but also potentiate siderophore activity by acting as iron shuttles. PMID:22458330

  18. Binding of aluminum to human serum transferrin, human serum albumin and rat serum proteins

    SciTech Connect

    El-Sebae, A.K.H.; Zeid, M.M.A.; Abdel-Rahman, F.H.; Saleh, M.A. . Environmental Chemistry and Toxicology Lab.)

    1994-01-01

    Human serum transferrin (HSTF), human serum albumin (HSA) and rat serum were compared for their interaction with AlCl[sub 3], in a Tris-HCl buffer solutions. The AlCl[sub 3] was tested in series of concentrations in the range of 50 [mu]M up to 500 [mu]M. HSTF, HSA and their 1:1 mixture and rat serum were incubated at 37 C with series of AlCl[sub 3] concentrations. The protein profile of the incubated solutions were compared to control using SDS-PAGE and FPLC tests. The results indicated that HSTF was more specifically responsive to AlCl[sub 3] showing a characteristic increase in it UV absorption, peak and area dimensions. Simultaneously, HSA was less affected, but it showed a significant shift with an increase in molecular weight accompanied with a change in its profile. The respective bands of transferrin and albumin in rat serum behaved similarly.

  19. Lower Plasma Soluble Transferrin Receptor Range in Healthy Indian Pediatric Cohort as Compared to Asian and Western Data.

    PubMed

    Bhatia, P; Siyaram, D; Deepshikha; Marathe, R; Dayal, D

    2017-09-01

    Soluble serum transferrin receptor is derived from erythroid transferrin receptor expressed on surface of developing erythroid cells. It can be detected in blood using sensitive ELISA methodology and blood levels reflect physiological iron dependent erythropoiesis state in bone marrow. Normal adult levels vary from 2 to 5 mg/l. However, pediatric studies are few and describe normal ranges to the tune of 1.0-3.0 mg/l, which are relatively lower than that of adults. In present study 40 healthy children (2-12 years) were evaluated to establish normal soluble transferrin receptor range. The mean transferrin receptor levels were 0.39 mg/l with a range of 0.17-2.1 mg/l. The levels were low as compared to mean levels described in other studies from West and our country (4.39 and 2.0 mg/l respectively). Since, no internationally standard method for reporting and testing for transferrin receptor levels are yet available, hence it is imperative to establish normal control ranges in different population cohorts, especially in pediatric age group, to better interpret their levels in diagnostic context.

  20. Determination of genetic transferrin variants in human serum by high-resolution capillary zone electrophoresis(†).

    PubMed

    Caslavska, Jitka; Joneli, Jeannine; Wanzenried, Ursula; Schiess, Jeannette; Lanz, Christian; Thormann, Wolfgang

    2014-07-01

    High-resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate-deficient transferrin in human serum. The assay comprises mixing of human serum with a Fe(III) -containing solution prior to analysis of the iron-saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, one BD and three CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high-resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate-deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.

  1. Transferrin receptors and the targeted delivery of therapeutic agents against cancer

    PubMed Central

    Daniels, Tracy R.; Bernabeu, Ezequiel; Rodríguez, José A.; Patel, Shabnum; Kozman, Maggie; Chiappetta, Diego A.; Holler, Eggehard; Ljubimova, Julia Y.; Helguera, Gustavo; Penichet, Manuel L.

    2012-01-01

    Background Traditional cancer therapy can be successful in destroying tumors, but can also cause dangerous side effects. Therefore, many targeted therapies are in development. The transferrin receptor (TfR) functions in cellular iron uptake through its interaction with transferrin. This receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and is efficiently internalized. This receptor can be targeted in two ways: 1) for the delivery of therapeutic molecules into malignant cells or 2) to block the natural function of the receptor leading directly to cancer cell death. Scope of review In the present article we discuss the strategies used to target the TfR for the delivery of therapeutic agents into cancer cells. We provide a summary of the vast types of anti-cancer drugs that have been delivered into cancer cells employing a variety of receptor binding molecules including Tf, anti-TfR antibodies, or TfR-binding peptides alone or in combination with carrier molecules including nanoparticles and viruses. Major conclusions Targeting the TfR has been shown to be effective in delivering many different therapeutic agents and causing cytotoxic effects in cancer cells in vitro and in vivo. General significance The extensive use of TfR for targeted therapy attests to the versatility of targeting this receptor for therapeutic purposes against malignant cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR for cancer therapy leading to an increase in the number of clinical trials of molecules targeting this receptor. PMID:21851850

  2. Hepatocyte Nuclear Factor 4α Controls Iron Metabolism and Regulates Transferrin Receptor 2 in Mouse Liver*

    PubMed Central

    Matsuo, Shunsuke; Ogawa, Masayuki; Muckenthaler, Martina U.; Mizui, Yumiko; Sasaki, Shota; Fujimura, Takafumi; Takizawa, Masayuki; Ariga, Nagayuki; Ozaki, Hiroaki; Sakaguchi, Masakiyo; Gonzalez, Frank J.; Inoue, Yusuke

    2015-01-01

    Iron is an essential element in biological systems, but excess iron promotes the formation of reactive oxygen species, resulting in cellular toxicity. Several iron-related genes are highly expressed in the liver, a tissue in which hepatocyte nuclear factor 4α (HNF4α) plays a critical role in controlling gene expression. Therefore, the role of hepatic HNF4α in iron homeostasis was examined using liver-specific HNF4α-null mice (Hnf4aΔH mice). Hnf4aΔH mice exhibit hypoferremia and a significant change in hepatic gene expression. Notably, the expression of transferrin receptor 2 (Tfr2) mRNA was markedly decreased in Hnf4aΔH mice. Promoter analysis of the Tfr2 gene showed that the basal promoter was located at a GC-rich region upstream of the transcription start site, a region that can be transactivated in an HNF4α-independent manner. HNF4α-dependent expression of Tfr2 was mediated by a proximal promoter containing two HNF4α-binding sites located between the transcription start site and the translation start site. Both the GC-rich region of the basal promoter and the HNF4α-binding sites were required for maximal transactivation. Moreover, siRNA knockdown of HNF4α suppressed TFR2 expression in human HCC cells. These results suggest that Tfr2 is a novel target gene for HNF4α, and hepatic HNF4α plays a critical role in iron homeostasis. PMID:26527688

  3. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding

    PubMed Central

    Feder, John N.; Penny, David M.; Irrinki, Alivelu; Lee, Vince K.; Lebrón, José A.; Watson, Nicole; Tsuchihashi, Zenta; Sigal, Elliott; Bjorkman, Pamela J.; Schatzman, Randall C.

    1998-01-01

    We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called HFE. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 → Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with β2-microglobulin (β2m) and prevents cell-surface expression. A second mutation that has no effect on β2m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37°C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/β2m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4°C, the added soluble complex of HFE/β2m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis. PMID:9465039

  4. Biochemical and Structural Characterization of Recombinant Human Serum Transferrin from Rice (Oryza sativa L.)

    PubMed Central

    Steere, Ashley N.; Bobst, Cedric E.; Zhang, Deshui; Pettit, Steve; Kaltashov, Igor A.; Huang, Ning; Mason, Anne B.

    2012-01-01

    The Fe3+ binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin™ (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin™) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin™ is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin™ is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin™ bind Fe3+ tightly yet reversibly. Although previously shown to be capable of delivering Fe3+ to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin™ might be suitable for consideration in biopharmaceutical applications. PMID:23010327

  5. Interaction of vanadium(IV) with human serum apo-transferrin.

    PubMed

    Mehtab, Sameena; Gonçalves, Gisela; Roy, Somnath; Tomaz, Ana Isabel; Santos-Silva, Teresa; Santos, Marino F A; Romão, Maria J; Jakusch, Tamás; Kiss, Tamás; Pessoa, João Costa

    2013-04-01

    The interaction of V(IV)O-salts as well as of a few V(IV)O(carrier)n complexes with human serum transferrin (hTF) is studied focusing on the determination of the nature and stoichiometry of the binding of V(IV)O(2+) to hTF, as well as whether the conformation of hTF upon binding to V(IV)O(2+) or to its complexes is changed. Circular dichroism (CD) spectra measured for solutions containing V(IV)O(2+) and apo-hTF, and V(IV)O-maltol and apo-hTF, clearly indicate that hTF-V(IV)O-maltol ternary species form with a V(IV)O:maltol stoichiometry of 1:1. For V(IV)O salts and several V(IV)O(carrier)n complexes (carrier ligand=maltolato, dhp, picolinato and dipicolinato) (Hdhp=1,2-dimethyl-3-hydroxy-4-pyridinone) the maximum number of V(IV)O(2+) bound per mole of hTF is determined to be ~2 or lower in all cases. The binding of V(IV)O to apo-hTF most certainly involves several amino acid residues of the Fe-binding site, and as concluded by urea gel electrophoresis experiments, the formation of (V(IV)O)2hTF species may occur with the closing of the hTF conformation as is the case in (Fe(III))2hTF, which is an essential feature for the transferrin receptor recognition. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Biochemical and structural characterization of recombinant human serum transferrin from rice (Oryza sativa L.).

    PubMed

    Steere, Ashley N; Bobst, Cedric E; Zhang, Deshui; Pettit, Steve C; Kaltashov, Igor A; Huang, Ning; Mason, Anne B

    2012-11-01

    The Fe(3+) binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin bind Fe(3+) tightly yet reversibly. Although previously shown to be capable of delivering Fe(3+) to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin might be suitable for consideration in biopharmaceutical applications. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Haemophilus influenzae can use human transferrin as a sole source for required iron.

    PubMed Central

    Herrington, D A; Sparling, P F

    1985-01-01

    Haemophilus influenzae grown on enriched medium containing protoporphyrin IX rather than hemin was iron starved by the addition of the chelator ethylenediamine di-o-hydroxyphenylacetic acid. Iron starvation could be overcome in each of 33 H. influenzae type b isolates by 30% Fe-saturated human transferrin but not by human lactoferrin. Among nontypeable H. influenzae, 28 of 35 isolates, including 2 of 3 systemic isolates, were able to utilize Fe-transferrin. None of 18 H. parainfluenzae isolates was able to use Fe-transferrin. Iron starvation of H. influenzae type b resulted in increased amounts of three membrane proteins of 94,000 to 98,000 daltons. Images PMID:3872264

  8. Iron and bismuth bound human serum transferrin reveals a partially-opened conformation in the N-lobe.

    PubMed

    Yang, Nan; Zhang, Hongmin; Wang, Minji; Hao, Quan; Sun, Hongzhe

    2012-01-01

    Human serum transferrin (hTF) binds Fe(III) tightly but reversibly, and delivers it to cells via a receptor-mediated endocytosis process. The metal-binding and release result in significant conformational changes of the protein. Here, we report the crystal structures of diferric-hTF (Fe(N)Fe(C)-hTF) and bismuth-bound hTF (Bi(N)Fe(C)-hTF) at 2.8 and 2.4 Å resolutions respectively. Notably, the N-lobes of both structures exhibit unique "partially-opened" conformations between those of the apo-hTF and holo-hTF. Fe(III) and Bi(III) in the N-lobe coordinate to, besides anions, only two (Tyr95 and Tyr188) and one (Tyr188) tyrosine residues, respectively, in contrast to four residues in the holo-hTF. The C-lobe of both structures are fully closed with iron coordinating to four residues and a carbonate. The structures of hTF observed here represent key conformers captured in the dynamic nature of the transferrin family proteins and provide a structural basis for understanding the mechanism of metal uptake and release in transferrin families.

  9. Iron and bismuth bound human serum transferrin reveals a partially-opened conformation in the N-lobe

    PubMed Central

    Yang, Nan; Zhang, Hongmin; Wang, Minji; Hao, Quan; Sun, Hongzhe

    2012-01-01

    Human serum transferrin (hTF) binds Fe(III) tightly but reversibly, and delivers it to cells via a receptor-mediated endocytosis process. The metal-binding and release result in significant conformational changes of the protein. Here, we report the crystal structures of diferric-hTF (FeNFeC-hTF) and bismuth-bound hTF (BiNFeC-hTF) at 2.8 and 2.4 Å resolutions respectively. Notably, the N-lobes of both structures exhibit unique “partially-opened” conformations between those of the apo-hTF and holo-hTF. Fe(III) and Bi(III) in the N-lobe coordinate to, besides anions, only two (Tyr95 and Tyr188) and one (Tyr188) tyrosine residues, respectively, in contrast to four residues in the holo-hTF. The C-lobe of both structures are fully closed with iron coordinating to four residues and a carbonate. The structures of hTF observed here represent key conformers captured in the dynamic nature of the transferrin family proteins and provide a structural basis for understanding the mechanism of metal uptake and release in transferrin families. PMID:23256035

  10. Structure and Dynamics of Drug Carriers and Their Interaction with Cellular Receptors: Focus on Serum Transferrin#

    PubMed Central

    Luck, Ashley N.; Mason, Anne B.

    2012-01-01

    Highly proliferative cells have a dramatically increased need for iron which results in the expression of an increased number of transferrin receptors (TFR). This insight makes the transferrin receptor on these cells an excellent candidate for targeted therapeutics. In this regard, it is critical to understand at a molecular level exactly how the TFR interacts with its ligand, hTF. Understanding of the hTF/TFR pathway could, in theory, maximize the use of this system for development of more effective small molecules or toxin-conjugates to specifically target cancer cells. Many strategies have been attempted with the objective of utilizing the hTF/TFR system to deliver drugs; these include conjugation of a toxin or drug to hTF or direct targeting of the TFR by antibodies. To date, in spite of all of the effort, there is a conspicuous absence of any successful candidate drugs reaching the clinic. We suggest that a lack of quantitative data to determine the basic biochemical properties of the drug carrier and the effects of drug-conjugation on the hTF-TFR interaction may have contributed to the failure to realize the full potential of this system. This review provides some guidelines for developing a more quantitative approach for evaluation of current and future hTF-drug conjugates. PMID:23183585

  11. Structure and dynamics of drug carriers and their interaction with cellular receptors: focus on serum transferrin.

    PubMed

    Luck, Ashley N; Mason, Anne B

    2013-07-01

    Highly proliferative cells have a dramatically increased need for iron which results in the expression of an increased number of transferrin receptors (TFR). This insight makes the transferrin receptor on these cells an excellent candidate for targeted therapeutics. In this regard, it is critical to understand at a molecular level exactly how the TFR interacts with its ligand, hTF. Understanding of the hTF/TFR pathway could, in theory, maximize the use of this system for development of more effective small molecules or toxin-conjugates to specifically target cancer cells. Many strategies have been attempted with the objective of utilizing the hTF/TFR system to deliver drugs; these include conjugation of a toxin or drug to hTF or direct targeting of the TFR by antibodies. To date, in spite of all of the effort, there is a conspicuous absence of any successful candidate drugs reaching the clinic. We suggest that a lack of quantitative data to determine the basic biochemical properties of the drug carrier and the effects of drug-conjugation on the hTF-TFR interaction may have contributed to the failure to realize the full potential of this system. This review provides some guidelines for developing a more quantitative approach for evaluation of current and future hTF-drug conjugates. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Transferrin receptor 2 and HFE regulate furin expression via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) signaling. Implications for transferrin-dependent hepcidin regulation

    PubMed Central

    Poli, Maura; Luscieti, Sara; Gandini, Valentina; Maccarinelli, Federica; Finazzi, Dario; Silvestri, Laura; Roetto, Antonella; Arosio, Paolo

    2010-01-01

    Background Impaired regulation of hepcidin in response to iron is the cause of genetic hemochromatosis associated with defects of HFE and transferrin receptor 2. However, the role of these proteins in the regulation of hepcidin expression is unclear. Design and Methods Hepcidin expression, SMAD and extracellular signal-regulated kinase (Erk) phosphorylation and furin expression were analyzed in hepatic HepG2 cells in which HFE and transferrin receptor 2 were down-regulated or expressed, or furin activity specifically inhibited. Furin expression was also analyzed in the liver of transferrin receptor 2 null mice. Results We showed that the silencing of HFE and transferrin receptor 2 reduced both Erk phosphorylation and furin expression, that the exogenous expression of the two enhanced the induction of phosphoErk1/2 and furin by holotransferrin, but that this did not occur when the pathogenic HFE mutant C282Y was expressed. Furin, phosphoErk1/2 and phosphoSMAD1/5/8 were down-regulated also in transferrin receptor 2-null mice. Treatment of HepG2 cells with an inhibitor of furin activity caused a strong suppression of hepcidin mRNA, probably due to the inhibition of bone morphogenic protein maturation. Conclusions The data indicate that transferrin receptor 2 and HFE are involved in holotransferrin-dependent signaling for the regulation of furin which involved Erk phosphorylation. Furin in turn may control hepcidin expression. PMID:20634490

  13. A simple method for obtaining transferrins from human plasma and porcine serum: preparations and properties.

    PubMed

    Wu, Lin; Wu, Jinhui; Zhang, Jian; Zhou, Yuanyuan; Ren, Guoyan; Hu, Yiqiao

    2008-05-01

    A simple method was described for the purification of serum transferrin (Tf) from human plasma and porcine serum with relative high yield and purity. The properties including purity, integrity, immunoreactivity and the receptor-binding ability of the proteins were studied by several assays, comprising spectrometry, SDS-PAGE, HPLC, Western blotting, urea electrophoresis, mass spectrometry and cytometry. Analysis from all the different aspects manifested that the proteins were of high purity. The two kinds of Tfs appeared to be iron-saturated as confirmed by their absorbance spectra and urea-PAGE mobility. The specific spectra of absorption of the two Tfs were both at around 465 nm. The relative molecular weights of human Tf (hTf) and porcine Tf (pTf) were determined by SDS-PAGE and further identified by MAIDI-TOF mass spectrometry with a result of 79,707 and 79,258, respectively. Immunoblotting assay showed that pTf could react with the anti-human Tf monoclonal antibody with a less level compared to hTf. FACS assays of their binding activities to Tf receptor-positive cell (K562 cell line) indicated that pTf could be recognized by the hTf receptor and internalized into cells, with a slightly less efficacy than hTf. All special property studies demonstrated that pTf was similar to hTf in physical and chemical characteristics, which gave a hint that pTf could substitute for hTf in some kinds of researches, such as using hTf as a carrier in drug targeting system.

  14. Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.

    PubMed

    Sorio, Daniela; De Palo, Elio Franco; Bertaso, Anna; Bortolotti, Federica; Tagliaro, Franco

    2017-02-01

    This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

  15. Effects of exercise on soluble transferrin receptor and other variables of the iron status

    PubMed Central

    Schumacher, Y; Schmid, A; Konig, D; Berg, A

    2002-01-01

    Background: Soluble transferrin receptor (sTfr) is a new marker of iron status and erythropoietic activity. It has been included in multivariable blood testing models for the detection of performance enhancing erythropoietin misuse in sport. Objective: To evaluate the effect of different types and volumes of physical activity on sTfr concentration, variables of iron status (ferritin, transferrin, iron, and protein), and haematological indices. Methods: Thirty nine subjects were divided into three groups: 1, untrained (n = 12); 2, moderately trained (n = 14); 3, highly trained (n = 13, seven men, six women). Groups 1 and 2 carried out two exercise tests: an incremental running test until exhaustion (test A) and a 45 minute constant speed running test at 70% VO2MAX (test B). Group 3 performed three days (women) or four days (men) of prolonged aerobic cycling exercise. The above variables together with haemoglobin and packed cell volume were analysed in venous blood samples before and after exercise. Changes in blood and plasma volume were estimated. Results: sTfr levels were slightly increased in trained and untrained subjects immediately after test A. Test B and aerobic exercise had no significant effect on sTfr. Ferritin levels were increased after the laboratory tests for trained and untrained subjects and after prolonged aerobic exercise in male cyclists. Transferrin was increased significantly in trained and untrained subjects after both laboratory tests, but remained unchanged after prolonged exercise. Plasma and blood volumes were decreased after the laboratory tests but increased after aerobic exercise. No differences in the variables were observed between trained and untrained subjects with respect to response to exercise. Conclusion: The changes in sTfr and the variables of iron status can be mainly attributed to exercise induced changes in volume. Taking these limitations into account, sTfr can be recommended as a marker of iron deficiency in athletes

  16. Structural change of N-glycan exposes hydrophobic surface of human transferrin.

    PubMed

    Nagae, Masamichi; Morita-Matsumoto, Kana; Arai, Seisuke; Wada, Ikuo; Matsumoto, Yuka; Saito, Kiyoshi; Hashimoto, Yasuhiro; Yamaguchi, Yoshiki

    2014-08-01

    Transferrin is an iron-transport protein which possesses N-glycans at Asn432 and Asn630 in humans. Transferrin glycoforms Tf-1 and Tf-2, previously identified in human cerebrospinal fluid, are defined as the lower and upper bands in gel electrophoresis, respectively. Importantly, the Tf-2/Tf-1 ratio is raised in idiopathic normal pressure hydrocephalus patients and is useful as a clinical marker. In order to gain insight into the relationship between transferrin glycoform and biological function, we performed comparative characterization of Tf-1, Tf-2 and serum transferrin (sTf). Mass spectrometric analyses confirmed that Tf-2 is modified with disialylated biantennary glycans at both of the two N-glycosylation sites, which are similar to the N-glycans of sTf. On the other hand, Tf-1 is site-specifically modified: Asn630 has biantennary agalacto-complex-type glycan with bisecting N-acetylglucosamine (GlcNAc) and core fucose while Asn432 is modified with complex/high mannose-type glycans and possibly single GlcNAc. Size exclusion chromatography and fluorescence correlation spectroscopy analysis revealed that the hydration volume of Tf-1 is slightly smaller than that of sTf. Our striking finding is that Tf-1 has an exposed hydrophobic surface as monitored by the fluorescence intensity and wavelength of a hydrophobic probe, 1-anilino-8-naphthalene sulfonate, whereas Tf-2 does not. These results suggest that the different N-glycan structure of Tf-1 lowers the apparent hydration volume and reveals a patch of hydrophobic surface on transferrin which is otherwise covered with sialoglycan in sTf and Tf-2. The carbohydrate deficiency in certain pathological conditions may also expose hydrophobic surface which may modulate the function and/or stability of transferrin. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Transferrin as a drug carrier: Cytotoxicity, cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells.

    PubMed

    Szwed, Marzena; Matusiak, Agnieszka; Laroche-Clary, Audrey; Robert, Jacques; Marszalek, Ilona; Jozwiak, Zofia

    2014-03-01

    Leukemias are one of most common malignancies worldwide. There is a substantial need for new chemotherapeutic drugs effective against this cancer. Doxorubicin (DOX), used for treatment of leukemias and solid tumors, is poorly efficacious when it is administered systemically at conventional doses. Therefore, several strategies have been developed to reduce the side effects of this anthracycline treatment. In this study we compared the effect of DOX and doxorubicin-transferrin conjugate (DOX-TRF) on human leukemia cell lines: chronic erythromyeloblastoid leukemia (K562), sensitive and resistant (K562/DOX) to doxorubicin, and acute lymphoblastic leukemia (CCRF-CEM). Experiments were also carried out on normal cells, peripheral blood mononuclear cells (PBMC). We analyzed the chemical structure of DOX-TRF conjugate by using mass spectroscopy. The in vitro growth-inhibition assay XTT, indicated that DOX-TRF is more cytotoxic for leukemia cells sensitive and resistant to doxorubicin and significantly less sensitive to normal cells compared to DOX alone. During the assessment of intracellular DOX-TRF accumulation it was confirmed that the tested malignant cells were able to retain the examined conjugate for longer periods of time than normal lymphocytes. Comparison of kinetic parameters showed that the rate of DOX-TRF efflux was also slower in the tested cells than free DOX. The results presented here should contribute to the understanding of the differences in antitumor activities of the DOX-TRF conjugate and free drug.

  18. Effects of human serum and apo-Transferrin on Staphylococcus epidermidis RP62A biofilm formation.

    PubMed

    She, Pengfei; Chen, Lihua; Qi, Yong; Xu, Huan; Liu, Yuan; Wang, Yangxia; Luo, Zhen; Wu, Yong

    2016-12-01

    Biofilm-associated Staphylococcus epidermidis infections present clinically important features due to their high levels of resistance to traditional antibiotics. As a part of human innate immune system, serum shows different degrees of protection against systemic S. epidermidis infection. We investigated the ability of human serum as well as serum component to inhibit the formation of, and eradication of mature S. epidermidis biofilms. In addition, the synergistic effect of vancomycin combined with apo-Transferrin was checked. Human serum exhibited significant antibiofilm activities against S. epidermidis at the concentration without affecting planktonic cell growth. However, there was no effect of human serum on established biofilms. By component separation, we observed that antibiofilm effect of serum components mainly due to the proteins could be damaged by heat inactivation (e.g., complement) or heat-stable proteins ≥100 kDa. In addition, serum apo-Transferrin showed modest antibiofilm effect, but without influence on S. epidermidis initial adhesion. And there was a synergistic antibiofilm interaction between vancomycin and apo-Transferrin against S. epidermidis. Our results indicate that serum or its components (heat-inactivated components or heat-stable proteins ≥100 kDa) could inhibits S. epidermidis biofilm formation. Besides, apo-Transferrin could partially reduce the biofilm formation at the concentration that does not inhibit planktonic cell growth. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  19. Serum soluble transferrin receptor concentration is an accurate estimate of the mass of tissue receptors.

    PubMed

    R'zik, S; Beguin, Y

    2001-06-01

    Serum levels of the soluble transferrin receptor (sTfR) vary depending on the erythropoietic activity and iron status. In vitro, sTfR shed in the incubation medium correlates well with cellular TfR, but this relationship has never been established in vivo. To determine the value of serum sTfR as a quantitative marker of the body mass of tissue TfR, we designed experiments to examine the correlation between serum sTfR and tissue TfR in rats with various degrees of erythropoietic activity or iron status. We studied changes in erythropoietic activity in normal rats as well as in animals experiencing hemolysis, phlebotomy-induced iron deficiency, transfusion- or thiamphenicol-induced erythroid aplasia, or inflammation. At the end of follow-up, ferrokinetic studies were performed and animals were sacrificed. Organs were isolated and homogenized to determine the total mass of tissue TfR from the sum of tissue solubilized TfR in the bone marrow, spleen, liver, and blood cells (direct method). An indirect method was developed to derive the corporeal mass of tissue TfR from a representative marrow sample. As expected, serum sTfR and total mass of tissue TfR varied as a function of iron status and erythropoiesis. Relative erythroid expansion in the spleen was greater than in the bone marrow. With the exception of phlebotomized animals, the indirect method correlated very well with direct measurements of the total mass of tissue TfR (r = 0.97, p < 0.0001). There was a close relationship between the total mass of tissue TfR and the total mass of serum sTfR (r = 0.79, p < 0.0001). Serum sTfR represented approximately 5-6% of the total mass of tissue TfR in most experimental situations, but this ratio was twice as high during iron-restricted erythropoiesis. In addition, the ratio could be higher or lower in nonsteady-state situations, because changes in tissue TfR occurred faster than those of serum sTfR. Serum sTfR represents a constant proportion of the total mass of tissue Tf

  20. Structure of the Membrane Proximal Oxioreductase Domain of Human Steap3, the Dominant Ferrireductase of the Erythroid Transferrin Cycle

    SciTech Connect

    Sendamarai, A.K.; Ohgami, R.S.; Fleming, M.D.; Lawrence, C.M.

    2009-05-27

    The daily production of 200 billion erythrocytes requires 20 mg of iron, accounting for nearly 80% of the iron demand in humans. Thus, erythroid precursor cells possess an efficient mechanism for iron uptake in which iron loaded transferrin (Tf) binds to the transferrin receptor (TfR) at the cell surface. The Tf:TfR complex then enters the endosome via receptor-mediated endocytosis. Upon endosomal acidification, iron is released from Tf, reduced to Fe{sup 2+} by Steap3, and transported across the endosomal membrane by divalent metal iron transporter 1. Steap3, the major ferrireductase in erythrocyte endosomes, is a member of a unique family of reductases. Steap3 is comprised of an N-terminal cytosolic oxidoreductase domain and a C-terminal heme-containing transmembrane domain. Cytosolic NADPH and a flavin are predicted cofactors, but the NADPH/flavin binding domain differs significantly from those in other eukaryotic reductases. Instead, Steap3 shows remarkable, although limited homology to FNO, an archaeal oxidoreductase. We have determined the crystal structure of the human Steap3 oxidoreductase domain in the absence and presence of NADPH. The structure reveals an FNO-like domain with an unexpected dimer interface and substrate binding sites that are well positioned to direct electron transfer from the cytosol to a heme moiety predicted to be fixed within the transmembrane domain. Here, we discuss possible gating mechanisms for electron transfer across the endosomal membrane.

  1. FRET reveals the organization of different receptor-ligand complexes (polymeric IgA-R and Transferrin-R) in endocytic membranes of polarized MDCK cells

    NASA Astrophysics Data System (ADS)

    Wallrabe, Horst K.; Barroso, Margarida

    2004-06-01

    FRET-based assay has been used to determine the organization of transferrin-receptor bound to holo-transferrin in basolateral endocytic membranes and compare it to the previously characterized clustered organization of polymeric IgA-receptor (pIgA-R) bound to pIgA-R ligand in apical endocytic membranes. In polarized MDCK-PTR cells, we have internalized holo-transferrin from the basolateral plasma membrane - labeled with donor and acceptor fluorophores. Transferrin-receptor-holo-transferrin complexes were imaged in the basolateral endocytic compartment using FRET confocal laser scanning microscopy in fixed and live MDCK polarized cells. A two-parameter FRET assay demonstrates whether complexes are randomly distributed or clustered: Acceptor's positive impact on E% signifies random distribution; E% being independent of acceptor fluorescence levels indicates clusters. A second parameter for clustering is E% being negatively dependent on D:A ratios. Our results indicating a clustered organization of transferrin-receptor-holo transferrin complexes fit the well-known homodimeric structure of transferrin-receptor.

  2. Transferrin Receptor 2 Dependent Alterations of Brain Iron Metabolism Affect Anxiety Circuits in the Mouse

    PubMed Central

    Pellegrino, Rosa Maria; Boda, Enrica; Montarolo, Francesca; Boero, Martina; Mezzanotte, Mariarosa; Saglio, Giuseppe; Buffo, Annalisa; Roetto, Antonella

    2016-01-01

    The Transferrin Receptor 2 (Tfr2) modulates systemic iron metabolism through the regulation of iron regulator Hepcidin (Hepc) and Tfr2 inactivation causes systemic iron overload. Based on data demonstrating Tfr2 expression in brain, we analysed Tfr2-KO mice in order to examine the molecular, histological and behavioural consequences of Tfr2 silencing in this tissue. Tfr2 abrogation caused an accumulation of iron in specific districts in the nervous tissue that was not accompanied by a brain Hepc response. Moreover, Tfr2-KO mice presented a selective overactivation of neurons in the limbic circuit and the emergence of an anxious-like behaviour. Furthermore, microglial cells showed a particular sensitivity to iron perturbation. We conclude that Tfr2 is a key regulator of brain iron homeostasis and propose a role for Tfr2 alpha in the regulation of anxiety circuits. PMID:27477597

  3. Transferrin receptor bearing cells in the peripheral blood of patients with rheumatoid arthritis.

    PubMed Central

    Salmon, M; Bacon, P A; Symmons, D P; Blann, A D

    1985-01-01

    Activated, proliferating lymphocytes are a feature of rheumatoid arthritis. They are present both in the synovial membrane and in the peripheral circulation. The expression of transferrin receptors(TFR) is a good marker of cellular proliferation. This study shows increased levels of circulating TFR-bearing lymphocytes in patients with rheumatoid arthritis (RA). The TFR+ population contains a disproportionately large number of T4+ cells, leading to a high T4:T8 ratio (5:1 in the TFR+ population, compared to 2:1 in the total circulating pool of lymphocytes). This reflects the pattern found in the rheumatoid synovium and suggests that lymphocyte activation in RA may be an extra-articular phenomenon. The TFR+ population also contains a range of non-T cells, including B cells, and a population bearing phenotypic similarities to natural killer (NK) cells. PMID:3002686

  4. Seasonal changes in haematology, lymphocyte transferrin receptors and intracellular iron in Ironman triathletes and untrained men.

    PubMed

    Broadbent, Suzanne

    2011-01-01

    We investigated whether 12 months of chronic endurance training would affect haematology, CD4(+) lymphocyte transferrin receptor (CD71) expression, CD4(+) intracellular iron and the incidence of upper respiratory tract illnesses (URTI) in Ironman triathletes compared with untrained men. Resting venous blood samples were taken from 15 Ironman triathletes (TR 30 ± 5 year) and 12 untrained men (UT 30 ± 6 year) every 4 weeks for 12 months. Erythrocyte, leukocyte and platelet concentration, haematocrit, haemoglobin (Hb) and mean corpuscular haemoglobin (MCHC) were measured with a full blood count. CD4(+) lymphocytes were analysed for changes in transferrin receptor (CD71) expression (CD4(+)CD71(+)), and intracellular iron (Fe(3+)), by flow cytometry. The TR group had significantly lower Hb, MCHC, and platelets for 10, 9 and 11 months, respectively; lower CD4(+)CD71(+) (3 months) and Fe(3+) (1 month), respectively; higher CD4(+)CD71(+) (1 month); a higher lymphocyte count for 4 months. There were no between-group differences in other variables. In both groups haematology and lymphocytes increased during spring, early summer and winter and decreased during late summer/late winter, with an inverse relationship between CD4(+)CD71(+) and Fe(3+). The TR group reported significantly fewer URTI than the UT. Low Hb and MCHC suggest an iron deficiency which may affect triathlete performance. Monthly changes in lymphocytes, CD4(+)CD71(+) and Fe(3+) suggested that spring, summer and late autumn are associated with CD4(+) proliferation. There may be seasonal relationships between haematology and lymphocyte function, independent of endurance training, possibly affecting performance but not the incidence of URTI.

  5. Human serum transferrin fibrils: nanomineralisation in bacteria and destruction of red blood cells.

    PubMed

    Mukherjee, Arindam; Barnett, Mark A; Venkatesh, V; Verma, Sandeep; Sadler, Peter J

    2015-01-02

    Fibrils formed by human serum transferrin [(1-3 μM) apo-Tf, partially iron-saturated (Fe0.6 -Tf) and holo-Tf (Fe2 -Tf) forms], from dilute bicarbonate solutions, were deposited on formvar surfaces and studied by electron microscopy. We observed that possible bacterial contamination appears to give rise to long, pea-pod-like (PPL) structures for Fe2 -Tf, attributable to the formation of polyhydroxybutyrate (PHB) storage granules, under the nutrient-limiting conditions used. These PPL structures contained periodic nanomineralisation sites susceptible to uranyl stain. Extended incubation of transferrin solutions (about four days) gave rise to extensive transferrin fibril structures. Optical microscopy and AFM studies showed that red blood cells (RBCs) readily adhere to these fibrils. Moreover, the fibrils appear to penetrate RBC membranes and to induce rapid cell destruction (within about 5 h). It is speculated that in situations in vivo where transferrin fibrils can form, such interactions might have adverse physiological consequences, and further studies could aid the understanding of related pathological events. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor

    SciTech Connect

    Davis, R.J.; Kuck, L.; Faucher, M.; Czech, M.P.

    1986-05-01

    Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of (/sup 125/I) diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 minutes. The effect is transient with (/sup 125/I) diferric transferrin binding returning to control values within 25 minutes. In contrast, PDGF and rIGF-I cause a prolonged stimulation of (/sup 125/I) diferric transferrin binding that could be observed up to 2 hours. The increase in the binding of (/sup 125/I) diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. EGF, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of (/sup 59/Fe) diferric transferrin by BALB/c 3T3 fibroblasts. Thus, the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.

  7. Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor

    PubMed Central

    Hultqvist, Greta; Syvänen, Stina; Fang, Xiaotian T; Lannfelt, Lars; Sehlin, Dag

    2017-01-01

    The blood-brain barrier (BBB) is an obstacle for antibody passage into the brain, impeding the development of immunotherapy and antibody-based diagnostics for brain disorders. In the present study, we have developed a brain shuttle for active transport of antibodies across the BBB by receptor-mediated transcytosis. We have thus recombinantly fused two single-chain variable fragments (scFv) of the transferrin receptor (TfR) antibody 8D3 to the light chains of mAb158, an antibody selectively binding to Aβ protofibrils, which are involved in the pathogenesis of Alzheimer's disease (AD). Despite the two TfR binders, a monovalent interaction with TfR was achieved due to the short linkers that sterically hinder bivalent binding to the TfR dimer. The design enabled efficient receptor-mediated brain uptake of the fusion protein. Two hours after administration, brain concentrations were 2-3% of the injected dose per gram brain, comparable to small molecular drugs and 80-fold higher than unmodified mAb158. After three days, fusion protein concentrations in AD transgenic mouse brains were 9-fold higher than in wild type mice, demonstrating high in vivo specificity. Thus, our innovative recombinant design markedly increases mAb158 brain uptake, which makes it a strong candidate for improved Aβ immunotherapy and as a PET radioligand for early diagnosis and evaluation of treatment effect in AD. Moreover, this approach could be applied to any target within the brain. PMID:28042336

  8. The erythroid function of transferrin receptor 2 revealed by Tmprss6 inactivation in different models of transferrin receptor 2 knockout mice

    PubMed Central

    Nai, Antonella; Pellegrino, Rosa M.; Rausa, Marco; Pagani, Alessia; Boero, Martina; Silvestri, Laura; Saglio, Giuseppe; Roetto, Antonella; Camaschella, Clara

    2014-01-01

    Transferrin receptor 2 (TFR2) is a transmembrane glycoprotein expressed in the liver and in the erythroid compartment, mutated in a form of hereditary hemochromatosis. Hepatic TFR2, together with HFE, activates the transcription of the iron-regulator hepcidin, while erythroid TFR2 is a member of the erythropoietin receptor complex. The TMPRSS6 gene, encoding the liver-expressed serine protease matriptase-2, is the main inhibitor of hepcidin and inactivation of TMPRSS6 leads to iron deficiency with high hepcidin levels. Here we evaluate the phenotype resulting from the genetic loss of Tmprss6 in Tfr2 total (Tfr2−/−) and liver-specific (Tfr2LCKO) knockout mice. Tmprss6−/−Tfr2−/− and Tmprss6−/−Tfr2LCKO mice have increased hepcidin levels and show iron-deficiency anemia like Tmprss6−/−mice. However, while Tmprss6−/−Tfr2LCKO are phenotypically identical to Tmprss6−/− mice, Tmprss6−/−Tfr2−/− mice have increased red blood cell count and more severe microcytosis than Tmprss6−/− mice. In addition hepcidin expression in Tmprss6−/−Tfr2−/− mice is higher than in the wild-type animals, but lower than in Tmprss6−/− mice, suggesting partial inhibition of the hepcidin activating pathway. Our results prove that hepatic TFR2 acts upstream of TMPRSS6. In addition Tfr2 deletion causes a relative erythrocytosis in iron-deficient mice, which likely attenuates the effect of over-expression of hepcidin in Tmprss6−/− mice. Since liver-specific deletion of Tfr2 in Tmprss6−/− mice does not modify the erythrocyte count, we speculate that loss of Tfr2 in the erythroid compartment accounts for the hematologic phenotype of Tmprss6−/−Tfr2−/− mice. We propose that TFR2 is a limiting factor for erythropoiesis, particularly in conditions of iron restriction. PMID:24658816

  9. Mechanisms by Which Salt Concentration Moderates the Dynamics of Human Serum Transferrin.

    PubMed

    Abdizadeh, Haleh; Atilgan, Ali Rana; Atilgan, Canan

    2017-05-11

    The dynamical and thermodynamic behavior of human transferrin (hTf) protein in saline aqueous solution of various concentrations is studied. hTf is an essential transport protein circulating iron in the blood and delivering it to tissues. It displays highly pH dependent cooperativity between the two lobes, each carrying an iron, and forms a tight complex with the receptor during endocytosis, eventually recycled to the serum after iron release. Molecular dynamics simulations are used to investigate the effects of the amount of salt on protein conformation and dynamics to analyze the structure-function relationship in free hTf at serum pH. To monitor the ionic strength dependence, four different ionic concentrations, 0, 50, 130, and 210 mM NaCl for two protonation states of the iron coordination site is considered. Two mechanisms by which salt affects hTf are disclosed. In the totally closed state where iron coordinating tyrosines are deprotonated, the addition of even 50 mM of salt alters the electrostatic potential distribution around the protein, opening energetic pathways for tyrosine protonation from nearby charged residues as a required first step for iron release. Once domain opening is observed, conformational plasticity renders the iron binding site more accessible by the solvent. At this second stage of iron release, R124 in the N-lobe is identified as kinetically significant anion binding site that accommodates chloride ions and allosterically communicates with the iron binding residues. Opening motions are maximized at 150 mM IS in the N-lobe, and at 210 mM in the C-lobe. The extra mobility in the latter is thought to preclude binding of hTf to its receptor. Thus, the physiological IS is optimal for exposing iron for release from hTf. However, the calculated binding affinities of iron show that even in the most open conformations, iron dissociation needs to be accompanied by chelators.

  10. Comparison of the Antiproliferative Activity of Two Antitumour Ruthenium(III) Complexes With Their Apotransferrin and Transferrin-Bound Forms in a Human Colon Cancer Cell Line

    PubMed Central

    Keppler, B. K.; Hartmann, M.; Messori, L.; Berger, M. R.

    1996-01-01

    Two ruthenium(III) complexes, namely trans-indazolium[tetrachlorobis(indazole)- ruthenate(III)], HInd[RuInd2Cl4] and trans-imidazolium[tetrachlorobis(imidazole)- ruthenate(III)], HIm[RuIm2Cl4] exhibit high anticancer activity in an autochthonous colorectal carcinoma model in rats. Recently, it has been shown that both complexes bind specifically to human serum apotransferrin and the resulting adducts have been studied through spectroscopic and chromatographic techniques with the ultimate goal of preparing adducts with good selectivity for cancer cells due to the fact that tumour cells express high amounts of transferrin receptors on their cell surface. In order to investigate whether the cellular uptake of the complexes was mediated by apotransferrin or transferrin, we compared the antiproliferative efficacy of HInd[RuInd2Cl4] and HIm[RuIm2Cl4] with its apotransferrin- and transferrin-bound form in the human colon cancer cell line SW707 using the microculture tetrazolium test (MTT). Our results show that especially the transferrin-bound forms exhibit high antiproliferative activity, which exceeds that of the free complex, indicating that this protein can act as a carrier of the ruthenium complexes into the tumor cell. PMID:18472789

  11. Aluminum access to the brain: A role for transferrin and its receptor

    SciTech Connect

    Roskams, A.J.; Connor, J.R. )

    1990-11-01

    The toxicity of aluminum in plant and animal cell biology is well established, although poorly understood. Several recent studies have identified aluminum as a potential, although highly controversial, contributory factor in the pathology of Alzheimer's disease, amyotrophic lateral sclerosis, and dialysis dementia. For example, aluminum has been found in high concentrations in senile plaques and neurofibrillary tangles, which occur in the brains of subjects with Alzheimer's disease. However, a mechanism for the entry of aluminum (Al{sup 3+}) into the cells of the central nervous system (CNS) has yet to be found. Here the authors describe a possible route of entry for aluminum into the cells of the CNS via the same high-affinity receptor-ligand system that has been postulated for iron (Fe{sup 3}) aluminum is able to gain access to the central nervous system under normal physiological conditions. Furthermore, these data suggest that the interaction between transferrin and its receptor may function as a general metal ion regulatory system in the CNS, extending beyond its postulated role in iron regulation.

  12. The second transferrin receptor regulates red blood cell production in mice

    PubMed Central

    Nai, Antonella; Lidonnici, Maria Rosa; Rausa, Marco; Mandelli, Giacomo; Pagani, Alessia; Silvestri, Laura; Ferrari, Giuliana

    2015-01-01

    Transferrin receptor 2 (TFR2) contributes to hepcidin regulation in the liver and associates with erythropoietin receptor in erythroid cells. Nevertheless, TFR2 mutations cause iron overload (hemochromatosis type 3) without overt erythroid abnormalities. To clarify TFR2 erythroid function, we generated a mouse lacking Tfr2 exclusively in the bone marrow (Tfr2BMKO). Tfr2BMKO mice have normal iron parameters, reduced hepcidin levels, higher hemoglobin and red blood cell counts, and lower mean corpuscular volume than normal control mice, a phenotype that becomes more evident in iron deficiency. In Tfr2BMKO mice, the proportion of nucleated erythroid cells in the bone marrow is higher and the apoptosis lower than in controls, irrespective of comparable erythropoietin levels. Induction of moderate iron deficiency increases erythroblasts number, reduces apoptosis, and enhances erythropoietin (Epo) levels in controls, but not in Tfr2BMKO mice. Epo-target genes such as Bcl-xL and Epor are highly expressed in the spleen and in isolated erythroblasts from Tfr2BMKO mice. Low hepcidin expression in Tfr2BMKO is accounted for by erythroid expansion and production of the erythroid regulator erythroferrone. We suggest that Tfr2 is a component of a novel iron-sensing mechanism that adjusts erythrocyte production according to iron availability, likely by modulating the erythroblast Epo sensitivity. PMID:25499454

  13. Radioprotective effect of transferrin targeted citicoline liposomes.

    PubMed

    Suresh Reddy, Jannapally; Venkateswarlu, Vobalaboina; Koning, Gerben A

    2006-01-01

    The high level of expression of transferrin receptors (Tf-R) on the surface of endothelial cells of the blood-brain-barrier (BBB) had been widely utilized to deliver drugs to the brain. The primary aim of this study was to use transferrin receptor mediated endocytosis as a pathway for the rational development of holo-transferrin coupled liposomes for drug targeting to the brain. Citicoline is a neuroprotective agent used clinically to treat for instance Parkinson disease, stroke, Alzheimer's disease and brain ischemia. Citicoline does not readily cross the BBB because of its strong polar nature. Hence, citicoline was used as a model drug. (Citicoline liposomes have been prepared using dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC) by dry lipid film hydration-extrusion method). The effect of the use of liposomes composed of DPPC or DSPC on their citicoline encapsulation efficiency and their stability in vitro were studied. Transferrin was coupled to liposomes by a technique which involves the prevention of scavenging diferric iron atoms of transferrin. The coupling efficiency of transferrin to the liposomes was studied. In vitro evaluation of transferrin-coupled liposomes was performed for their radioprotective effect in radiation treated cell cultures. In this study, OVCAR-3 cells were used as a model cell type over-expressing the Tf-R and human umbilical vein endothelial cells (HUVEC) as BBB endothelial cell model. The average diameter of DPPC and DSPC liposomes were 138 +/- 6.3 and 79.0 +/- 3.2 nm, respectively. The citicoline encapsulation capacity of DPPC and DSPC liposomes was 81.8 +/- 12.8 and 54.9 +/- 0.04 microg/micromol of phospholipid, respectively. Liposomes prepared from DSPC showed relatively better stability than DPPC liposomes at 37 degrees C and in the presence of serum. Hence, DSPC liposomes were used for transferrin coupling and an average of 46-55 molecules of transferrin were present per liposome. Free citicoline

  14. Diagnosis of Iron Deficiency in Inflammatory Bowel Disease by Transferrin Receptor-Ferritin Index.

    PubMed

    Abitbol, Vered; Borderie, Didier; Polin, Vanessa; Maksimovic, Fanny; Sarfati, Gilles; Esch, Anouk; Tabouret, Tessa; Dhooge, Marion; Dreanic, Johann; Perkins, Geraldine; Coriat, Romain; Chaussade, Stanislas

    2015-07-01

    Iron deficiency is common in patients with inflammatory bowel disease (IBD), but can be difficult to diagnose in the presence of inflammation because ferritin is an acute phase reactant. The transferrin receptor-ferritin index (TfR-F) has a high sensitivity and specificity for iron deficiency diagnosis in chronic diseases. The diagnostic efficacy of TfR-F is little known in patients with IBD. The aim of the study was to assess the added value of TfR-F to iron deficiency diagnosis in a prospective cohort of patients with IBD.Consecutive IBD patients were prospectively enrolled. Patients were excluded in case of blood transfusion, iron supplementation, or lack of consent. IBD activity was assessed on markers of inflammation (C-reactive protein, endoscopy, fecal calprotectin). Hemoglobin, ferritin, vitamin B9 and B12, Lactate dehydrogenase, haptoglobin, and soluble transferrin receptor (sTfR) were assayed. TfR-F was calculated as the ratio sTfR/log ferritin. Iron deficiency was defined by ferritin <30 ng/mL or TfR-F >2 in the presence of inflammation.One-hundred fifty patients with median age 38 years (16-78) and Crohn disease (n = 105), ulcerative colitis (n = 43), or unclassified colitis (n = 2) were included. Active disease was identified in 45.3%. Anemia was diagnosed in 28%. Thirty-six patients (24%) had ferritin <30 ng/mL. Thirty-two patients (21.3%) had ferritin levels from 30 to 100 ng/ml and inflammation: 2 had vitamin B12 deficiency excluding TfR-F analysis, 13 of 30 (43.3%) had TfR-F >2. Overall, iron deficiency was diagnosed in 32.7% of the patients.TfR-F in addition to ferritin <30 ng/mL criterion increased by 36% diagnosis rates of iron deficiency. TfR-F appeared as a useful biomarker that could help physicians to diagnose true iron deficiency in patients with active IBD.

  15. Transferrin receptor-2 gene and non-C282Y homozygous patients with hemochromatosis.

    PubMed

    Aguilar-Martinez, P; Esculié-Coste, C; Bismuth, M; Giansily-Blaizot, M; Larrey, D; Schved, J F

    2001-01-01

    More than 80% of the patients affected by hereditary hemochromatosis, a common inherited iron disorder, are homozygotes for the 845G --> A (C282Y) mutation of the HFE gene. However, depending on the population, 10-20% of hereditary hemochromatosis can be linked either to other HFE genotypes, particularly the compound heterozygous state for C282Y and the 187 C --> G (H63D) mutation, or to mutations of new other genes. Recently, Camaschella et al. (Nat. Genet. 25, 14-15, 2000) identified a stop mutation (exon 6 nt 750 C --> T, Y250X) on the transferrin receptor-2 (TFR2) gene in two unrelated Sicilian families with hereditary hemochromatosis. The TFR2 gene is a transferrin receptor gene homologue that seems to be involved in iron metabolism. Moreover, one of the patients described by Camaschella et al. was a H63D homozygote. H63D homozygosity can be associated with various phenotypes from asymptomatic subjects to patients with a typical form of hereditary hemochromatosis. Thus, the Y250X mutation could be the molecular defect responsible for hereditary hemochromatosis in subjects with atypical HFE genotypes. We have searched for the Y250X mutation in 63 unrelated French subjects. Forty-three had a diagnosis of hereditary hemochromatosis based on classical criteria. This group included 12 H63D homozygotes, 3 C282Y heterozygotes, and 3 patients with none of the two most prevalent HFE mutants. These 18 patients had no other HFE sequence change and were subsequently subjected to DNA sequencing of the 15 last exons and flanking sequences of the TFR2 gene. The 25 remaining hereditary hemochromatosis patients who were tested for the Y250X mutant were compound heterozygotes for the C282Y and H63D mutations. Finally, we also tested for this TFR2 mutation 20 H63D homozygotes with milder manifestations of iron overload and no acquired cause of iron overload. None of the 63 tested subjects had the Y250X mutation. Concurrently, none of the 18 hereditary hemochromatosis patients

  16. Interaction of imatinib mesylate with human serum transferrin: The comparative spectroscopic studies

    NASA Astrophysics Data System (ADS)

    Śliwińska-Hill, Urszula

    2017-02-01

    Imatinib mesylate (Imt) is a tyrosine kinase inhibitor mainly used in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph + CML). Human serum transferrin is the most abundant serum protein responsible for the transport of iron ions and many endogenous and exogenous ligands. In this study the mechanism of interactions between the imatinib mesylate and all states of transferrin (apo-Tf, Htf and holo-Tf) has been investigated by fluorescence, ultraviolet-visible (UV-vis), circular dichroism (CD) and zeta potential spectroscopic methods. Based on the experimental results it was proved that under physiological conditions the imatinib mesylate binds to the each form of transferrin with a binding constant c.a. 105 M- 1. The thermodynamic parameters indicate that hydrogen bonds and van der Waals were involved in the interaction of apo-Tf with the drug and hydrophobic and ionic strength participate in the reaction of Htf and holo-Tf with imatinib mesylate. Moreover, it was shown that common metal ions, Zn2 + and Ca2 + strongly influenced apo-Tf-Imt binding constant. The CD studies showed that there are no conformational changes in the secondary structure of the proteins. No significant changes in secondary structure of the proteins upon binding with the drug and instability of apo-Tf-Imt system are the desirable effects from pharmacological point of view.

  17. Interaction of imatinib mesylate with human serum transferrin: The comparative spectroscopic studies.

    PubMed

    Śliwińska-Hill, Urszula

    2017-02-15

    Imatinib mesylate (Imt) is a tyrosine kinase inhibitor mainly used in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph+CML). Human serum transferrin is the most abundant serum protein responsible for the transport of iron ions and many endogenous and exogenous ligands. In this study the mechanism of interactions between the imatinib mesylate and all states of transferrin (apo-Tf, Htf and holo-Tf) has been investigated by fluorescence, ultraviolet-visible (UV-vis), circular dichroism (CD) and zeta potential spectroscopic methods. Based on the experimental results it was proved that under physiological conditions the imatinib mesylate binds to the each form of transferrin with a binding constant c.a. 10(5)M(-1). The thermodynamic parameters indicate that hydrogen bonds and van der Waals were involved in the interaction of apo-Tf with the drug and hydrophobic and ionic strength participate in the reaction of Htf and holo-Tf with imatinib mesylate. Moreover, it was shown that common metal ions, Zn(2+) and Ca(2+) strongly influenced apo-Tf-Imt binding constant. The CD studies showed that there are no conformational changes in the secondary structure of the proteins. No significant changes in secondary structure of the proteins upon binding with the drug and instability of apo-Tf-Imt system are the desirable effects from pharmacological point of view.

  18. Study of maternal influences on fetal iron status at term using cord blood transferrin receptors

    PubMed Central

    Sweet, D; Savage, G; Tubman, T; Lappin, T; Halliday, H

    2001-01-01

    AIMS—To determine effects of maternal iron depletion and smoking on iron status of term babies using serum transferrin receptors (STfR) and their ratio to ferritin (TfR-F index) in cord blood.
METHODS—Iron, ferritin, STfR, and haemoglobin (Hb) concentration were measured and TfR-F index calculated in 67 cord /maternal blood pairs. Twenty six mothers were iron depleted (ferritin <10 µg/l) and 28 were smokers.
RESULTS—Maternal iron depletion was associated with decreased cord ferritin (113 v 171 µg/l) and Hb (156 v 168 g/l) but no change in STfR or TfR-F index. Smoking was associated with increased cord Hb (168 v 157 g/l) and TfR-F index (4.1 v 3.4), and decreased ferritin (123 v 190 µg/l). Cord TfR-F index and Hb were positively correlated (r = 0.48).
CONCLUSIONS—Maternal iron depletion is associated with reduced fetal iron stores but no change in free iron availability. Smoking is associated with increased fetal iron requirements for erythropoiesis.

 PMID:11124923

  19. Reference limits and behaviour of serum transferrin receptor in children 6-10 years of age.

    PubMed

    Danise, P; Maconi, M; Morelli, G; Di Palma, A; Rescigno, G; Esposito, C; Avino, D; Talento, B

    2008-08-01

    Serum transferrin receptor (sTfR) originates mostly from erythroblasts and lesser from reticulocytes. The usefulness of sTfR has been implicated in several clinical situations, mainly as a marker of accelerated erythropoiesis or iron deficiency. The assessment of sTfR may be useful in the period of rapid growth during infancy, childhood and adolescence. We evaluated sTfR and the other quantitative and qualitative parameters of the erythropoiesis (Hb, MCV, CHr, Ret-He) and of the iron storage (serum ferritin, sTfR/ferritin index) in a total of 916 children aged 6-10 years. Children were divided into three groups: (A) healthy children, (B) with storage iron deficiency (serum ferritin < 12 microg/l) and (C) Beta trait carriers (HbA2 > 3.3). We determined reference intervals by sex and by age in healthy children. sTfR showed a slight but statistically significant age related increase but did not show significant sex differences. We compared sTfR and the other parameters investigated in the three groups of children. sTfR is not a decisive parameter that can be utilized alone in discriminating the border-line situations between normal and pathologic ones but can help in completing the panel of tests in iron deficiency and in thalassaemia Beta trait carriers.

  20. Effect of malaria on soluble transferrin receptor levels in Tanzanian infants.

    PubMed

    Menendez, C; Quinto, L L; Kahigwa, E; Alvarez, L; Fernandez, R; Gimenez, N; Schellenberg, D; Aponte, J J; Tanner, M; Alonso, P L

    2001-08-01

    The diagnosis of iron deficiency anemia in malaria endemic areas is complicated by the influence of the infection on the laboratory tests conventionally used to assess iron status. Determination of soluble transferrin receptor (sTfR) levels has been shown to be a sensitive indicator of iron deficiency in adults and is not affected by a range of infectious and inflammatory conditions. The utility of sTfR levels in the diagnosis of iron deficiency in malaria endemic areas remains unresolved. Three hundred and fourteen infants in a rural area of southern Tanzania living under conditions of intense and perennial malaria transmission were studied to determine the utility of sTfR plasma levels in the assessment of iron deficiency anemia. Independent of the presence of anemia, malaria parasitemia was associated with a significant increase in sTfR plasma levels that were even higher than those found in iron deficiency anemia. We conclude that the measurement of sTfR levels does not have a role in the diagnosis of iron deficiency anemia in young children exposed to malaria infection.

  1. Double diagnostic meaning of serum transferrin receptor in hemodialysis patients: two case reports.

    PubMed

    Passanante, S; Diquattro, M; Greco, V; Li Cavoli, G; Bono, L; Palma, B; Scola, S; Faraci, C; Rotolo, U; Menozzi, I

    2004-01-01

    Hemodialysis patients on maintenance erythropoietin need an adequate supply of iron to optimize therapy and achieve and maintain target levels of hemoglobin. Evaluation of iron stores and early detection of iron deficiency are essential for management of erythropoiesis in chronic renal failure, but there is still no single biochemical or hematological parameter that is sensitive or specific enough to completely describe the distribution of iron in the body. Serum transferrin receptor (sTfR) is a marker of iron that is available for erythropoiesis. We selected 2 clinical cases in which hemodialysis patients were receiving maintenance erythropoietin. To suggest how sTfR can be used in its double diagnostic meaning according to the clinical context of the patient, sTfR was evaluated in one case as a marker of iron deficiency and in the other as a marker of erythropoiesis. The association of sTfR with hematological parameters of iron-deficient erythropoiesis (reticulocyte hemoglobin content, percentage of hypochromic erythrocytes, ratio of reticulocyte hemoglobin content to hemoglobin content) and parameters of stimulated erythropoiesis (absolute reticulocyte count, immature reticulocyte fraction) increases the accuracy of sTfR in its double diagnostic power.

  2. Percentile estimates for transferrin receptor in normal infants 9-15 mo of age.

    PubMed

    Yeung, G S; Zlotkin, S H

    1997-08-01

    To establish percentile estimates of transferrin receptor (TfR) for healthy infants, plasma TfR was measured in 485 healthy infants 9-15 mo of age from Edmonton, Toronto, Montreal, and Halifax. Education and income of the sample families were reflective of the average family based on the 1991 census estimates. The mean (+/-SD) plasma TfR concentration was 4.4 +/- 1.1 mg/L. As expected in the infant population, there were no differences in TfR concentrations as a result of sex, and within this small age range there was no significant change across age. Furthermore, the TfR concentration in plasma was not associated with hemoglobin, serum ferritin, or free erythrocyte protoporphyrin. TfR has been shown to be a sensitive, quantitative measure of tissue iron deficiency not affected by inflammation and is potentially important in the diagnosis of iron deficiency, but there is a lack of normative data, particularly in infants, who are at highest risk of iron deficiency. If TfR proves useful in the diagnosis of iron deficiency, the current data will be useful as a reference standard for healthy infants.

  3. The assessment of frequency of iron deficiency in athletes from the transferrin receptor-ferritin index.

    PubMed

    Malczewska, J; Szczepańska, B; Stupnicki, R; Sendecki, W

    2001-03-01

    The transferrin receptor-ferritin index (sTfR/logFerr) was determined in 131 male and 121 female athletes in order to assess the frequency of iron deficiency (threshold value of that index taken as 1.8). Blood was drawn for determining morphological indices as well as sTfR, ferritin, iron, total iron binding capacity (TIBC), and haptoglobin. A significantly (p <.01) higher incidence of iron deficiency was observed in women (26%) than in men (11%). The iron deficiency was latent, since no subject was found to be anemic. The plasma iron was significantly lower and TIBC higher (p <.001) in both iron-deficient subgroups than in the non-deficient ones. This confirmed the latent character of iron deficiency. Some hematological indices (Hb, MCH, MCHC, MCV) were significantly lower in iron-deficient female athletes than in male athletes, which suggested a more profound iron deficiency in the former. The sTfR/logFerr index might thus be useful in detecting iron deficiency in athletes, especially in those with erythropoiesis disorders, since physical loads may affect the widely used ferritin levels.

  4. Serum transferrin receptor in children: usefulness for determinating the nature of anemia in infection.

    PubMed

    Angeles Vázquez López, M; Molinos, Francisco Lendinez; Carmona, Moisés Leyva; Morales, Amparo Carracedo; Muñoz Vico, Francisco Javier; Muñoz, Juan López; Muñoz Hoyos, Antonio

    2006-12-01

    To know the variations of serum transferrin receptor (sTfR) and its indices depending on the status of body iron and the presence of infection in children, to evaluate their usefulness for recognizing the nature of anemia in infection, and to know the role of erythropoietic activity in these conditions. Three hundred and sixty-eight children between 1 and 10 years were included: 206 healthy children; 60 iron deficient anemic children (IDA); 102 with anemia and infectious disease, 58 of them meeting criteria for IDA. We measured hemoglobin, red cell indices, reticulocytes, transferrin saturation, serum ferritin, erythrocyte protoporphyrin, serum erythropoietin, and sTfR. Statistic method: ANOVA test, multiple linear regression, and ROC curve. sTfR, sTfR/ferritin ratio, and sTfR-logferritin index values were found to increase significantly in IDA children. These values were significantly lower in infectious anemia than iron deficiency states. Serum erythropoietin only was elevated significantly in iron deficiency states. In children without infection, mean corpuscular hemoglobin, erythrocyte protoporphirin, erythropoietin logarithm, and total-iron-binding-capacity logarithm predicted 81% of sTfR variability. sTfR and its indices showed a very high sensitivity and specificity for recognizing iron deficiency states. In children with IDA and infection sensitivity for sTfR/ferritin ratio was low (area under the curve: 0.71; 95% confidence interval: 0.64-0.88). For discriminating the nature of anemia in infection the cut-off point obtained for sTfR, sTfR/ferritin ratio, and sTfR-F index were 3, 70, and 1.8, respectively, and their sensitivity and specificity were also very high. sTfR, sTfR/ferritin ratio, and sTfR-F index are useful parameters for recognizing iron deficiency and the nature of anemia in infection. In IDA+infection, sTfR/ferritin ratio should not be recommended in the diagnosis of iron deficiency. In iron deficiency, erythropoietic activity has a secondary

  5. Serum transferrin receptor is a marker of iron deficiency in patients included in programs for preoperative autologous blood donation.

    PubMed

    Dimonte, Donato; Pepe, Maria; Silletti, Immacolata; Cazzato, Luciano; Fiore, Rosa

    2002-12-01

    Recently programs for preoperative autologous blood donation (PABD) have expanded to reduce the need for allogenic blood transfusion. Nevertheless, the ability of the patients's bone marrow to replace the red blood cells (RBCs) mass reduced by phlebotomies determines the efficacy of PABD. In mild anemia, known as iron-deficient erythropoiesis (IDE) or iron deficiency without anemia, precipitated by PABD, the marrow response is suboptimal and needs adjuvant therapy. The aim of this study was to evaluate the use of the serum transferrin receptor (sTfR) for the assessment of IDE in patients undergoing PABD. Two autologous blood units from 50 consecutive patients scheduled for elective orthopedic surgery were collected preoperatively. Serial measurements of RBCs, haematocrit (Hct), haemoglobin (Hb), serum iron, serum ferritin, reticulocyte count, reticulocyte maturity index (RMI), endogenous erythropoietin (EPO) and sTfR were performed throughout the phlebotomy program. RBC, Hct, Hb and serum iron significantly decreased although within the normal range. There was no change in serum ferritin levels. Reticulocytes, RMI and EPO significantly increased as did sTfR which significantly exceeds the normal range. These results demonstrate that the sTfR is a reliable laboratory marker for detecting mild anemia or IDE. In patients undergoing PABD increased sTfR levels may suggest a treatment with recombinant human EPO (rh-EPO) or iron to improve the bone marrow performance.

  6. Transferrin receptor 2: Continued expression in mouse liver in the face of iron overload and in hereditary hemochromatosis

    PubMed Central

    Fleming, Robert E.; Migas, Mary C.; Holden, Christopher C.; Waheed, Abdul; Britton, Robert S.; Tomatsu, Shunji; Bacon, Bruce R.; Sly, William S.

    2000-01-01

    Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. Liver disease resulting from iron toxicity is the major cause of death in HH. Hepatic iron loading in HH is progressive despite down-regulation of the classical transferrin receptor (TfR). Recently a human cDNA highly homologous to TfR was identified and reported to encode a protein (TfR2) that binds holotransferrin and mediates uptake of transferrin-bound iron. We independently identified a full-length murine EST encoding the mouse orthologue of the human TfR2. Although homologous to murine TfR in the coding region, the TfR2 transcript does not contain the iron-responsive elements found in the 3′ untranslated sequence of TfR mRNA. To determine the potential role for TfR2 in iron uptake by liver, we investigated TfR and TfR2 expression in normal mice and murine models of dietary iron overload (2% carbonyl iron), dietary iron deficiency (gastric parietal cell ablation), and HH (HFE −/−). Northern blot analyses demonstrated distinct tissue-specific patterns of expression for TfR and TfR2, with TfR2 expressed highly only in liver where TfR expression is low. In situ hybridization demonstrated abundant TfR2 expression in hepatocytes. In contrast to TfR, TfR2 expression in liver was not increased in iron deficiency. Furthermore, hepatic expression of TfR2 was not down-regulated with dietary iron loading or in the HFE −/− model of HH. From these observations, we propose that TfR2 allows continued uptake of Tf-bound iron by hepatocytes even after TfR has been down-regulated by iron overload, and this uptake contributes to the susceptibility of liver to iron loading in HH. PMID:10681454

  7. A chimeric LDL receptor containing the cytoplasmic domain of the transferrin receptor is degraded by PCSK9.

    PubMed

    Holla, Øystein L; Strøm, Thea Bismo; Cameron, Jamie; Berge, Knut Erik; Leren, Trond P

    2010-02-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the extracellular domain of the low density lipoprotein receptor (LDLR) at the cell surface, and disrupts the normal recycling of the LDLR. However, the exact mechanism by which the LDLR is re-routed for lysosomal degradation remains to be determined. To clarify the role of the cytoplasmic domain of the LDLR for re-routing to the lysosomes, we have studied the ability of PCSK9 to degrade a chimeric receptor which contains the extracellular and transmembrane domains of the LDLR and the cytoplasmic domain of the transferrin receptor. These studies were performed in CHO T-REx cells stably transfected with a plasmid encoding the chimeric receptor and a novel assay was developed to study the effect of PCSK9 on the LDLR in these cells. Localization, function and stability of the chimeric receptor were similar to that of the wild-type LDLR. The addition of purified gain-of-function mutant D374Y-PCSK9 to the culture medium of stably transfected CHO T-REx cells showed that the chimeric receptor was degraded, albeit to a lower extent than the wild-type LDLR. In addition, a mutant LDLR, which has the three lysines in the intracellular domain substituted with arginines, was also degraded by D374Y-PCSK9. Thus, the mechanism for the PCSK9-mediated degradation of the LDLR does not appear to involve an interaction between the endosomal sorting machinery and LDLR-specific motifs in the cytoplasmic domain. Moreover, ubiquitination of lysines in the cytoplasmic domain does not appear to play a critical role in the PCSK9-mediated degradation of the LDLR.

  8. Reticulocyte haemoglobin content vs. soluble transferrin receptor and ferritin index in iron deficiency anaemia accompanied with inflammation.

    PubMed

    Marković, M; Majkić-Singh, N; Ignjatović, S; Singh, S

    2007-10-01

    Ferritin concentration, as a parameter of iron status that is commonly used in the diagnosis of iron deficiency anaemia (IDA), often has limited values if the iron deficiency is accompanied by inflammatory disease. This study evaluated the value of reticulocyte haemoglobin content (CHr) and soluble transferrin receptor-ferritin index (sTfR/F) in the diagnosis of IDA and differential diagnosis of IDA and anaemia of chronic disease. The study included 66 nonanaemic individuals as controls, 86 patients with IDA divided into noninflammatory and inflammatory subgroups, and 32 patients with anaemia of chronic disease. Blood count, iron, transferrin saturation, total iron binding capacity, ferritin, C-reactive protein, sTfR and CHr were determined. Receiver operator characteristic curve analysis showed very high discriminating power for CHr, soluble transferrin receptor (sTfR) and sTfR/F in the diagnosis of IDA. In patients with anaemia of chronic disease these parameters showed no significant difference from the control. CHr and sTfR enabled recognition of iron deficiency and were not affected by acute phase reaction. They are sensitive markers of body iron status with additional value to conventional tests for the detection of iron deficiency.

  9. Effects of siderophores on the growth of Pseudomonas aeruginosa in human serum and transferrin.

    PubMed Central

    Ankenbauer, R; Sriyosachati, S; Cox, C D

    1985-01-01

    A combination of the siderophores produced by Pseudomonas aeruginosa, pyochelin and pyoverdin, dramatically stimulates the growth of this bacterium in medium containing human transferrin. The amount of growth stimulation observed when each siderophore was added alone was only slightly less than the amount observed with the combination. Siderophore-defective mutants of strain PAO1 were isolated to test the effects of siderophore production on growth in transferrin and human serum. The pyoverdin-proficient (Pvd+), pyochelin-deficient (Pch-) strain (IA5) grows just as well as the parent (PAO1), which produces both siderophores. On the other hand, the Pvd- Pch+ strain (211-5) has severely retarded growth, similar to that demonstrated by a mutant lacking production of both siderophores (IA1), but has an accelerated log phase compared with strain IA1 at the later stages of the growth curve. However, the Pvd- Pch+ strain (211-5) had no observable advantage over the Pvd- Pch- strain, IA1, during incubation in human serum. The inability of P. aeruginosa strains to produce pyochelin in glucose-minimal medium may explain the poor growth of 211-5 in this medium and in human serum. The 211-5 strain grows much better than the IA1 strain in the medium that allows pyochelin synthesis, but it still does not grow as well as the Pvd+ Pch- strain (IA5). Therefore, pyoverdin appears to be the most important siderophore for growth in human serum. PMID:3159677

  10. An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells

    PubMed Central

    Ng, Patrick P.; Dela Cruz, Jay S.; Sorour, David N.; Stinebaugh, James M.; Shin, Seung-Uon; Shin, Daniel S.; Morrison, Sherie L.; Penichet, Manuel L.

    2002-01-01

    We have developed an antibody fusion protein (anti-rat TfR IgG3-Av) with the ability to deliver different molecules into cancer cells. It consists of avidin genetically fused to the CH3 region of a human IgG3 specific for the rat transferrin receptor. It forms strong, noncovalent interactions with biotinylated molecules such as glucose oxidase and β-galactosidase, and delivers them into the rat myeloma cell line Y3-Ag1.2.3 through receptor-mediated endocytosis. Importantly, the β-galactosidase retains activity after internalization. Furthermore, we have unexpectedly discovered that anti-rat TfR IgG3-Av, but not a recombinant anti-rat TfR IgG3 or a nonspecific IgG3-Av, possesses proapoptotic activities against Y3-Ag1.2.3 and the rat T cell lymphoma cell line C58 (NT) D.1.G.OVAR.1. These activities were not observed in two rat cell lines of nonhematopoietic lineage (bladder carcinoma BC47 and gliosarcoma 9L). Anti-human TfR IgG3-Av also demonstrated proapoptotic activity against the human erythroleukemia cell line K562. Studies showed that anti-rat TfR IgG3-Av exists as a dimer, suggesting that cross-linking of the surface transferrin receptor may be responsible for the cytotoxic activity. These findings demonstrate that it is possible to transform an antibody specific for a growth factor receptor that does not exhibit inhibitory activity into a drug with significant intrinsic cytotoxic activity against selected cells by fusing it with avidin. The antitumor activity may be enhanced by delivering biotinylated therapeutics into cancer cells. Further development of this technology may lead to effective therapeutics for in vivo eradication of hematological malignancies, and ex vivo purging of cancer cells in autologous transplantation. PMID:12149472

  11. The small-molecule iron transport inhibitor ferristatin/NSC306711 promotes degradation of the transferrin receptor.

    PubMed

    Horonchik, Lior; Wessling-Resnick, Marianne

    2008-07-21

    Iron delivery by transferrin (Tf) is accomplished through clathrin-mediated endocytosis of Tf receptors. The small molecule NSC306711 inhibits iron uptake from the Tf-TfR pathway. Here we show that the drug's mechanism of action is to induce internalization and degradation of unoccupied Tf receptors through an unexpected endocytic pathway. Unlike classical clathrin-mediated Tf receptor endocytosis, internalization promoted by NSC306711 is independent of clathrin and dynamin, and is sensitive to the cholesterol-depleting agents filipin and nystatin. The finding of this cholesterol-dependent Tf receptor internalization pathway through use of the small-molecule inhibitor sheds light on the pleiotropic nature of membrane trafficking dynamics and adds a complex dimension to our understanding of receptor regulation. Because of its unusual properties to inhibit iron uptake, we refer to NSC306711 as "ferristatin."

  12. Functional consequences of transferrin receptor-2 mutations causing hereditary hemochromatosis type 3

    PubMed Central

    Joshi, Ricky; Shvartsman, Maya; Morán, Erica; Lois, Sergi; Aranda, Jessica; Barqué, Anna; de la Cruz, Xavier; Bruguera, Miquel; Vagace, José Manuel; Gervasini, Guillermo; Sanz, Cristina; Sánchez, Mayka

    2015-01-01

    Hereditary hemochromatosis (HH) type 3 is an autosomal recessive disorder of iron metabolism characterized by excessive iron deposition in the liver and caused by mutations in the transferrin receptor 2 (TFR2) gene. Here, we describe three new HH type 3 Spanish families with four TFR2 mutations (p.Gly792Arg, c.1606-8A>G, Gln306*, and Gln672*). The missense variation p.Gly792Arg was found in homozygosity in two adult patients of the same family, and in compound heterozygosity in an adult proband that also carries a novel intronic change (c.1606-8A>G). Two new nonsense TFR2 mutations (Gln306* and Gln672*) were detected in a pediatric case. We examine the functional consequences of two TFR2 variants (p.Gly792Arg and c.1606-8A>G) using molecular and computational methods. Cellular protein localization studies using immunofluorescence demonstrated that the plasma membrane localization of p.Gly792Arg TFR2 is impaired. Splicing studies in vitro and in vivo reveal that the c.1606-8A>G mutation leads to the creation of a new acceptor splice site and an aberrant TFR2 mRNA. The reported mutations caused HH type 3 by protein truncation, altering TFR2 membrane localization or by mRNA splicing defect, producing a nonfunctional TFR2 protein and a defective signaling transduction for hepcidin regulation. TFR2 genotyping should be considered in adult but also in pediatric cases with early-onset of iron overload. PMID:26029709

  13. Functional consequences of transferrin receptor-2 mutations causing hereditary hemochromatosis type 3.

    PubMed

    Joshi, Ricky; Shvartsman, Maya; Morán, Erica; Lois, Sergi; Aranda, Jessica; Barqué, Anna; de la Cruz, Xavier; Bruguera, Miquel; Vagace, José Manuel; Gervasini, Guillermo; Sanz, Cristina; Sánchez, Mayka

    2015-05-01

    Hereditary hemochromatosis (HH) type 3 is an autosomal recessive disorder of iron metabolism characterized by excessive iron deposition in the liver and caused by mutations in the transferrin receptor 2 (TFR2) gene. Here, we describe three new HH type 3 Spanish families with four TFR2 mutations (p.Gly792Arg, c.1606-8A>G, Gln306*, and Gln672*). The missense variation p.Gly792Arg was found in homozygosity in two adult patients of the same family, and in compound heterozygosity in an adult proband that also carries a novel intronic change (c.1606-8A>G). Two new nonsense TFR2 mutations (Gln306* and Gln672*) were detected in a pediatric case. We examine the functional consequences of two TFR2 variants (p.Gly792Arg and c.1606-8A>G) using molecular and computational methods. Cellular protein localization studies using immunofluorescence demonstrated that the plasma membrane localization of p.Gly792Arg TFR2 is impaired. Splicing studies in vitro and in vivo reveal that the c.1606-8A>G mutation leads to the creation of a new acceptor splice site and an aberrant TFR2 mRNA. The reported mutations caused HH type 3 by protein truncation, altering TFR2 membrane localization or by mRNA splicing defect, producing a nonfunctional TFR2 protein and a defective signaling transduction for hepcidin regulation. TFR2 genotyping should be considered in adult but also in pediatric cases with early-onset of iron overload.

  14. Targeting transferrin receptors at the blood-brain barrier improves the uptake of immunoliposomes and subsequent cargo transport into the brain parenchyma.

    PubMed

    Johnsen, Kasper Bendix; Burkhart, Annette; Melander, Fredrik; Kempen, Paul Joseph; Vejlebo, Jonas Bruun; Siupka, Piotr; Nielsen, Morten Schallburg; Andresen, Thomas Lars; Moos, Torben

    2017-09-04

    Drug delivery to the brain is hampered by the presence of the blood-brain barrier, which excludes most molecules from freely diffusing into the brain, and tightly regulates the active transport mechanisms that ensure sufficient delivery of nutrients to the brain parenchyma. Harnessing the possibility of delivering neuroactive drugs by way of receptors already present on the brain endothelium has been of interest for many years. The transferrin receptor is of special interest since its expression is limited to the endothelium of the brain as opposed to peripheral endothelium. Here, we investigate the possibility of delivering immunoliposomes and their encapsulated cargo to the brain via targeting of the transferrin receptor. We find that transferrin receptor-targeting increases the association between the immunoliposomes and primary endothelial cells in vitro, but that this does not correlate with increased cargo transcytosis. Furthermore, we show that the transferrin receptor-targeted immunoliposomes accumulate along the microvessels of the brains of rats, but find no evidence for transcytosis of the immunoliposome. Conversely, the increased accumulation correlated both with increased cargo uptake in the brain endothelium and subsequent cargo transport into the brain. These findings suggest that transferrin receptor-targeting is a relevant strategy of increasing drug exposure to the brain.

  15. Cytokines derived from activated human mononuclear cells markedly stimulate transferrin secretion by cultured Sertoli cells.

    PubMed

    Hoeben, E; Van Damme, J; Put, W; Swinnen, J V; Verhoeven, G

    1996-02-01

    There is considerable evidence that Sertoli cell function is controlled not only by hormones, but also by locally produced growth factors and cytokines. To gain more insight into the nature and effects of cytokines potentially involved in the control of Sertoli cell function, we incubated rat Sertoli cells with media conditioned by activated human peripheral blood mononuclear cells. Such media (PBMC-CM) are known to be an extremely rich source of a variety of cytokines. It was demonstrated that PMBC-CM and protein fractions derived from them stimulate Sertoli cell transferrin secretion and messenger RNA production more potently then peritubular cell-conditioned medium or FIRT (a combination of FSH, insulin, retinol, and testosterone). Transferrin secretion expressed per mg cell DNA was stimulated approximately 5-fold by peritubular cell-conditioned medium or FIRT and nearly 20-fold by PBMC-CM. The effects of PBMC-CM were accompanied by a limited increase in cAMP and a noticeable rise in cGMP. Affinity chromatography on a column coated with an antiserum directed against interleukin-1 beta (IL-1 beta) showed that part of the activity in the PBMC-CM was related to IL-1 beta. The remainder of the activity was largely retained by an affinity column coated with an antiserum that recognizes IL-6 and a number of other known and unknown cytokines. Purified IL-1 beta provoked a 2- to 3-fold stimulation of Sertoli cell transferrin secretion. More limited stimulatory effects were observed with IL-6. Neither of these cytokines or their combination approached the degree of stimulation observed with crude PBMC-CM, suggesting that other cytokines are involved. It is concluded that the mixture of cytokines present in PBMC-CM is a more powerful stimulator of Sertoli cell transferrin secretion than any other agonist known at the present time. IL-1 and IL-6 may be responsible for part of the observed effects, but one or more other cytokines are probably involved.

  16. Identification of a kinetically significant anion binding (KISAB) site in the N-lobe of human serum transferrin.

    PubMed

    Byrne, Shaina L; Steere, Ashley N; Chasteen, N Dennis; Mason, Anne B

    2010-05-18

    Human serum transferrin (hTF) binds two ferric iron ions which are delivered to cells in a transferrin receptor (TFR) mediated process. Critical to the delivery of iron to cells is the binding of hTF to the TFR and the efficient release of iron orchestrated by the interaction. Within the endosome, iron release from hTF is also aided by lower pH, the presence of anions, and a chelator yet to be identified. We have recently shown that three of the four residues comprising a loop in the N-lobe (Pro142, Lys144, and Pro145) are critical to the high-affinity interaction of hTF with the TFR. In contrast, Arg143 in this loop does not participate in the binding isotherm. In the current study, the kinetics of iron release from alanine mutants of each of these four residues (placed into both diferric and monoferric N-lobe backgrounds) have been determined +/- the TFR. The R143A mutation greatly retards the rate of iron release from the N-lobe in the absence of the TFR but has considerably less of an effect in its presence. Our data definitively show that Arg143 serves as a kinetically significant anion binding (KISAB) site that is, by definition, sensitive to salt concentration and critical to the conformational change necessary to induce iron release from the N-lobe of hTF (in the absence of the TFR). This is the first identification of an authentic KISAB site in the N-lobe of hTF. The effect of the single R143A mutation on the kinetic profile of iron release provides a dramatic illustration of the dynamic nature of hTF.

  17. Different binding affinities of Pb2+ and Cu2+ to glycosylation variants of human serum transferrin interfere with the detection of carbohydrate-deficient transferrin.

    PubMed

    Luo, Lian-Zhong; Jin, Hong-Wei; Huang, Lin; Huang, He-Qing

    2011-12-01

    Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse, and for diagnosis of chronic alcohol, abuse is often determined using isoelectric focusing (IEF) and chromatographic techniques. To allow this method to be used for the diagnosis of alcohol abuse, inferences of various physical and chemical factors with the detection of CDT have been investigated. However, few reports have focused thus far on whether different metal ions have different binding affinities to CDT and HTf variants or further interfere in the detection of CDT. Here, in order to figure out whether and how metal ions such as Pb(2+) and Cu(2+) bind to holo-human serum transferrin (holo-HTf) and further interfere in CDT detection, the binding characteristics and the binding parameters of holo-HTf with metal ions such as Pb(2+) and Cu(2+) were investigated using UV-visible spectroscopy, Fluorescence spectroscopy, and ICP-MS. Moreover, whether the metal ions such as Pb(2+) and Cu(2+) will reduce the diagnostic accuracy of CDT in clinic was investigated using IEF. The present study demonstrates that Pb(2+) and Cu(2+) have different binding affinities to holo-HTf variants and produce different changes in the relative amounts of each glycosylation isoforms of HTf. Accordingly, the glycosylation chains of HTf will affect the binding affinities of glycosylation isoforms with Pb(2+) and Cu(2+), causing further interferences in CDT detection.

  18. Supernatant from a cloned helper T cell stimulates resting B cells to express transferrin and IL-2 receptors.

    PubMed

    Diu, A; Leclercq, L; Dautry-Varsat, A; Theze, J

    1987-07-01

    We describe the properties of the supernatant from a murine cloned helper T cell (clone 52.3) which is able to polyclonally activate most resting B cells in the absence of any additional stimulus. We hypothesize that an activity which we call BCAF (B-cell-activating factor(s] exists in our supernatant which can activate resting B cells alone or in conjunction with other lymphokines. In the present report, we investigate changes in the surface antigen pattern induced on resting B cells by BCAF-containing supernatant. Analysis of the cells by flow cytometry shows that transferrin receptor and IL-2 receptor expression increase on a large fraction of B cells after 2 days of activation by the T-helper-cell clone supernatant. Monoclonal anti-transferrin receptor antibody inhibits cell division but does not affect blastogenesis, while IL-2 has no effect in our experimental system. Our present results confirm that BCAF-containing supernatants can act on most resting B cells and replace helper T cells in inducing B-cell activation and proliferation.

  19. α-Taxilin interacts with sorting nexin 4 and participates in the recycling pathway of transferrin receptor.

    PubMed

    Sakane, Hiroshi; Horii, Yukimi; Nogami, Satoru; Kawano, Yoji; Kaneko-Kawano, Takako; Shirataki, Hiromichi

    2014-01-01

    Membrane traffic plays a crucial role in delivering proteins and lipids to their intracellular destinations. We previously identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. α-Taxilin is overexpressed in tumor tissues and interacts with polymerized tubulin, but the precise function of α-taxilin remains unclear. Receptor proteins on the plasma membrane are internalized, delivered to early endosomes and then either sorted to the lysosome for degradation or recycled back to the plasma membrane. In this study, we found that knockdown of α-taxilin induced the lysosomal degradation of transferrin receptor (TfnR), a well-known receptor which is generally recycled back to the plasma membrane after internalization, and impeded the recycling of transferrin. α-Taxilin was immunoprecipitated with sorting nexin 4 (SNX4), which is involved in the recycling of TfnR. Furthermore, knockdown of α-taxilin decreased the number and length of SNX4-positive tubular structures. We report for the first time that α-taxilin interacts with SNX4 and plays a role in the recycling pathway of TfnR.

  20. Temporal manipulation of transferrin-receptor-1-dependent iron uptake identifies a sensitive period in mouse hippocampal neurodevelopment.

    PubMed

    Fretham, S J B; Carlson, E S; Wobken, J; Tran, P V; Petryk, A; Georgieff, M K

    2012-08-01

    Iron is a necessary substrate for neuronal function throughout the lifespan, but particularly during development. Early life iron deficiency (ID) in humans (late gestation through 2-3 yr) results in persistent cognitive and behavioral abnormalities despite iron repletion. Animal models of early life ID generated using maternal dietary iron restriction also demonstrate persistent learning and memory deficits, suggesting a critical requirement for iron during hippocampal development. Precise definition of the temporal window for this requirement has been elusive due to anemia and total body and brain ID inherent to previous dietary restriction models. To circumvent these confounds, we developed transgenic mice that express tetracycline transactivator regulated, dominant negative transferrin receptor (DNTfR1) in hippocampal neurons, disrupting TfR1 mediated iron uptake specifically in CA1 pyramidal neurons. Normal iron status was restored by doxycycline administration. We manipulated the duration of ID using this inducible model to examine long-term effects of early ID on Morris water maze learning, CA1 apical dendrite structure, and defining factors of critical periods including parvalbmin (PV) expression, perineuronal nets (PNN), and brain-derived neurotrophic factor (BDNF) expression. Ongoing ID impaired spatial memory and resulted in disorganized apical dendrite structure accompanied by altered PV and PNN expression and reduced BDNF levels. Iron repletion at P21, near the end of hippocampal dendritogenesis, restored spatial memory, dendrite structure, and critical period markers in adult mice. However, mice that remained hippocampally iron deficient until P42 continued to have spatial memory deficits, impaired CA1 apical dendrite structure, and persistent alterations in PV and PNN expression and reduced BDNF despite iron repletion. Together, these findings demonstrate that hippocampal iron availability is necessary between P21 and P42 for development of normal

  1. Serum transferrin receptors: Distribution and diagnostic performance in pre-school children.

    PubMed

    Chouliaras, Giorgos L; Premetis, Evangelos; Tsiftis, George; Drosatou, Panayiota; Papassotiriou, Ioannis; Stamoulakatou, Alexandra; Lycopoulou, Lilia

    2009-01-01

    Soluble transferrin receptors have gained interest in the field of diagnosing anemias. Reference ranges differ according to the method used for the quantification of sTfR. We aim to explore the distributional properties and diagnostic performance of sTfR in pre-school healthy children as well as in children with beta-thalassemia carriers, iron deficiency with normal hematological phenotype (ID) and iron deficiency anemia (IDA). Circulating sTfR as well as biochemical and hematological indices were determined in 521 pre-school children and four groups (normal children, beta-thalassemia traits, ID and IDA) were formed. Diagnostic performance and distribution of sTfR according to age and in relation to several parameters were evaluated in every group. Three hundred eighty one children (261 normal, 60 beta-thalassemia traits, 44 ID and 16 IDA) aged 1-6 years were included. We found that distribution of sTfR differed significantly among the four groups (Kruskal Wallis p<0.001) with children in the normal group exhibiting lower concentrations compared to all other. A negative correlation between sTfR and age occurred in the normal (beta=-0.12, p<0.001) and the ID groups (beta=-0.13, p=0.035). In the beta-thal and IDA groups sTfR is correlated to HbA(2) (beta=0.34, p=0.001) and ferritin (Spearman's rho=-0.6, p=0.014) respectively. An area under the curve equal to 0.63 was achieved by sTfR in distinguishing between normal and ID children. Sensitivity and specificity were 70.5% and 50% respectively at a cut-off of 2.5 mg/l. Levels of sTfR are negatively correlated to age in pre-school children while dyserythropoietic procedures like beta-thal, ID, and IDA significantly affect them. These findings indicated that the accuracy of sTfR in diagnosing ID from normal children is limited. Standardization will allow the use of formulas that combine sTfR and ferritin which are of greater diagnostic value than sTfR alone.

  2. [Value of soluble transferrin receptor in the diagnosis of iron deficiency in children].

    PubMed

    Wang, Ya-Ping; Shao, Jie; Zhuang, Xue-Ling

    2011-07-01

    To study the prevalence of iron deficiency in children between 6 months and 7 years and to study the diagnostic value of soluble transferrin receptor (sTfR) for iron deficiency in the children. A total of 502 healthy children between 6 months and 7 years from Hangzhou City of Zhejiang Province were enrolled. Serum sTfR, serum ferritin (SF), serum iron (SI), total iron blinding capacity (TIBC), zinc protoporphyrin (ZPP), Hb, MCV and CRP levels were measured. The prevalence rate of iron deficiency was 19.5% in children at ages of 6 months to 7 years. The prevalence rate of iron deficiency was the highest in infants (≤1 year old; 34.7%), followed by in toddlers (1-3 years old; 19.4%) and preschoolers (3-7 years old; 14.0%). The mean serum sTfR level in infants (2.02±0.73 mg/L) was significantly higher than that in toddlers (1.68±0.40 mg/L) and preschoolers (1.67±0.29 mg/L) (P<0.05).The best cut-off value of serum sTfR for the diagnosis of iron deficiency was 2.02 mg/L in infants (sensitivity: 70.3%, specificity: 82.2%). The best cut-off value was 1.85 mg/L in toddlers (sensitivity: 71.7%; specificity: 86.4%), and that was 1.85 mg/L in preschoolers (sensitivity: 77.8%; specificity: 88.6%). Serum sTfR was correlated with SF (r=0.107, P<0.05), TIBC (r=0.276, P<0.01), TS (r=-0.139, P<0.05), ZPP (r=0.175, P<0.01) and MCV (r=-0.140, P<0.01). Iron deficiency is more prevalent in infants ≤1 year old. The mean serum level and the cut-off value of sTfR in infants are higher than in toddlers and preschoolers. Serum sTfR is an effective index for the diagnosis of iron deficiency in children, especially in infants≤ 1 year old.

  3. Glutaminolysis and Transferrin Regulate Ferroptosis.

    PubMed

    Gao, Minghui; Monian, Prashant; Quadri, Nosirudeen; Ramasamy, Ravichandran; Jiang, Xuejun

    2015-07-16

    Ferroptosis has emerged as a new form of regulated necrosis that is implicated in various human diseases. However, the mechanisms of ferroptosis are not well defined. This study reports the discovery of multiple molecular components of ferroptosis and its intimate interplay with cellular metabolism and redox machinery. Nutrient starvation often leads to sporadic apoptosis. Strikingly, we found that upon deprivation of amino acids, a more rapid and potent necrosis process can be induced in a serum-dependent manner, which was subsequently determined to be ferroptosis. Two serum factors, the iron-carrier protein transferrin and amino acid glutamine, were identified as the inducers of ferroptosis. We further found that the cell surface transferrin receptor and the glutamine-fueled intracellular metabolic pathway, glutaminolysis, played crucial roles in the death process. Inhibition of glutaminolysis, the essential component of ferroptosis, can reduce heart injury triggered by ischemia/reperfusion, suggesting a potential therapeutic approach for treating related diseases.

  4. Alteration of Serum Concentrations of Manganese, Iron, Ferritin, and Transferrin Receptor Following Exposure to Welding Fumes Among Career Welders

    PubMed Central

    Lu, Ling; Zhang, Long-lian; Li, G. Jane; Guo, Wenrui; Liang, Wannian; Zheng, Wei

    2014-01-01

    This study was performed to determine airborne manganese levels during welding practice and to establish the relationship between long-term, low-level exposure to manganese and altered serum concentrations of manganese, iron, and proteins associated with iron metabolism in career welders. Ninety-seven welders (average age of 36 years) who have engaged in electric arc weld in a vehicle manufacturer were recruited as the exposed group. Welders worked 7–8 h per day with employment duration of 1–33 years. Control subjects consisted of 91 employees (average age of 35 years) in the same factory but not in the welding profession. Ambient manganese levels in welders’ breathing zone were the highest inside the vehicle (1.5 ± 0.7 mg/m3), and the lowest in the center of the workshop (0.2 ± 0.05 mg/m3). Since the filter size was 0.8 μm, it is possible that these values may be likely an underestimation of the true manganese levels. Serum levels of manganese and iron in welders were about three-fold (p < 0.01) and 1.2-fold (p < 0.01), respectively, higher than those of controls. Serum concentrations of ferritin and transferrin were increased among welders, while serum transferrin receptor levels were significantly decreased in comparison to controls. Linear regression analyses revealed a lack of association between serum levels of manganese and iron. However, serum concentrations of iron and ferritin were positively associated with years of welder experience (p < 0.05). Moreover, serum transferrin receptor levels were inversely associated with serum manganese concentrations (p < 0.05). These findings suggest that exposure to welding fume among welders disturbs serum homeostasis of manganese, iron, and the proteins associated with iron metabolism. Serum manganese may serve as a reasonable biomarker for assessment of recent exposure to airborne manganese. PMID:15713346

  5. The usefulness of soluble transferrin receptor in the diagnosis and treatment of iron deficiency anemia in children

    PubMed Central

    Yoon, Se Hoon; Kim, Dong Sup; Yu, Seung Taek; Shin, Sae Ron

    2015-01-01

    Purpose Soluble transferrin receptor (sTfR) is a truncated extracellular form of the membrane transferrin receptor produced by proteolysis. Concentrations of serum sTfR are related to iron status and erythropoiesis in the body. We investigated whether serum sTfR levels can aid in diagnosis and treatment of iron deficiency anemia (IDA) in children. Methods Ninety-eight patients with IDA were enrolled and were classified according to age at diagnosis. Group 1 comprised 78 children, aged 6-59 months, and group 2 comprised 20 adolescents, aged 12-16 years. Results In group 1, patients' serum sTfR levels correlated negatively with mean corpuscular volume; hemoglobin (Hb), ferritin, and serum iron levels; and transferrin saturation and positively with total iron binding capacity (TIBC) and red cell distribution width. In group 2, patients' serum sTfR levels did not correlate with ferritin levels and TIBC, but had a significant relationship with other iron indices. Hb and serum sTfR levels had a significant inverse relationship in both groups; however, in group 1, there was no correlation between Hb and serum ferritin levels. In 30 patients of group 1, serum sTfR levels were significantly decreased with an increase in Hb levels after iron supplementation for 1 month. Conclusion Serum sTfR levels significantly correlated with other diagnostic iron parameters of IDA and inversely correlated with an increase in Hb levels following iron supplementation. Therefore, serum sTfR levels can be a useful marker for the diagnosis and treatment of IDA in children. PMID:25729394

  6. Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway.

    PubMed

    Schaffert, David H; Okholm, Anders H; Sørensen, Rasmus S; Nielsen, Jesper S; Tørring, Thomas; Rosen, Christian B; Kodal, Anne Louise B; Mortensen, Michael R; Gothelf, Kurt V; Kjems, Jørgen

    2016-05-01

    DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Blood-brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody.

    PubMed

    Pardridge, William M

    2015-02-01

    Biologic drugs are large molecules that do not cross the blood- brain barrier (BBB). Brain penetration is possible following the re-engineering of the biologic drug as an IgG fusion protein. The IgG domain is a MAb against an endogenous BBB receptor such as the transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the fused biologic drug into the brain via receptor-mediated transport on the endogenous BBB TfR. This review discusses TfR isoforms, models of BBB transport of transferrin and TfRMAbs, and the genetic engineering of TfRMAb fusion proteins, including BBB penetrating IgG-neurotrophins, IgG-decoy receptors, IgG-lysosomal enzyme therapeutics and IgG-avidin fusion proteins, as well as BBB transport of bispecific antibodies formed by fusion of a therapeutic antibody to a TfRMAb targeting antibody. Also discussed are quantitative aspects of the plasma pharmacokinetics and brain uptake of TfRMAb fusion proteins, as compared to the brain uptake of small molecules, and therapeutic applications of TfRMAb fusion proteins in mouse models of neural disease, including Parkinson's disease, stroke, Alzheimer's disease and lysosomal storage disorders. The review covers the engineering of TfRMAb-avidin fusion proteins for BBB targeted delivery of biotinylated peptide radiopharmaceuticals, low-affinity TfRMAb Trojan horses and the safety pharmacology of chronic administration of TfRMAb fusion proteins. The BBB delivery of biologic drugs is possible following re-engineering as a fusion protein with a molecular Trojan horse such as a TfRMAb. The efficacy of this technology will be determined by the outcome of future clinical trials.

  8. A molecular docking study of the interactions between human transferrin and seven metallocene dichlorides.

    PubMed

    Güette-Fernández, Jorge R; Meléndez, Enrique; Maldonado-Rojas, Wilson; Ortega-Zúñiga, Carlos; Olivero-Verbel, Jesus; Parés-Matos, Elsie I

    2017-08-01

    Human Transferrin (hTf) is a metal-binding protein found in blood plasma and is well known for its role in iron delivery. With only a 30% of its capacity for Fe(+3) binding, this protein has the potential ability to transport other metal ions or organometallic compounds from the blood stream to all cell tissues. In this perspective, recent studies have described seven metallocene dichlorides (Cp2M(IV)Cl2, M(IV)=V, Mo, W, Nb, Ti, Zr, Hf) suitable as anticancer drugs and less secondary effects than cisplatin. However, these studies have not provided enough data to clearly explain how hTf binds and transports these organometallic compounds into the cells. Thus, a computational docking study with native apo-hTf using Sybyl-X 2.0 program was conducted to explore the binding modes of these seven Cp2M(IV)Cl2 after their optimization and minimization using Gaussian 09. Our model showed that the first three Cp2M(IV)Cl2 (M(IV)=V, Mo, W) can interact with apo-hTf on a common binding site with the amino acid residues Leu-46, Ile-49, Arg-50, Leu-66, Asp-69, Ala-70, Leu-72, Ala-73, Pro-74 and Asn-75, while the next four Cp2M(IV)Cl2 (M(IV)=Nb, Ti, Zr, Hf) showed different binding sites, unknown until now. A decreasing order in the total score (equal to -log Kd) was observed from these docking studies: W (5.4356), Mo (5.2692), Nb (5.1672), V (4.5973), Ti (3.6529), Zr (2.0054) and Hf (1.8811). High and significant correlation between the affinity of these seven ligands (metallocenes) for apo-hTf and their bond angles CpMCp (r=0.94, p<0.01) and Cl-M-Cl (r=0.95, p<0.01) were observed, thus indicating the important role that these bond angles can play in ligand-protein interactions. Fluorescence spectra of apo-hTf, measured at pH 7.4, had a decrease in the fluorescence emission spectrum with increasing concentration of Cp2M(IV)Cl2. Experimental data has a good correlation between KA (r=0.84, p=0.027) and Kd (r=0.94, p=0.0014) values and the calculated total scores obtained from our

  9. SNAP23/25 and VAMP2 mediate exocytic event of transferrin receptor-containing recycling vesicles

    PubMed Central

    Kubo, Keiji; Kobayashi, Minako; Nozaki, Shohei; Yagi, Chikako; Hatsuzawa, Kiyotaka; Katoh, Yohei; Shin, Hye-Won; Takahashi, Senye; Nakayama, Kazuhisa

    2015-01-01

    ABSTRACT We recently showed that Rab11 is involved not only in formation of recycling vesicles containing the transferrin (Tfn)–transferrin receptor (TfnR) complex at perinuclear recycling endosomes but also in tethering of recycling vesicles to the plasma membrane (PM) in concert with the exocyst tethering complex. We here aimed at identifying SNARE proteins responsible for fusion of Tfn–TfnR-containing recycling vesicles with the PM, downstream of the exocyst. We showed that exocyst subunits, Sec6 and Sec8, can interact with SNAP23 and SNAP25, both of which are PM-localizing Qbc-SNAREs, and that depletion of SNAP23 and/or SNAP25 in HeLa cells suppresses fusion of Tfn–TfnR-containing vesicles with the PM, leading to accumulation of the vesicles at the cell periphery. We also found that VAMP2, an R-SNARE, is colocalized with endocytosed Tfn on punctate endosomal structures, and that its depletion in HeLa cells suppresses recycling vesicle exocytosis. These observations indicate that fusion of recycling vesicles with the PM downstream of the exocyst is mediated by SNAP23/25 and VAMP2, and provide novel insight into non-neuronal roles of VAMP2 and SNAP25. PMID:26092867

  10. Enhanced transferrin receptor expression by proinflammatory cytokines in enterocytes as a means for local delivery of drugs to inflamed gut mucosa.

    PubMed

    Harel, Efrat; Rubinstein, Abraham; Nissan, Aviram; Khazanov, Elena; Nadler Milbauer, Mirela; Barenholz, Yechezkel; Tirosh, Boaz

    2011-01-01

    Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with adverse effects related to drug distribution into non-diseased tissues, a situation which attracts a rational design of a targeted treatment confined to the inflamed mucosa. Upon activation of immune cells, transferrin receptor (TfR) expression increases at their surface. Because TfR is expressed in all cell types we hypothesized that its cell surface levels are regulated also in enterocytes. We, therefore, compared TfR expression in healthy and inflamed human colonic mucosa, as well as healthy and inflamed colonic mucosa of the DNBS-induced rat model. TfR expression was elevated in the colonic mucosa of IBD patients in both the basolateral and apical membranes of the enterocytes. Increased TfR expression was also observed in colonocytes of the induced colitis rats. To explore the underlying mechanism CaCo-2 cells were treated with various proinflammatory cytokines, which increased both TfR expression and transferrin cellular uptake in a mechanism that did not involve hyper proliferation. These findings were then exploited for the design of targetable carrier towards inflamed regions of the colon. Anti-TfR antibodies were conjugated to nano-liposomes. As expected, iron-starved Caco-2 cells internalized anti-TfR immunoliposomes better than controls. Ex vivo binding studies to inflamed mucosa showed that the anti-TfR immunoliposomes accumulated significantly better in the mucosa of DNBS-induced rats than the accumulation of non-specific immunoliposomes. It is concluded that targeting mucosal inflammation can be accomplished by nano-liposomes decorated with anti-TfR due to inflammation-dependent, apical, elevated expression of the receptor.

  11. The unique kinetics of iron release from transferrin: the role of receptor, lobe-lobe interactions, and salt at endosomal pH.

    PubMed

    Byrne, Shaina L; Chasteen, N Dennis; Steere, Ashley N; Mason, Anne B

    2010-02-12

    Transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal. Human serum transferrin (hTF) carries one iron in each of two similar lobes. Understanding the detailed mechanism of iron release from each lobe of hTF during receptor-mediated endocytosis has been extremely challenging because of the active participation of the transferrin receptor (TFR), salt, a chelator, lobe-lobe interactions, and the low pH within the endosome. Our use of authentic monoferric hTF (unable to bind iron in one lobe) or diferric hTF (with iron locked in one lobe) provided distinct kinetic end points, allowing us to bypass many of the previous difficulties. The capture and unambiguous assignment of all kinetic events associated with iron release by stopped-flow spectrofluorimetry, in the presence and in the absence of the TFR, unequivocally establish the decisive role of the TFR in promoting efficient and balanced iron release from both lobes of hTF during one endocytic cycle. For the first time, the four microscopic rate constants required to accurately describe the kinetics of iron removal are reported for hTF with and without the TFR. Specifically, at pH 5.6, the TFR enhances the rate of iron release from the C-lobe (7-fold to 11-fold) and slows the rate of iron release from the N-lobe (6-fold to 15-fold), making them more equivalent and producing an increase in the net rate of iron removal from Fe(2)hTF. Calculated cooperativity factors, in addition to plots of time-dependent species distributions in the absence and in the presence of the TFR, clearly illustrate the differences. Accurate rate constants for the pH and salt-induced conformational changes in each lobe precisely delineate how delivery of iron within the physiologically relevant time frame of 2 min might be accomplished. Copyright 2009 Elsevier Ltd. All rights reserved.

  12. Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa

    PubMed Central

    Serrano-Luna, Jesús

    2015-01-01

    Iron is the fourth most abundant element on Earth and the most abundant metal in the human body. This element is crucial for life because almost all organisms need iron for several biological activities. This is the case with pathogenic organisms, which are at the vanguard in the battle with the human host for iron. The latest regulates Fe concentration through several iron-containing proteins, such as transferrin. The transferrin receptor transports iron to each cell that needs it and maintains it away from pathogens. Parasites have developed several strategies to obtain iron as the expression of specific transferrin receptors localized on plasma membrane, internalized through endocytosis. Signal transduction pathways related to the activation of the receptor have functional importance in proliferation. The study of transferrin receptors and other proteins with action in the signaling networks is important because these proteins could be used as therapeutic targets due to their specificity or to differences with the human counterpart. In this work, we describe proteins that participate in signal transduction processes, especially those that involve transferrin endocytosis, and we compare these processes with those found in T. brucei, T. cruzi, Leishmania spp., and E. histolytica parasites. PMID:26090431

  13. Enhanced delivery of IL-1 receptor antagonist to the central nervous system as a novel anti–transferrin receptor-IL-1RA fusion reverses neuropathic mechanical hypersensitivity

    PubMed Central

    Webster, Carl I.; Hatcher, Jon; Burrell, Matthew; Thom, George; Thornton, Peter; Gurrell, Ian; Chessell, Iain

    2016-01-01

    Abstract Neuropathic pain is a major unmet medical need, with only 30% to 35% of patients responding to the current standard of care. The discovery and development of novel therapeutics to address this unmet need have been hampered by poor target engagement, the selectivity of novel molecules, and limited access to the relevant compartments. Biological therapeutics, either monoclonal antibodies (mAbs) or peptides, offer a solution to the challenge of specificity as the intrinsic selectivity of these kinds of molecules is significantly higher than traditional medicinal chemistry–derived approaches. The interleukin-1 receptor system within the spinal cord has been implicated in the amplification of pain signals, and its central antagonism provides relief of neuropathic pain. Targeting the IL-1 system in the spinal cord with biological drugs, however, raises the even greater challenge of delivery to the central compartment. Targeting the transferrin receptor with monoclonal antibodies has proved successful in traversing the endothelial cell–derived blood–brain barrier and delivering proteins to the central nervous system. In this study, we describe a novel construct exemplifying an engineered solution to overcome these challenges. We have generated a novel anti–transferrin receptor-interleukin-1 receptor antagonist fusion that transports to the central nervous system and delivers efficacy in a model of nerve ligation–induced hypersensitivity. Approaches such as these provide promise for novel and selective analgesics that target the central compartment. PMID:28009628

  14. Transfusion of human volunteers with older, stored red blood cells produces extravascular hemolysis and circulating non-transferrin-bound iron.

    PubMed

    Hod, Eldad A; Brittenham, Gary M; Billote, Genia B; Francis, Richard O; Ginzburg, Yelena Z; Hendrickson, Jeanne E; Jhang, Jeffrey; Schwartz, Joseph; Sharma, Shruti; Sheth, Sujit; Sireci, Anthony N; Stephens, Hannah L; Stotler, Brie A; Wojczyk, Boguslaw S; Zimring, James C; Spitalnik, Steven L

    2011-12-15

    Transfusions of RBCs stored for longer durations are associated with adverse effects in hospitalized patients. We prospectively studied 14 healthy human volunteers who donated standard leuko-reduced, double RBC units. One unit was autologously transfused "fresh" (3-7 days of storage), and the other "older" unit was transfused after 40 to 42 days of storage. Of the routine laboratory parameters measured at defined times surrounding transfusion, significant differences between fresh and older transfusions were only observed in iron parameters and markers of extravascular hemolysis. Compared with fresh RBCs, mean serum total bilirubin increased by 0.55 mg/dL at 4 hours after transfusion of older RBCs (P = .0003), without significant changes in haptoglobin or lactate dehydrogenase. In addition, only after the older transfusion, transferrin saturation increased progressively over 4 hours to a mean of 64%, and non-transferrin-bound iron appeared, reaching a mean of 3.2μM. The increased concentrations of non-transferrin-bound iron correlated with enhanced proliferation in vitro of a pathogenic strain of Escherichia coli (r = 0.94, P = .002). Therefore, circulating non-transferrin-bound iron derived from rapid clearance of transfused, older stored RBCs may enhance transfusion-related complications, such as infection.

  15. Transfusion of human volunteers with older, stored red blood cells produces extravascular hemolysis and circulating non–transferrin-bound iron

    PubMed Central

    Brittenham, Gary M.; Billote, Genia B.; Francis, Richard O.; Ginzburg, Yelena Z.; Hendrickson, Jeanne E.; Jhang, Jeffrey; Schwartz, Joseph; Sharma, Shruti; Sheth, Sujit; Sireci, Anthony N.; Stephens, Hannah L.; Stotler, Brie A.; Wojczyk, Boguslaw S.; Zimring, James C.; Spitalnik, Steven L.

    2011-01-01

    Transfusions of RBCs stored for longer durations are associated with adverse effects in hospitalized patients. We prospectively studied 14 healthy human volunteers who donated standard leuko-reduced, double RBC units. One unit was autologously transfused “fresh” (3-7 days of storage), and the other “older” unit was transfused after 40 to 42 days of storage. Of the routine laboratory parameters measured at defined times surrounding transfusion, significant differences between fresh and older transfusions were only observed in iron parameters and markers of extravascular hemolysis. Compared with fresh RBCs, mean serum total bilirubin increased by 0.55 mg/dL at 4 hours after transfusion of older RBCs (P = .0003), without significant changes in haptoglobin or lactate dehydrogenase. In addition, only after the older transfusion, transferrin saturation increased progressively over 4 hours to a mean of 64%, and non–transferrin-bound iron appeared, reaching a mean of 3.2μM. The increased concentrations of non–transferrin-bound iron correlated with enhanced proliferation in vitro of a pathogenic strain of Escherichia coli (r = 0.94, P = .002). Therefore, circulating non–transferrin-bound iron derived from rapid clearance of transfused, older stored RBCs may enhance transfusion-related complications, such as infection. The trial was registered with www.clinicaltrials.gov as #NCT01319552. PMID:22021369

  16. Human serum transferrin: is there a link among autism, high oxalate levels, and iron deficiency anemia?

    PubMed

    Luck, Ashley N; Bobst, Cedric E; Kaltashov, Igor A; Mason, Anne B

    2013-11-19

    It has been previously suggested that large amounts of oxalate in plasma could play a role in autism by binding to the bilobal iron transport protein transferrin (hTF), thereby interfering with iron metabolism by inhibiting the delivery of iron to cells. By examining the effect of the substitution of oxalate for the physiologically utilized synergistic carbonate anion in each lobe of hTF, we sought to provide a molecular basis for or against such a role. Our work clearly shows both qualitatively (6 M urea gels) and quantitatively (kinetic analysis by stopped-flow spectrofluorimetry) that the presence of oxalate in place of carbonate in each binding site of hTF does indeed greatly interfere with the removal of iron from each lobe (in the absence and presence of the specific hTF receptor). However, we also clearly demonstrate that once the iron is bound within each lobe of hTF, neither anion can displace the other. Additionally, as verified by urea gels and electrospray mass spectrometry, formation of completely homogeneous hTF-anion complexes requires that all iron must first be removed and hTF then reloaded with iron in the presence of either carbonate or oxalate. Significantly, experiments described here show that carbonate is the preferred binding partner; i.e., even if an equal amount of each anion is available during the iron loading process, the hTF-carbonate complex is formed.

  17. Human serum transferrin: Is there a link between autism, high oxalate and iron deficiency anemia?

    PubMed Central

    Luck, Ashley N.; Bobst, Cedric E.; Kaltashov, Igor A.; Mason, Anne B.

    2013-01-01

    It has been previously suggested that high amounts of oxalate in plasma could play a role in autism by binding to the bilobal iron transport protein transferrin (hTF) thereby interfering with iron metabolism by inhibiting iron delivery to cells. By examining the effect of the substitution of oxalate for the physiologically utilized synergistic carbonate anion in each lobe of hTF we sought to provide a molecular basis for or against such a role. Our work clearly shows both qualitatively (6 M urea gels) and quantitatively (kinetic analysis by stop flow spectrofluorimetry) that the presence of oxalate in place of carbonate in each binding site of hTF does indeed greatly interfere with iron removal from each lobe (both in the absence and presence of the specific hTF receptor). However, we also clearly demonstrate that once the iron is bound within each lobe of hTF, neither anion can displace the other. Additionally, as verified by urea gels and electrospray mass spectrometry, formation of completely homogeneous hTF-anion complexes requires that all iron must first be removed and hTF then reloaded with iron in the presence of either carbonate or oxalate. Of significance, experiments described herein show that carbonate is the preferred binding partner, i.e., even if an equal amount of each anion is available during the iron loading process the hTF-carbonate complex is formed. PMID:24152109

  18. Observation of interactions of human serum components with transferrin by affinity capillary electrophoresis.

    PubMed

    Taga, Atsushi; Maruyama, Rie; Yamamoto, Yuka; Honda, Susumu

    2008-01-22

    Interaction of human transferrin (TF) with human serum components was investigated by affinity capillary electrophoresis. It was found that any peaks of human serum protein fractions did not give migration time change on addition of intact TF to running buffer (50mM phosphate buffer, pH 7.5), whereas two peaks belonging to alpha-globulin fraction showed marked acceleration upon addition of desialylated TF. These results provide strong evidence that the sialic acid residue in TF masks its binding ability to serum proteins. The association constants of desialylated TF to these interactive components, estimated based on the double reciprocal plot of migration time change vs. glycoprotein concentration, were at a high level of 10(7)M(-1). TF is well known as a ferric ion transfer protein, and hence formation of this protein might be changed by ferric ion. The presence of iron(II) played no essential role in this interaction, though its influence was not negligible.

  19. Differential requirement for N-ethylmaleimide-sensitive factor in endosomal trafficking of transferrin receptor from anterograde trafficking of vesicular stomatitis virus glycoprotein G.

    PubMed

    Fan, Jiannan; Zhou, Xiaoxu; Wang, Yanli; Kuang, Cuifang; Sun, Yonghong; Liu, Xu; Toomre, Derek; Xu, Yingke

    2017-01-01

    N-ethylmaleimide-sensitive fusion factor (NSF) is an ATPase that plays a crucial role in vesicular transport. Here, we examined the effects of NSF knockdown on Golgi structure and different vesicle trafficking pathways in mammalian cells. NSF knockdown caused Golgi fragmentation and abolished transferrin receptor exocytosis, defects that were rescued by RNAi-resistant NSF. Strikingly, NSF deficiency in HeLa cells barely affected cell viability, anterograde trafficking of vesicular stomatitis virus glycoprotein G and transferrin endocytosis. These results confirm the central role of NSF in Golgi structure and reveal differential requirement of NSF for exocytic recycling and constitutive trafficking pathways. © 2016 Federation of European Biochemical Societies.

  20. Mutational analysis of C-lobe ligands of human serum transferrin: insights into the mechanism of iron release.

    PubMed

    Mason, Anne B; Halbrooks, Peter J; James, Nicholas G; Connolly, Susan A; Larouche, Julia R; Smith, Valerie C; MacGillivray, Ross T A; Chasteen, N Dennis

    2005-06-07

    Each homologous lobe of human serum transferrin (hTF) has one Fe(3+) ion bound by an aspartic acid, a histidine, two tyrosine residues, and two oxygens from the synergistic anion, carbonate. Extensive characterization of these ligands in the N-terminal lobe has been carried out. Despite sharing the same set of ligands, there is a substantial amount of evidence that the N- and C-lobes are inequivalent. Studies of full-length hTF have shown that iron release from each lobe is kinetically distinguishable. To simplify the assessment of mutations in the C-lobe, we have created mutant hTF molecules in which the N-lobe binds iron with high affinity or not at all. Mutations targeting the C-lobe liganding residues have been introduced into these hTF constructs. UV-visible spectral, kinetic, and EPR studies have been undertaken to assess the effects of each mutation and to allow direct comparison to the N-lobe. As found for the N-lobe, the presence of Y517 in the C-lobe (equivalent to Y188 in the N-lobe) is absolutely essential for the binding of iron. Unlike the N-lobe, however, mutation of Y426 (equivalent to Y95) does not produce a stable complex with iron. For the mutants that retain the ability to bind iron (D392S and H585A), the rates of release are considerably slower than those measured for equivalent mutations in the N-lobe at both pH 7.4 and pH 5.6. Equilibrium binding experiments with HeLa S(3) cells indicate that recombinant hTF, in which Y426 or H585 is mutated, favor a closed or nearly closed conformation while those with mutations of the D392 or Y517 ligands appear to promote an open conformation. The differences in the effects of mutating the liganding residues in the two lobes and the subtle indications of cooperativity between lobes point to the importance of the transferrin receptor in effecting iron release from the C-lobe. Significantly, the equilibrium binding experiments also indicate that, regardless of which lobe contains the iron, the free energy of

  1. Escape from bacterial iron piracy through rapid evolution of transferrin

    PubMed Central

    Barber, Matthew F.; Elde, Nels C.

    2015-01-01

    Iron sequestration provides an innate defense termed nutritional immunity, leading pathogens to scavenge iron from hosts. Although the molecular basis of this battle for iron is established, its potential as a force for evolution at host-pathogen interfaces is unknown. We show that the iron transport protein transferrin is engaged in ancient and ongoing evolutionary conflicts with TbpA, a transferrin surface receptor from bacteria. Single substitutions in transferrin at rapidly evolving sites reverse TbpA binding, providing a mechanism to counteract bacterial iron piracy among great apes. Furthermore, the C2 transferrin polymorphism in humans evades TbpA variants from Haemophilus influenzae, revealing a functional basis for standing genetic variation. These findings identify a central role for nutritional immunity in the persistent evolutionary conflicts between primates and bacterial pathogens. PMID:25504720

  2. Targeting anti-transferrin receptor antibody (OX26) and OX26-conjugated liposomes to brain capillary endothelial cells using in situ perfusion.

    PubMed

    Gosk, Sara; Vermehren, Charlotte; Storm, Gert; Moos, Torben

    2004-11-01

    Brain capillary endothelial cells (BCECs) express transferrin receptors. The uptake of a potential drug vector (OX26, or anti-transferrin receptor antibody IgG2a) conjugated to polyethyleneglycol-coated liposomes by BCECs was studied using in situ perfusion in 18-day-old rats in which the uptake of OX26 is almost twice as high as in the adult rat. Using radio-labeling, the uptake of OX26 by BCECs after 15-minute perfusion was approximately 16 times higher than that of nonimmune IgG2a (Ni-IgG2a). OX26 and OX26-conjugated liposomes selectively distributed to BCECs, leaving choroid plexus epithelium, neurons, and glia unlabeled. Ni-IgG2a and unconjugated liposomes did not reveal any labeling of BCECs. The labeling of BCECs by OX26 was profoundly higher than that of transferrin. Perfusion with albumin for 15 minutes did not reveal any labeling of neurons or glia, thus confirming the integrity of the blood-brain barrier. The failure to label neurons and glia shows that OX26 and OX26-conjugated liposomes did not pass through BCECs. The expression of transferrin receptors by endothelial cells selective to the brain qualifies OX26 as a candidate for blood-to-endothelium transport. A specifically designed formulation of liposomes may allow for their degradation within BCECs, leading to subsequent transport of liposomal cargo further into the brain.

  3. Acetaldehyde/alcohol dehydrogenase-2 (EhADH2) and clathrin are involved in internalization of human transferrin by Entamoeba histolytica.

    PubMed

    Reyes-López, Magda; Bermúdez-Cruz, Rosa María; Avila, Eva E; de la Garza, Mireya

    2011-01-01

    Transferrin (Tf) is a host glycoprotein capable of binding two ferric-iron ions to become holotransferrin (holoTf), which transports iron in to all cells. Entamoeba histolytica is a parasitic protozoan able to use holoTf as a sole iron source in vitro. The mechanism by which this parasite scavenges iron from holoTf is unknown. An E. histolytica holoTf-binding protein (EhTfbp) was purified by using an anti-human transferrin receptor (TfR) monoclonal antibody. EhTfbp was identified by MS/MS analysis and database searches as E. histolytica acetaldehyde/alcohol dehydrogenase-2 (EhADH2), an iron-dependent enzyme. Both EhTfbp and EhADH2 bound holoTf and were recognized by the anti-human TfR antibody, indicating that they correspond to the same protein. It was found that the amoebae internalized holoTf through clathrin-coated pits, suggesting that holoTf endocytosis could be important for the parasite during colonization and invasion of the intestinal mucosa and liver.

  4. Incorporation of 5-hydroxytryptophan into transferrin and its receptor allows assignment of the pH induced changes in intrinsic fluorescence when iron is released

    PubMed Central

    James, Nicholas G.; Byrne, Shaina L.; Mason, Anne B.

    2008-01-01

    Human serum transferrin (hTF) is a bilobal glycoprotein that transports iron to cells. At neutral pH, diferric hTF binds with nM affinity to the transferrin receptor (TFR) on the cell surface. The complex is taken into the cell where, at the acidic pH of the endosome (~pH 5.6), iron is released. Since iron coordination strongly quenches the intrinsic tryptophan fluorescence of hTF, the increase in the fluorescent signal reports the rate constant(s) of iron release. At pH 5.6, the TFR considerably enhances iron release from the C-lobe (with little effect on iron release from the N-lobe). The recombinant soluble TFR is a dimer with 11 tryptophan residues per monomer. In the hTF/TFR complex these residues could contribute to and compromise the readout ascribed to iron release from hTF. We report that compared to FeC hTF alone, the increase in the fluorescent signal from the preformed complex of FeC hTF and the TFR at pH 5.6 is significantly quenched (75%). To dissect the contributions of hTF and the TFR to the change in fluorescence, 5-hydroxytryptophan was incorporated into each using our mammalian expression system. Selective excitation of the samples at 280 or 315 nm shows that the TFR contributes little or nothing to the increase in fluorescence when ferric iron is released from FeC hTF. Quantum yield determinations of TFR, FeC hTF and the FeC hTF/TFR complex strongly support our interpretation of the kinetic data. PMID:19103311

  5. Incorporation of 5-hydroxytryptophan into transferrin and its receptor allows assignment of the pH induced changes in intrinsic fluorescence when iron is released.

    PubMed

    James, Nicholas G; Byrne, Shaina L; Mason, Anne B

    2009-03-01

    Human serum transferrin (hTF) is a bilobal glycoprotein that transports iron to cells. At neutral pH, diferric hTF binds with nM affinity to the transferrin receptor (TFR) on the cell surface. The complex is taken into the cell where, at the acidic pH of the endosome ( approximately pH 5.6), iron is released. Since iron coordination strongly quenches the intrinsic tryptophan fluorescence of hTF, the increase in the fluorescent signal reports the rate constant(s) of iron release. At pH 5.6, the TFR considerably enhances iron release from the C-lobe (with little effect on iron release from the N-lobe). The recombinant soluble TFR is a dimer with 11 tryptophan residues per monomer. In the hTF/TFR complex these residues could contribute to and compromise the readout ascribed to iron release from hTF. We report that compared to Fe(C) hTF alone, the increase in the fluorescent signal from the preformed complex of Fe(C) hTF and the TFR at pH 5.6 is significantly quenched (75%). To dissect the contributions of hTF and the TFR to the change in fluorescence, 5-hydroxytryptophan was incorporated into each using our mammalian expression system. Selective excitation of the samples at 280 or 315 nm shows that the TFR contributes little or nothing to the increase in fluorescence when ferric iron is released from Fe(C) hTF. Quantum yield determinations of TFR, Fe(C) hTF and the Fe(C) hTF/TFR complex strongly support our interpretation of the kinetic data.

  6. Transferrin-bearing maghemite nano-constructs for biomedical applications

    NASA Astrophysics Data System (ADS)

    Piraux, H.; Hai, J.; Gaudisson, T.; Ammar, S.; Gazeau, F.; El Hage Chahine, J. M.; Hémadi, M.

    2015-05-01

    Superparamagnetic nanoparticles (NPs) are widely used in biomedicine for hyperthermia and magnetic resonance imagery. Targeting them to specific cancerous cells is, therefore, of a great value for therapy and diagnostic. Transferrin and its receptor constitute the major iron-acquisition system in human. The former crosses the plasma membrane within a few minutes by receptor-mediated endocytosis. Thus, transferrin can be a valuable vector for the delivery of NPs to specific cells and across the blood brain barrier. For such a purpose, three different sizes of maghemite NPs (5, 10, and 15 nm) were synthesized by the polyol method, coated with 3-aminopropyltriethoxysilane, and coupled to transferrin by amide bonds. The number of transferrins per nanoparticle was determined. Raw nanoparticles and the "transferrin-nanoparticle" constructs were characterized. The magnetic properties and the colloidal stability of raw NPs and transferrin-NP constructs were measured and analyzed in relation to their inorganic core size variation. They all proved to be good candidates for nanoparticle targeting for biomedical application.

  7. The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia

    PubMed Central

    Perez Bay, Andres E; Schreiner, Ryan; Mazzoni, Francesca; Carvajal-Gonzalez, Jose M; Gravotta, Diego; Perret, Emilie; Lehmann Mantaras, Gullermo; Zhu, Yuan-Shan; Rodriguez-Boulan, Enrique J

    2013-01-01

    Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis. PMID:23749212

  8. Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.

    PubMed

    Mukaiyama, Hiroyuki; Giga-Hama, Yuko; Tohda, Hideki; Takegawa, Kaoru

    2009-11-01

    The effect of medium supplementation on heterologous production of human serum transferrin (hTF) in the fission yeast Schizosaccharomyces pombe has been investigated. The productivity of recombinant hTF was low in wild-type S. pombe cells. To overcome this impediment, culture media supplements were screened for their ability to improve secretion of hTF. Casamino acids (CAA), which have been reported to increase heterologous protein productivity in Pichia pastoris, improved the secretion hTF by more than fourfold. An anion surfactant deoxycholate or polyethylene glycol also improved the secretion hTF. Interestingly, dextran sodium sulfate (DSS), a poly-anion surfactant, was found to enhance production of secreted hTF better than any other supplement tested. Addition of DSS in the presence of 2% CAA exhibited a synergistic effect on increasing hTF secretion, resulting in an increase of about sevenfold relative to conventional conditions. Cell growth was not found to be affected by the addition of DSS or CAA. DSS may act as a surfactant and may also facilitate the anchoring of liposomes, and these properties may contribute to efficient secretion or exocytosis through the plasma membrane.

  9. N-lobe versus C-lobe complexation of bismuth by human transferrin.

    PubMed Central

    Sun, H; Li, H; Mason, A B; Woodworth, R C; Sadler, P J

    1999-01-01

    Interactions of recombinant N-lobe of human serum transferrin (hTF/2N) with Bi3+, a metal ion widely used in medicine, have been investigated by both UV and NMR spectroscopy. The bicarbonate-independent stability constant for Bi3+ binding (K*) to hTF/2N was determined to be log K* 18.9+/-0.2 in 5 mM bicarbonate/10 mM Hepes buffer at 310 K, pH7.4. The presence of Fe3+ in the C-lobe of intact hTF perturbed Bi3+ binding to the N-lobe, whereas binding of Bi3+ to the C-lobe was unaffected by the presence of Fe3+ in the N-lobe. Reactions of Bi3+ (as bismuth nitrilotriacetate or ranitidine bismuth citrate) with hTF/2N in solutions containing 10 mM bicarbonate induced specific changes to high-field 1H-NMR peaks. The 1H co-ordination shifts induced by Bi3+ were similar to those induced by Fe3+ and Ga3+, suggesting that Bi3+ binding causes similar structural changes to those induced by hTF/2N. 13C-NMR data showed that carbonate binds to hTF/2N concomitantly with Bi3+. PMID:9854031

  10. Interaction of [V(IV) O(acac)2 ] with Human Serum Transferrin and Albumin.

    PubMed

    Correia, Isabel; Chorna, Ielyzaveta; Cavaco, Isabel; Roy, Somnath; Kuznetsov, Maxim L; Ribeiro, Nádia; Justino, Gonçalo; Marques, Fernanda; Santos-Silva, Teresa; Santos, Marino F A; Santos, Hugo M; Capelo, José L; Doutch, James; Pessoa, João Costa

    2017-08-17

    [VO(acac)2 ] is a remarkable vanadium compound and has potential as a therapeutic drug. It is important to clarify how it is transported in blood, but the reports addressing its binding to serum proteins have been contradictory. We use several spectroscopic and mass spectrometric techniques (ESI and MALDI-TOF), small-angle X-ray scattering and size exclusion chromatography (SEC) to characterize solutions containing [VO(acac)2 ] and either human serum apotransferrin (apoHTF) or albumin (HSA). DFT and modeling protein calculations are carried out to disclose the type of binding to apoHTF. The measured circular dichroism spectra, SEC and MALDI-TOF data clearly prove that at least two VO-acac moieties may bind to apoHTF, most probably forming [V(IV) O(acac)(apoHTF)] complexes with residues of the HTF binding sites. No indication of binding of [VO(acac)2 ] to HSA is obtained. We conclude that V(IV) O-acac species may be transported in blood by transferrin. At very low complex concentrations speciation calculations suggest that [(VO)(apoHTF)] species form. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Immunoregulation by low density lipoproteins in man. Inhibition of mitogen-induced T lymphocyte proliferation by interference with transferrin metabolism.

    PubMed Central

    Cuthbert, J A; Lipsky, P E

    1984-01-01

    Human low density lipoprotein (LDL, d = 1.020-1.050 g/ml) inhibits mitogen-stimulated T lymphocyte DNA synthesis. Because both LDL and transferrin bind to specific cell surface receptors and enter cells by the similar means of receptor-mediated endocytosis, and because transferrin is necessary for lymphocyte DNA synthesis, we investigated the possibility that LDL may inhibit mitogen-stimulated lymphocyte responses by interfering with transferrin metabolism. LDL inhibited mitogen-stimulated lymphocyte [3H]thymidine incorporation in a concentration-dependent manner. The degree of inhibition was most marked in serum-free cultures, but was also observed in serum-containing cultures. The addition of transferrin not only augmented mitogen-induced lymphocyte [3H]thymidine incorporation in serum-free medium but also completely reversed the inhibitory effect of LDL in both serum-free and serum-containing media. Similar results were obtained when lymphocyte proliferation was assayed by counting the number of cells in culture. Transferrin also reversed the inhibition of lymphocyte responses caused by very low density lipoproteins and by cholesterol. The ability of transferrin to reverse the inhibitory effect of lipoproteins was specific, in that native but not denatured transferrin was effective whereas a variety of other proteins were ineffective. These results indicate that LDL inhibits mitogen-stimulated lymphocyte responses by interfering with transferrin metabolism. LDL only inhibited lymphocyte responses after a 48-h incubation if present from the initiation of the culture. By contrast, transferrin reversed inhibition when added after 24 h of the 48-h incubation. LDL did not inhibit lymphocyte responses by nonspecifically associating with transferrin. In addition, the acquisition of specific lymphocyte transferrin receptors was not blocked by LDL. Moreover, transferrin did not prevent the binding and uptake of fluorescent-labeled LDL by activated lymphocytes

  12. Serum Hepcidin and Soluble Transferrin Receptor in the Assessment of Iron Metabolism in Children on a Vegetarian Diet.

    PubMed

    Ambroszkiewicz, Jadwiga; Klemarczyk, Witold; Mazur, Joanna; Gajewska, Joanna; Rowicka, Grażyna; Strucińska, Małgorzata; Chełchowska, Magdalena

    2017-03-24

    The aim of this study was to assess the effect of vegetarian diet on iron metabolism parameters paying special attention to serum hepcidin and soluble transferrin receptor (sTfR) concentrations in 43 prepubertal children (age range 4.5-9.0 years) on vegetarian and in 46 children on omnivorous diets. There were no significant differences according to age, weight, height, and body mass index (BMI) between vegetarian and omnivorous children. Vegetarians had similar intake of iron and vitamin B12 and a significantly higher intake of vitamin C (p < 0.05) compared with non-vegetarians. Hematologic parameters and serum iron concentrations were within the reference range in both groups of children. Serum transferrin levels were similar in all subjects; however, ferritin concentrations were significantly (p < 0.01) lower in vegetarians than in omnivores. In children on a vegetarian diet, median hepcidin levels were lower (p < 0.05) but sTfR concentrations significantly higher (p < 0.001) compared with omnivorous children. In the multivariate regression model, we observed associations between hepcidin level and ferritin concentration (β = 0.241, p = 0.05) in the whole group of children as well as between hepcidin concentration and CRP level (β = 0.419, p = 0.047) in vegetarians. We did not find significant associations with concentration of sTfR and selected biochemical, anthropometric, and dietary parameters in any of the studied groups of children. As hematologic parameters and iron concentrations in vegetarians and omnivores were comparable and ferritin level was lower in vegetarians, we suggest that inclusion of novel markers, in particular sTfR (not cofounded by inflammation) and hepcidin, can better detect subclinical iron deficiency in children following vegetarian diets.

  13. Direct thermodynamic and kinetic measurements of Fe²⁺ and Zn²⁺ binding to human serum transferrin.

    PubMed

    Terpstra, Tyson; McNally, Justin; Han, Thi-Hong-Lien; Ha-Duong, Nguyet-Thanh; El-Hage-Chahine, Jean-Michel; Bou-Abdallah, Fadi

    2014-07-01

    Human serum transferrin (hTf) is a single-chain bilobal glycoprotein that efficiently delivers iron to mammalian cells by endocytosis via the transferrin/transferrin receptor system. While extensive studies have been directed towards the study of ferric ion binding to hTf, ferrous ion interactions with the protein have never been firmly investigated owing to the rapid oxidation of Fe(II) to Fe(III) and the difficulty in maintaining a fully anaerobic environment. Here, the binding of Fe(2+) and Zn(2+) ions to hTf has been studied under anaerobic and aerobic conditions, respectively, in the presence and absence of bicarbonate by means of isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The ITC data indicate the presence of one class of strong binding sites with dissociation constants of 25.2 nM for Fe(2+) and 6.7 nM for Zn(2+) and maximum binding stoichiometries of 1 Zn(2+) (or 1 Fe(2+)) per hTf molecule. With either metal, the binding interaction was achieved by both favorable enthalpy and entropy changes (ΔH(0)~-12 kJ/mol and ΔS(0)~106 J/mol·K for Fe(2+) and ΔH(0)~-18 kJ/mol and ΔS(0)~97 J/mol·K for Zn(2+)). The large and positive entropy values are most likely due to the change in the hydration of the protein and the metal ions upon interaction. Rapid kinetics stopped-flow fluorescence spectroscopy revealed two different complexation mechanisms with different degrees of conformational changes upon metal ion binding. Our results are discussed in terms of a plausible scenario for iron dissociation from transferrin by which the highly stable Fe(3+)-hTf complex might be reduced to the more labile Fe(2+) ion before iron is released to the cytosol. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1

    PubMed Central

    Daniels-Wells, Tracy R.; Helguera, Gustavo; Rodríguez, José A.; Leoh, Lai Sum; Erb, Michael A.; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L.

    2012-01-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin. PMID:23085102

  15. Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.

    PubMed

    Daniels-Wells, Tracy R; Helguera, Gustavo; Rodríguez, José A; Leoh, Lai Sum; Erb, Michael A; Diamante, Graciel; Casero, David; Pellegrini, Matteo; Martínez-Maza, Otoniel; Penichet, Manuel L

    2013-02-01

    We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.

  16. Expression of functional human transferrin in stably transfected Drosophila S2 cells.

    PubMed

    Lim, Hye Jung; Kim, Yeon Kyu; Hwang, Dong Soo; Cha, Hyung Joon

    2004-01-01

    Human transferrin (hTf) is a serum glycoprotein involved in Fe3+ transport. Here, a plasmid encoding the hTf gene fused with a hexahistidine (His6) epitope tag under Drosophila metallothionein promoter (pMT) was stably transfected into Drosophila melanogaster S2 cells as a nonlytic plasmid-based system. Following 3 days of copper sulfate induction, transfected S2 cells were found to secrete hTf into serum-free culture medium at a competitively high expression level of 40.8 microg/mL, producing 6.8 microg/mL/day in a 150-mL spinner flask culture. Purification of secreted recombinant hTf using immobilized metal affinity chromatography (IMAC) yielded 95.5% pure recombinant hTf with a recovery of 32%. According to MALDI-TOF mass spectrometry analysis, purified S2 cell-derived His6-tagged recombinant hTf had a molecular weight (76.4 kDa) smaller than that of native apo-hTf (78.0 kDa). 2-Dimensional gel electrophoresis patterns showed recombinant hTf had a simpler and less acidic profile compared to that of native hTf. These data suggest recombinant hTf was incompletely (noncomplex) glycosylated and lacked sialic acids on N-glycans. However, this difference in N-glycan structure compared to native hTf had no effect on the iron-binding activity of recombinant hTf. The present data show that a plasmid-based stable transfection S2 cell system can be successfully employed as an alternative for producing secreted functional recombinant hTf.

  17. Anti-transferrin receptor-modified amphotericin B-loaded PLA–PEG nanoparticles cure Candidal meningitis and reduce drug toxicity

    PubMed Central

    Tang, Xiaolong; Liang, Yong; Zhu, Yongqiang; Xie, Chunmei; Yao, Aixia; Chen, Li; Jiang, Qinglin; Liu, Tingting; Wang, Xiaoyu; Qian, Yunyun; Wei, Jia; Ni, Wenxuan; Dai, Jingjing; Jiang, Zhenyou; Hou, Wei

    2015-01-01

    Fatal fungal infections in central nervous system (CNS) can occur through hematogenous spread or direct extension. At present, hydrophobic amphotericin B (AMB) is the most effective antifungal drug in clinical trials. However, AMB is hydrophobic and therefore penetrates poorly into the CNS, and therapeutic levels of AMB are hard to achieve. The transferrin receptor (TfR/CD71) located at the blood–brain barrier mediates transferrin transcytosis. In order to enhance the receptor-mediated delivery of AMB into CNS with therapeutic level, an anti-TfR antibody (OX26)-modified AMB-loaded PLA (poly[lactic acid])–PEG (polyethylene glycol)-based micellar drug delivery system was constructed. The prepared OX26-modified AMB-loaded nanoparticles (OX26-AMB-NPs) showed significant reduction of CNS fungal burden and an increase of mouse survival time. In conclusion, OX26-AMB-NPs represent a promising novel drug delivery system for intracerebral fungal infection. PMID:26491294

  18. Single-Particle Tracking Shows that a Point Mutation in the Carnivore Parvovirus Capsid Switches Binding between Host-Specific Transferrin Receptors

    PubMed Central

    Lee, Donald W.; Allison, Andrew B.; Bacon, Kaitlyn B.

    2016-01-01

    Determining how viruses infect new hosts via receptor-binding mechanisms is important for understanding virus emergence. We studied the binding kinetics of canine parvovirus (CPV) variants isolated from raccoons—a newly recognized CPV host—to different carnivore transferrin receptors (TfRs) using single-particle tracking. Our data suggest that CPV may utilize adhesion-strengthening mechanisms during TfR binding and that a single mutation in the viral capsid at VP2 position 300 can profoundly alter receptor binding and infectivity. PMID:26889026

  19. The Significance of Serum Transferrin Receptor Levels in the Diagnosis of the Coexistence of Anemia of Chronic Disease and Iron Deficiency Anemia

    PubMed Central

    Yokus, Osman; Yilmaz, Bilal; Albayrak, Murat; Balcik, Ozlem Sahin; Helvaci, Mehmet Rami; Sennaroglu, Engin

    2011-01-01

    Objective: Iron deficiency anemia is the most common cause of microcytic anemia throughout the world. Ferritin levels are good indicators of iron stores; however, levels may increase irrespective of iron stores in cases of chronic disease. Therefore, it is difficult to diagnose iron deficiency anemia coexisting with anemia of chronic disease. Materials and Methods: To determine the level of transferrin receptor in subjects, 30 patients with iron deficiency anemia, 30 patients with anemia of chronic disease and 30 patients with both diseases were included in the study. Results: Mean serum transferrin receptor levels were 5.99±2.98 mg/L in the iron deficiency anemia group, 1.90±1.15 mg/L in the anemia of chronic disease group and 3.07±0.90 mg/L in the combination group. Comparing groups with each other revealed significant differences (p<0.05). Conclusion: It is concluded that the assessment of serum transferrin receptor levels is a useful method for the diagnosis of iron deficiency anemia in patients. PMID:25610152

  20. The significance of serum transferrin receptor levels in the diagnosis of the coexistence of anemia of chronic disease and iron deficiency anemia.

    PubMed

    Yokus, Osman; Yilmaz, Bilal; Albayrak, Murat; Balcik, Ozlem Sahin; Helvaci, Mehmet Rami; Sennaroglu, Engin

    2011-04-01

    Iron deficiency anemia is the most common cause of microcytic anemia throughout the world. Ferritin levels are good indicators of iron stores; however, levels may increase irrespective of iron stores in cases of chronic disease. Therefore, it is difficult to diagnose iron deficiency anemia coexisting with anemia of chronic disease. To determine the level of transferrin receptor in subjects, 30 patients with iron deficiency anemia, 30 patients with anemia of chronic disease and 30 patients with both diseases were included in the study. Mean serum transferrin receptor levels were 5.99±2.98 mg/L in the iron deficiency anemia group, 1.90±1.15 mg/L in the anemia of chronic disease group and 3.07±0.90 mg/L in the combination group. Comparing groups with each other revealed significant differences (p<0.05). It is concluded that the assessment of serum transferrin receptor levels is a useful method for the diagnosis of iron deficiency anemia in patients.

  1. Soluble transferrin receptor levels are positively associated with insulin resistance but not with the metabolic syndrome or its individual components.

    PubMed

    Suárez-Ortegón, Milton Fabian; McLachlan, Stela; Wild, Sarah H; Fernández-Real, José Manuel; Hayward, Caroline; Polašek, Ozren

    2016-10-01

    The metabolic syndrome (MetS) is known to be associated with elevated serum ferritin levels. The possible association with other Fe markers has been less well studied. We aimed to investigate the cross-sectional association of soluble transferrin receptor (sTfR) and ferritin levels with the MetS components, insulin resistance and glycosylated Hb (HbA1C). The sample consisted of 725 adults, aged 19-93 years (284 men, 151 premenopausal and 290 postmenopausal women), from the Croatian island of Vis. Serum sTfR and ferritin levels were measured by immunoturbidimetry and electrochemiluminescence assays, respectively. The MetS was defined using modified international consensus criteria. Logistic and linear regression analyses were conducted to investigate the associations adjusting for age, fibrinogen, smoking status, alcohol consumption and BMI. Prevalence of the MetS was 48·7 %. Standardised values of ferritin were positively associated with all of the MetS components (except high blood pressure and waist circumference) in men (P0·05). sTfR levels could be spuriously elevated in subjects with insulin resistance and without association with the MetS or its components. We conclude that different markers of Fe metabolism are not consistently associated with cardiometabolic risk.

  2. Quantitative assessment of erythropoiesis and functional classification of anemia based on measurements of serum transferrin receptor and erythropoietin.

    PubMed

    Beguin, Y; Clemons, G K; Pootrakul, P; Fillet, G

    1993-02-15

    We evaluated the quantitative value of a simple model of erythropoiesis, based on the basic assumptions that the red blood cell (RBC) mass determines erythropoietin (Epo) production, which in turn stimulates erythropoietic activity. The RBC mass was quantitated by direct isotopic measurement (RCM), Epo production by serum Epo levels, and erythropoiesis by the ferrokinetic measurement of the erythron transferrin uptake (ETU), the serum transferrin receptor (TfR) level, and the reticulocyte (retic) index, and was completed by an evaluation of overall marrow erythron cellularity. We studied a total of 195 subjects, including 31 normal individuals, 38 patients with polycythemia, and 126 patients with various forms of anemia. Instead of only quantitating Epo and erythropoiesis in absolute terms, we also evaluated them in relation to the degree of anemia or polycythemia, and expressed the results as a ratio of observed values to values predicted from the regression equations between hematocrit (Hct) on the one hand, and Epo, TfR, and ETU on the other, obtained in a carefully selected subpopulation. The slope of the regression of TfR (as well as ETU) versus Hct was very similar to the slope of the regression of Epo versus Hct. Average EPO and TfR (as well as ETU) values predicted from the regression equations were quite comparable to observed values in most groups of subjects, with exceptions predictable from knowledge of the pathophysiology of these hematologic disorders. We identified four major patterns of erythropoiesis, ie, normal, hyperdestruction (with variants of hemolysis or ineffective erythropoiesis), intrinsic marrow hypoproliferation, and defective Epo production. Dissecting out groups of patients showed much greater heterogeneity than when patients were analyzed by group. This was particularly true in the case of a hypoproliferative component being combined with hyperdestruction, giving what we called a "mixed disorder of erythropoiesis." We conclude that

  3. YTRF is the conserved internalization signal of the transferrin receptor, and a second YTRF signal at position 31-34 enhances endocytosis.

    PubMed

    Collawn, J F; Lai, A; Domingo, D; Fitch, M; Hatton, S; Trowbridge, I S

    1993-10-15

    By functional analysis of mutant human transferrin receptors (TR) expressed in chicken embryo fibroblasts, we previously identified a tetrapeptide sequence, Y20TRF23, within the 61-residue cytoplasmic tail as the signal for high-efficiency endocytosis (Collawn, J. F., Stangel, M., Kuhn, L. A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J.A. (1990) Cell 63, 1061-1072). It has been inferred from other studies, however, that the TR internalization signal was localized to a much larger region, residues 7 through 26 (Girones, N., Alvarez, E., Seth, A., Lin, I-M., Latour, D.A., and Davis, R.J. (1991) J. Biol. Chem. 266, 19006-19012). Additionally, Tyr20 was reported to not be conserved in the Chinese hamster cytoplasmic tail sequence (Alvarez, E., Girones, N., and Davis, R.J. (1990) Biochem. J. 267, 31-35). In the studies reported here, we examined the effect of insertion of an extra copy of a YTRF sequence at three different locations within the human TR cytoplasmic domain and show that the insertion of another YTRF signal at position 31-34 in the wild-type TR, but not the other two locations, increases the rate of endocytosis 2-fold. Furthermore, introduction of YTRF at position 31-34 in an internalization-defective mutant receptor restores endocytosis to wild-type levels, indicating that YTRF signals at either positions 20-23 or 31-34 are necessary and sufficient to promote TR internalization and function in an independent and additive manner. We also report the complete primary structure of the Chinese hamster TR deduced from its cDNA sequence and show that the Tyr20 as well as the complete YTRF motif is conserved.

  4. Overexpressed or intraperitoneally injected human transferrin prevents photoreceptor degeneration in rd10 mice.

    PubMed

    Picard, Emilie; Jonet, Laurent; Sergeant, Claire; Vesvres, Marie-Hélène; Behar-Cohen, Francine; Courtois, Yves; Jeanny, Jean-Claude

    2010-12-08

    Retinal degeneration has been associated with iron accumulation in age-related macular degeneration (AMD), and in several rodent models that had one or several iron regulating protein impairments. We investigated the iron concentration and the protective role of human transferrin (hTf) in rd10 mice, a model of retinal degeneration. The proton-induced X-ray emission (PIXE) method was used to quantify iron in rd10 mice 2, 3, and 4 weeks after birth. We generated mice with the β-phosphodiesterase mutation and hTf expression by crossbreeding rd10 mice with TghTf mice (rd10/hTf mice). The photoreceptor loss and apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling in 3-week-old rd10/hTf mice and compared with 3-week-old rd10 mice. The neuroprotective effect of hTf was analyzed in 5-day-old rd10 mice treated by intraperitoneal administration with hTf for up to 25 days. The retinal hTf concentrations and the thickness of the outer nuclear layer were quantified in all treated mice at 25 days postnatally. PIXE analysis demonstrated an age-dependent iron accumulation in the photoreceptors of rd10 mice. The rd10/hTf mice had the rd10 mutation, expressed high levels of hTf, and showed a significant decrease in photoreceptor death. In addition, rd10 mice intraperitoneally treated with hTf resulted in the retinal presence of hTf and a dose-dependent reduction in photoreceptor degeneration. Our results suggest that iron accumulation in the retinas of rd10 mutant mice is associated with photoreceptor degeneration. For the first time, the enhanced survival of cones and rods in the retina of this model has been demonstrated through overexpression or systemic administration of hTf. This study highlights the therapeutic potential of Tf to inhibit iron-induced photoreceptor cell death observed in degenerative diseases such as retinitis pigmentosa and age-related macular degeneration.

  5. Overexpressed or intraperitoneally injected human transferrin prevents photoreceptor degeneration in rd10 mice

    PubMed Central

    Jonet, Laurent; Sergeant, Claire; Vesvres, Marie-Hélène; Behar-Cohen, Francine; Courtois, Yves; Jeanny, Jean-Claude

    2010-01-01

    Purpose Retinal degeneration has been associated with iron accumulation in age-related macular degeneration (AMD), and in several rodent models that had one or several iron regulating protein impairments. We investigated the iron concentration and the protective role of human transferrin (hTf) in rd10 mice, a model of retinal degeneration. Methods The proton-induced X-ray emission (PIXE) method was used to quantify iron in rd10 mice 2, 3, and 4 weeks after birth. We generated mice with the β-phosphodiesterase mutation and hTf expression by crossbreeding rd10 mice with TghTf mice (rd10/hTf mice). The photoreceptor loss and apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling in 3-week-old rd10/hTf mice and compared with 3-week-old rd10 mice. The neuroprotective effect of hTf was analyzed in 5-day-old rd10 mice treated by intraperitoneal administration with hTf for up to 25 days. The retinal hTf concentrations and the thickness of the outer nuclear layer were quantified in all treated mice at 25 days postnatally. Results PIXE analysis demonstrated an age-dependent iron accumulation in the photoreceptors of rd10 mice. The rd10/hTf mice had the rd10 mutation, expressed high levels of hTf, and showed a significant decrease in photoreceptor death. In addition, rd10 mice intraperitoneally treated with hTf resulted in the retinal presence of hTf and a dose-dependent reduction in photoreceptor degeneration. Conclusions Our results suggest that iron accumulation in the retinas of rd10 mutant mice is associated with photoreceptor degeneration. For the first time, the enhanced survival of cones and rods in the retina of this model has been demonstrated through overexpression or systemic administration of hTf. This study highlights the therapeutic potential of Tf to inhibit iron-induced photoreceptor cell death observed in degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. PMID:21179240

  6. Identification of new binding sites of human transferrin incubated with organophosphorus agents via Q Exactive LC-MS/MS.

    PubMed

    Sun, Fengjuan; Ding, Junjie; Yu, Huilan; Gao, Runli; Wang, Hongmei; Pei, Chengxin

    2016-06-01

    Organophosphorus agents (OPs) like sarin, VX, or soman could inhibit acetylcholinesterase activity and cause poisoning. OPs could bind many proteins, such as butyrylcholinesterase and albumin, and the adducts formed could identify the exposure. In this paper, we studied human transferrin, which was one of the proteins that could be labeled by OPs. Pure human transferrin was incubated with an overdose of organophosphorus agents, including sarin, soman, VX, tabun, cyclosarin, ethyl tabun, and propyl tabun, and then additional OPs was removed through dialysis. Trypsin was used to cleave the OP-treated proteins and Q Exactive liquid chromatography tandem mass spectrometry (Q Exactive LC-MS/MS) was used to identify them. The present study set out to accomplish two goals. The first goal was to find a good method for identifying multiple binding sites on a given protein through Q Exactive LC-MS/MS. The second goal was to investigate the labeled peptides when transferrin was incubated with a numerous molar excess of OPs. Results showed that tyrosine, lysine, and serine formed covalent bonds with OPs. Twenty OP-labeled sites were found: ten tyrosine sites (including two reported sites), seven lysine sites, and three serine sites. Characteristic fragments for labeled-tyrosine and labeled-lysine adducts were summarized in detail. In conclusion, the method by Q Exactive LC-MS/MS using in this present work is a good way to diagnose exposure to OPs accurately when the binding sites of OPs are uncertain. Novel modified peptides and the characteristic ions found in this work could help investigators assess exposure to OPs.

  7. Selective separation of ferric and non-ferric forms of human transferrin by capillary micellar electrokinetic chromatography.

    PubMed

    Nowak, Paweł; Śpiewak, Klaudyna; Nowak, Julia; Brindell, Małgorzata; Woźniakiewicz, Michał; Stochel, Grażyna; Kościelniak, Paweł

    2014-05-09

    The previously published method allowing the separation of non-ferric (iron-free) and ferric (iron-saturated) forms of human serum transferrin via capillary electrophoresis has been further developed. Using a surface response methodology and a three-factorial Doehlert design we have established a new optimized running buffer composition: 50mM Tris-HCl, pH 8.5, 22.5% (v/v) methanol, 17.5mM SDS. As a result, two previously unobserved monoferric forms of protein have been separated and identified, moreover, the loss of ferric ions from transferrin during electrophoretic separation has been considerably reduced by methanol, and the method selectivity has been yet increased resulting in a total separation of proteins exerting only subtle or none difference in mass-to-charge ratio. The new method has allowed us to monitor the gradual iron saturation of transferrin by mixing the iron-free form of protein with the buffers with different concentrations of ferric ions. It revealed continuously changing contribution of monoferric forms, characterized by different affinities of two existing iron binding sites on N- and C-lobes of protein, respectively. Afterwards, the similar experiment has been conducted on-line, i.e. inside the capillary, comparing the effectiveness of two possible modes of the reactant zones mixing: diffusion mediated and electrophoretically mediated ones. Finally, the total time of separation has been decreased down to 4min, taking the advantage from a short-end injection strategy and maintaining excellent selectivity. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Establishment of a miniaturized enzyme-linked immunosorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate.

    PubMed

    Spies, Peter; Chen, Guo Jun; Gygax, Daniel

    2008-11-01

    A miniaturized enzyme-linked immunosorbent assay (ELISA) with a reaction volume of 5 microl for human transferrin quantification has successfully been developed using an intelligent multifunctional analytical plate (IMAPlate 5RC96), the first miniature analytical platform capable of manually performing parallel liquid transfer, reaction, and analysis. This is the first article to validate the platform for the ELISA application. The data obtained from the standards in this miniaturized ELISA can well be fitted by a one-site binding reaction mode, the coefficient of variation (CV) of the whole plate for an artificial sample (spiking a known concentration of human transferrin into the assay diluent) is 7.0%, and the mean recovery is between 94 and 114% (n=96), comparable to the values from conventional ELISA in a 96-well format plate. The IMAPlate 5RC96-based miniaturized ELISA not only can reduce sample and reagent consumption to 5% of the conventional ELISA but also can shorten the reaction time. Combined with the advantages brought by miniaturization, the easy-to-handle, parallel, and simultaneous liquid transfer features of the IMAPlate 5RC96 provide a completely new lab tool for manually performing high-throughput ELISA. Our results demonstrate that the IMAPlate 5RC96 is a convenient, robust, high-throughput lab device feasible for miniaturized ELISA in an ordinary laboratory.

  9. Erythrocytic Iron Deficiency Enhances Susceptibility to Plasmodium chabaudi Infection in Mice Carrying a Missense Mutation in Transferrin Receptor 1.

    PubMed

    Lelliott, Patrick M; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

    2015-11-01

    The treatment of iron deficiency in areas of high malaria transmission is complicated by evidence which suggests that iron deficiency anemia protects against malaria, while iron supplementation increases malaria risk. Iron deficiency anemia results in an array of pathologies, including reduced systemic iron bioavailability and abnormal erythrocyte physiology; however, the mechanisms by which these pathologies influence malaria infection are not well defined. In the present study, the response to malaria infection was examined in a mutant mouse line, Tfrc(MRI24910), identified during an N-ethyl-N-nitrosourea (ENU) screen. This line carries a missense mutation in the gene for transferrin receptor 1 (TFR1). Heterozygous mice exhibited reduced erythrocyte volume and density, a phenotype consistent with dietary iron deficiency anemia. However, unlike the case in dietary deficiency, the erythrocyte half-life, mean corpuscular hemoglobin concentration, and intraerythrocytic ferritin content were unchanged. Systemic iron bioavailability was also unchanged, indicating that this mutation results in erythrocytic iron deficiency without significantly altering overall iron homeostasis. When infected with the rodent malaria parasite Plasmodium chabaudi adami, mice displayed increased parasitemia and succumbed to infection more quickly than their wild-type littermates. Transfusion of fluorescently labeled erythrocytes into malaria parasite-infected mice demonstrated an erythrocyte-autonomous enhanced survival of parasites within mutant erythrocytes. Together, these results indicate that TFR1 deficiency alters erythrocyte physiology in a way that is similar to dietary iron deficiency anemia, albeit to a lesser degree, and that this promotes intraerythrocytic parasite survival and an increased susceptibility to malaria in mice. These findings may have implications for the management of iron deficiency in the context of malaria. Copyright © 2015, American Society for Microbiology

  10. Evaluating the effectiveness of transferrin receptor-1 (TfR1) as a magnetic resonance reporter gene.

    PubMed

    Pereira, Sofia M; Herrmann, Anne; Moss, Diana; Poptani, Harish; Williams, Steve R; Murray, Patricia; Taylor, Arthur

    2016-05-01

    Magnetic resonance (MR) reporter genes have the potential for tracking the biodistribution and fate of cells in vivo, thus allowing the safety, efficacy and mechanisms of action of cell-based therapies to be comprehensively assessed. In this study, we evaluate the effectiveness of the iron importer transferrin receptor-1 (TfR1) as an MR reporter gene in the model cell line CHO-K1. Overexpression of the TfR1 transgene led to a reduction in the levels of endogenous TfR1 mRNA, but to a 60-fold increase in total TfR1 protein levels. Although the mRNA levels of ferritin heavy chain-1 (Fth1) did not change, Fth1 protein levels increased 13-fold. The concentration of intracellular iron increased significantly, even when cells were cultured in medium that was not supplemented with iron and the amount of iron in the extracellular environment was thus at physiological levels. However, we found that, by supplementing the cell culture medium with ferric citrate, a comparable degree of iron uptake and MR contrast could be achieved in control cells that did not express the TfR1 transgene. Sufficient MR contrast to enable the cells to be detected in vivo following their administration into the midbrain of chick embryos was obtained irrespective of the reporter gene. We conclude that TfR1 is not an effective reporter and that, to track the biodistribution of cells with MR imaging in the short term, it is sufficient to simply culture cells in the presence of ferric citrate. Copyright © 2016 The Authors Contrast Media & Molecular Imaging Published by John Wiley & Sons Ltd.

  11. Erythrocytic Iron Deficiency Enhances Susceptibility to Plasmodium chabaudi Infection in Mice Carrying a Missense Mutation in Transferrin Receptor 1

    PubMed Central

    Lelliott, Patrick M.; McMorran, Brendan J.; Foote, Simon J.

    2015-01-01

    The treatment of iron deficiency in areas of high malaria transmission is complicated by evidence which suggests that iron deficiency anemia protects against malaria, while iron supplementation increases malaria risk. Iron deficiency anemia results in an array of pathologies, including reduced systemic iron bioavailability and abnormal erythrocyte physiology; however, the mechanisms by which these pathologies influence malaria infection are not well defined. In the present study, the response to malaria infection was examined in a mutant mouse line, TfrcMRI24910, identified during an N-ethyl-N-nitrosourea (ENU) screen. This line carries a missense mutation in the gene for transferrin receptor 1 (TFR1). Heterozygous mice exhibited reduced erythrocyte volume and density, a phenotype consistent with dietary iron deficiency anemia. However, unlike the case in dietary deficiency, the erythrocyte half-life, mean corpuscular hemoglobin concentration, and intraerythrocytic ferritin content were unchanged. Systemic iron bioavailability was also unchanged, indicating that this mutation results in erythrocytic iron deficiency without significantly altering overall iron homeostasis. When infected with the rodent malaria parasite Plasmodium chabaudi adami, mice displayed increased parasitemia and succumbed to infection more quickly than their wild-type littermates. Transfusion of fluorescently labeled erythrocytes into malaria parasite-infected mice demonstrated an erythrocyte-autonomous enhanced survival of parasites within mutant erythrocytes. Together, these results indicate that TFR1 deficiency alters erythrocyte physiology in a way that is similar to dietary iron deficiency anemia, albeit to a lesser degree, and that this promotes intraerythrocytic parasite survival and an increased susceptibility to malaria in mice. These findings may have implications for the management of iron deficiency in the context of malaria. PMID:26303393

  12. The Content of Reticulocyte Hemoglobin and Serum Concentration of the Soluble Transferrin Receptor for Diagnostics of Anemia in Chronically Hemodialyzed Patients.

    PubMed

    Kurzawa, Teresa; Owczarek, Aleksander; Strzelczyk, Joanna K; Gołąbek, Karolina; Wiczkowski, Andrzej

    2016-01-01

    Chronic renal disease constitutes a serious worldwide clinical problem. An important issue arising early during the treatment of renal failure is anemia. Patients in the end-stage of renal disease chronically treated with hemodialysis frequently suffer from anemia with iron deficiency. The aim of the study was to evaluate the usefulness of determining the reticulocyte hemoglobin content and serum concentration of soluble transferrin receptor in the detection of anemia caused by iron deficiency in comparison with the classic markers of iron circulation in serum in chronic dialysis patients with ESRD. 66 sets of hematologic results and iron turnover rates were analyzed, sampled from hemodialyzed patients (test group), as well as 34 sets of the same results taken from healthy people (control group). Statistically significant variables were found and a stepwise backward discriminant analysis was performed for them. The results showed that dialyzed patients have a significantly lower serum concentration of hemoglobin, CHr, HCT, TSAT, Fe and TIBC and significantly higher serum concentration of sTfR, ferritin and C-reactive protein compared to the control group. Based on the results of discriminant analysis, we proposed a scheme for assessing the risk of anemia. The concentrations of hemoglobin, soluble transferrin receptor, iron in the serum and C-reactive protein turned out to be the most useful for diagnostic purposes. Moreover, the concentration of soluble transferrin receptor confirmed its high diagnostic value in the detection of iron deficiency-based anemia in patients undergoing dialysis for chronic renal failure at the end-stage compared to conventional iron turnover ratios in the serum.

  13. Transferrin receptor-targeted vitamin E TPGS micelles for brain cancer therapy: preparation, characterization and brain distribution in rats.

    PubMed

    Sonali; Agrawal, Poornima; Singh, Rahul Pratap; Rajesh, Chellappa V; Singh, Sanjay; Vijayakumar, Mahalingam R; Pandey, Bajrangprasad L; Muthu, Madaswamy Sona

    2016-06-01

    The effective treatment of brain cancer is hindered by the poor transport across the blood-brain barrier (BBB) and the low penetration across the blood-tumor barrier (BTB). The objective of this work was to formulate transferrin-conjugated docetaxel (DTX)-loaded d-alpha-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS) micelles for targeted brain cancer therapy. The micelles with and without transferrin conjugation were prepared by the solvent casting method and characterized for their particle size, polydispersity, drug encapsulation efficiency, drug loading, in vitro release study and brain distribution study. Particle sizes of prepared micelles were determined at 25 °C by dynamic light scattering technique. The external surface morphology was determined by transmission electron microscopy analysis and atomic force microscopy. The encapsulation efficiency was determined by spectrophotometery. In vitro release studies of micelles and control formulations were carried out by dialysis bag diffusion method. The particle sizes of the non-targeted and targeted micelles were <20 nm. About 85% of drug encapsulation efficiency was achieved with micelles. The drug release from transferrin-conjugated micelles was sustained for >24 h with 50% of drug release. The in vivo results indicated that transferrin-targeted TPGS micelles could be a promising carrier for brain targeting due to nano-sized drug delivery, solubility enhancement and permeability which provided an improved and prolonged brain targeting of DTX in comparison to the non-targeted micelles and marketed formulation.

  14. An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium.

    PubMed

    Jensen, Mark P; Gorman-Lewis, Drew; Aryal, Baikuntha; Paunesku, Tatjana; Vogt, Stefan; Rickert, Paul G; Seifert, Soenke; Lai, Barry; Woloschak, Gayle E; Soderholm, L

    2011-06-26

    Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway--receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu(C)Fe(N)Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.

  15. An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium.

    SciTech Connect

    Jensen, M. P.; Gorman-Lewis, D.; Aryal, B. P.; Paunesku, T.; Vogt, S.; Rickert, P. G.; Seifert, S.; Lai, B.; Woloschak, G. E.; Soderholm, L.

    2011-08-01

    Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small-angle X-ray scattering, receptor binding assays and synchrotron X-ray fluorescence microscopy, we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway -- receptor-mediated endocytosis of the iron transport protein serum transferrin; however, only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small-angle scattering show that only transferrin with plutonium bound in the protein's C-terminal lobe (C-lobe) and iron bound in the N-terminal lobe (N-lobe) (Pu{sub c}Fe{sub N}Tf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal-binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin's two lobes act to restrict, but not eliminate, cellular Pu uptake.

  16. Adjusting soluble transferrin receptor concentrations for inflammation: Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project.

    PubMed

    Rohner, Fabian; Namaste, Sorrel Ml; Larson, Leila M; Addo, O Yaw; Mei, Zuguo; Suchdev, Parminder S; Williams, Anne M; Sakr Ashour, Fayrouz A; Rawat, Rahul; Raiten, Daniel J; Northrop-Clewes, Christine A

    2017-07-01

    Background: Iron deficiency is thought to be one of the most prevalent micronutrient deficiencies globally, but an accurate assessment in populations who are frequently exposed to infections is impeded by the inflammatory response, which causes iron-biomarker alterations.Objectives: We assessed the relation between soluble transferrin receptor (sTfR) concentrations and inflammation and malaria in preschool children (PSC) (age range: 6-59 mo) and women of reproductive age (WRA) (age range: 15-49 y) and investigated adjustment algorithms to account for these effects.Design: Cross-sectional data from the Biomarkers Reflecting the Inflammation and Nutritional Determinants of Anemia (BRINDA) project from 11,913 PSC in 11 surveys and from 11,173 WRA in 7 surveys were analyzed individually and combined with the use of a meta-analysis. The following 3 adjustment approaches were compared with estimated iron-deficient erythropoiesis (sTfR concentration >8.3 mg/L): 1) the exclusion of individuals with C-reactive protein (CRP) concentrations >5 mg/L or α-1-acid glycoprotein (AGP) concentrations >1 g/L, 2) the application of arithmetic correction factors, and 3) the use of regression approaches.Results: The prevalence of elevated sTfR concentrations incrementally decreased as CRP and AGP deciles decreased for PSC and WRA, but the effect was more pronounced for AGP than for CRP. Depending on the approach used to adjust for inflammation, the estimated prevalence of iron-deficient erythropoiesis decreased by 4.4-14.6 and 0.3-9.5 percentage points in PSC and WRA, respectively, compared with unadjusted values. The correction-factor approach yielded a more modest reduction in the estimated prevalence of iron-deficient erythropoiesis than did the regression approach. Mostly, adjustment for malaria in addition to AGP did not significantly change the estimated prevalence of iron-deficient erythropoiesis.Conclusions: sTfR may be useful to assess iron-deficient erythropoiesis, but

  17. Adjusting soluble transferrin receptor concentrations for inflammation: Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project

    PubMed Central

    Larson, Leila M; Mei, Zuguo; Sakr Ashour, Fayrouz A; Rawat, Rahul; Raiten, Daniel J; Northrop-Clewes, Christine A

    2017-01-01

    Background: Iron deficiency is thought to be one of the most prevalent micronutrient deficiencies globally, but an accurate assessment in populations who are frequently exposed to infections is impeded by the inflammatory response, which causes iron-biomarker alterations. Objectives: We assessed the relation between soluble transferrin receptor (sTfR) concentrations and inflammation and malaria in preschool children (PSC) (age range: 6–59 mo) and women of reproductive age (WRA) (age range: 15–49 y) and investigated adjustment algorithms to account for these effects. Design: Cross-sectional data from the Biomarkers Reflecting the Inflammation and Nutritional Determinants of Anemia (BRINDA) project from 11,913 PSC in 11 surveys and from 11,173 WRA in 7 surveys were analyzed individually and combined with the use of a meta-analysis. The following 3 adjustment approaches were compared with estimated iron-deficient erythropoiesis (sTfR concentration >8.3 mg/L): 1) the exclusion of individuals with C-reactive protein (CRP) concentrations >5 mg/L or α-1-acid glycoprotein (AGP) concentrations >1 g/L, 2) the application of arithmetic correction factors, and 3) the use of regression approaches. Results: The prevalence of elevated sTfR concentrations incrementally decreased as CRP and AGP deciles decreased for PSC and WRA, but the effect was more pronounced for AGP than for CRP. Depending on the approach used to adjust for inflammation, the estimated prevalence of iron-deficient erythropoiesis decreased by 4.4–14.6 and 0.3–9.5 percentage points in PSC and WRA, respectively, compared with unadjusted values. The correction-factor approach yielded a more modest reduction in the estimated prevalence of iron-deficient erythropoiesis than did the regression approach. Mostly, adjustment for malaria in addition to AGP did not significantly change the estimated prevalence of iron-deficient erythropoiesis. Conclusions: sTfR may be useful to assess iron-deficient erythropoiesis

  18. Ferritin and Soluble Transferrin Receptors in Type 2 Diabetic and Non-diabetic Post-menopausal Women in Dhaka, Bangladesh.

    PubMed

    Md Ruhul, A; Sharmin, H; Luthfor, A; Farzana, S; Liaquat, A

    2010-12-01

    This cross-sectional comparative study was aimed at investigating the iron status of a group of post-menopausal women with and without diabetes. Thirty-five post-menopausal women in each group were selected purposively from among patients attending the out-patient department of Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM), a specialist hospital, and two of its satellite clinics, all in Dhaka. Patients were enrolled based on their existing records. The subjects were matched on age, menstrual status and fasting status at blood draw. Ferritin, serum soluble transferrin receptors (sTfR) and fasting plasma glucose were measured by standard methods. Dietary information was collected by a specific food frequency questionnaire. No significant difference in plasma ferritin [62.02 ng/ml, (range: 4.68-288.89) vs 54.25 ng/ml (range: 4.58-137.17); p=0.28] was observed between the groups. But a higher level of plasma sTfR was found in diabetic women [(21.12 nmol/l (range: 7.91-39.79) vs 17.63 nmol/l (range: 10.30-110.00); p<0.01]. TFR-F index showed no difference between diabetic and control (p=0.25). Significantly a lower hemoglobin level [10.58±0.67 g/dl vs11.76±1.5 g/dl; p<0.01] was detected in diabetic women. Plasma sTfR (log) did not show any significant association with the dietary parameters and iron indices. No significant association between fasting glucose, ferritin and sTfR was seen except for haemoglobin (r=0.39, p=0.05). Total iron intake recorded was more than the requirement, and was significantly higher in control group [38.11mg/day (range: 19.83-105.63) vs 56.65 mg/day (range: 29.75-109.54); p<0.01)]. More than 97 % of total iron was of plant origin. No differences in heme iron [0.85 mg/day (range: 0.09-4.07) vs. 0.96 mg/day (range: 0.04-4.34), p= 0.17] and vitamin C intake was observed between the groups. Iron indices of non-diabetic women were within the normal range. A higher level of sTfR and a

  19. N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth)cells using the LdMNPV expression system

    Treesearch

    One Choi; Noboru Tomiya; Jung H. Kim; James M. Slavicek; Michael J. Betenbaugh; Yuan C. Lee

    2003-01-01

    N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then...

  20. Response of soluble transferrin receptor and iron-related parameters to iron supplementation in elite, iron-depleted, nonanemic female athletes.

    PubMed

    Pitsis, George C; Fallon, Kieran E; Fallon, Susan K; Fazakerley, Ruth

    2004-09-01

    To assess the effect of short-term iron supplementation on soluble transferrin receptor (sTfR) and the soluble transferrin receptor/log ferritin ratio in a group of elite, iron-depleted, nonanemic female athletes and to assess the relationship between soluble transferrin receptor and serum ferritin in a group of elite, iron-depleted female athletes. A prospective, interventional study and an observation at 1 point in time. The primary inclusion criteria for each component of the study was serum ferritin less than 30 ng/mL and hemoglobin greater than 12 g/dL. The exclusion criteria were use of iron supplementation within 1 month of the initial assessment, acute or chronic infectious or inflammatory disease, hematological disorders, and recent musculoskeletal injury. The Department of Sports Medicine at the Australian Institute of Sport. For the prospective study, 51, and for the observational study, 85 elite, iron-depleted (serum ferritin <30 ng/mL), nonanemic (hemoglobin <12 g/dL for the prospective study only) female athletes. Athletes were participants at the elite level in netball, basketball, gymnastics, rowing, volleyball, track and field, soccer, sailing, and cricket. Ingestion of 1 iron tablet (325 mg dried ferrous sulfate, equivalent to 105 mg elemental iron) plus one 500-mg tablet of vitamin C each morning for 60 days. Following supplementation, the following changes were statistically significant. Serum ferritin increased (19.7 to 37.4 ng/mL; P < 0.00005), serum transferrin decreased (3.34 to 3.16 g/L; P = 0.023), sTfR decreased (3.46 to 3.16 mg/L; P = 0.006), and sTfR/log ferritin index decreased (1.34 to 1.00; P < 0.00005). If it is assumed that iron stores are depleted when serum ferritin is less than 12 ng/mL and that sTfR could be expected to be elevated below that level of serum ferritin, the following figures can be calculated for the value of sTfR in relation to absent iron stores: sensitivity, 33%; specificity, 96%; positive predictive value, 50

  1. Insect transferrins: multifunctional proteins.

    PubMed

    Geiser, Dawn L; Winzerling, Joy J

    2012-03-01

    Many studies have been done evaluating transferrin in insects. Genomic analyses indicate that insects could have more than one transferrin. However, the most commonly studied insect transferrin, Tsf1, shows greatest homology to mammalian blood transferrin. Aspects of insect transferrin structure compared to mammalian transferrin and the roles transferrin serves in insects are discussed in this review. Insect transferrin can have one or two lobes, and can bind iron in one or both. The iron binding ligands identified for the lobes of mammalian blood transferrin are generally conserved in the lobes of insect transferrins that have an iron binding site. Available information supports that the form of dietary iron consumed influences the regulation of insect transferrin. Although message is expressed in several tissues in many insects, fat body is the likely source of hemolymph transferrin. Insect transferrin is a vitellogenic protein that is down-regulated by Juvenile Hormone. It serves a role in transporting iron to eggs in some insects, and transferrin found in eggs appears to be endowed from the female. In addition to the roles of transferrin in iron delivery, this protein also functions to reduce oxidative stress and to enhance survival of infection. Future studies in Tsf1 as well as the other insect transferrins that bind iron are warranted because of the roles of transferrin in preventing oxidative stress, enhancing survival to infections and delivering iron to eggs for development. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Nutritional immunity. Escape from bacterial iron piracy through rapid evolution of transferrin.

    PubMed

    Barber, Matthew F; Elde, Nels C

    2014-12-12

    Iron sequestration provides an innate defense, termed nutritional immunity, leading pathogens to scavenge iron from hosts. Although the molecular basis of this battle for iron is established, its potential as a force for evolution at host-pathogen interfaces is unknown. We show that the iron transport protein transferrin is engaged in ancient and ongoing evolutionary conflicts with TbpA, a transferrin surface receptor from bacteria. Single substitutions in transferrin at rapidly evolving sites reverse TbpA binding, providing a mechanism to counteract bacterial iron piracy among great apes. Furthermore, the C2 transferrin polymorphism in humans evades TbpA variants from Haemophilus influenzae, revealing a functional basis for standing genetic variation. These findings identify a central role for nutritional immunity in the persistent evolutionary conflicts between primates and bacterial pathogens.

  3. Targeted Delivery of Amoxicillin to C. trachomatis by the Transferrin Iron Acquisition Pathway.

    PubMed

    Hai, Jun; Serradji, Nawal; Mouton, Ludovic; Redeker, Virginie; Cornu, David; El Hage Chahine, Jean-Michel; Verbeke, Philippe; Hémadi, Miryana

    2016-01-01

    Weak intracellular penetration of antibiotics makes some infections difficult to treat. The Trojan horse strategy for targeted drug delivery is among the interesting routes being explored to overcome this therapeutic difficulty. Chlamydia trachomatis, as an obligate intracellular human pathogen, is responsible for both trachoma and sexually transmitted diseases. Chlamydia develops in a vacuole and is therefore protected by four membranes (plasma membrane, bacterial inclusion membrane, and bacterial membranes). In this work, the iron-transport protein, human serum-transferrin, was used as a Trojan horse for antibiotic delivery into the bacterial vacuole. Amoxicillin was grafted onto transferrin. The transferrin-amoxicillin construct was characterized by mass spectrometry and absorption spectroscopy. Its affinity for transferrin receptor 1, determined by fluorescence emission titration [KaffTf-amox = (1.3 ± 1.0) x 108], is very close to that of transferrin [4.3 x 108]. Transmission electron and confocal microscopies showed a co-localization of transferrin with the bacteria in the vacuole and were also used to evaluate the antibiotic capability of the construct. It is significantly more effective than amoxicillin alone. These promising results demonstrate targeted delivery of amoxicillin to suppress Chlamydia and are of interest for Chlamydiaceae and maybe other intracellular bacteria therapies.

  4. Targeted Delivery of Amoxicillin to C. trachomatis by the Transferrin Iron Acquisition Pathway

    PubMed Central

    Hai, Jun; Serradji, Nawal; Mouton, Ludovic; Redeker, Virginie; Cornu, David; El Hage Chahine, Jean-Michel

    2016-01-01

    Weak intracellular penetration of antibiotics makes some infections difficult to treat. The Trojan horse strategy for targeted drug delivery is among the interesting routes being explored to overcome this therapeutic difficulty. Chlamydia trachomatis, as an obligate intracellular human pathogen, is responsible for both trachoma and sexually transmitted diseases. Chlamydia develops in a vacuole and is therefore protected by four membranes (plasma membrane, bacterial inclusion membrane, and bacterial membranes). In this work, the iron-transport protein, human serum-transferrin, was used as a Trojan horse for antibiotic delivery into the bacterial vacuole. Amoxicillin was grafted onto transferrin. The transferrin-amoxicillin construct was characterized by mass spectrometry and absorption spectroscopy. Its affinity for transferrin receptor 1, determined by fluorescence emission titration [KaffTf-amox = (1.3 ± 1.0) x 108], is very close to that of transferrin [4.3 x 108]. Transmission electron and confocal microscopies showed a co-localization of transferrin with the bacteria in the vacuole and were also used to evaluate the antibiotic capability of the construct. It is significantly more effective than amoxicillin alone. These promising results demonstrate targeted delivery of amoxicillin to suppress Chlamydia and are of interest for Chlamydiaceae and maybe other intracellular bacteria therapies. PMID:26919720

  5. Thermodynamics and Mechanisms of the Interactions between Ultrasmall Fluorescent Gold Nanoclusters and Human Serum Albumin, γ-Globulins, and Transferrin: A Spectroscopic Approach.

    PubMed

    Yin, Miao-Miao; Dong, Ping; Chen, Wen-Qi; Xu, Shi-Ping; Yang, Li-Yun; Jiang, Feng-Lei; Liu, Yi

    2017-05-30

    Noble metal nanoclusters (NCs) show great promise as nanoprobes for bioanalysis and cellular imaging in biological applications due to ultrasmall size, good photophysical properties, and excellent biocompatibility. In order to achieve a comprehensive understanding of possible biological implications, a series of spectroscopic measurements were conducted under different temperatures to investigate the interactions of Au NCs (∼1.7 nm) with three model plasmatic proteins (human serum albumin (HSA), γ-globulins, and transferrin). It was found that the fluorescence quenching of HSA and γ-globulins triggered by Au NCs was due to dynamic quenching mechanism, while the fluorescence quenching of transferrin by Au NCs was a result of the formation of a Au NC-transferrin complex. The apparent association constants of the Au NCs bound to HSA, γ-globulins, and transferrin demonstrated no obvious difference. Thermodynamic studies demonstrated that the interaction between Au NCs and HSA (or γ-globulins) was driven by hydrophobic forces, while the electrostatic interactions played predominant roles in the adsorption process for transferrin. Furthermore, it was proven that Au NCs had no obvious interference in the secondary structures of these three kinds of proteins. In turn, these three proteins had a minor effect on the fluorescence intensity of Au NCs, which made fluorescent Au NCs promising in biological applications owing to their chemical and photophysical stability. In addition, by comparing the interactions of small molecules, Au NCs, and large nanomaterials with serum albumin, it was found that the binding constants were gradually increased with the increase of particle size. This work has elucidated the interaction mechanisms between nanoclusters and proteins, and shed light on a new interaction mode different from the protein corona on the surface of nanoparticles, which will highly contribute to the better design and applications of fluorescent nanoclusters.

  6. Inequivalent contribution of the five tryptophan residues in the C-lobe of human serum transferrin to the fluorescence increase when iron is released.

    PubMed

    James, Nicholas G; Byrne, Shaina L; Steere, Ashley N; Smith, Valerie C; MacGillivray, Ross T A; Mason, Anne B

    2009-04-07

    Human serum transferrin (hTF), with two Fe3+ binding lobes, transports iron into cells. Diferric hTF preferentially binds to a specific receptor (TFR) on the surface of cells, and the complex undergoes clathrin dependent receptor-mediated endocytosis. The clathrin-coated vesicle fuses with an endosome where the pH is lowered, facilitating iron release from hTF. On a biologically relevant time scale (2-3 min), the factors critical to iron release include pH, anions, a chelator, and the interaction of hTF with the TFR. Previous work, in which the increase in the intrinsic fluorescence signal was used to monitor iron release from the hTF/TFR complex, established that the TFR significantly enhances the rate of iron release from the C-lobe of hTF. In the current study, the role of the five C-lobe Trp residues in reporting the fluorescence change has been evaluated (+/-sTFR). Only four of the five recombinant Trp --> Phe mutants produced well. A single slow rate constant for iron release is found for the monoferric C-lobe (FeC hTF) and the four Trp mutants in the FeC hTF background. The three Trp residues equivalent to those in the N-lobe differed from the N-lobe and each other in their contributions to the fluorescent signal. Two rate constants are observed for the FeC hTF control and the four Trp mutants in complex with the TFR: k(obsC1) reports conformational changes in the C-lobe initiated by the TFR, and k(obsC2) is ascribed to iron release. Excitation at 295 nm (Trp only) and at 280 nm (Trp and Tyr) reveals interesting and significant differences in the rate constants for the complex.

  7. Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis

    PubMed Central

    Lampe, Marko; Pierre, Fabienne; Al-Sabah, Suleiman; Krasel, Cornelius; Merrifield, Christien J.

    2014-01-01

    The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein–coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)–based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs. PMID:25079691

  8. Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis.

    PubMed

    Lampe, Marko; Pierre, Fabienne; Al-Sabah, Suleiman; Krasel, Cornelius; Merrifield, Christien J

    2014-10-01

    The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein-coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)-based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs.

  9. Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether

    PubMed Central

    1995-01-01

    Our previous results indicated that the plasma membrane of cultured normal rat kidney fibroblastic cell is compartmentalized for diffusion of receptor molecules, and that long-range diffusion is the result of successive intercompartmental jumps (Sako, Y. and Kusumi, A. 1994. J. Cell Biol. 125:1251-1264). In the present study, we characterized the properties of intercompartmental boundaries by tagging transferrin receptor (TR) with either 210-nm-phi latex or 40-nm-phi colloidal gold particles, and by dragging the particle-TR complexes laterally along the plasma membrane using laser tweezers. Approximately 90% of the TR- particle complexes showed confined-type diffusion with a microscopic diffusion coefficient (Dmicro) of approximately 10(-9) cm2/s and could be dragged past the intercompartmental boundaries in their path by laser tweezers at a trapping force of 0.25 pN for gold-tagged TR and 0.8 pN for latex-tagged TR. At lower dragging forces between 0.05 and 0.1 pN, particle-TR complexes tended to escape from the laser trap at the boundaries, and such escape occurred in both the forward and backward directions of dragging. The average distance dragged was half of the confined distance of TR, which further indicates that particle- TR complexes escape at the compartment boundaries. Since variation in the particle size (40 and 210 nm, the particles are on the extracellular surface of the plasma membrane) hardly affects the diffusion rate and behavior of the particle-TR complexes at the compartment boundaries, and since treatment with cytochalasin D or vinblastin affects the movements of TR (Sako and Kusumi as cited above), argument has been advanced that the boundaries are present in the cytoplasmic domain. Rebound of the particle-TR complexes when they escape from the laser tweezers at the compartment boundaries suggests that the boundaries are elastic structures. These results are consistent with the proposal that the compartment boundaries consist of membrane

  10. Transferrin-mediated cellular iron delivery.

    PubMed

    Luck, Ashley N; Mason, Anne B

    2012-01-01

    Essential to iron homeostasis is the transport of iron by the bilobal protein human serum transferrin (hTF). Each lobe (N- and C-lobe) of hTF forms a deep cleft which binds a single Fe(3+). Iron-bearing hTF in the blood binds tightly to the specific transferrin receptor (TFR), a homodimeric transmembrane protein. After undergoing endocytosis, acidification of the endosome initiates the release of Fe(3+) from hTF in a TFR-mediated process. Iron-free hTF remains tightly bound to the TFR at acidic pH; following recycling back to the cell surface, it is released to sequester more iron. Efficient delivery of iron is critically dependent on hTF/TFR interactions. Therefore, identification of the pH-specific contacts between hTF and the TFR is crucial. Recombinant protein production has enabled deconvolution of this complex system. The studies reviewed herein support a model in which pH-induced interrelated events control receptor-stimulated iron release from each lobe of hTF. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Transferrin-Mediated Cellular Iron Delivery

    PubMed Central

    Luck, Ashley N.; Mason, Anne B.

    2015-01-01

    Essential to iron homeostasis is the transport of iron by the bilobal protein human serum transferrin (hTF). Each lobe (N- and C-lobe) of hTF forms a deep cleft which binds a single Fe3+. Iron-bearing hTF in the blood binds tightly to the specific transferrin receptor (TFR), a homodimeric transmembrane protein. After undergoing endocytosis, acidification of the endosome initiates the release of Fe3+ from hTF in a TFR-mediated process. Iron-free hTF remains tightly bound to the TFR at acidic pH; following recycling back to the cell surface, it is released to sequester more iron. Efficient delivery of iron is critically dependent on hTF/TFR interactions. Therefore, identification of the pH-specific contacts between hTF and the TFR is crucial. Recombinant protein production has enabled deconvolution of this complex system. The studies reviewed herein support a model in which pH-induced interrelated events control receptor-stimulated iron release from each lobe of hTF. PMID:23046645

  12. Efficacy of doxorubicin-transferrin conjugate in apoptosis induction in human leukemia cells through reactive oxygen species generation.

    PubMed

    Szwed, Marzena; Laroche-Clary, Audrey; Robert, Jacques; Jozwiak, Zofia

    2016-04-01

    Doxorubicin (DOX) is a small molecular cytotoxic agent that can be transferred efficiently to cancer cells by nanocarriers. This anthracycline antibiotic serves as an effective anti-neoplastic drug against both hematological and solid malignancies. Here, we set out to assess the capacity of a novel doxorubicin - transferrin conjugate (DOX-TRF) to provoke apoptosis in human normal and leukemia cells through free radicals produced via a redox cycle of doxorubicin (DOX) when released from its conjugate. After DOX-TRF exposure, we determined the time-course of apoptotic and necrotic events, the generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential, as well as alterations in cytochrome c levels and intracellular calcium concentrations in human leukemia-derived cell lines (CCRF-CEM, K562 and its doxorubicin-resistant derivative K562/DOX) and normal peripheral blood-derived mononuclear cells (PBMC). We found that DOX-TRF can induce apoptosis in all leukemia-derived cell lines tested, which was associated with morphological changes and decreases in mitochondrial membrane potential. In comparison to free DOX treated cells, we observed a time-dependency between a higher level of ROS generation and a higher drop in mitochondrial membrane potential, particularly in the doxorubicin-resistant cell line. In addition, we found that the apoptotic cell death induced by DOX-TRF was directly associated with a release of cytochrome c from the mitochondria and an increase in intracellular calcium level in all human leukemia-derived cell lines tested. Our data indicate that DOX-TRF is considerably more cytotoxic to human leukemia cells than free DOX. In addition, we show that DOX-TRF can effectively produce free radicals, which are directly involved in apoptosis induction.

  13. STUDIES ON THE TRANSFERRINS OF ADULT SERUM, CORD SERUM, AND CEREBROSPINAL FLUID

    PubMed Central

    Parker, W. Carey; Bearn, Alexander G.

    1962-01-01

    Nine of the twelve known variants of human transferrin have been resolved by the action of neuraminidase into stepwise patterns of four additional slower moving components whose relative intensities depended upon the concentration of enzyme. These components appeared to represent the stepwise removal of the four sialic acid residues from the transferrin molecule, and at large enzyme concentrations, almost all of the transferrin was reduced to the position of the slowest moving component. In contrast, the electrophoretic mobilities of haptoglobin, ceruloplasmin, and α2-macroglobulin showed a gradual decrease with increasing neuraminidase concentration. The transferrins of chimpanzees, rhesus and cynomolgus monkeys, and cattle were resolved by neuraminidase into two slower moving components. These experiments suggested that the primate and cattle transferrins contained only two sialic acid residues accessible to the enzyme. Transferrins C, B2, and D1 and a cynomolgus monkey transferrin were purified from serum by starch block electrophoresis and cellulose chromatography. Ultracentrifugal analysis could detect no difference in sedimentation rate between transferrin C, the primate transferrin, and neuraminidase-treated transferrin C. The human transferrins showed no variation in amino acid composition, but the cynomolgus transferrin was approximately 20 per cent higher in serine content and 50 per cent lower in glucosamine than human transferrin C. Reactions of antigenic identity were obtained among five human transferrin variants but a reaction of only partial identity was obtained between transferrin C and the cynomolgus transferrin. The transferrin pattern of cord blood showed a prominent band in the position of transferrin C, accompanied by four faint slower moving bands which coincided with the four transferrin components produced by the action of neuraminidase on transferrin C. The transferrin pattern of cerebrospinal fluid in individuals homozygous for serum

  14. An iron-dependent and transferrin-mediated cellular uptake pathway for plutonium

    PubMed Central

    Jensen, Mark P.; Gorman-Lewis, Drew; Aryal, Baikuntha; Paunesku, Tatjana; Vogt, Stefan; Rickert, Paul G.; Seifert, Soenke; Lai, Barry; Woloschak, Gayle E.; Soderholm, L.

    2012-01-01

    Plutonium is a toxic synthetic element with no natural biological function, but it is strongly retained by humans when ingested. Using small angle X-ray scattering, receptor binding assays, and synchrotron X-ray fluorescence microscopy we find that rat adrenal gland (PC12) cells can acquire plutonium in vitro through the major iron acquisition pathway, receptor-mediated endocytosis of the iron transport protein serum transferrin; however only one form of the plutonium-transferrin complex is active. Low-resolution solution models of plutonium-loaded transferrins derived from small angle scattering demonstrate that only transferrin with plutonium bound in the protein’s C-terminal lobe and iron bound in the N-lobe (PuCFeNTf) adopts the proper conformation for recognition by the transferrin receptor protein. Although the metal binding site in each lobe contains the same donors in the same configuration and both lobes are similar, the differences between transferrin’s two lobes act to restrict, but not eliminate, cellular Pu uptake. PMID:21706034

  15. Non-invasive (89)Zr-Transferrin PET Shows Improved Tumor Targeting Compared to (18)F-FDG PET in MYC-overexpressing Human Triple Negative Breast Cancer.

    PubMed

    Henry, Kelly E; Dilling, Thomas R; Abdel-Atti, Dalya; Edwards, Kimberly J; Evans, Michael J; Lewis, Jason S

    2017-08-28

    The current standard for breast positron emission tomography (PET) imaging is (18)F-fluorodeoxyglucose ((18)F-FDG). The heterogeneity of (18)F-FDG uptake in breast cancer limits its utility, varying greatly among receptor status, histopathological subtypes, and proliferation markers. (18)F-FDG PET often exhibits non-specific internalization and low specificity and sensitivity, especially with tumors < 1 cm(3) MYC is a protein involved in oncogenesis and is overexpressed in triple negative breast cancer (TNBC). Increased surface expression of transferrin receptor (TfR) is a downstream event of MYC upregulation, and has been validated as a clinically relevant target for molecular imaging. Transferrin (Tf) labeled with zirconium-89 ((89)Zr) has successfully identified MYC status in many cancer subtypes preclinically, and been shown to predict response and changes in oncogene status via treatment with small molecule inhibitors that target MYC and PI3K signaling pathways. We hypothesized that (89)Zr-Tf PET will non-invasively detect MYC and TfR and improve upon the current standard of (18)F-FDG PET for MYC-overexpressing TNBC. Methods: In this study, (89)Zr-Tf and (18)F-FDG imaging were compared in preclinical models of TNBC. TNBC cells (MDA-MB-157, MBA-MB-231, and Hs578T) were treated with bromodomain-containing protein 4 (BRD4) inhibitors JQ1 and OTX015 (0.5-1 μM). Cell proliferation, gene expression, and protein expression were assayed to explore the effects of these inhibitors on MYC and TfR. Results: Head-to-head comparison showed that (89)Zr-Tf targets TNBC tumors significantly better (P < 0.05 - 0.001) than (18)F-FDG through PET imaging and biodistribution studies in MDA-MB-231 and MDA-MB-157 xenografts and a patient-derived xenograft model of TNBC. MYC and TfR gene expression were decreased upon treatment with BRD4 inhibitors and c-MYC small interfering RNA (siRNA) (P < 0.01 - 0.001 for responding cell lines) compared to vehicle-treatment. MYC and TfR protein

  16. Complexation of Cm(III) with the recombinant N-lobe of human serum transferrin studied by time-resolved laser fluorescence spectroscopy (TRLFS).

    PubMed

    Bauer, N; Smith, V C; MacGillivray, R T A; Panak, P J

    2015-01-28

    The complexation of Cm(III) with the recombinant N-lobe of human serum transferrin (hTf/2N) is investigated in the pH range from 4.0 to 11.0 using TRLFS. At pH ≥ 7.4 a Cm(III) hTf/2N species is formed with Cm(III) bound at the Fe(III) binding site. The results are compared with Cm(III) transferrin interaction at the C-lobe and indicate the similarity of the coordination environment of the C- and N-terminal binding sites with four amino acid residues of the protein, two H2O molecules and three additional ligands (e.g. synergistic anions such as carbonate) in the first coordination sphere. Measurements at c(carbonate)tot = 0.23 mM (ambient carbonate concentration) and c(carbonate)tot = 25 mM (physiological carbonate concentration) show that an increase of the total carbonate concentration suppresses the formation of the Cm(III) hTf/2N species significantly. Additionally, the three Cm(III) carbonate species Cm(CO3)(+), Cm(CO3)2(-) and Cm(CO3)3(3-) are formed successively with increasing pH. In general, carbonate complexation is a competing reaction for both Cm(III) complexation with transferrin and hTf/2N but the effect is significantly higher for the half molecule. At c(carbonate)tot = 0.23 mM the complexation of Cm(III) with transferrin and hTf/2N starts at pH ≥ 7.4. At physiological carbonate concentration the Cm(III) transferrin species II forms at pH ≥ 7.0 whereas the Cm(III) hTf/2N species is not formed until pH > 10.0. Hence, our results reveal significant differences in the complexation behavior of the C-terminal site of transferrin and the recombinant N-lobe (hTf/2N) towards trivalent actinides.

  17. Staphylococcus aureus transporters Hts, Sir, and Sst capture iron liberated from human transferrin by Staphyloferrin A, Staphyloferrin B, and catecholamine stress hormones, respectively, and contribute to virulence.

    PubMed

    Beasley, Federico C; Marolda, Cristina L; Cheung, Johnson; Buac, Suzana; Heinrichs, David E

    2011-06-01

    Staphylococcus aureus is a frequent cause of bloodstream, respiratory tract, and skin and soft tissue infections. In the bloodstream, the iron-binding glycoprotein transferrin circulates to provide iron to cells throughout the body, but its iron-binding properties make it an important component of innate immunity. It is well established that siderophores, with their high affinity for iron, in many instances can remove iron from transferrin as a means to promote proliferation of bacterial pathogens. It is also established that catecholamine hormones can interfere with the iron-binding properties of transferrin, thus allowing infectious bacteria access to this iron pool. The present study demonstrates that S. aureus can use either of two carboxylate-type siderophores, staphyloferrin A and staphyloferrin B, via the transporters Hts and Sir, respectively, to access the transferrin iron pool. Growth of staphyloferrin-producing S. aureus in serum or in the presence of holotransferrin was not enhanced in the presence of catecholamines. However, catecholamines significantly enhanced the growth of staphyloferrin-deficient S. aureus in human serum or in the presence of human holotransferrin. It was further demonstrated that the Sst transporter was essential for this activity as well as for the utilization of bacterial catechol siderophores. The substrate binding protein SstD was shown to interact with ferrated catecholamines and catechol siderophores, with low to submicromolar affinities. Experiments involving mice challenged intravenously with wild-type S. aureus and isogenic mutants demonstrated that the combination of Hts, Sir, and Sst transport systems was required for full virulence of S. aureus.

  18. Receptor-based differences in human aortic smooth muscle cell membrane stiffness

    NASA Technical Reports Server (NTRS)

    Huang, H.; Kamm, R. D.; So, P. T.; Lee, R. T.

    2001-01-01

    Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (P<0.001). Mechanical characteristics of vascular smooth muscle cells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.

  19. Receptor-based differences in human aortic smooth muscle cell membrane stiffness

    NASA Technical Reports Server (NTRS)

    Huang, H.; Kamm, R. D.; So, P. T.; Lee, R. T.

    2001-01-01

    Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (P<0.001). Mechanical characteristics of vascular smooth muscle cells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.

  20. Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking.

    PubMed

    Solis, Gonzalo P; Hülsbusch, Nikola; Radon, Yvonne; Katanaev, Vladimir L; Plattner, Helmut; Stuermer, Claudia A O

    2013-09-01

    The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.

  1. Human presynaptic receptors.

    PubMed

    Schlicker, Eberhard; Feuerstein, Thomas

    2017-04-01

    Presynaptic receptors are sites at which transmitters, locally formed mediators or hormones inhibit or facilitate the release of a given transmitter from its axon terminals. The interest in the identification of presynaptic receptors has faded in recent years and it may therefore be justified to give an overview of their occurrence in the autonomic and central nervous system; this review will focus on presynaptic receptors in human tissues. Autoreceptors are presynaptic receptors at which a given transmitter restrains its further release, though in some instances may also increase its release. Inhibitory autoreceptors represent a typical example of a negative feedback; they are tonically activated by the respective endogenous transmitter and/or are constitutively active. Autoreceptors also play a role under pathophysiological conditions, e.g. by limiting the massive noradrenaline release occurring during congestive heart failure. They can be used for therapeutic purposes; e.g., the α2-adrenoceptor antagonist mirtazapine is used as an antidepressant and the inverse histamine H3 receptor agonist pitolisant has been marketed as a new drug for the treatment of narcolepsy in 2016. Heteroreceptors are presynaptic receptors at which transmitters from adjacent neurons, locally formed mediators (e.g. endocannabinoids) or hormones (e.g. adrenaline) can inhibit or facilitate transmitter release; they may be subject to an endogenous tone. The constipating effect of the sympathetic nervous system or of the antihypertensive drug clonidine is related to the activation of inhibitory α2-adrenoceptors on postganglionic parasympathetic neurons. Part of the stimulating effect of adrenaline on the sympathetic nervous system during stress is related to its facilitatory effect on noradrenaline release via β2-adrenoceptors.

  2. Spectral and metal-binding properties of three single-point tryptophan mutants of the human transferrin N-lobe.

    PubMed Central

    He, Q Y; Mason, A B; Lyons, B A; Tam, B M; Nguyen, V; MacGillivray, R T; Woodworth, R C

    2001-01-01

    Human serum transferrin N-lobe (hTF/2N) contains three conserved tryptophan residues, Trp(8), Trp(128) and Trp(264), located in three different environments. The present report addresses the different contributions of the three tryptophan residues to the UV-visible, fluorescence and NMR spectra of hTF/2N and the effect of the mutations at each tryptophan residue on the iron-binding properties of the protein. Trp(8) resides in a hydrophobic box containing a cluster of three phenylalanine side chains and is H bonded through the indole N to an adjacent water cluster lying between two beta-sheets containing Trp(8) and Lys(296) respectively. The fluorescence of Trp(8) may be quenched by the benzene rings. The apparent increase in the rate of iron release from the Trp(8)-->Tyr mutant could be due to the interference of the mutation with the H-bond linkage resulting in an effect on the second shell network. The partial quenching in the fluorescence of Trp(128) results from the nearby His(119) residue. Difference-fluorescence spectra reveal that any protein containing Trp(128) shows a blue shift upon binding metal ion, and the NMR signal of Trp(128) broadens out and disappears upon the binding of paramagnetic metals to the protein. These data imply that Trp(128) is a major fluorescent and NMR reporter group for metal binding, and possibly for cleft closure in hTF/2N. Trp(264) is located on the surface of the protein and does not connect to any functional residues. This explains the facts that Trp(264) is the major contributor to both the absorbance and fluorescence spectra, has a strong NMR signal and the mutation at Trp(264) has little effect on the iron-binding and release behaviours of the protein. PMID:11171122

  3. Uptake of Al3+ into the N-lobe of human serum transferrin.

    PubMed Central

    Kubal, G; Mason, A B; Sadler, P J; Tucker, A; Woodworth, R C

    1992-01-01

    We have studied the binding of Al3+ to human serum apotransferrin (80 kDa) and recombinant N-lobe human apotransferrin (40 kDa) in 0.1 M-sodium bicarbonate solution at a pH meter reading in 2H2O (pH*) of 8.8 using 1H n.m.r. spectroscopy. The results show that for the intact protein, preferential binding of Al3+ to the N-lobe occurs. Molecular modelling combined with an analysis of ring-current-induced shifts suggest that n.m.r. spectroscopy can be used to probe hinge bending processes which accompany metal uptake in solution. PMID:1497609

  4. Evolutionary Reconstructions of the Transferrin Receptor of Caniforms Supports Canine Parvovirus Being a Re-emerged and Not a Novel Pathogen in Dogs

    PubMed Central

    Kaelber, Jason T.; Demogines, Ann; Harbison, Carole E.; Allison, Andrew B.; Goodman, Laura B.; Ortega, Alicia N.; Sawyer, Sara L.; Parrish, Colin R.

    2012-01-01

    Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host. PMID:22570610

  5. Transferrin surface-modified PLGA nanoparticles-mediated delivery of a proteasome inhibitor to human pancreatic cancer cells.

    PubMed

    Frasco, Manuela F; Almeida, Gabriela M; Santos-Silva, Filipe; Pereira, Maria do Carmo; Coelho, Manuel A N

    2015-04-01

    The aim of this study was to develop a drug delivery system based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles for an efficient and targeted action of the proteasome inhibitor bortezomib against pancreatic cancer cells. The PLGA nanoparticles were formulated with a poloxamer, and further surface-modified with transferrin for tumor targeting. The nanoparticles were characterized as polymer carriers of bortezomib, and the cellular uptake and growth inhibitory effects were evaluated in pancreatic cells. Cellular internalization of nanoparticles was observed in normal and cancer cells, but with higher uptake by cancer cells. The sustained release of the loaded bortezomib from PLGA nanoparticles showed cytotoxic effects against pancreatic normal and cancer cells. Noteworthy differential cytotoxicity was attained by transferrin surface-modified PLGA nanoparticles since significant cell growth inhibition by delivered bortezomib was only observed in cancer cells. These findings demonstrate that the ligand transferrin enhanced the targeted delivery of bortezomib-loaded PLGA nanoparticles to pancreatic cancer cells. These in vitro results highlight the transferrin surface-modified PLGA nanoparticles as a promising system for targeted delivery of anticancer drugs.

  6. Comparative binding, endocytosis, and biodistribution of antibodies and antibody-coated carriers for targeted delivery of lysosomal enzymes to ICAM-1 versus transferrin receptor

    PubMed Central

    Papademetriou, Jason; Garnacho, Carmen; Serrano, Daniel; Bhowmick, Tridib; Schuchman, Edward H.; Muro, Silvia

    2012-01-01

    Targeting lysosomal enzymes to receptors involved in transport into and across cells holds promise to enhance peripheral and brain delivery of enzyme replacement therapies for lysosomal storage disorders. Receptors being explored include those associated with clathrin-mediated pathways, yet other pathways seem also viable. Well characterized examples are that of transferrin receptor (TfR) and intercellular adhesion molecule 1 (ICAM-1), involved in iron transport and leukocyte extravasation, respectively. TfR and ICAM-1 support ERT delivery via clathrin- vs. cell adhesion molecule-mediated mechanisms, displaying different valency and size restrictions. To comparatively assess this, we used antibodies vs. larger multivalent antibody-coated carriers and evaluated TfR vs. ICAM-1 binding and endocytosis in endothelial cells, as well as in vivo biodistribution and delivery of a model lysosomal enzyme required in peripheral organs and brain: acid sphingomyelinase (ASM), deficient in types A–B Niemann Pick disease. We found similar binding of antibodies to both receptors under control conditions, with enhanced binding to activated endothelium for ICAM-1, yet only anti-TfR induced endocytosis efficiently. Contrarily, antibody-coated carriers showed enhanced binding, engulfment, and endocytosis for ICAM-1. In mice, anti-TfR enhanced brain targeting over anti-ICAM, with an opposite outcome in the lungs, while carriers enhanced ICAM-1 targeting over TfR in both organs. Both targeted carriers enhanced ASM delivery to the brain and lungs vs. free ASM, with greater enhancement for anti-ICAM carriers. Therefore, targeting TfR or ICAM-1 improves lysosomal enzyme delivery. Yet, TfR targeting may be more efficient for smaller conjugates or fusion proteins, while ICAM-1 targeting seems superior for multivalent carrier formulations. PMID:22968581

  7. The Next Generation Non-competitive Active Polyester Nanosystems for Transferrin Receptor-mediated Peroral Transport Utilizing Gambogic Acid as a Ligand

    PubMed Central

    Saini, P.; Ganugula, R.; Arora, M.; Kumar, M. N. V. Ravi

    2016-01-01

    The current methods for targeted drug delivery utilize ligands that must out-compete endogenous ligands in order to bind to the active site facilitating the transport. To address this limitation, we present a non-competitive active transport strategy to overcome intestinal barriers in the form of tunable nanosystems (NS) for transferrin receptor (TfR) utilizing gambogic acid (GA), a xanthanoid, as its ligand. The NS made using GA conjugated poly(lactide-co-glycolide) (PLGA) have shown non-competitive affinity to TfR evaluated in cell/cell-free systems. The fluorescent PLGA-GA NS exhibited significant intestinal transport and altered distribution profile compared to PLGA NS in vivo. The PLGA-GA NS loaded with cyclosporine A (CsA), a model peptide, upon peroral dosing to rodents led to maximum plasma concentration of CsA at 6 h as opposed to 24 h with PLGA-NS with at least 2-fold higher levels in brain at 72 h. The proposed approach offers new prospects for peroral drug delivery and beyond. PMID:27388994

  8. α6 Integrin (α6high)/Transferrin Receptor (CD71)low Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells

    PubMed Central

    Metral, Elodie; Bechetoille, Nicolas; Demarne, Frédéric; Rachidi, Walid; Damour, Odile

    2017-01-01

    The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype. PMID:28134816

  9. Serum erythropoietin and its relation with soluble transferrin receptor in patients with different types of anaemia in a locally defined reference population.

    PubMed

    Roque, M E; Sandoval, M J; Aggio, M C

    2001-10-01

    Serum erythropoietin (Epo) and soluble transferrin receptor (sTR) were measured in a locally defined reference population (n=100): healthy volunteers (n=50); iron- deficiency anaemia (n=41) and haemolytic anaemia (n=9) (beta-thalassaemia, n = 4; autoimmune, n=5). Our data demonstrated an inverse relationship between erythroid activity and Epo levels. The regression line between Ln Epo and haemoglobin (Hb) was highly significant: P < 0.0001, r2=0.8275, Ln Epo=8.5346-0.04275 Hb, confidence limit 95%. The mean observed/predicted (O/P) ratio of Ln (Epo) was 1.01 +/- 0.11. We demonstrated that the serum Epo concentration in this particular population correlated consistently with clinical measures of erythropoietic activity. sTR, a new index of erythropoiesis, varied from 16.1 to 148 nmol/l, mean 62.0 nmol/l in the anaemic patients' group. The relationship between Ln Epo and Ln sTR was highly significant: P < 0.0001. We conclude that locally defined regression analyses are crucial for correct data interpretation and can indicate whether or not Epo production is appropriate or inappropriate. Serial determinations of sTR could help in the assessment of response to therapeutic doses of Epo.

  10. α6 Integrin (α6(high))/Transferrin Receptor (CD71)(low) Keratinocyte Stem Cells Are More Potent for Generating Reconstructed Skin Epidermis Than Rapid Adherent Cells.

    PubMed

    Metral, Elodie; Bechetoille, Nicolas; Demarne, Frédéric; Rachidi, Walid; Damour, Odile

    2017-01-27

    The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6(high)/CD71(low)) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6(high)/CD71(low) cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6(high)/CD71(low) phenotype.

  11. Combined measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein by an inexpensive, sensitive, and simple sandwich enzyme-linked immunosorbent assay technique.

    PubMed

    Erhardt, Juergen G; Estes, John E; Pfeiffer, Christine M; Biesalski, Hans K; Craft, Neal E

    2004-11-01

    The measurement of vitamin A (VA) and iron status is very important in the assessment of nutritional deficiencies. The objective of this research was to develop a sandwich ELISA technique for the simultaneous measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein (CRP) as indicators for VA and iron status. The inclusion of CRP as marker of infection allows for more accurate interpretation of VA and iron status. This is accomplished in a 30-microL serum or plasma sample using an ELISA with different capture and detection antibodies and different dilutions of the sample. Commercially available clinical serum controls were used for calibration purposes. The developed assays were compared to commercially available traditional tests. Regression coefficients comparing both assays were better than 0.84 (P < 0.001). Using a limited sample set, the sandwich ELISA assay produced very similar specificity and sensitivity compared to traditional methods when common cutoff values were applied. Intra- and interassay variability was between 5 and 14% for all tests. The cost of the materials for all 5 measurements decreases to less than $1/sample if a large number of samples is analyzed. Due to the low cost, high throughput, and comparability to traditional tests, this procedure has several advantages for assessing VA and iron status in population surveys.

  12. Real-Time Specific Light-Up Sensing of Transferrin Receptor: Image-Guided Photodynamic Ablation of Cancer Cells through Controlled Cytomembrane Disintegration.

    PubMed

    Zhang, Ruoyu; Feng, Guangxue; Zhang, Chong-Jing; Cai, Xiaolei; Cheng, Xiamin; Liu, Bin

    2016-05-03

    Transferrin receptor (TfR) represents a unique target for specific imaging of cancer cells and targeted delivery of therapeutic reagents. Detection and qualification of TfR is thus of great importance for cancer diagnosis and therapy. In this contribution, a light-up probe TPETH-2T7 was developed by conjugating a red-emissive photosensitizer with aggregation-induced emission (AIE) characteristics to a TfR-targeting peptide T7. The probe is almost nonemissive by itself, but it gives turn-on fluorescence in the presence of TfR with a detection limit of 0.45 μg/mL. Cellular experiments show that the probe specifically binds to TfR-overexpressed cancer cells. Real-time imaging results reveal that the probe stains the MDA-MB-231 cell membrane in 30 min, which is followed by probe internalization. Experiments on image-guided photodynamic cancer ablation show that the therapeutic performance is better when TPETH-2T7 is localized on the cell membrane as compared to that being internalized into cells. Confocal laser scanning microscopy (CLSM) study reveals that cytomembrane disintegration allows quick ablation of MDA-MB-231 cells.

  13. Transferrin Iron Starvation Therapy for Lethal Bacterial and Fungal Infections

    PubMed Central

    Lin, Lin; Pantapalangkoor, Paul; Tan, Brandon; Bruhn, Kevin W.; Ho, Tiffany; Nielsen, Travis; Skaar, Eric P.; Zhang, Yaofang; Bai, Ruipeng; Wang, Amy; Doherty, Terence M.; Spellberg, Brad

    2014-01-01

    New strategies to treat antibiotic-resistant infections are urgently needed. We serendipitously discovered that stem cell conditioned media possessed broad antimicrobial properties. Biochemical, functional, and genetic assays confirmed that the antimicrobial effect was mediated by supra-physiological concentrations of transferrin. Human transferrin inhibited growth of gram-positive (Staphylococcus aureus), gram-negative (Acinetobacter baumannii), and fungal (Candida albicans) pathogens by sequestering iron and disrupting membrane potential. Serial passage in subtherapeutic transferrin concentrations resulted in no emergence of resistance. Infected mice treated with intravenous human transferrin had improved survival and reduced microbial burden. Finally, adjunctive transferrin reduced the emergence of rifampin-resistant mutants of S. aureus in infected mice treated with rifampin. Transferrin is a promising, novel antimicrobial agent that merits clinical investigation. These results provide proof of principle that bacterial infections can be treated in vivo by attacking host targets (ie, trace metal availability) rather than microbial targets. PMID:24446527

  14. TIBC, UIBC and Transferrin

    MedlinePlus

    ... test to evaluate people suspected of having either iron deficiency or iron overload. These two tests are used ... transferrin sites are used to transport iron. In iron deficiency, the iron level is low but the TIBC ...

  15. Revisiting nanoparticle technology for blood-brain barrier transport: Unfolding at the endothelial gate improves the fate of transferrin receptor-targeted liposomes.

    PubMed

    Johnsen, Kasper Bendix; Moos, Torben

    2016-01-28

    An unmet need exists for therapeutic compounds to traverse the brain capillary endothelial cells that denote the blood-brain barrier (BBB) to deliver effective treatment to the diseased brain. The use of nanoparticle technology for targeted delivery to the brain implies that targeted liposomes encapsulating a drug of interest will undergo receptor-mediated uptake and transport through the BBB with a subsequent unfolding of the liposomal content inside the brain, hence revealing drug release to adjacent drug-demanding neurons. As transferrin receptors (TfRs) are present on brain capillary endothelial, but not on endothelial cells elsewhere in the body, the use of TfR-targeted liposomes - colloidal particulates with a phospholipid bilayer membrane - remains the most relevant strategy to obtain efficient drug delivery to the brain. However, many studies have failed to provide sufficient quantitative data to proof passage of the BBB and significant appearance of drugs inside the brain parenchyma. Here, we critically evaluate the current evidence on the use of TfR-targeted liposomes for brain drug delivery based on a thorough investigation of all available studies within this research field. We focus on issues with respect to experimental design and data analysis that may provide an explanation to conflicting reports, and we discuss possible explanations for the current lack of sufficient transcytosis across the BBB for implementation in the design of TfR-targeted liposomes. We finally provide a list of suggestions for strategies to obtain substantial uptake and transport of drug carriers at the BBB with a concomitant transport of therapeutics into the brain.

  16. Iron, transferrin and myelinogenesis

    NASA Astrophysics Data System (ADS)

    Sergeant, C.; Vesvres, M. H.; Devès, G.; Baron, B.; Guillou, F.

    2003-09-01

    Transferrin (Tf), the iron binding protein of vertebrates serum, is known to be synthesized by oligodendrocytes (Ols) in the central nervous system. It has been postulated that Tf is involved in Ols maturation and myelinogenesis. This link is particularly important in the understanding of a severe human pathology: the multiple sclerosis, which remains without efficient treatment. We generated transgenic mice containing the complete human Tf gene and extensive regulatory sequences from the 5 ' and 3 ' untranslated regions that specifically overexpress Tf in Ols. Brain cytoarchitecture of the transgenic mice appears to be normal in all brain regions examined, total myelin content is increased by 30% and motor coordination is significantly improved when compared with non-transgenic littermates. Tf role in the central nervous system may be related to its affinity for metallic cations. Normal and transgenic mice were used for determination of trace metals (iron, copper and zinc) and minerals (potassium and calcium) concentration in cerebellum and corpus callosum. The freeze-dried samples were prepared to allow proton-induced X-ray emission and Rutherford backscattering spectrometry analyses with the nuclear microprobe in Bordeaux. Preliminary results were obtained and carbon distribution was revealed as a very good analysis to distinguish precisely the white matter region. A comparison of metallic and mineral elements contents in brain between normal and transgenic mice shows that iron, copper and zinc levels remained constant. This result provides evidence that effects of Tf overexpression in the brain do not solely relate to iron transport.

  17. Combined radiolabel-binding and immunocytochemical evaluation of receptor–ligand interactions. Studies of transferrin receptors on activated lymphocytes

    PubMed Central

    Galbraith, Gillian M. P.; Galbraith, Robert M.

    1981-01-01

    A protocol that involved both immunohistological and radiolabel-binding procedures was devised for the study of transferrin–receptor interactions. This composite approach yielded considerably more information than did either technique used alone, and also provided a simple means for exclusion of several common potential sources of error. PMID:6277297

  18. Cross sectional, comparative study of serum erythropoietin, transferrin receptor, ferritin levels and other hematological indices in normal pregnancies and iron deficiency anemia during pregnancy.

    PubMed

    Sharma, Jai B; Bumma, Sirisha D; Saxena, Renu; Kumar, Sunesh; Roy, Kallol K; Singh, Neeta; Vanamail, P

    2016-08-01

    To test the correlation of the serum erythropoietin levels, serum transferrrin receptor levels and serum ferritin levels along with other hematological parameters in normal pregnant and anemic pregnant patients. In a prospective study, 120 pregnant women were recruited between 18 and 36 weeks of gestation; 53 normal pregnant patients, 67 anemic pregnant patients, in which, 17 had mild, 30 had moderate anemia, 20 had severe anemia. A blood sample was taken. The various hematological parameters, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), total iron binding capacity (TIBC), serum ferritin, percentage saturation of iron, serum erythropoietin (SEPO) levels, serum transferrin receptors (STfRS) were performed. For statistics, Student's 't' test, Pearson's Chi test, Mann Whitney test and Bartlett test were used as per data. MCV was significantly reduced in anemic pregnancies as compared to non-anemic pregnancies (80.2±9.6 vs 94.12±9.8fl, p=0.001), MCHC was also reduced in them (30.2±3.38% vs 34.2±2.33%, p=0.176), TIBC was significantly increased in anemic pregnancies (343.31±28.54% vs 322.88±23.84%, p=0.001), serum ferritin was significantly reduced (24.9±10.48μg/L vs 31.03±9.98μg/L, p=0.001), percentage saturation of iron was also reduced (53.85±13.21% vs 62.04±15.79%, p=0.0024), serum erythropoietin levels were significantly higher in anemic women (26.24±26.61mU/ml vs 18.12±19.08mU/ml, p=0.064). The levels were significantly higher in severe anemia (46.5±46.8mU/ml than in moderate anemia 27.4±28.1mU/ml and mild anemia 22.8±22.8mU/ml. Serum transferrin receptors were significantly higher in anemic pregnancies than in non-anemic pregnancies (1.40±0.0802μg/ml vs 1.08±0.641μg/ml, p=0.019) with rise being higher in severe anemia (2.28±0.986μg/ml) than in moderate (1.4±0.816μg/ml) and mild anemia (1.16±0.702μg/ml). Various hematological parameters especially sTfR, serum erythropoietin, serum

  19. Characterization of transferrin binding proteins 1 and 2 in invasive type b and nontypeable strains of Haemophilus influenzae.

    PubMed Central

    Gray-Owen, S D; Schryvers, A B

    1995-01-01

    Haemophilus influenzae has the ability to obtain iron from human transferrin via two bacterial cell surface transferrin binding proteins, Tbp1 and Tbp2. Although a wide array of strains have been shown to express these receptor proteins, two studies have recently identified a series of isolates which appeared to lack the ability to bind transferrin. Included in this group were the members of a cryptic genospecies of nontypeable biotype IV strains which appear to possess a tropism for female urogenital tissues and are major etiologic agents of neonatal and postpartum bacteremia due to H. influenzae. The present study employed oligonucleotide primers specific for genes encoding the Tbp proteins of a type b biotype I strain of H. influenzae to probe the genomic DNAs of isolates from the previous studies. The tbpA and tbpB genes which encode Tbp1 and Tbp2, respectively, were detected in all of the strains tested either by PCR amplification directly or by Southern hybridization analysis. All of the strains displayed a transferrin binding phenotype, and affinity isolation of receptor proteins with transferrin-conjugated Sepharose recovered Tbp1 and/or Tbp2 from 11 of 14 strains, including 2 of the nontypeable biotype IV strains. In addition, all of the strains were capable of growing on human transferrin specifically, indicating that the mechanism of iron assimilation from transferrin is functional and is not siderophore mediated. These results confirm the presence of tbp genes in all of the invasive H. influenzae isolates characterized to date, suggesting that Tbp-mediated iron acquisition is important in disease which initiates from either the respiratory or urogenital mucosa. PMID:7558284

  20. Depleted iron stores and iron deficiency anemia associated with reduced ferritin and hepcidin and elevated soluble transferrin receptors in a multiethnic group of preschool-age children.

    PubMed

    Weiler, Hope A; Jean-Philippe, Sonia; Cohen, Tamara R; Vanstone, Catherine A; Agellon, Sherry

    2015-09-01

    Iron deficiency anemia is prevalent in subgroups of the Canadian population. The objective of this study was to examine iron status and anemia in preschool-age children. Healthy children (n = 430, 2-5 years old, Montreal, Quebec, Canada) were sampled from randomly selected daycares. Anthropometry, demographics, and diet were assessed. Biochemistry included hemoglobin, ferritin, soluble transferrin receptors (sTfR), ferritin index, markers of inflammation (C-reactive protein, interleukin 6 (IL-6), and tumour necrosis factor alpha (TNFα)), and hepcidin. Iron deficiency and anemia cutoffs conformed to the World Health Organization criteria. Differences among categories were tested using mixed-model ANOVA or χ(2) tests. Children were 3.8 ± 1.0 years of age, with a body mass index z score of 0.48 ± 0.97, and 51% were white. Adjusted intakes of iron indicated <1% were at risk for deficiency. Hemoglobin was higher in white children, whereas ferritin was higher with greater age and female sex. Inflammatory markers and hepcidin did not vary with any demographic variable. The prevalence of iron deficiency was 16.5% (95% confidence interval (CI), 13.0-20.0). Three percent (95% CI, 1.4-4.6) of children had iron deficiency anemia and 12.8% (95% CI, 9.6-16.0) had unexplained anemia. Children with iron deficiency, with and without anemia, had lower plasma ferritin and hepcidin but higher sTfR, ferritin index, and IL-6, whereas those with unexplained anemia had elevated TNFα. We conclude that iron deficiency anemia is not very common in young children in Montreal. While iron deficiency without anemia is more common than iron deficiency with anemia, the correspondingly reduced circulating hepcidin would have enabled heightened absorption of dietary iron in support of erythropoiesis.

  1. Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking

    PubMed Central

    Solis, Gonzalo P.; Hülsbusch, Nikola; Radon, Yvonne; Katanaev, Vladimir L.; Plattner, Helmut; Stuermer, Claudia A. O.

    2013-01-01

    The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1–decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA–mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin–transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca2+ switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca2+ repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved. PMID:23825023

  2. Enhanced sandwich immunoassay using antibody-functionalized magnetic iron-oxide nanoparticles for extraction and detection of soluble transferrin receptor on a photonic crystal biosensor.

    PubMed

    Peterson, Ross D; Chen, Weili; Cunningham, Brian T; Andrade, Juan E

    2015-12-15

    Iron deficiency anemia (IDA) has detrimental effects on individuals and societies worldwide. A standard sandwich assay (SA) for the detection of soluble transferrin receptor (sTfR), a biomarker of IDA, on a photonic crystal (PC) biosensor was established, but it was susceptible to non-specific signals from complex matrixes. In this study, iron-oxide nanoparticles (fAb-IONs) were used as magnetic immuno-probes to bind sTfR and minimize non-specific signals, while enhancing detection on the PC biosensor. This inverse sandwich assay (IA) method completely bound sTfR with low variability (<4% RSD) in buffer and allowed for its accurate and precise detection in sera (Liquichek™ control sera) on the PC biosensor using two certified ELISAs as reference methods. A linear dose-response curve was elicited at the fAb-IONs concentration in which the theoretical binding ratio (sTfR:fAb-IONs) was calculated to be <1 on the IA. The LoDs for sTfR in the SA and IA were similar (P>0.05) at 14 and 21 μg/mL, respectively. The inherent imprecision of the IA and reference ELISAs was σ(δ)=0.45 µg/mL and the mean biases for Liquichek™ 1, 2 and 3 were 0.18, 0.19 and -0.04 µg/mL, respectively. Whereas the inherent imprecision of the SA and reference ELISAs was σ(δ)=0.52 µg/mL and the biases for Liquichek™ 1, 2 and 3 were 0.66, 0.14 and -0.67 µg/mL, respectively. Thus, unlike the SA, the IA method measures sTfR with the same bias as the reference ELISAs. Combined magnetic separation and detection of nutrition biomarkers on PC biosensors represents a facile method for their accurate and reliable quantification in complex matrixes.

  3. Serum Soluble Transferrin Receptor Concentrations Are Elevated in Congolese Children with Glucose-6-Phosphate Dehydrogenase Variants, but Not Sickle Cell Variants or α-Thalassemia.

    PubMed

    Barker, Mikaela K; Henderson, Amanda M; Naguib, Karimah; Vercauteren, Suzanne M; Devlin, Angela M; Albert, Arianne Y; Bahizire, Esto; Tugirimana, Pierrot L; Akilimali, Pierre Z; Boy, Erick; Green, Tim J; Karakochuk, Crystal D

    2017-09-01

    Background: Anemia is common in Congolese children, and inherited blood disorders may be a contributing cause. The presence of sickle cell variants, X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency and α-thalassemia, has been previously reported. G6PD A- deficiency is characterized by the co-inheritance of G6PD 376 and 202 variants and is common in sub-Saharan Africa.Objective: We aimed to measure the associations between inherited blood disorders and hemoglobin, ferritin, and soluble transferrin receptor (sTfR) concentrations in Congolese children.Methods: Venous blood was collected from 744 children aged 6-59 mo from 2 provinces. We measured biomarkers of nutritional and inflammation status and malaria. Pyrosequencing was used to detect sickle cell variants. Polymerase chain reaction was used to detect G6PD variants and α-thalassemia deletions.Results: Overall, 11% of children had a sickle cell variant, 19% of boys were G6PD A- hemizygotes, 12% and 10% of girls were G6PD A- hetero- or homozygotes, respectively, and 12% of children had α-thalassemia. Multivariable linear regression models (adjusted for age, province, altitude, malaria, and biomarkers of nutritional and inflammation status) showed that G6PD A- hemizygous boys and G6PD 376 homozygous girls had higher sTfR concentrations [geometric mean ratios (95% CIs): 1.20 (1.03, 1.39) and 1.25 (1.02, 1.53), respectively] than children with no G6PD variants. Hemoglobin and ferritin concentrations were not independently associated with any of the inherited blood disorder genotypes.Conclusions: We found that 2 G6PD variant genotypes were associated with elevated sTfR concentrations, which limits the accuracy of sTfR as a biomarker of iron status in this population. © 2017 American Society for Nutrition.

  4. Transferrin receptor-targeted pH-sensitive micellar system for diminution of drug resistance and targetable delivery in multidrug-resistant breast cancer

    PubMed Central

    Gao, Wei; Ye, Guihua; Duan, Xiaochuan; Yang, Xiaoying; Yang, Victor C

    2017-01-01

    The emergence of drug resistance is partially associated with overproduction of transferrin receptor (TfR). To overcome multidrug resistance (MDR) and achieve tumor target delivery, we designed a novel biodegradable pH-sensitive micellar system modified with HAIYPRH, a TfR ligand (7pep). First, the polymers poly(l-histidine)-coupled polyethylene glycol-2000 (PHIS-PEG2000) and 7pep-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (7pep-DSPE-PEG2000) were synthesized, and the mixed micelles were prepared by blending of PHIS-PEG2000 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG2000) or 7pep-DSPE-PEG2000 (7-pep HD micelles). The micelles exhibited good size uniformity, high encapsulation efficiency, and a low critical micelle concentration. By changing the polymer ratio in the micellar formulation, the pH response range was specially tailored to pH ~6.0. When loaded with antitumor drug doxorubicin (DOX), the micelle showed an acid pH-triggering drug release profile. The cellular uptake and cytotoxicity study demonstrated that 7-pep HD micelles could significantly enhance the intracellular level and antitumor efficacy of DOX in multidrug-resistant cells (MCF-7/Adr), which attributed to the synergistic effect of poly(l-histidine)-triggered endolysosom escape and TfR-mediated endocytosis. Most importantly, the in vivo imaging study confirmed the target-ability of 7-pep HD micelles to MDR tumor. These findings indicated that 7-pep HD micelles would be a promising drug delivery system in the treatment of drug-resistant tumors. PMID:28223798

  5. Utility of Access Soluble Transferrin Receptor (sTfR) and sTfR/log Ferritin Index in Diagnosing Iron Deficiency Anemia.

    PubMed

    Shin, Dong Hoon; Kim, Hyun Soo; Park, Min Jeong; Suh, In Bum; Shin, Kyu Sung

    2015-01-01

    The Access(®) soluble transferrin receptor (sTfR) is considered the world's first automated chemiluminescence immunoassay. In this study, the diagnostic utility of this and other tests for serum iron were evaluated by studying their interrelationships with inflammation. A total of 367 patients with anemia (iron deficiency anemia [IDA], 157; anemia of chronic disease [ACD], 210) and 80 normal controls were subjected to a battery of diagnostic tests, including complete blood cell count, serum iron, total iron-binding capacity (TIBC), C-reactive protein (CRP), ferritin, sTfR, and hepcidin. The accuracy of test parameters was determined by the area under the receiver operating characteristic curve (AUC). Patients falling within the ferritin grey zone (10-100 ng/ml) were evaluated separately, given that such individuals are typically difficult to detect and manage in actual clinical practice. CRP was used to assess the correlation between the aforementioned markers of iron and inflammation. The single most accurate diagnostic test used to differentiate IDA from ACD was serum ferritin (AUC 0.989). However, sTfR assay outperformed other tests in the ferritin grey zone (AUC 0.931), and the sTfR/log ferritin index was the most reliable parameter in both scenarios (AUC 0.994 and 0.962, respectively). Ferritin, TIBC, and hepcidin showed the highest correlation with CRP, whereas sTfR displayed the lowest. The Access sTfR and sTfR/log ferritin index enabled highly accurate diagnosis of IDA in the ferritin grey zone. This is an easy-to-use automated chemiluminescence immunoassay, amenable to routine use in hospitals. © 2015 by the Association of Clinical Scientists, Inc.

  6. Neptunium uptake by serum transferrin.

    PubMed

    Llorens, Isabelle; Den Auwer, Christophe; Moisy, Philippe; Ansoborlo, Eric; Vidaud, Claude; Funke, Harld

    2005-04-01

    Although of major impact in terms of biological and environmental hazards, interactions of actinide cations with biological molecules are only partially understood. Human serum transferrin (Tf) is one of the major iron carriers in charge of iron regulation in the cell cycle and consequently contamination by actinide cations is a critical issue of nuclear toxicology. Combined X-ray absorption spectroscopy (XAS) and near infrared absorption spectrometry were used to characterize a new complex between Tf and Np (IV) with the synergistic nitrilotriacetic acid (NTA) anion. Description of the neptunium polyhedron within the iron coordination site is given.

  7. Comparison of colorimetry and electrothermal atomic absorption spectroscopy for the quantification of non-transferrin bound iron in human sera.

    PubMed

    Jittangprasert, Piyada; Wilairat, Prapin; Pootrakul, Pensri

    2004-12-01

    This paper describes a comparison of two analytical techniques, one employing bathophenanthrolinedisulfonate (BPT), a most commonly-used reagent for Fe (II) determination, as chromogen and an electrothermal atomic absorption spectroscopy (ETAAS) for the quantification of non-transferrin bound iron (NTBI) in sera from thalassemic patients. Nitrilotriacetic acid (NTA) was employed as the ligand for binding iron from low molecular weight iron complexes present in the serum but without removing iron from the transferrin protein. After ultrafiltration the Fe (III)-NTA complex was then quantified by both methods. Kinetic study of the rate of the Fe (II)-BPT complex formation for various excess amounts of NTA ligand was also carried out. The kinetic data show that a minimum time duration (> 60 minutes) is necessary for complete complex formation when large excess of NTA is used. Calibration curves given by colorimetric and ETAAS methods were linear over the range of 0.15-20 microM iron (III). The colorimetric and ETAAS methods exhibited detection limit (3sigma) of 0.13 and 0.14 microM, respectively. The NTBI concentrations from 55 thalassemic serum samples measured employing BPT as chromogen were statistically compared with the results determined by ETAAS. No significant disagreement at 95% confidence level was observed. It is, therefore, possible to select any one of these two techniques for determination of NTBI in serum samples of thalassemic patients. However, the colorimetric procedure requires a longer analysis time because of a slow rate of exchange of NTA ligand with BPT, leading to the slow rate of formation of the colored complex.

  8. [13C]Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride.

    PubMed Central

    He, Q Y; Mason, A B; Tam, B M; MacGillivray, R T; Woodworth, R C

    1999-01-01

    The N-lobe of human serum transferrin (hTF/2N) and single point mutants in which each of the five methionine residues was individually mutated have been produced in a mammalian tissue-culture expression system. Since the five methionine residues are well distributed in the transferrin N-lobe, (13)C NMR of the [epsilon-(13)C]methionine-labelled proteins has been used to monitor conformational changes of the protein during metal binding. All five methionine residues have been assigned [Beatty, Cox, Frenkiel, Tam, Mason, MacGillivray, Sadler and Woodworth (1996) Biochemistry 35, 7635-7642]. The tentative two-dimensional NMR assignment for two of the five methionine residues, namely Met(26) and Met(109), has been corrected. A series of NMR spectra for the complexes of (13)C-Met-labelled hTF/2N with six different metal ions, Fe(III), Cu(II), Cr(III), Co(III), Ga(III) and In(III), demonstrate that the conformational change of the protein upon metal binding can be observed by means of the changes in the NMR chemical shifts associated with certain methionine residues, regardless of whether diamagnetic or paramagnetic metals are used. Changing any of the methionine residues should have minimal effects on transferrin function, since structural analysis shows that none of these residues contacts functional amino acids or has any obvious role in iron uptake or release. In fact, UV-visible spectra show little perturbation of the electronic spectra of any of the mutants. Nevertheless, the M109L mutant (Met(109)-->Leu) releases iron at half the rate of the wild-type N-lobe, and chloride shows a significantly greater retarding effect on the rate of iron release from all five mutants. All the methionine mutants (especially in the apo form) show a poor solubility in Hepes buffer lacking anions such as bicarbonate. These findings imply a more general effect of anion binding to surface residues than previously realized. PMID:10585877

  9. [Transferrin in various hematological syndrome].

    PubMed

    Gepner-Woźniewska, M; Sitarska, E; Kluciński, W; Konopka, L; Roszkowski, S

    1977-01-01

    In 100 patients with various haematological syndromes and in 22 healthy subjects serum transferrin was determined by the method of radial immunodiffusion of Mancini. The results were correlated with total iron-binding capacity, iron concentration, beta1 globulin and albumin levels. A statistically significant rise in serum transferrin concentration was demonstrated only in patients with sideropenic anaemia. In the remaining groups of patients transferrin concentration was decreased without regard to associated disturbances of iron metabolism and iron level. The comparison of the total iron-binding capacity with transferrin level determined by radial immunodiffusion may suggest presence of iron-binding proteins other than transferrin.

  10. Alteration at translational but not transcriptional level of transferrin receptor expression following manganese exposure at the blood–CSF barrier in vitro

    PubMed Central

    Li, G. Jane; Zhao, Qiuqu; Zheng, Wei

    2014-01-01

    Manganese exposure alters iron homeostasis in blood and cerebrospinal fluid (CSF), possibly by acting on iron transport mechanisms localized at the blood–brain barrier and/or blood–CSF barrier. This study was designed to test the hypothesis that manganese exposure may change the binding affinity of iron regulatory proteins (IRPs) to mRNAs encoding transferrin receptor (TfR), thereby influencing iron transport at the blood–CSF barrier. A primary culture of choroidal epithelial cells was adapted to grow on a permeable membrane sandwiched between two culture chambers to mimic blood–CSF barrier. Trace 59Fe was used to determine the transepithelial transport of iron. Following manganese treatment (100 µM for 24 h), the initial flux rate constant (Ki) of iron was increased by 34%, whereas the storage of iron in cells was reduced by 58%, as compared to controls. A gel shift assay demonstrated that manganese exposure increased the binding of IRP1 and IRP2 to the stem loop-containing mRNAs. Consequently, the cellular concentrations of TfR proteins were increased by 84% in comparison to controls. Assays utilizing RT-PCR, quantitative real-time reverse transcriptase-PCR, and nuclear run off techniques showed that manganese treatment did not affect the level of heterogeneous nuclear RNA (hnRNA) encoding TfR, nor did it affect the level of nascent TfR mRNA. However, manganese exposure resulted in a significantly increased level of TfR mRNA and reduced levels of ferritin mRNA. Taken together, these results suggest that manganese exposure increases iron transport at the blood–CSF barrier; the effect is likely due to manganese action on translational events relevant to the production of TfR, but not due to its action on transcriptional, gene expression of TfR. The disrupted protein–TfR mRNA interaction in the choroidal epithelial cells may explain the toxicity of manganese at the blood–CSF barrier. PMID:15893546

  11. The Homozygous Hemoglobin EE Genotype and Chronic Inflammation Are Associated with High Serum Ferritin and Soluble Transferrin Receptor Concentrations among Women in Rural Cambodia.

    PubMed

    Karakochuk, Crystal D; Whitfield, Kyly C; Rappaport, Aviva I; Barr, Susan I; Vercauteren, Suzanne M; McLean, Judy; Prak, Sophonneary; Hou, Kroeun; Talukder, Aminuzzaman; Devenish, Robyn; Green, Timothy J

    2015-12-01

    Ferritin and soluble transferrin receptor (sTfR) concentrations are commonly used to assess iron deficiency (ID); however, they are influenced by multiple factors. We assessed associations between numerous variables and both ferritin and sTfR concentrations in Cambodian women and compared ID prevalence through the use of study-generated correction factors (CFs) for ferritin with those from a published meta-analysis. Venous blood from 450 women (aged 18-45 y) was assessed for hemoglobin (Hb), ferritin, sTfR, retinol binding protein, folate, vitamin B-12, C-reactive protein, α-1 acid glycoprotein (AGP), and genetic Hb disorders. Linear regression was used to calculate geometric mean ratios (95% CIs) for ferritin and sTfR concentrations. The variant Hb EE genotype was associated with 50% (14%, 96%) and 51% (37%, 66%) higher geometric mean ferritin and sTfR concentrations, respectively, than was the normal Hb AA genotype; a 1-g/L increase in AGP was associated with 99% (50%, 162%) and 48% (33%, 64%) higher concentrations in the same variables, respectively. ID prevalence in nonpregnant women (n = 420) was 2% (n = 9) with the use of ferritin <15 μg/L and 18% (n = 79) with the use of sTfR >8.3 mg/L as criteria. ID prevalence with the use of sTfR was higher in women with the Hb EE genotype (n = 17; 55%) than in those with the Hb AA genotype (n = 20; 10%); and in women with the Hb AA genotype and chronic inflammation (n = 10; 18%) than in that group of women without chronic inflammation (n = 10; 7%) (P < 0.05). No differences in ID prevalence were found with the use of ferritin between women with Hb EE and AA genotypes (P = 1.0) or by chronic inflammation status (P = 0.32). There were no differences in mean ferritin concentrations among all 450 women when study-generated CFs were compared with those from the meta-analysis (P = 0.87). Compared with sTfR, ferritin concentrations appear to reflect more accurately true ID in rural Cambodian women. The CFs from a published

  12. Serum soluble transferrin receptor concentrations in US preschool children and non-pregnant women of childbearing age from the National Health and Nutrition Examination Survey 2003-2010.

    PubMed

    Mei, Zuguo; Pfeiffer, Christine M; Looker, Anne C; Flores-Ayala, Rafael C; Lacher, David A; Mirel, Lisa B; Grummer-Strawn, Laurence M

    2012-10-09

    Serum soluble transferrin receptor (sTfR) is recommended as a sensitive and accurate measure of iron deficiency (ID) in populations when only a single indicator can be used. The lack of assay standardization and of representative data on the distribution of sTfR in at-risk populations currently limits its utility. Using data from NHANES 2003-2010, we examined the distribution of sTfR and developed assay-specific cutoff values for defining elevated sTfR in 2 US populations groups: children aged 1-5 y (n=2820) and non-pregnant women aged 15-49 y (n=6575). On average, children had higher geometric mean sTfR concentrations (4.09 mg/l; 95% CI: 4.04-4.14) than non-pregnant women (3.31 mg/l; 95% CI: 3.26-3.35) (p<0.001). Among children, those aged 1-2 y (compared to those aged 3-5 y), boys (compared to girls), and non-Hispanic black (NHB) children (compared to non-Hispanic white (NHW) and Mexican-American (MA) children) had higher sTfR concentrations. Among non-pregnant women, adolescents (15-19 y) had higher sTfR concentrations than adults aged 20-34 y but not compared to adults aged 35-49 y; NHB women (compared to NHW and MA women) and multiparous women (compared to nulliparous women) had higher sTfR concentrations. The derived cutoff values (97.5th percentile in a defined healthy reference population) for defining elevated sTfR in the US were 6.00 mg/l for children 1-5 y and 5.33 mg/l for non-pregnant women 15-49 y. A different sTfR cutoff value may be needed in children and non-pregnant women to define ID. Published by Elsevier B.V.

  13. Alteration at translational but not transcriptional level of transferrin receptor expression following manganese exposure at the blood-CSF barrier in vitro

    SciTech Connect

    Li, G. Jane; Zhao Qiuqu; Zheng Wei . E-mail: wzheng@purdue.edu

    2005-06-01

    Manganese exposure alters iron homeostasis in blood and cerebrospinal fluid (CSF), possibly by acting on iron transport mechanisms localized at the blood-brain barrier and/or blood-CSF barrier. This study was designed to test the hypothesis that manganese exposure may change the binding affinity of iron regulatory proteins (IRPs) to mRNAs encoding transferrin receptor (TfR), thereby influencing iron transport at the blood-CSF barrier. A primary culture of choroidal epithelial cells was adapted to grow on a permeable membrane sandwiched between two culture chambers to mimic blood-CSF barrier. Trace {sup 59}Fe was used to determine the transepithelial transport of iron. Following manganese treatment (100 {mu}M for 24 h), the initial flux rate constant (K {sub i}) of iron was increased by 34%, whereas the storage of iron in cells was reduced by 58%, as compared to controls. A gel shift assay demonstrated that manganese exposure increased the binding of IRP1 and IRP2 to the stem loop-containing mRNAs. Consequently, the cellular concentrations of TfR proteins were increased by 84% in comparison to controls. Assays utilizing RT-PCR, quantitative real-time reverse transcriptase-PCR, and nuclear run off techniques showed that manganese treatment did not affect the level of heterogeneous nuclear RNA (hnRNA) encoding TfR, nor did it affect the level of nascent TfR mRNA. However, manganese exposure resulted in a significantly increased level of TfR mRNA and reduced levels of ferritin mRNA. Taken together, these results suggest that manganese exposure increases iron transport at the blood-CSF barrier; the effect is likely due to manganese action on translational events relevant to the production of TfR, but not due to its action on transcriptional, gene expression of TfR. The disrupted protein-TfR mRNA interaction in the choroidal epithelial cells may explain the toxicity of manganese at the blood-CSF barrier.

  14. Insights into endosomal maturation of human holo-transferrin in the enteric parasite Entamoeba histolytica: essential roles of Rab7A and Rab5 in biogenesis of giant early endocytic vacuoles.

    PubMed

    Verma, Kuldeep; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2015-12-01

    The pathogenic amoeba Entamoeba histolytica is one of the causative agents of health hazards in tropical countries. It causes amoebic dysentery, colitis and liver abscesses in human. Iron is one of the essential nutritional resources for survival and chronic infection caused by the amoeba. The parasite has developed multiple ways to import, sequester and utilize iron from various iron-binding proteins from its host. In spite of its central role in pathogenesis, the mechanism of iron uptake by the parasite is largely unknown. Here, we carried out a systematic study to understand the role of some of the amoebic homologues of mammalian endocytic Rab GTPases (Rab5 and Rab21, Rab7A and Rab7B) in intracellular transport of human holo-transferrin by the parasite. Flow cytometry and quantitative microscopic image analysis revealed that Rab5 and Rab7A are required for the biogenesis of amoebic giant endocytic vacuoles (GEVs) and regulate the early phase of intracellular trafficking of transferrin. Rab7B is involved in the late phase, leading to the degradation of transferrin in the amoebic lysosome-like compartments. Using time-lapse fluorescence imaging in fixed trophozoites, we determined the kinetics of the vesicular transport of transferrin through Rab5-, Rab7A- and Rab7B-positive compartments. The involvement of Rab7A in the early phase of endocytosis by the parasite marks a significant divergence from its host in terms of spatiotemporal regulation by the Rab GTPases.

  15. Studies on the binding of fulvic acid with transferrin by spectroscopic analysis

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-feng; Yang, Guang; Dong, Yu; Zhao, Yan-qin; Sun, Xiao-ran; Chen, Lei; Chen, Hong-bo

    2015-02-01

    Transferrin has shown potential in the delivery of anticancer drugs into primarily proliferating cancer cells that over-express transferrin receptors. Fulvic acid has a wide range of biological and pharmacological activities which caused widespread concerns, the interaction of fulvic acid with human serum transferrin (Tf) has great significance for gaining a deeper insight about anticancer activities of fulvic acid. In this study, the mechanism of interaction between fulvic acid and Tf, has been investigated by using fluorescence quenching, thermodynamics, synchronous fluorescence and circular dichroism (CD) under physiological condition. Our results have shown that fulvic acid binds to Tf and form a new complex, and the calculated apparent association constants are 5.04 × 108 M-1, 5.48 × 107 M-1, 7.38 × 106 M-1 from the fluorescence quenching at 288 K, 298 K, and 310 K. The thermodynamic parameters indicate that hydrogen bonding and weak van der Waals are involved in the interaction between fulvic acid and Tf. The binding of fulvic acid to Tf causes the α-helix structure content of the protein to reduce, and resulting that peptide chains of Tf become more stretched. Our results have indicated a mechanism of the interaction between fulvic acid and Tf, which may provide information for possible design of methods to deliver drug molecules via transferrin to target tissues and cells effectively.

  16. Transferrin is required for early T-cell differentiation.

    PubMed

    Macedo, M Fatima; de Sousa, Maria; Ned, Renee M; Mascarenhas, Claudia; Andrews, Nancy C; Correia-Neves, Margarida

    2004-08-01

    Transferrin, the major plasma iron carrier, mediates iron entry into cells through interaction with its receptor. Several in vitro studies have demonstrated that transferrin plays an essential role in lymphocyte division, a role attributed to its iron transport function. In the present study we used hypotransferrinaemic (Trf(hpx/hpx)) mice to investigate the possible involvement of transferrin in T lymphocyte differentiation in vivo. The absolute number of thymocytes was substantially reduced in Trf(hpx/hpx) mice, a result that could not be attributed to increased apoptosis. Moreover, the proportions of the four major thymic subpopulations were maintained and the percentage of dividing cells was not reduced. A leaky block in the differentiation of CD4(-) CD8(-) CD3(-) CD44(-) CD25(+) (TN3) into CD4(-) CD8(-) CD3(-) CD44(-) CD25(-) (TN4) cells was observed. In addition, a similar impairment of early thymocyte differentiation was observed in mice with reduced levels of transferrin receptor. The present study demonstrates, for the first time, that transferrin itself or a pathway triggered by the interaction of transferrin with its receptor is essential for normal early T-cell differentiation in vivo.

  17. Using Soluble Transferrin Receptor and Taking Inflammation into Account When Defining Serum Ferritin Cutoffs Improved the Diagnosis of Iron Deficiency in a Group of Canadian Preschool Inuit Children from Nunavik

    PubMed Central

    Turgeon O'Brien, Huguette; Blanchet, Rosanne; Gagné, Doris; Vézina, Carole

    2016-01-01

    The prevalence of iron depletion, iron deficient erythropoiesis (IDE), and iron deficiency anemia (IDA) was assessed in preschool Inuit children using soluble transferrin receptor (sTfR) and traditional indicators of iron status while disregarding or taking inflammation into account when defining SF cutoffs. Iron depletion was defined as follows: (1) SF < 15 μg/L regardless of the C-reactive protein (CRP) level and (2) SF < 15 or <50 μg/L with CRP ≤ 5 or >5 mg/L, respectively. IDE corresponded to iron depletion combined with total iron binding capacity > 72 μmol/L and/or transferrin saturation < 16%. Iron depletion and IDE affected almost half of the children when accounting for inflammation, compared to one-third when the SF cutoff was defined regardless of CRP level (P < 0.0001). The prevalence of IDE adjusted for inflammation (45.1%) was very similar to the prevalence observed when sTfR was used as a sole marker of IDE (47.4%). The prevalence of anemia was 15%. The prevalence of IDA (IDE + hemoglobin < 110 g/L) was higher when accounting for than when disregarding inflammation (8.0% versus 6.2%, P = 0.083). Using sTfR and different SF cutoffs for children with versus without inflammation improved the diagnosis of iron depletion and IDE. Our results confirm that Inuit children are at particularly high risk for iron deficiency. PMID:27382488

  18. Investigation on the interaction between tamoxifen and human holo-transferrin: determination of the binding mechanism by fluorescence quenching, resonance light scattering and circular dichroism methods.

    PubMed

    Sarzehi, Sareh; Chamani, Jamshidkhan

    2010-11-01

    The interaction between tamoxifen (TMX) and human serum transferrin (HTF) was for the first time studied at varying pH values by fluorescence spectroscopy, circular dichroism (CD) and resonance light scattering (RLS). The fluorescence spectroscopy experiments were performed in order to study conformational changes, possibly due to a discrete reorganization of tryptophan residues during TMX-HTF binding at varying ligand concentrations, as well as quenching properties of the drug-serum transferrin complex and the differentiation between static and dynamic quenching. The binding affinity and number of binding sites were obtained for the TMX-HTF interaction at different pH. Second derivative fluorescence spectroscopy was employed for monitoring the complex and characterizing the transitions taking place in the environments of tyrosine and tryptophan (mainly tryptophans) in proteins. The variation of the K(SV) value suggested that hydrophobic and electrostatic interactions were the predominant intermolecular forces stabilizing the complex. The RLS technique was utilized to determine the protein type, and to investigate the effect of anticancer drugs on its determination. This is the first report of its kind. An explanation is also given of the enhancement in RLS intensity, which attributed to the new complex formation between TMX and the protein a self-aggregation process and the formation of a precipitate HTF occurred and a micelle came into being when the amount of TMX was augmented. The great increase of polarizability was one of the important factors for the enhancement of RLS and the formation of complexes. The results from synchronous fluorescence spectroscopy showed that the micro-environment around tryptophan and tyrosine demonstrated a faint red shift. The circular dichroism data revealed that the presence of TMX decreased the α-helix content of HTF and induced the remarkable unfolding of the polypeptides of the protein. This confirmed certain micro

  19. Binding patterns of vanadium ions with different valence states to human serum transferrin studied by HPLC/high-resolution ICP-MS.

    PubMed

    Nagaoka, Megumi Hamano; Yamazaki, Takeshi; Maitani, Tamio

    2002-09-06

    Vanadium (V) is an essential metal for mammals and has different valence states. In blood, V is bound to serum transferrin (Tf), a glycoprotein which has two metal-binding sites, and carbonate is generally required for the binding. In this study, the binding patterns of V(III), V(IV), and V(V) to human serum Tf (hTf) were analyzed using an HPLC system equipped with an anion-exchange column and directly connected to a high-resolution inductively coupled plasma-mass spectrometer for metal detection (51V). In affinity to hTf, the three ions were ranked V(III)>V(IV)>V(V) in the presence of bicarbonate and V(III) reverse congruent V(IV)>V(V) in the absence. Intermediates in the "open forms" binding to the respective sites were detected at the initial stage. V(IV) and V(V) were bound to the N-lobe site in the "closed form" and "open form," respectively. In the absence of bicarbonate, V ions with respective valence states were bound to hTf in the "open form." In terms of binding to hTf, tri-valent V was most favorable in the presence of bicarbonate.

  20. Study on effect of lomefloxacin on human holo-transferrin in the presence of essential and nonessential amino acids: Spectroscopic and molecular modeling approaches.

    PubMed

    Marouzi, Somaye; Sharifi Rad, Atena; Beigoli, Sima; Teimoori Baghaee, Parisa; Assaran Darban, Reza; Chamani, Jamshidkhan

    2017-04-01

    The purpose of this study was to determine how lomefloxacin (LMF) interacts with human holo-transferrin (HTF) in the presence of two kinds of essential and nonessential amino acids. The investigations were carried out by fluorescence spectroscopy, zeta potential and molecular modeling techniques under imitated physiological conditions. We were able to determine the number of binding sites, the drug binding affinity to HTF in the presence of essential and nonessential amino acids and the quenching source of HTF. The interaction between HTF with LMF suggested that the microenvironment of the Trp residues was altered causing a strong static fluorescence quenching in the binary and ternary systems. The results pointed at the formation of a complex in the binary and ternary systems which caused an enhancement of the RLS intensity that was analyzed using synchronous fluorescence spectroscopy. The density functional theory (DFT) was employed to determine the amino acid residues on HTF that interacted with LMF. Also, Steric and van der Waals forces as well as the contribution of small amounts of hydrogen bonds were stronger or Tyr 71 in chain (b) than for 128 Trp in chain (a) of HTF.

  1. Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin1

    PubMed Central

    Steere, Ashley N.; Roberts, Samantha E.; Byrne, Shaina L.; Chasteen, N. Dennis; Bobst, Cedric E.; Kaltashov, Igor A; Smith, Valerie C.; MacGillivray, Ross T. A.; Mason, Anne B.

    2010-01-01

    Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated FeChTF. The new bridge sequence of this construct, designated FeCTEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe. PMID:20064616

  2. Pulmonary transvascular flux of transferrin

    SciTech Connect

    Cooper, J.A.; Malik, A.B. )

    1989-11-01

    We compared the pulmonary transvascular fluxes of transferrin and albumin in the intact sheep lung. Anesthetized sheep were prepared with lung lymph fistulas. The vascular blood pool was marked with {sup 99m}Tc-erythrocytes, autologous transferrin was labeled with {sup 113m}In, and albumin was labeled with {sup 125}I. Samples of blood, plasma, lymph, and lung were obtained up to 180 min after tracer infusion. Lymph tissue radioactivities were corrected for the intravascular component and expressed as extravascular-to-plasma concentration ratios. Clearance of transferrin and albumin from the plasma space followed a two-compartment model. The clearance rate constant was 2.1 {plus minus} 0.1 x 10(-3) min for albumin and 2.4 {plus minus} 0.1 x 10(-3) min for transferrin (P less than 0.05). Lymph-to-plasma ratios for albumin and transferrin were not different. However, the extravascular-to-plasma ratio for albumin was greater than transferrin (P less than 0.05). The lymph and lung data were deconvoluted for the plasma input function and fit to a two-compartment model. The results indicate that albumin and transferrin have similar permeabilities across the vascular barrier but have different pulmonary circulation to lymph kinetics because the extravascular volume of distribution of albumin is greater than transferrin.

  3. Pharmaceutical formulation affects titanocene transferrin interactions.

    PubMed

    Buettner, Katherine M; Snoeberger, Robert C; Batista, Victor S; Valentine, Ann M

    2011-10-07

    Since the discovery of the anticancer activity of titanocene dichloride (TDC), many derivatives have been developed and evaluated. MKT4, a soluble, water-stable formulation of TDC, was used for both Phase I and Phase II human clinical trials. This formulation is investigated here by using (1)H and (13)C NMR, FT-ICR mass spectrometry, and UV/vis-detected pH-dependent speciation. DFT calculations are also utilized to assess the likelihood of proposed species. Human serum transferrin has been identified as a potential vehicle for the Ti anticancer drugs; these studies examine whether and how formulation of TDC as MKT4 may influence its interactions, both thermodynamic and kinetic, with human serum transferrin by using UV/vis absorption and fluorescence quenching. MKT4 binds differently than TDC to transferrin, showing different kinetics of binding as well as a different molar absorptivity of binding (7500 M(-1) cm(-1) per site). Malate, used in the buffer for MKT4 administration, acts as a synergistic anion for Ti binding, shifting the tyrosine to Ti charge transfer energy and decreasing the molar absorptivity to 5000 M(-1) cm(-1) per site. These differences may have had consequences after the change from TDC to MKT4 in human clinical trials.

  4. Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens.

    PubMed

    Cornelissen, C N; Sparling, P F

    1994-12-01

    The mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenic Neisseria species and Haemophilus species. Two transferrin-binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB-dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin-binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter-species genetic exchange.

  5. Noninvasive Measurement of mTORC1 Signaling with (89)Zr-Transferrin.

    PubMed

    Truillet, Charles; Cunningham, John T; Parker, Matthew F L; Huynh, Loc T; Conn, Crystal S; Ruggero, Davide; Lewis, Jason S; Evans, Michael J

    2017-06-15

    Purpose: mTOR regulates many normal physiological processes and when hyperactive can drive numerous cancers and human diseases. However, it is very challenging to detect and quantify mTOR signaling noninvasively in clinically relevant animal models of disease or man. We hypothesized that a nuclear imaging tool measuring intracellular mTOR activity could address this unmet need.Experimental Design: Although the biochemical activity of mTOR is not directly amenable to nuclear imaging probe development, we show that the transferrin receptor can be used to indirectly measure intracellular changes in mTOR activity.Results: After verifying that the uptake of radiolabeled transferrin (the soluble ligand of the transferrin receptor) is stimulated by active mTORC1 in vitro, we showed that (89)Zr-labeled transferrin (Tf) can measure mTORC1 signaling dynamics in normal and cancerous mouse tissues with PET. Finally, we show that (89)Zr-Tf can detect the upregulation of mTORC1 by tumor cells to escape the antitumor effects of a standard-of-care antiandrogen, which is to our knowledge the first example of applying PET to interrogate the biology of treatment resistant cancer.Conclusions: In summary, we have developed the first quantitative assay to provide a comprehensive measurement of mTOR signaling dynamics in vivo, in specific normal tissues, and during tumor development in genetically engineered animal models using a nuclear imaging tool that is readily translatable to man. Clin Cancer Res; 23(12); 3045-52. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. Determination of LMF Binding Site on a HSA-PPIX Complex in the Presence of Human Holo Transferrin from the Viewpoint of Drug Loading on Proteins

    PubMed Central

    Sattar, Zohreh; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2014-01-01

    Holo transferrin (TF) and the natural complex of human serum albumin and protoporphyrin IX (HSA-PPIX) are two serum carrier proteins that can interact with each other. Such an interaction may alter their binding sites. In this study, fluorescence spectroscopy, as well as zeta potential and molecular modeling techniques, have been used to compare the complexes (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF. The Ka1, Ka2, values of (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF were 1.1×105 M−1, 9.7×106 M−1, and 2.0×104 M−1, 1.8×105 M−1, respectively, and the n1, n2 values were respectively 1.19, 1.53 and 1.17, 1.65. The second derivative of the Trp emission scan of (HSA-PPIX)-LMF exhibited one negative band at 310 nm, whereas for the [(HSA-PPIX)-TF]-LMF system, we observed one negative band at 316 nm indicating an increase in polarity around Trp. The effect of TF on the conformation of (HSA-PPIX)-TF was analyzed using three-dimensional fluorescence spectroscopy. The phase diagram indicated that the presence of a second binding site on HSA and TF was due to the existence of intermediate structures. Zeta potential analysis showed that the presence of TF increased the positive charges of the HSA-PPIX system. Site marker experiments revealed that the binding site of LMF to HSA-PPIX changed from Sudlow's site IIA to Sudlow's site IIIB in the presence of TF. Moreover, molecular modeling studies suggested the sub-domain IIIB in HSA as the candidate place for the formation of the binding site of LMF on the (HSA-PPIX)-TF complex. PMID:24392106

  7. Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin.

    PubMed

    Steere, Ashley N; Roberts, Samantha E; Byrne, Shaina L; Dennis Chasteen, N; Bobst, Cedric E; Kaltashov, Igor A; Smith, Valerie C; MacGillivray, Ross T A; Mason, Anne B

    2010-07-01

    Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated Fe(C)hTF. The new bridge sequence of this construct, designated Fe(C)TEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe. Copyright 2010 Elsevier Inc. All rights reserved.

  8. Use of spectroscopic, zeta potential and molecular dynamic techniques to study the interaction between human holo-transferrin and two antagonist drugs: comparison of binary and ternary systems.

    PubMed

    Kabiri, Mona; Amiri-Tehranizadeh, Zeinab; Baratian, Ali; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2012-03-12

    For the first time, the binding of ropinirole hydrochloride (ROP) and aspirin (ASA) to human holo-transferrin (hTf) has been investigated by spectroscopic approaches (fluorescence quenching, synchronous fluorescence, time-resolved fluorescence, three-dimensional fluorescence, UV-vis absorption, circular dichroism, resonance light scattering), as well as zeta potential and molecular modeling techniques, under simulated physiological conditions. Fluorescence analysis was used to estimate the effect of the ROP and ASA drugs on the fluorescence of hTf as well as to define the binding and quenching properties of binary and ternary complexes. The synchronized fluorescence and three-dimensional fluorescence spectra demonstrated some micro-environmental and conformational changes around the Trp and Tyr residues with a faint red shift. Thermodynamic analysis displayed the van der Waals forces and hydrogen bonds interactions are the major acting forces in stabilizing the complexes. Steady-state and time-resolved fluorescence data revealed that the fluorescence quenching of complexes are static mechanism. The effect of the drugs aggregating on the hTf resulted in an enhancement of the resonance light scattering (RLS) intensity. The average binding distance between were computed according to the forster non-radiation energy transfer theory. The circular dichroism (CD) spectral examinations indicated that the binding of the drugs induced a conformational change of hTf. Measurements of the zeta potential indicated that the combination of electrostatic and hydrophobic interactions between ROP, ASA and hTf formed micelle-like clusters. The molecular modeling confirmed the experimental results. This study is expected to provide important insight into the interaction of hTf with ROP and ASA to use in various toxicological and therapeutic processes.

  9. A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS).

    PubMed

    Feng, Liuxing; Zhang, Dan; Wang, Jun; Shen, Dairui; Li, Hongmei

    2015-07-16

    Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with (34)S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and (34)S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (m(sp)/m(sam)) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5-3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Binding of trivalent chromium to serum transferrin is sufficiently rapid to be physiologically relevant.

    PubMed

    Deng, Ge; Wu, Kristi; Cruce, Alex A; Bowman, Michael K; Vincent, John B

    2015-02-01

    Transferrin, the major iron transport protein in the blood, also transports trivalent chromium in vivo. Recent in vitro studies have, however, suggested that the binding of chromic ions to apotransferrin is too slow to be biologically relevant. Nevertheless, the in vitro studies have generally failed to adequately take physiological bicarbonate concentrations into account. In aqueous buffer (with ambient (bi)carbonate concentrations), the binding of chromium to transferrin is too slow to be physiologically relevant, taking days to reach equilibrium with the protein's associated conformational changes. However, in the presence of 25mM (bi)carbonate, the concentration in human blood, chromic ions bind rapidly and tightly to transferrin. Details of the kinetics of chromium binding to human serum transferrin and conalbumin (egg white transferrin) in the presence of bicarbonate and other major potential chromium ligands are described and are consistent with transferrin being the major chromic ion transporter from the blood to tissues.

  11. Neural and oligodendrocyte progenitor cells: transferrin effects on cell proliferation

    PubMed Central

    Silvestroff, Lucas; Franco, Paula Gabriela; Pasquini, Juana María

    2013-01-01

    NSC (neural stem cells)/NPC (neural progenitor cells) are multipotent and self-renew throughout adulthood in the SVZ (subventricular zone) of the mammalian CNS (central nervous system). These cells are considered interesting targets for CNS neurodegenerative disorder cell therapies, and understanding their behaviour in vitro is crucial if they are to be cultured prior to transplantation. We cultured the SVZ tissue belonging to newborn rats under the form of NS (neurospheres) to evaluate the effects of Tf (transferrin) on cell proliferation. The NS were heterogeneous in terms of the NSC/NPC markers GFAP (glial fibrillary acidic protein), Nestin and Sox2 and the OL (oligodendrocyte) progenitor markers NG2 (nerve/glia antigen 2) and PDGFRα (platelet-derived growth factor receptor α). The results of this study indicate that aTf (apoTransferrin) is able to increase cell proliferation of SVZ-derived cells in vitro, and that these effects were mediated at least in part by the TfRc1 (Tf receptor 1). Since OPCs (oligodendrocyte progenitor cells) represent a significant proportion of the proliferating cells in the SVZ-derived primary cultures, we used the immature OL cell line N20.1 to show that Tf was able to augment the proliferation rate of OPC, either by adding aTf to the culture medium or by overexpressing rat Tf in situ. The culture medium supplemented with ferric iron, together with aTf, increased the DNA content, while ferrous iron did not. The present work provides data that could have a potential application in human cell replacement therapies for neurodegenerative disease and/or CNS injury that require the use of in vitro amplified NPCs. PMID:23368675

  12. Prevalence of Anaemia and Evaluation of Transferrin Receptor (sTfR) in the Diagnosis of Iron Deficiency in the Hospitalized Elderly Patients: Anaemia Clinical Studies in Chile.

    PubMed

    López-Sierra, Mauricio; Calderón, Susana; Gómez, Jorge; Pilleux, Lilian

    2012-01-01

    Iron constitutes the most prevalent nutritional deficiency worldwide. In Chile, anaemia epidemiological data is scarce, evaluating mainly children and women. Our objective was to determine prevalence of anaemia in an inpatient elderly population (≥60 years) and assess the usefulness of sTfR levels analyzed by other authors as a good predictor in the differential diagnosis of iron deficiency anaemia and anaemia of chronic disease. Method. We studied medical patients admitted at Hospital of Valdivia (HV), Chile, in a 2month period. World Health Organization criteria were used for anaemia. Results. 391 patients were hospitalized, average age 62.5 years, 247 elderly and 99 of which had anaemia. Anaemia was normocytic in 88.8%, and we observed: low serum iron in 46.3%, low ferritin 10.1%, high TIBC 2%, low % transferrin saturation (Tsat) 40%, and high sTfR 25%. Conclusions. As a first figure known in Chile, the prevalence of anaemia in the elderly inpatient was 40.1%. Our findings encourage us to promote the implementation of sTfR determination in the clinical setting to analyze the state of erythropoiesis in patients with chronic diseases wich commonly occurs in elderly.

  13. Comparing the interaction of cyclophosphamide monohydrate to human serum albumin as opposed to holo-transferrin by spectroscopic and molecular modeling methods: evidence for allocating the binding site.

    PubMed

    Tousi, Shirin Hamed-Akbari; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2010-12-01

    The interaction between cyclophosphamide monohydrate with human serum albumin (HSA) and human serum transferrin (hTf) was studied with UV absorption, fluorescence and circular dichroism (CD) spectroscopies as well as molecular modeling. Based on the fluorescence quenching results, it was determined that HSA and hTf had two classes of apparent binding constants and binding sites at physiological conditions. The K(SV1), K(SV2), n(1) and n(2) values for HSA were found to be 8.6 x 10(8) Lmol(-1), 6.34 x 10(8) Lmol(-1), 0.7 and 0.8, respectively, and the corresponding results for hTf were 6.08 x 10(7) Lmol(-1), 4.65 x 10(7) Lmol(-1), 1.3 and 2.6, respectively. However, the binding affinity of cyclophosphamide monohydrate to HSA was more significant than to hTf. Circular dichroism results demonstrated that the binding of cyclophosphamide to HSA and hTf induced secondary changes in the structure and that the a-helix content became altered into b-sheet, turn and random coil forms. The participation of tyrosyl and tryptophan residues of proteins was also estimated in the drug-HSA and hTf complexes by synchronous fluorescence. The micro-environment of the HSA and hTf fluorophores was transferred to hydrophobic and hydrophilic conditions, respectively. The distance r between donor and acceptor was obtained by the Forster energy according to fluorescence resonance energy transfer (FRET) and found to be 1.84 nm and 1.73 nm for HSA and hTf, respectively. This confirmed the existence of static quenching for both proteins in the presence of cyclophosphamide monohydrate. Site marker competitive displacement experiments demonstrated that cyclophosphamide bound with high affinity to Site II, sub-domain IIIA of HSA, and for hTf, the C-lobe constituted the binding site. Furthermore, a study of molecular modeling showed that cyclophosphamide situated in domain II in HSA was bound through hydrogen bonding with Arg 257 and Ser 287, and that cyclophosphamide was situated in the C-lobe in h

  14. Structure of diferric hen serum transferrin at 2.8 A resolution.

    PubMed

    Guha Thakurta, Piyali; Choudhury, Debi; Dasgupta, Rakhi; Dattagupta, J K

    2003-10-01

    Hen serum transferrin in its diferric form (hST) has been isolated, purified and the three-dimensional structure determined by X-ray crystallography at 2.8 A resolution. The final refined structure of hST, comprising 5232 protein atoms, two Fe(3+) cations, two CO(3)(2-) anions, 54 water molecules and one fucose moiety, has an R factor of 21.5% and an R(free) of 26.9% for all data. The structure has been compared with the three-dimensional structure of hen ovotransferrin (hOT) and also with structures of some other transferrins, viz. rabbit serum transferrin (rST) and human lactoferrin (hLF). The overall conformation of the hST molecule is essentially the same as that of other transferrins. However, the relative orientation of the two lobes, which is related to the species-specific receptor-recognition property of transferrins, has been found to be different in hST from that in hOT, rST and hLF. On the basis of superposition of the N lobes, rotations of 5.8, 16.9 and 11.3 degrees are required to bring the C lobes of hOT, rST and hLF, respectively, into coincidence with that of hST. A number of additional hydrogen bonds between the two domains in the N and C lobes have been identified in the structure of hST compared with that of hOT, which indicate a greater compactness of the lobes of hST than those of hOT. Being products of the same gene, hST and hOT have 100% sequence identity and differ only in the attached carbohydrate moiety. On the other hand, despite having similar functions, hST and rST have only 51% sequence similarity. However, the nature of the interdomain interactions of hST are closer to rST than to hOT. A putative carbohydrate-binding site has been identified in the N lobe of hST at Asn52 and a fucose molecule could be modelled at the site. The variations in interdomain and interlobe interactions in hST, together with altered lobe orientation with respect to hOT, rST and hLF, which are the representatives of the other subfamily of transferrins, are

  15. Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism.

    PubMed

    Lane, Darius J R; Chikhani, Sherin; Richardson, Vera; Richardson, Des R

    2013-06-01

    Although ascorbate has long been known to stimulate dietary iron (Fe) absorption and non-transferrin Fe uptake, the role of ascorbate in transferrin Fe uptake is unknown. Transferrin is a serum Fe transport protein supplying almost all cellular Fe under physiological conditions. We sought to examine ascorbate's role in this process, particularly as cultured cells are typically ascorbate-deficient. At typical plasma concentrations, ascorbate significantly increased (59)Fe uptake from transferrin by 1.5-2-fold in a range of cells. Moreover, ascorbate enhanced ferritin expression and increased (59)Fe accumulation in ferritin. The lack of effect of cycloheximide or the cytosolic aconitase inhibitor, oxalomalate, on ascorbate-mediated (59)Fe uptake from transferrin indicate increased ferritin synthesis or cytosolic aconitase activity was not responsible for ascorbate's activity. Experiments with membrane-permeant and -impermeant ascorbate-oxidizing reagents indicate that while extracellular ascorbate is required for stimulation of (59)Fe uptake from (59)Fe-citrate, only intracellular ascorbate is needed for transferrin (59)Fe uptake. Additionally, experiments with l-ascorbate analogs indicate ascorbate's reducing ene-diol moiety is necessary for its stimulatory activity. Importantly, neither N-acetylcysteine nor buthionine sulfoximine, which increase or decrease intracellular glutathione, respectively, affected transferrin-dependent (59)Fe uptake. Thus, ascorbate's stimulatory effect is not due to a general increase in cellular reducing capacity. Ascorbate also did not affect expression of transferrin receptor 1 or (125)I-transferrin cellular flux. However, transferrin receptors, endocytosis, vacuolar-type ATPase activity and endosomal acidification were required for ascorbate's stimulatory activity. Therefore, ascorbate is a novel modulator of the classical transferrin Fe uptake pathway, acting via an intracellular reductive mechanism.

  16. Human dopamine receptor and its uses

    DOEpatents

    Civelli, Olivier; Van Tol, Hubert Henri-Marie

    1999-01-01

    The present invention is directed toward the isolation, characterization and pharmacological use of the human D4 dopamine receptor. The nucleotide sequence of the gene corresponding to this receptor and alleleic variant thereof are provided by the invention. The invention also includes recombinant eukaryotic expression constructs capable of expressing the human D4 dopamine receptor in cultures of transformed eukaryotic cells. The invention provides cultures of transformed eukaryotic cells which synthesize the human D4 dopamine receptor, and methods for characterizing novel psychotropic compounds using such cultures.

  17. Human olfactory receptor responses to odorants

    PubMed Central

    Mainland, Joel D; Li, Yun R; Zhou, Ting; Liu, Wen Ling L; Matsunami, Hiroaki

    2015-01-01

    Although the human olfactory system is capable of discriminating a vast number of odors, we do not currently understand what chemical features are encoded by olfactory receptors. In large part this is due to a paucity of data in a search space covering the interactions of hundreds of receptors with billions of odorous molecules. Of the approximately 400 intact human odorant receptors, only 10% have a published ligand. Here we used a heterologous luciferase assay to screen 73 odorants against a clone library of 511 human olfactory receptors. This dataset will allow other researchers to interrogate the combinatorial nature of olfactory coding. PMID:25977809

  18. Xenobiotic receptor humanized mice and their utility.

    PubMed

    Scheer, Nico; Roland Wolf, C

    2013-02-01

    The nuclear receptors pregnane X receptor, constitutive androstane receptor, and peroxisome proliferator-activated receptor alpha have important endogenous functions and are also involved in the induction of drug-metabolizing enzymes and transporters in response to exogenous xenobiotics. Though not belonging to the same protein family, the Per-Sim-ARNT domain receptor aryl hydrocarbon receptor functionally overlaps with the three nuclear receptors in many aspects and is therefore included in this review. Significant species differences in ligand affinity and biological responses as a result of activation of these receptors have been described. Several xenobiotic receptor humanized mice have been created to overcome these species differences and to provide in vivo models that are more predictive for human responses. This review provides an overview of the different xenobiotic receptor humanized mouse models described to date and will summarize how these models can be applied in basic research and improve drug discovery and development. Some of the key applications in the evaluation of drug induction, drug-drug interactions, nongenotoxic carcinogenicity, other toxicity, or efficacy studies are described. We also discuss relevant considerations in the interpretation of such data and potential future directions for the use of xenobiotic receptor humanized mice.

  19. The Importance of the Stem Cell Marker Prominin-1/CD133 in the Uptake of Transferrin and in Iron Metabolism in Human Colon Cancer Caco-2 Cells

    PubMed Central

    Benoit, Jean-Pierre; Garcion, Emmanuel

    2011-01-01

    As the pentaspan stem cell marker CD133 was shown to bind cholesterol and to localize in plasma membrane protrusions, we investigated a possible function for CD133 in endocytosis. Using the CD133 siRNA knockdown strategy and non-differentiated human colon cancer Caco-2 cells that constitutively over-expressed CD133, we provide for the first time direct evidence for a role of CD133 in the intracellular accumulation of fluorescently labeled extracellular compounds. Assessed using AC133 monoclonal antibody, CD133 knockdown was shown to improve Alexa488-transferrin (Tf) uptake in Caco-2 cells but had no impact on FITC-dextran or FITC-cholera-toxin. Absence of effect of the CD133 knockdown on Tf recycling established a role for CD133 in inhibiting Tf endocytosis rather than in stimulating Tf exocytosis. Use of previously identified inhibitors of known endocytic pathways and the positive impact of CD133 knockdown on cellular uptake of clathrin-endocytosed synthetic lipid nanocapsules supported that CD133 impact on endocytosis was primarily ascribed to the clathrin pathway. Also, cholesterol extraction with methyl-β-cyclodextrine up regulated Tf uptake at greater intensity in the CD133high situation than in the CD133low situation, thus suggesting a role for cholesterol in the inhibitory effect of CD133 on endocytosis. Interestingly, cell treatment with the AC133 antibody down regulated Tf uptake, thus demonstrating that direct extracellular binding to CD133 could affect endocytosis. Moreover, flow cytometry and confocal microscopy established that down regulation of CD133 improved the accessibility to the TfR from the extracellular space, providing a mechanism by which CD133 inhibited Tf uptake. As Tf is involved in supplying iron to the cell, effects of iron supplementation and deprivation on CD133/AC133 expression were investigated. Both demonstrated a dose-dependent down regulation here discussed to the light of transcriptional and post-transciptional effects. Taken

  20. Bovine prolactin elevates hTF expression directed by a tissue-specific goat β-casein promoter through prolactin receptor-mediated STAT5a activation.

    PubMed

    Jiang, Shizhong; Ren, Zhaorui; Xie, Fei; Yan, Jingbin; Huang, Shuzhen; Zeng, Yitao

    2012-11-01

    Prolactin promotes the expression of exogenous human transferrin gene in the milk of transgenic mice. To elucidate this, a recombinant plasmid of bovine prolactin plus human transferrin vector was co-transfected into cultured murine mammary gland epithelial cells. Prolactin-receptor antagonist and shRNA corresponding to prolactin-receptor mRNA were added into the cell culture mixture to investigate the relations between prolactin-receptor and human transferrin expression after bovine prolactin inducement. Levels of human transferrin in the supernatants were increased under the presentation of bovine prolactin (from 1,076 ± 115 to 1,886 ± 114 pg/ml). With the treatment of prolactin-receptor antagonist or shRNA, human transferrin in cells was declined (1,886 ± 113 vs. 1,233 ± 85 pg/ml or 1,114 ± 75 pg/ml, respectively). An inverse correlation was found between the dosage of prolactin-receptor antagonist and expression level of human transferrin. Real-time qRT-PCR analysis showed that the relative level of signal transducer and activator of transcription 5a (STAT5a) transcript in transfected cells correlated with expression levels of human transferrin in the supernatant of the same cells. Bovine prolactin thus improved the expression of human transferrin through such a possible mechanism that bovine prolactin activated STAT5a transcription expression via combined with prolactin-receptor and suggest a potential utility of the bovine prolactin for efficient expression of valuable pharmaceutical proteins in mammary glands of transgenic animals.

  1. Acetylcholine receptors in the human retina

    SciTech Connect

    Hutchins, J.B.; Hollyfield, J.G.

    1985-11-01

    Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand TH-propylbenzilylcholine mustard (TH-PrBCM) to label muscarinic receptors. TH- or SVI-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that TH-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer of the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina.

  2. Disaggregation of amyloid plaque in brain of Alzheimer's disease transgenic mice with daily subcutaneous administration of a tetravalent bispecific antibody that targets the transferrin receptor and the Abeta amyloid peptide.

    PubMed

    Sumbria, Rachita K; Hui, Eric Ka-Wai; Lu, Jeff Zhiqiang; Boado, Ruben J; Pardridge, William M

    2013-09-03

    Anti-amyloid antibodies (AAA) are under development as new therapeutics that disaggregate the amyloid plaque in brain in Alzheimer's disease (AD). However, the AAAs are large molecule drugs that do not cross the blood-brain barrier (BBB), in the absence of BBB disruption. In the present study, an AAA was re-engineered for receptor-mediated transport across the BBB via the endogenous BBB transferrin receptor (TfR). A single chain Fv (ScFv) antibody form of an AAA was fused to the carboxyl terminus of each heavy chain of a chimeric monoclonal antibody (mAb) against the mouse TfR, and this produced a tetravalent bispecific antibody designated the cTfRMAb-ScFv fusion protein. Unlike a conventional AAA, which has a plasma half-time of weeks, the cTfRMAb-ScFv fusion protein is cleared from plasma in mice with a mean residence time of about 3 h. Therefore, a novel protocol was developed for the treatment of one year old presenilin (PS)-1/amyloid precursor protein (APP) AD double transgenic PSAPP mice, which were administered daily subcutaneous (sc) injections of 5 mg/kg of the cTfRMAb-ScFv fusion protein for 12 consecutive weeks. At the end of the treatment, brain amyloid plaques were quantified with confocal microscopy using both Thioflavin-S staining and immunostaining with the 6E10 antibody against Abeta amyloid fibrils. Fusion protein treatment caused a 57% and 61% reduction in amyloid plaque in the cortex and hippocampus, respectively. No increase in plasma immunoreactive Abeta amyloid peptide, and no cerebral microhemorrhage, was observed. Chronic daily sc treatment of the mice with the fusion protein caused no immune reactions and only a low titer antidrug antibody response. In conclusion, re-engineering AAAs for receptor-mediated BBB transport allows for reduction in brain amyloid plaque without cerebral microhemorrhage following daily sc treatment for 12 weeks.

  3. Structural Allostery and Binding of the Transferring Receptor Complex

    SciTech Connect

    Xu,G.; Liu, R.; Zak, O.; Aisen, P.; Chance, M.

    2005-01-01

    The structural allostery and binding interface for the human serum transferrin (Tf){center_dot}transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions. The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.

  4. Soluble form of transferrin receptor-1 level is associated with the age at first diagnosis and the risk of therapeutic intervention and iron overloading in patients with non-transfusion-dependent thalassemia.

    PubMed

    Ricchi, Paolo; Meloni, Antonella; Costantini, Silvia; Spasiano, Anna; Di Matola, Tiziana; Pepe, Alessia; Cinque, Patrizia; Filosa, Aldo

    2017-09-01

    We retrospectively evaluated the relationship between serum transferrin receptor-1 (sTfR1) and some fundamental events in the life and the management (the age at diagnosis, the age at the first red blood cells transfusion, the age at splenectomy, and the overall need of chelation therapy) of 111 patients with non-transfusion-dependent thalassemia (NTDT) subdivided in four genetic entities: patients with homozygous or compound heterozygous state for β-thalassemia, patients with triplicated α genotype associated with β heterozygosity, patients with deletional HbH, and patients with the combination of a β defect plus a β chain variant. We found that the group with homozygous or compound heterozygous state for β-thalassemia had the highest sTfR1 levels and that the presence of increased sTfR1 levels (>5 times normal) was associated with a complex and severe history of disease requiring splenectomy, occasional red blood cells transfusions, and early start and continuous iron chelation therapy.The complexity in the management of NTDT patients is an emerging issue due to the wide heterogeneity of clinical behavior. Our data indicate that the measurement of sTfR1 levels, a common laboratory test, could contribute to correctly stratify disease history and the iron chelation strategy in NTDT patients.

  5. A useful relationship between the presence of extramedullary erythropoeisis and the level of the soluble form of the transferrin receptor in a large cohort of adult patients with thalassemia intermedia: a prospective study.

    PubMed

    Ricchi, Paolo; Ammirabile, Massimiliano; Costantini, Silvia; Di Matola, Tiziana; Verna, Roberto; Diano, Alvaro; Foglia, Maria Carmela; Spasiano, Anna; Cinque, Patrizia; Prossomariti, Luciano

    2012-06-01

    In thalassemia intermedia (TI), the increase in bone marrow hemopoietic activity frequently leads to extramedullary erythropoeisis (EMH), but its relationship with the soluble form of transferrin receptor (sTfR) which fully reflects the marrow erythropoietic activity, has not yet been explored. From January 2007 to December 2010, all TI patients attending at our center were prospectively enrolled to undergo sTfR assay and MRI or CT (if claustrophobic) scan evaluation for the presence of paraspinal EMH. A total of 59 patients with TI were studied; EMH involved 23 (39%) patients; overall, the concentration of sTfR varied from 2.6 to 20.6 (mean = 8.7) mg/L, but in splenectomized group and in unsplenectomized group, it varied from 4.2 to 17.8 (mean ± SD = 9.86 ± 3.33) mg/L and from 2.6 to 20.6 (mean ± SD = 7.25 ± 3.9) mg/L, respectively with a statistically significant intergroup difference (p < 0.01). The cutoff point at 8.6 mg/L using the ROC curve showed a sensitivity of 78.3% and a specificity of 72.2%, in predicting EMH but, in unsplenectomized subgroup, they raised to 100% and 90.9%, respectively. These data showed that in TI the level of sTfR could represent a predictive factor of EMH particularly in patients with spleen.

  6. A spectroscopic and molecular modeling study of sinomenine binding to transferrin.

    PubMed

    Du, Hongyan; Xiang, Junfeng; Zhang, Yazhou; Tang, Yalin

    2007-03-15

    Sinomenine, an herbal ingredient isolated from Sinomenium acutum, is used for the amelioration of arthritis. It has been found that this molecule could bind to human serum transferrin (Tf), the iron (III) transport protein in the blood, by using fluorescence, circular dichroism (CD) spectroscopy, and molecular modeling methods. The results provide possible usage of transferrin to transport sinomenine.

  7. Determination of carbohydrate-deficient transferrin in human serum by capillary zone electrophoresis: evaluation of assay performance and quality assurance over a 10-year period in the routine arena.

    PubMed

    Joneli, Jeannine; Wanzenried, Ursula; Schiess, Jeannette; Lanz, Christian; Caslavska, Jitka; Thormann, Wolfgang

    2013-06-01

    The performance of high-resolution CZE for determination of carbohydrate-deficient transferrin (CDT) in human serum based on internal and external quality data gathered over a 10-year period is reported. The assay comprises mixing of serum with a Fe(III) ion-containing solution prior to analysis of the iron saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. CDT values obtained with a human serum of a healthy individual and commercial quality control sera are shown to vary less than 10%. Values of a control from a specific lot were found to slowly decrease as function of time (less than 10% per year). Furthermore, due to unknown reasons, gradual changes in the monitored pattern around pentasialo-transferrin were detected, which limit the use of commercial control sera of the same lot to less than 2 years. Analysis of external quality control sera revealed correct classification of the samples over the entire 10-year period. Data obtained compare well with those of HPLC and CZE assays of other laboratories. The data gathered over a 10-year period demonstrate the robustness of the high-resolution CZE assay. This is the first account of a CZE-based CDT assay with complete internal and external quality assessment over an extended time period.

  8. Induction of GST-P-positive proliferative lesions facilitating lipid peroxidation with possible involvement of transferrin receptor up-regulation and ceruloplasmin down-regulation from the early stage of liver tumor promotion in rats.

    PubMed

    Mizukami, Sayaka; Ichimura, Ryohei; Kemmochi, Sayaka; Taniai, Eriko; Shimamoto, Keisuke; Ohishi, Takumi; Takahashi, Miwa; Mitsumori, Kunitoshi; Shibutani, Makoto

    2010-04-01

    To elucidate the role of metal-related molecules in hepatocarcinogenesis, we examined immunolocalization of transferrin receptor (Tfrc), ceruloplasmin (Cp) and metallothionein (MT)-1/2 in relation to liver cell foci positive for glutathione-S-transferase placental form (GST-P) in the early stage of tumor promotion by fenbendazole (FB), phenobarbital, piperonyl butoxide or thioacetamide in a rat two-stage hepatocarcinogenesis model. To estimate the involvement of oxidative stress responses to the promotion, immunolocalization of 4-hydroxy-2-nonenal, malondialdehyde and acrolein was similarly examined. Our findings showed that MT-1/2 immunoreactivity was not associated with the cellular distribution of GST-P and proliferating cell nuclear antigen, suggesting no role of MT-1/2 in hepatocarcinogenesis. We also found enhanced expression of Tfrc after treatment with strong tumor-promoting chemicals. With regard to Cp, the population showing down-regulation was increased in the GST-P-positive foci in relation to tumor promotion. Up-regulation of Tfrc and down-regulation of Cp was maintained in GST-P-positive neoplastic lesions induced after long-term promotion with FB, suggesting the expression changes occurring downstream of the signaling pathway involved in the formation of GST-P-positive lesions. Furthermore, enhanced accumulation of lipid peroxidation end products was observed in the GST-P-positive foci by promotion. Post-initiation treatment with peroxisome proliferator-activated receptor alpha agonists did not enhance any such distribution changes in GST-P-negative foci. The results thus suggest that facilitation of lipid peroxidation is involved in the induction of GST-P-positive lesions by tumor promotion from an early stage, and up-regulation of Tfrc and down-regulation of Cp may be a signature of enhanced oxidative cellular stress in these lesions.

  9. Complex of transferrin with ruthenium for medical applications

    DOEpatents

    Richards, Powell; Srivastava, Suresh C.; Meinken, George E.

    1984-05-15

    A novel Ruthenium-transferrin complex, prepared by reacting iron-free human transferrin dissolved in a sodium acetate solution at pH 7 with ruthenium by heating at about 40.degree. C. for about 2 hours, and purifying said complex by means of gel chromotography with pH 7 sodium acetate as eluent. The mono- or di-metal complex produced can be used in nuclear medicine in the diagnosis and/or treatment of tumors and abscesses. Comparative results with Ga-67-citrate, which is the most widely used tumor-localizing agent in nuclear medicine, indicate increased sensitivity of detection and greater tumor uptake with the Ru-transferrin complex.

  10. Complex of transferrin with ruthenium for medical applications

    DOEpatents

    Richards, P.; Srivastava, S.C.; Meinken, G.E.

    1984-05-15

    A novel ruthenium-transferrin complex is disclosed which is prepared by reacting iron-free human transferrin dissolved in a sodium acetate solution at pH 7 with ruthenium by heating at about 40 C for about 2 hours. The complex is purified by means of gel chromotography with pH 7 sodium acetate as eluent. The mono- or di-metal complex produced can be used in nuclear medicine in the diagnosis and/or treatment of tumors and abscesses. Comparative results with Ga-67-citrate, which is the most widely used tumor-localizing agent in nuclear medicine, indicate increased sensitivity of detection and greater tumor uptake with the Ru-transferrin complex. No Drawings

  11. Leukocyte chemoattractant receptors in human disease pathogenesis.

    PubMed

    Zabel, Brian A; Rott, Alena; Butcher, Eugene C

    2015-01-01

    Combinations of leukocyte attractant ligands and cognate heptahelical receptors specify the systemic recruitment of circulating cells by triggering integrin-dependent adhesion to endothelial cells, supporting extravasation, and directing specific intratissue localization via gradient-driven chemotaxis. Chemoattractant receptors also control leukocyte egress from lymphoid organs and peripheral tissues. In this article, we summarize the fundamental mechanics of leukocyte trafficking, from the evolution of multistep models of leukocyte recruitment and navigation to the regulation of chemoattractant availability and function by atypical heptahelical receptors. To provide a more complete picture of the migratory circuits involved in leukocyte trafficking, we integrate a number of nonchemokine chemoattractant receptors into our discussion. Leukocyte chemoattractant receptors play key roles in the pathogenesis of autoimmune diseases, allergy, inflammatory disorders, and cancer. We review recent advances in our understanding of chemoattractant receptors in disease pathogenesis, with a focus on genome-wide association studies in humans and the translational implications of mechanistic studies in animal disease models.

  12. The effect of glycosylation on the transferrin structure: A molecular dynamic simulation analysis.

    PubMed

    Ghanbari, Z; Housaindokht, M R; Bozorgmehr, M R; Izadyar, M

    2016-09-07

    Transferrins have been defined by the highly cooperative binding of iron and a carbonate anion to form a Fe-CO3-Tf ternary complex. As such, the layout of the binding site residues affects transferrin function significantly; In contrast to N-lobe, C-lobe binding site of the transferrin structure has been less characterized and little research which surveyed the interaction of carbonate with transferrin in the C-lobe binding site has been found. In the present work, molecular dynamic simulation was employed to gain access into the molecular level understanding of carbonate binding site and their interactions in each lobe. Residues responsible for carbonate binding of transferrin structure were pointed out. In addition, native human transferrin is a glycoprotein that two N-linked complex glycan chains located in the C-lobe. Usually, in the molecular dynamic simulation for simplifying, glycan is removed from the protein structure. Here, we explore the effect of glycosylation on the transferrin structure. Glycosylation appears to have an effect on the layout of the binding site residue and transferrin structure. On the other hand, sometimes the entire transferrin formed by separated lobes that it allows the results to be interpreted in a straightforward manner rather than more parameters required for full length protein. But, it should be noted that there are differences between the separated lobe and full length transferrin, hence, a comparative analysis by the molecular dynamic simulation was performed to investigate such structural variations. Results revealed that separation in C-lobe caused a significant structural variation in comparison to N-lobe. Consequently, the separated lobes and the full length one are different, showing the importance of the interlobe communication and the impact of the lobes on each other in the transferrin structure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Use of chromatofocusing to detect a transferrin variant in serum of alcoholic subjects.

    PubMed

    Storey, E L; Mack, U; Powell, L W; Halliday, J W

    1985-09-01

    We describe a technique for detecting an abnormal (pl 5.7) transferrin component in serum, which appears after prolonged heavy consumption of alcohol. Serum transferrin was purified by chromatography on DEAE-Affi-Gel Blue and analyzed by chromatofocusing on an ion-exchange column (Mono P). The abnormal transferrin component was detected in 17 of 20 patients (85%) with a history or prolonged consumption of alcohol (100 g per day), and in control subjects who ingested up to 80 g of alcohol per day for seven days, but not in 14 normal control subjects or 14 patients with liver disease unrelated to alcohol. The variant consistently disappeared from the serum within three weeks of cessation of alcohol consumption. It is apparently produced by desialylation of ordinary human transferrin. We find that chromatofocusing on an ion-exchange column is a sensitive and reliable technique for its identification and conclude that detection of this desialylated transferrin indicates recent prolonged alcohol ingestion.

  14. Improved differential diagnosis of anemia of chronic disease and iron deficiency anemia: a prospective multicenter evaluation of soluble transferrin receptor and the sTfR/log ferritin index.

    PubMed

    Skikne, Barry S; Punnonen, Kari; Caldron, Paul H; Bennett, Michael T; Rehu, Mari; Gasior, Gail H; Chamberlin, Janna S; Sullivan, Linda A; Bray, Kurtis R; Southwick, Paula C

    2011-11-01

    Anemia of chronic disease (ACD) and iron deficiency anemia (IDA) are the most prevalent forms of anemia and often occur concurrently. Standard tests of iron status used in differential diagnosis are affected by inflammation, hindering clinical interpretation. In contrast, soluble transferrin receptor (sTfR) indicates iron deficiency and is unaffected by inflammation. Objectives of this prospective multicenter clinical trial were to evaluate and compare the diagnostic accuracy of sTfR and the sTfR/log ferritin index (sTfR Index) for differential diagnosis using the automated Access(®) sTfR assay (Beckman Coulter) and sTfR Index. We consecutively enrolled 145 anemic patients with common disorders associated with IDA and ACD. Subjects with IDA or ACD + IDA had significantly higher sTfR and sTfR Index values than subjects with ACD (P < 0.0001). ROC curves produced the following cutoffs for sTfR: 21 nmol/L (or 1.55 mg/L), and the sTfR Index: 14 (using nmol/L) (or 1.03 using mg/L). The sTfR Index was superior to sTfR (AUC 0.87 vs. 0.74, P < 0.0001). Use of all three parameters in combination more than doubled the detection of IDA, from 41% (ferritin alone) to 92% (ferritin, sTfR, sTfR Index). Use of sTfR and the sTfR Index improves detection of IDA, particularly in situations where routine markers provide equivocal results. Findings demonstrate a significant advantage in the simultaneous determination of ferritin, sTfR and sTfR Index. Obtaining a ferritin level alone may delay diagnosis of combined IDA and ACD.

  15. Effects of inflammation and Plasmodium falciparum infection on soluble transferrin receptor and plasma ferritin concentration in different age groups: a prospective longitudinal study in Côte d'Ivoire.

    PubMed

    Righetti, Aurélie A; Wegmüller, Rita; Glinz, Dominik; Ouattara, Mamadou; Adiossan, Lukas G; N'Goran, Eliézer K; Utzinger, Jürg; Hurrell, Richard F

    2013-06-01

    Iron deficiency (ID) is a major cause of anemia, along with other nutritional, parasitic, and genetic factors. Accurate biomarkers are needed to estimate the relative contribution of ID to anemia. Soluble transferrin receptor (sTfR) is thought to be unaffected by inflammation. The objectives were to determine the difference in sTfR and plasma ferritin (PF) concentrations among infants (6-23 mo of age), school-age children (6-8 y of age), and women (15-25 y of age) with and without inflammation and with and without Plasmodium falciparum infection and to assess the effect of adjusting sTfR and PF for inflammation or for P. falciparum infection on the estimated prevalence of ID. The data were derived from a 14-mo prospective longitudinal survey on anemia, which was conducted in the Taabo area, south-central Côte d'Ivoire. At baseline, sTfR concentration was significantly higher in infants and school-age children with either inflammation or P. falciparum infection than in control individuals without inflammation or without P. falciparum infection. Individuals with inflammation had significantly higher PF concentrations than did subjects without inflammation. Adjustments in sTfR concentrations for inflammation or P. falciparum infection in infants and school-age children resulted in significantly lower ID prevalence. Adjustment of PF for inflammation and Plasmodium infection resulted in a higher ID prevalence in infants and women. In Ivorian infants and school-age children, ID prevalence was considerably lower after adjustment of sTfR for inflammation. However, as the prevalence estimates for ID differed widely if based on sTfR or PF, caution is still needed when estimating ID prevalence in areas with a high prevalence of inflammation or malaria. This trial was registered at controlled-trials.com as ISRCTN02181959.

  16. Human receptors for sweet and umami taste

    PubMed Central

    Li, Xiaodong; Staszewski, Lena; Xu, Hong; Durick, Kyle; Zoller, Mark; Adler, Elliot

    2002-01-01

    The three members of the T1R class of taste-specific G protein-coupled receptors have been hypothesized to function in combination as heterodimeric sweet taste receptors. Here we show that human T1R2/T1R3 recognizes diverse natural and synthetic sweeteners. In contrast, human T1R1/T1R3 responds to the umami taste stimulus l-glutamate, and this response is enhanced by 5′-ribonucleotides, a hallmark of umami taste. The ligand specificities of rat T1R2/T1R3 and T1R1/T1R3 correspond to those of their human counterparts. These findings implicate the T1Rs in umami taste and suggest that sweet and umami taste receptors share a common subunit. PMID:11917125

  17. A mechanism for iron uptake by transferrin.

    PubMed

    Pakdaman, R; El Hage Chahine, J M

    1996-03-15

    Iron uptake by transferrin from iron nitrilotriacetate (FeNAc3) in the presence of bicarbonate has been investigated in the pH range 6.5-8. Apotransferrin, in interaction with bicarbonate, extracts iron from FeNAc3, without the formation of an intermediate protein-iron-ligand mixed complex (iron-exchange-equilibrium constant, K1=1 +/- 0.05; direct second-order-rate constant, k1=8.0x10(4) +/- 0.5x10(4)M(-1)s(-1)., reverse second-order-rate constant, k-1=7.5x10(4) +/- 0.5x10(4)M(-1)s(-1). The newly formed iron-protein complex loses a single proton (proton-dissociation constant, Ka=16 +/- 1.5nM) and then undergoes a modification of its conformation followed by loss of two or three protons (first-order-rate constant, K2=2.80 +/- 0.10s-1). This includes a new modification in the conformation (first-order-rate constant, K2=6.2x10(2) +/- 0.3x10(-2)s(-1). This second modification in conformation controls the rate of iron uptake by the N-site of the protein and is followed by loss of one proton (K3a=6.80 nM). Finally, the holoprotein or the monoferric transferrin in its final equilibrated state is produced by a third modification in the conformation that occurs after approximately 3000 s. Iron uptake by the N-site does not occur when the apotransferrin interacts with bicarbonate. Nevertheless, it occurs with the monoferric transferrin, in which iron is bound to the C-site, in its final state of equilibrium by a mechanism similar to that of iron uptake by the C-site of apotransferrin. These modifications in the conformation of the protein occur after iron uptake by the C-site and may be important for the recognition of the protein by its receptor prior to iron delivery by endocytosis.

  18. Applying 89Zr-Transferrin To Study the Pharmacology of Inhibitors to BET Bromodomain Containing Proteins

    PubMed Central

    2016-01-01

    Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that 89Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with 89Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors. PMID:26725682

  19. Monocyte transferrin-iron uptake in hereditary hemochromatosis

    SciTech Connect

    Sizemore, D.J.; Bassett, M.L.

    1984-05-01

    Transferrin-iron uptake by peripheral blood monocytes was studied in vitro to test the hypothesis that the relative paucity of mononuclear phagocyte iron loading in hereditary hemochromatosis results from a defect in uptake of iron from transferrin. Monocytes from nine control subjects and 17 patients with hemochromatosis were cultured in the presence of 59Fe-labelled human transferrin. There was no difference in 59Fe uptake between monocytes from control subjects and monocytes from patients with hemochromatosis who had been treated by phlebotomy and who had normal body iron stores. However, 59Fe uptake by monocytes from iron-loaded patients with hemochromatosis was significantly reduced compared with either control subjects or treated hemochromatosis patients. It is likely that this was a secondary effect of iron loading since iron uptake by monocytes from treated hemochromatosis patients was normal. Assuming that monocytes in culture reflect mononuclear phagocyte iron metabolism in vivo, this study suggests that the relative paucity of mononuclear phagocyte iron loading in hemochromatosis is not related to an abnormality in transferrin-iron uptake by these cells.

  20. Beta adrenergic receptors in human cavernous tissue

    SciTech Connect

    Dhabuwala, C.B.; Ramakrishna, C.V.; Anderson, G.F.

    1985-04-01

    Beta adrenergic receptor binding was performed with /sup 125/I iodocyanopindolol on human cavernous tissue membrane fractions from normal tissue and transsexual procedures obtained postoperatively, as well as from postmortem sources. Isotherm binding studies on normal fresh tissues indicated that the receptor density was 9.1 fmoles/mg. with a KD of 23 pM. Tissue stored at room temperature for 4 to 6 hours, then at 4C in saline solution for 19 to 20 hours before freezing showed no significant changes in receptor density or affinity, and provided evidence for the stability of postmortem tissue obtained within the same time period. Beta receptor density of 2 cavernous preparations from transsexual procedures was not significantly different from normal control tissues, and showed that high concentrations of estrogen received by these patients had no effect on beta adrenergic receptor density. Displacement of /sup 125/iodocyanopindolol by 5 beta adrenergic agents demonstrated that 1-propranolol had the greatest affinity followed by ICI 118,551, zinterol, metoprolol and practolol. When the results of these displacement studies were subjected to Scatfit, non- linear regression line analysis, a single binding site was described. Based on the relative potency of the selective beta adrenergic agents it appears that these receptors were of the beta 2 subtype.

  1. Imaging dopamine receptors in the human brain by position tomography

    SciTech Connect

    Wagner, H.N. Jr.; Burns, H.D.; Dannals, R.F.; Wong, D.F.; Langstrom, B.; Duelfer, T.; Frost, J.J.; Ravert, H.T.; Links, J.M.; Rosenbloom, S.B.

    1983-01-01

    Neurotransmitter receptors may be involved in a number of neuropsychiatric disease states. The ligand 3-N-(/sup 11/C)methylspiperone, which preferentially binds to dopamine receptors in vivo, was used to image the receptors by positron emission tomography scanning in baboons and in humans. This technique holds promise for noninvasive clinical studies of dopamine receptors in humans.

  2. The impact of highly hydrophobic material on the structure of transferrin and its ability to bind iron.

    PubMed

    Drug, E; Fadeev, L; Gozin, M

    2011-05-30

    Transferrin is a blood-plasma glycoprotein, which is responsible for ferric-ion delivery and which functions as the most important ferric pool in the body. The reversible complexation process of Fe(3+) ions is associated with conformational changes of the three-dimensional structure of the transferrin. This conformational dynamics is attributed to a partial unfolding of the N-lobe of the protein and could be described as a transition between the holo to the apo forms of the transferrin. The aim of the present work is to demonstrate the unprecedented ability of the transferrin to solubilize various polycyclic aromatic hydrocarbons in physiological solution and to explore the impact of these materials on the structure and functionality of the transferrin. The synthesis and characterization of novel materials, consisting of complexes between human transferrin and hydrophobic high-carbon-content compounds, is reported here for the first time. Furthermore, it is shown that the preparation of these complexes from holo-transferrin leads to an irreversible loss of the ferric ions from the protein. Analytical studies of these novel complexes may shed a light on the mechanism by which transferrin could lose its ability to bind and thus to transport and store iron. These findings clearly demonstrate a possible damaging impact of various hydrophobic pollutants, which can enter an organism by inhalation or ingestion, on the functionality of the transferrin.

  3. Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells.

    PubMed

    Reuter, Lauri J; Shahbazi, Mohammad-Ali; Mäkilä, Ermei M; Salonen, Jarno J; Saberianfar, Reza; Menassa, Rima; Santos, Hélder A; Joensuu, Jussi J; Ritala, Anneli

    2017-06-21

    The encapsulation of drugs to nanoparticles may offer a solution for targeted delivery. Here, we set out to engineer a self-assembling targeting ligand by combining the functional properties of human transferrin and fungal hydrophobins in a single fusion protein. We showed that human transferrin can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of-concept for the functionalization of hydrophobin coatings with transferrin as a targeting ligand.

  4. Carbamate Insecticides Target Human Melatonin Receptors.

    PubMed

    Popovska-Gorevski, Marina; Dubocovich, Margarita L; Rajnarayanan, Rajendram V

    2017-02-20

    Carbaryl (1-naphthyl methylcarbamate) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) are among the most toxic insecticides, implicated in a variety of diseases including diabetes and cancer among others. Using an integrated pharmacoinformatics based screening approach, we have identified these insecticides to be structural mimics of the neurohormone melatonin and were able to bind to the putative melatonin binding sites in MT1 and MT2 melatonin receptors in silico. Carbaryl and carbofuran then were tested for competition with 2-[(125)I]-iodomelatonin (300 pM) binding to hMT1 or hMT2 receptors stably expressed in CHO cells. Carbaryl and carbofuran showed higher affinity for competition with 2-[(125)I]-iodomelatonin binding to the hMT2 compared to the hMT1 melatonin receptor (33 and 35-fold difference, respectively) as predicted by the molecular modeling. In the presence of GTP (100 μM), which decouples the G-protein linked receptors to modulate signaling, the apparent efficacy of carbaryl and carbofuran for 2-[(125)I]-iodomelatonin binding for the hMT1 melatonin receptor was not affected but significantly decreased for the hMT2 melatonin receptor compatible with receptor antagonist/inverse agonist and agonist efficacy, respectively. Altogether, our data points to a potentially new mechanism through which carbamate insecticides carbaryl and carbofuran could impact human health by altering the homeostatic balance of key regulatory processes by directly binding to melatonin receptors.

  5. Differential transferrin expression in placentae from normal and abnormal pregnancies: a pilot study

    PubMed Central

    Kralova, Alena; Svetlikova, Marta; Madar, Jindrich; Ulcova-Gallova, Zdena; Bukovsky, Antonin; Peknicova, Jana

    2008-01-01

    Background The placenta is an important site for iron metabolism in humans. It transfers iron from the mother to the fetus. One of the major iron transport proteins is transferrin, which is a blood plasma protein crucial for iron uptake. Its localization and expression may be one of the markers to distinguish placental dysfunction. Methods In the experimental study we used antibody preparation, mass spectrometric analysis, biochemical and immunocytochemical methods for characterization of transferrin expression on the human choriocarcinoma cell line JAR (JAR cells), placental lysates, and cryostat sections. Newly designed monoclonal antibody TRO-tf-01 to human transferrin was applied on human placentae from normal (n = 3) and abnormal (n = 9) pregnancies. Results Variations of transferrin expression were detected in villous syncytiotrophoblast, which is in direct contact with maternal blood. In placentae from normal pregnancies, the expression of transferrin in the syncytium was significantly lower (p < 0.001) when compared to placentae from abnormal ones (gestational diabetes, pregnancy induced hypertension, drug abuse). Conclusion These observations suggest that in the case of abnormal pregnancies, the fetus may require higher levels of transferrin in order to prevent iron depletion due to the stress from the placental dysfunction. PMID:18597674

  6. Androgen receptor in human endothelial cells

    PubMed Central

    Torres-Estay, Verónica; Carreño, Daniela V; San Francisco, Ignacio F; Sotomayor, Paula; Godoy, Alejandro S; Smith, Gary J

    2015-01-01

    Androgen receptor (AR) is a ligand-inducible transcription factor, and a member of the steroid-thyroid-retinoid receptor superfamily, that mediates the biological effects of androgens in a wide range of physiological and pathological processes. AR expression was identified in vascular cells nearly 20 years ago, and recent research has shown that AR mediates a variety of actions of androgens in endothelial and vascular smooth muscle cells. In this mini-review, we review evidence indicating the importance of AR in human endothelial cell (HUVEC) homeostatic and pathogenic processes. Although a role for AR in the modulation of HUVEC biology is evident, the molecular mechanisms by which AR regulates HUVEC homeostasis and disease processes are not fully understood. Understanding these mechanisms could provide critical insights into the processes of pathogenesis of diseases ranging from cardiovascular disease to cancer that are major causes of human morbidity and mortality. PMID:25563353

  7. Adrenergic receptors in human fetal liver membranes

    SciTech Connect

    Falkay, G.; Kovacs, L. )

    1990-01-01

    The adrenergic receptor binding capacities in human fetal and adult livers were measured to investigate the mechanism of the reduced alpha-1 adrenoreceptor response of the liver associated with a reciprocal increase in beta-adrenoreceptor activity in a number of conditions. Alpha-1 and beta-adrenoreceptor density were determined using {sup 3}H-prazosin and {sup 3}H-dihydroalprenolol, respectively, as radioligand. Heterogeneous populations of beta-adrenoreceptors were found in fetal liver contrast to adult. Decreased alpha-1 and increased beta-receptor density were found which may relate to a decreased level in cellular differentiation. These findings may be important for the investigation of perinatal hypoglycemia of newborns after treatment of premature labor with beta-mimetics. This is the first demonstration of differences in the ratio of alpha-1 and beta-adrenoceptors in human fetal liver.

  8. Conjugation of transferrin to azide-modified CdSe/ZnS core-shell quantum dots using cyclooctyne click chemistry.

    PubMed

    Schieber, Christine; Bestetti, Alessandra; Lim, Jet Phey; Ryan, Anneke D; Nguyen, Tich-Lam; Eldridge, Robert; White, Anthony R; Gleeson, Paul A; Donnelly, Paul S; Williams, Spencer J; Mulvaney, Paul

    2012-10-15

    Twinkle twinkle quantum dot: Conjugation of biomolecules to azide-modified quantum dots (QDs) through a bifunctional linker, using strain-promoted azide-alkyne cycloaddition with the QD and a squaramide linkage to the biomolecule (see scheme). Transferrin-conjugated QDs were internalized by transferrin-receptor expressing HeLa cells.

  9. IL-21 Receptor Expression in Human Tendinopathy

    PubMed Central

    Campbell, Abigail L.; Smith, Nicola C.; Reilly, James H.; Kerr, Shauna C.; Leach, William J.; Fazzi, Umberto G.; Rooney, Brian P.; Murrell, George A. C.; Millar, Neal L.

    2014-01-01

    The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy. PMID:24757284

  10. Hormone Receptor Expression in Human Fascial Tissue

    PubMed Central

    Fede, C.; Albertin, G.; Petrelli, L.; Sfriso, M.M.; Biz, C.; De Caro, R.

    2016-01-01

    Many epidemiologic, clinical, and experimental findings point to sex differences in myofascial pain in view of the fact that adult women tend to have more myofascial problems with respect to men. It is possible that one of the stimuli to sensitization of fascial nociceptors could come from hormonal factors such as estrogen and relaxin, that are involved in extracellular matrix and collagen remodeling and thus contribute to functions of myofascial tissue. Immunohistochemical and molecular investigations (real-time PCR analysis) of relaxin receptor 1 (RXFP1) and estrogen receptor-alpha (ERα) localization were carried out on samples of human fascia collected from 8 volunteers patients during orthopedic surgery (all females, between 42 and 70 yrs, divided into pre- and post-menopausal groups), and in fibroblasts isolated from deep fascia, to examine both protein and RNA expression levels. We can assume that the two sex hormone receptors analyzed are expressed in all the human fascial districts examined and in fascial fibroblasts culture cells, to a lesser degree in the post-menopausal with respect to the pre-menopausal women. Hormone receptor expression was concentrated in the fibroblasts, and RXFP1 was also evident in blood vessels and nerves. Our results are the first demonstrating that the fibroblasts located within different districts of the muscular fasciae express sex hormone receptors and can help to explain the link between hormonal factors and myofascial pain. It is known, in fact, that estrogen and relaxin play a key role in extracellular matrix remodeling by inhibiting fibrosis and inflammatory activities, both important factors affecting fascial stiffness and sensitization of fascial nociceptors. PMID:28076930

  11. The Intracellular Trafficking Pathway of Transferrin

    PubMed Central

    Mayle, Kristine M.; Le, Alexander M.; Kamei, Daniel T.

    2011-01-01

    Background Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. Scope of Review We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). Major Conclusions Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. General Significance Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. PMID:21968002

  12. Purification of transferrins and lactoferrin using DEAE affi-gel blue.

    PubMed

    Chung, M C; Chan, S L; Shimizu, S

    1991-01-01

    1. A simple method for purifying transferrins and lactoferrin is described. 2. The method consists of a preliminary step of dye-ligand chromatography using DEAE Affi-Gel Blue as the gel matrix at pH 7.5. In this chromatographic step, the transferrins and lactoferrin were readily separated from the bulk of the other proteins by start buffer elution. 3. Differences in the chromatographic behaviour of the various serum transferrins (monkey, human, rabbit, pig, chicken and duck) and ovotransferrin upon DEAE Affi-Gel Blue chromatography can be attributed to differences in the anionic charge of the transferrins in 0.02 M potassium phosphate buffer, pH 7.5, thus resulting in the differential retardation of these protein molecules by the gel matrix. 4. The result of DEAE Affi-Gel Blue chromatography of human lactoferrin is different from that for the transferrins. This may possibly reflect the differences in the strength of interaction between lactoferrin and transferrin with this gel matrix.

  13. Bombesin-like peptide receptors in human bronchial epithelial cells.

    PubMed

    Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A

    1996-01-01

    Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

  14. Crystal structures of the human adiponectin receptors

    PubMed Central

    Tanabe, Hiroaki; Fujii, Yoshifumi; Hosaka, Toshiaki; Motoyama, Kanna; Ikeda, Mariko; Wakiyama, Motoaki; Terada, Takaho; Ohsawa, Noboru; Hato, Masakatsu; Ogasawara, Satoshi; Hino, Tomoya; Murata, Takeshi; Iwata, So; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yamauchi, Toshimasa; Kadowaki, Takashi; Yokoyama, Shigeyuki

    2015-01-01

    Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases AMPK and PPAR activities, respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G protein-coupled receptor (GPCR)s. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9- and 2.4-Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of GPCRs, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may play a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather than the C-terminal flexible tail, of the receptors. The present information will facilitate the understanding of novel structure-function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes. PMID:25855295

  15. Human blood-brain barrier insulin receptor.

    PubMed

    Pardridge, W M; Eisenberg, J; Yang, J

    1985-06-01

    A new model system for characterizing the human brain capillary, which makes up the blood-brain barrier (BBB) in vivo, is described in these studies and is applied initially to the investigation of the human BBB insulin receptor. Autopsy brains were obtained from the pathologist between 22-36 h postmortem and were used to isolate human brain microvessels which appeared intact on both light and phase microscopy. The microvessels were positive for human factor 8 and for a BBB-specific enzyme marker, gamma-glutamyl transpeptidase. The microvessels avidly bound insulin with a high-affinity dissociation constant, KD = 1.2 +/- 0.5 nM. The human brain microvessels internalized insulin based on acid-wash assay, and 75% of insulin was internalized at 37 degrees C. The microvessels transported insulin to the medium at 37 degrees C with a t1/2 = approximately 70 min. Little of the 125I-insulin was metabolized by the microvessels under these conditions based on the elution profile of the medium extract over a Sephadex G-50 column. Plasma membranes were obtained from the human brain microvessels and these membranes were enriched in membrane markers such as gamma-glutamyl transpeptidase or alkaline phosphatase. The plasma membranes bound 125I-insulin with and ED50 = 10 ng/ml, which was identical to the 50% binding point in intact microvessels. The human BBB plasma membranes were solubilized in Triton X-100 and were adsorbed to a wheat germ agglutinin Sepharose affinity column, indicating the BBB insulin receptor is a glycoprotein. Affinity cross-linking of insulin to the plasma membranes revealed a 127K protein that specifically binds insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Dopamine receptors in human gastrointestinal mucosa

    SciTech Connect

    Hernandez, D.E.; Mason, G.A.; Walker, C.H.; Valenzuela, J.E.

    1987-12-21

    Dopamine is a putative enteric neurotransmitter that has been implicated in exocrine secretory and motility functions of the gastrointestinal tract of several mammalian species including man. This study was designed to determine the presence of dopamine binding sites in human gastric and duodenal mucosa and to describe certain biochemical characteristics of these enteric receptor sites. The binding assay was performed in triplicate with tissue homogenates obtained from healthy volunteers of both sexes using /sup 3/H-dopamine as a ligand. The extent of nonspecific binding was determined in the presence of a 100-fold excess of unlabeled dopamine. Scatchard analysis performed with increasing concentrations of /sup 3/H-dopamine (20-500 nM) revealed a single class of saturable dopamine binding sites in gastric and duodenal mucosa. The results of this report demonstrate the presence of specific dopamine receptors in human gastric and duodenal mucosa. These biochemical data suggest that molecular abnormalities of these receptor sites may be operative in the pathogenesis of important gastrointestinal disorders. 33 references, 2 figures.

  17. Human specific loss of olfactory receptor genes

    PubMed Central

    Gilad, Yoav; Man, Orna; Pääbo, Svante; Lancet, Doron

    2003-01-01

    Olfactory receptor (OR) genes constitute the basis for the sense of smell and are encoded by the largest mammalian gene superfamily of >1,000 genes. In humans, >60% of these are pseudogenes. In contrast, the mouse OR repertoire, although of roughly equal size, contains only ≈20% pseudogenes. We asked whether the high fraction of nonfunctional OR genes is specific to humans or is a common feature of all primates. To this end, we have compared the sequences of 50 human OR coding regions, regardless of their functional annotations, to those of their putative orthologs in chimpanzees, gorillas, orangutans, and rhesus macaques. We found that humans have accumulated mutations that disrupt OR coding regions roughly 4-fold faster than any other species sampled. As a consequence, the fraction of OR pseudogenes in humans is almost twice as high as in the non-human primates, suggesting a human-specific process of OR gene disruption, likely due to a reduced chemosensory dependence relative to apes. PMID:12612342

  18. Enhanced blood-brain barrier transmigration using a novel transferrin embedded fluorescent magneto-liposome nanoformulation

    NASA Astrophysics Data System (ADS)

    Ding, Hong; Sagar, Vidya; Agudelo, Marisela; Pilakka-Kanthikeel, Sudheesh; Subba Rao Atluri, Venkata; Raymond, Andrea; Samikkannu, Thangavel; Nair, Madhavan P.

    2014-02-01

    The blood-brain barrier (BBB) is considered as the primary impediment barrier for most drugs. Delivering therapeutic agents to the brain is still a big challenge to date. In our study, a dual mechanism, receptor mediation combined with external non-invasive magnetic force, was incorporated into ferrous magnet-based liposomes for BBB transmigration enhancement. The homogenous magnetic nanoparticles (MNPs), with a size of ˜10 nm, were synthesized and confirmed by TEM and XRD respectively. The classical magnetism assay showed the presence of the characteristic superparamagnetic property. These MNPs encapsulated in PEGylated fluorescent liposomes as magneto-liposomes (MLs) showed mono-dispersion, ˜130 ± 10 nm diameter, by dynamic laser scattering (DLS) using the lipid-extrusion technique. Remarkably, a magnetite encapsulation efficiency of nearly 60% was achieved. Moreover, the luminescence and hydrodynamic size of the MLs was stable for over two months at 4 ° C. Additionally, the integrity of the ML structure remained unaffected through 120 rounds of circulation mimicking human blood fluid. After biocompatibility confirmation by cytotoxicity evaluation, these fluorescent MLs were further embedded with transferrin and applied to an in vitro BBB transmigration study in the presence or absence of external magnetic force. Comparing with magnetic force- or transferrin receptor-mediated transportation alone, their synergy resulted in 50-100% increased transmigration without affecting the BBB integrity. Consequently, confocal microscopy and iron concentration in BBB-composed cells further confirmed the higher cellular uptake of ML particles due to the synergic effect. Thus, our multifunctional liposomal magnetic nanocarriers possess great potential in particle transmigration across the BBB and may have a bright future in drug delivery to the brain.

  19. Enhanced blood-brain barrier transmigration using a novel transferrin embedded fluorescent magneto-liposome nanoformulation.

    PubMed

    Ding, Hong; Sagar, Vidya; Agudelo, Marisela; Pilakka-Kanthikeel, Sudheesh; Atluri, Venkata Subba Rao; Raymond, Andrea; Samikkannu, Thangavel; Nair, Madhavan P

    2014-02-07

    The blood-brain barrier (BBB) is considered as the primary impediment barrier for most drugs. Delivering therapeutic agents to the brain is still a big challenge to date. In our study, a dual mechanism, receptor mediation combined with external non-invasive magnetic force, was incorporated into ferrous magnet-based liposomes for BBB transmigration enhancement. The homogenous magnetic nanoparticles (MNPs), with a size of ∼10 nm, were synthesized and confirmed by TEM and XRD respectively. The classical magnetism assay showed the presence of the characteristic superparamagnetic property. These MNPs encapsulated in PEGylated fluorescent liposomes as magneto-liposomes (MLs) showed mono-dispersion, ∼130 ± 10 nm diameter, by dynamic laser scattering (DLS) using the lipid-extrusion technique. Remarkably, a magnetite encapsulation efficiency of nearly 60% was achieved. Moreover, the luminescence and hydrodynamic size of the MLs was stable for over two months at 4 ° C. Additionally, the integrity of the ML structure remained unaffected through 120 rounds of circulation mimicking human blood fluid. After biocompatibility confirmation by cytotoxicity evaluation, these fluorescent MLs were further embedded with transferrin and applied to an in vitro BBB transmigration study in the presence or absence of external magnetic force. Comparing with magnetic force- or transferrin receptor-mediated transportation alone, their synergy resulted in 50-100% increased transmigration without affecting the BBB integrity. Consequently, confocal microscopy and iron concentration in BBB-composed cells further confirmed the higher cellular uptake of ML particles due to the synergic effect. Thus, our multifunctional liposomal magnetic nanocarriers possess great potential in particle transmigration across the BBB and may have a bright future in drug delivery to the brain.

  20. Transferrin Impacts Bacillus thuringiensis Biofilm Levels

    PubMed Central

    Brown, Elrica; Taplin, Martha; Garcia, Angel; Williams-Mapp, Baracka

    2016-01-01

    The present study examined the impact of transferrin on Bacillus thuringiensis biofilms. Three commercial strains, an environmental strain (33679), the type strain (10792), and an isolate from a diseased insect (700872), were cultured in iron restricted minimal medium. All strains produced biofilm when grown in vinyl plates at 30°C. B. thuringiensis 33679 had a biofilm biomass more than twice the concentration exhibited by the other strains. The addition of transferrin resulted in slightly increased growth yields for 2 of the 3 strains tested, including 33679. In contrast, the addition of 50 μg/mL of transferrin resulted in an 80% decrease in biofilm levels for strain 33679. When the growth temperature was increased to 37°C, the addition of 50 μg/mL of transferrin increased culture turbidity for only strain 33679. Biofilm levels were again decreased in strain 33679 at 37°C. Growth of B. thuringiensis cultures in polystyrene resulted in a decrease in overall growth yields at 30°C, with biofilm levels significantly decreased for 33679 in the presence of transferrin. These findings demonstrate that transferrin impacts biofilm formation in select strains of B. thuringiensis. Identification of these differences in biofilm regulation may be beneficial in elucidating potential virulence mechanisms among the differing strains. PMID:28025643

  1. Transferrin Impacts Bacillus thuringiensis Biofilm Levels.

    PubMed

    Garner, Bianca; Brown, Elrica; Taplin, Martha; Garcia, Angel; Williams-Mapp, Baracka

    2016-01-01

    The present study examined the impact of transferrin on Bacillus thuringiensis biofilms. Three commercial strains, an environmental strain (33679), the type strain (10792), and an isolate from a diseased insect (700872), were cultured in iron restricted minimal medium. All strains produced biofilm when grown in vinyl plates at 30°C. B. thuringiensis 33679 had a biofilm biomass more than twice the concentration exhibited by the other strains. The addition of transferrin resulted in slightly increased growth yields for 2 of the 3 strains tested, including 33679. In contrast, the addition of 50 μg/mL of transferrin resulted in an 80% decrease in biofilm levels for strain 33679. When the growth temperature was increased to 37°C, the addition of 50 μg/mL of transferrin increased culture turbidity for only strain 33679. Biofilm levels were again decreased in strain 33679 at 37°C. Growth of B. thuringiensis cultures in polystyrene resulted in a decrease in overall growth yields at 30°C, with biofilm levels significantly decreased for 33679 in the presence of transferrin. These findings demonstrate that transferrin impacts biofilm formation in select strains of B. thuringiensis. Identification of these differences in biofilm regulation may be beneficial in elucidating potential virulence mechanisms among the differing strains.

  2. On the evolutionary significance and metal-binding characteristics of a monolobal transferrin from Ciona intestinalis

    PubMed Central

    Tinoco, Arthur D.; Peterson, Cynthia W.; Lucchese, Baldo; Doyle, Robert P.; Valentine, Ann M.

    2008-01-01

    Transferrins are a family of proteins that bind and transport Fe(III). Modern transferrins are typically bilobal and are believed to have evolved from an ancient gene duplication of a monolobal form. A novel monolobal transferrin, nicatransferrin (nicaTf), was identified in the primitive ascidian species Ciona intestinalis that possesses the characteristic features of the proposed ancestral Tf protein. In this work, nicaTf was expressed in Pichia pastoris. Extensive solution studies were performed on nicaTf, including UV-vis, fluorescence, CD, EPR and NMR spectroscopies, and electrospray time-of-flight mass spectrometry. The expressed protein is nonglycosylated, unlike the protein isolated from the organism. This property does not affect its ability to bind Fe(III). However, Fe(III)-bound nicaTf displays important spectral differences from other Fe(III)-bound transferrins, which are likely the consequence of differences in metal coordination. Coordination differences could also account for the weaker affinity of nicaTf for Fe(III) (log K = 18.5) compared with bilobal human serum transferrin (HsTf) (log K = 22.5 and 21.4). The Fe–nicaTf complex is not labile, as indicated by slow metal removal kinetics by the high-affinity chelator tiron at pH 7.4. The protein alternatively binds up to one equivalent of Ti(IV) or V(V), which suggests that it may transport nonferric metals. These solution studies provide insight into the structure and function of the primitive monolobal transferrin of C. intestinalis for comparison with higher order bilobal transferrins. They suggest that a major advantage for the evolution of modern transferrins, dominantly of bilobal form, is stronger Fe(III) affinity because of cooperativity. PMID:18287008

  3. Receptors for human γG Globulin on human neutrophils

    PubMed Central

    Messner, R. P.; Jelinek, J.

    1970-01-01

    Cell surface receptors for human γG antibodies directed against bacterial antigens were demonstrated on human neutrophils using an in vitro bacteriocidal-phagocytic assay. These results were confirmed by adherence of sensitized erythrocytes to monolayers of neutrophils or monocytes. Erythrocytes sensitized indirectly with antibacterial γG antibodies after passive sensitization with bacterial antigens adhered to both neutrophils and monocytes. Erythrocytes sensitized directly with conventional anti-D γG antibodies adhered only to monocytes, while those sensitized with the hyperimmune anti-CD γG antibody Ripley adhered to both monocytes and neutrophils. Adherence of anti-Rh or antibacterial γG antibodies to monocytes and neutrophils could be inhibited by whole γG, myeloma globulins of the γ1 or γ3 subclasses, or Fc fragments, but not by Fab fragment. These results indicate that receptors for the Fc portion of human γG antibodies exist on both neutrophils and monocytes, and that γG antibodies differ in their ability to attach to these two cell types. Differences in the behavior of the γG antibodies studied may be related to differences in the density of antibodies on the erythrocyte surface and receptors on the phagocytic cells. Images PMID:4991439

  4. NOD-like receptors and human diseases.

    PubMed

    Rosenstiel, Philip; Till, Andreas; Schreiber, Stefan

    2007-04-01

    NOD-like receptors are cytosolic proteins that contain a central nucleotide-binding oligomerization domain (NACHT), an N-terminal effector-binding domain and C-terminal leucine-rich repeats (LRRs). NOD-like receptors have been implicated as ancient cellular sentinels mediating protective immune responses against intracellular pathogens. Recent studies have described the genetic association of polymorphisms in NOD-like receptor genes with complex chronic inflammatory barrier diseases, such as Crohn's disease and asthma and with rare auto-inflammatory syndromes including familial cold urticaria, Muckle-Wells syndrome and Blau syndrome. Whereas genetic variability in NLRs may have been an important element to provide plasticity to antigen recognition and host defense in the past, recent changes in the lifestyle of industrialized societies (e.g. hygiene ("cold-chain-hypothesis"), nutrition, or antibiotics) may have turned ancient genetic variability into disease-causing mutations. The review focuses on NLR function in the molecular pathophysiology of human inflammatory disorders.

  5. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  6. Mosquito transferrin, an acute-phase protein that is up-regulated upon infection

    PubMed Central

    Yoshiga, Toyoshi; Hernandez, Vida P.; Fallon, Ann M.; Law, John H.

    1997-01-01

    When treated with heat-killed bacterial cells, mosquito cells in culture respond by up-regulating several proteins. Among these is a 66-kDa protein (p66) that is secreted from cells derived from both Aedes aegypti and Aedes albopictus. p66 was degraded by proteolysis and gave a virtually identical pattern of peptide products for each mosquito species. The sequence of one peptide (31 amino acids) was determined and found to have similarity to insect transferrins. By using conserved regions of insect transferrin sequences, degenerate oligonucleotide PCR primers were designed and used to isolate a cDNA clone encoding an A. aegypti transferrin. The encoded protein contained a signal sequence that, when cleaved, would yield a mature protein of 68 kDa. It contained the 31-amino acid peptide, and the 3′ end exactly matched a cDNA encoding a polypeptide that is up-regulated when A. aegypti encapsulates filarial worms [Beerntsen, B. T., Severson, D. W. & Christensen, B. M. (1994) Exp. Parasitol. 79, 312–321]. This transferrin, like those of two other insect species, has conserved iron-binding residues in the N-terminal lobe but not in the C-terminal lobe, which also has large deletions in the polypeptide chain, compared with transferrins with functional C-terminal lobes. The hypothesis is developed that this transferrin plays a role similar to vertebrate lactoferrin in sequestering iron from invading organisms and that degradation of the structure of the C-terminal lobe might be a mechanism for evading pathogens that elaborate transferrin receptors to tap sequestered iron. PMID:9356450

  7. Bitter taste receptor polymorphisms and human aging.

    PubMed

    Campa, Daniele; De Rango, Francesco; Carrai, Maura; Crocco, Paolina; Montesanto, Alberto; Canzian, Federico; Rose, Giuseppina; Rizzato, Cosmeri; Passarino, Giuseppe; Barale, Roberto

    2012-01-01

    Several studies have shown that genetic factors account for 25% of the variation in human life span. On the basis of published molecular, genetic and epidemiological data, we hypothesized that genetic polymorphisms of taste receptors, which modulate food preferences but are also expressed in a number of organs and regulate food absorption processing and metabolism, could modulate the aging process. Using a tagging approach, we investigated the possible associations between longevity and the common genetic variation at the three bitter taste receptor gene clusters on chromosomes 5, 7 and 12 in a population of 941 individuals ranging in age from 20 to 106 years from the South of Italy. We found that one polymorphism, rs978739, situated 212 bp upstream of the TAS2R16 gene, shows a statistically significant association (p = 0.001) with longevity. In particular, the frequency of A/A homozygotes increases gradually from 35% in subjects aged 20 to 70 up to 55% in centenarians. These data provide suggestive evidence on the possible correlation between human longevity and taste genetics.

  8. Laminin receptor on human breast carcinoma cells.

    PubMed Central

    Terranova, V P; Rao, C N; Kalebic, T; Margulies, I M; Liotta, L A

    1983-01-01

    Human MCF-7 breast carcinoma cells possess a receptor-like moiety on their surface that has a high binding affinity (Kd = 2 nM) for laminin, a glycoprotein localized in basement membranes. Laminin preferentially stimulates (8-fold) MCF-7 cells to attach to type IV (basement membrane) collagen, whereas fibronectin stimulates attachment only 2-fold for these cells on type I collagen. The attachment properties of two other human breast carcinoma cell lines to type IV collagen were also studied. The attachment of ZR-75-1 cells was stimulated 4-fold by laminin and 5-fold by fibronectin, whereas T47-D cell attachment was stimulated 2-fold by laminin and 7-fold by fibronectin. By employing protease-derived fragments of laminin, the major domains of the laminin molecule that participate in MCF-7 cell attachment to type IV collagen were identified. The whole laminin molecule has the configuration of a four-armed cross with three short arms and one long arm. A major cell-binding domain was found to reside near the intersection point of the short arms, and the type IV collagen-binding domain was associated with the globular end regions of the short arms. The receptor for laminin on the surface of these tumor cells may be involved in the initial interaction of tumor cells via laminin with the vascular basement membrane to facilitate invasion and subsequent promotion of metastasis. Images PMID:6300843

  9. Bitter Taste Receptor Polymorphisms and Human Aging

    PubMed Central

    Carrai, Maura; Crocco, Paolina; Montesanto, Alberto; Canzian, Federico; Rose, Giuseppina; Rizzato, Cosmeri

    2012-01-01

    Several studies have shown that genetic factors account for 25% of the variation in human life span. On the basis of published molecular, genetic and epidemiological data, we hypothesized that genetic polymorphisms of taste receptors, which modulate food preferences but are also expressed in a number of organs and regulate food absorption processing and metabolism, could modulate the aging process. Using a tagging approach, we investigated the possible associations between longevity and the common genetic variation at the three bitter taste receptor gene clusters on chromosomes 5, 7 and 12 in a population of 941 individuals ranging in age from 20 to 106 years from the South of Italy. We found that one polymorphism, rs978739, situated 212 bp upstream of the TAS2R16 gene, shows a statistically significant association (p = 0.001) with longevity. In particular, the frequency of A/A homozygotes increases gradually from 35% in subjects aged 20 to 70 up to 55% in centenarians. These data provide suggestive evidence on the possible correlation between human longevity and taste genetics. PMID:23133589

  10. Transferrin serves as a mediator to deliver organometallic ruthenium(II) anticancer complexes into cells.

    PubMed

    Guo, Wei; Zheng, Wei; Luo, Qun; Li, Xianchan; Zhao, Yao; Xiong, Shaoxiang; Wang, Fuyi

    2013-05-06

    We report herein a systematic study on interactions of organometallic ruthenium(II) anticancer complex [(η(6)-arene)Ru(en)Cl](+) (arene = p-cymene (1) or biphenyl (2), en = ethylenediamine) with human transferrin (hTf) and the effects of the hTf-ligation on the bioavailability of these complexes with cisplatin as a reference. Incubated with a 5-fold excess of complex 1, 2, or cisplatin, 1 mol of diferric hTf (holo-hTf) attached 0.62 mol of 1, 1.01 mol of 2, or 2.14 mol of cisplatin. Mass spectrometry revealed that both ruthenium complexes coordinated to N-donors His242, His273, His578, and His606, whereas cisplatin bound to O donors Tyr136 and Tyr317 and S-donor Met256 in addition to His273 and His578 on the surface of both apo- and holo-hTf. Moreover, cisplatin could bind to Thr457 within the C-lobe iron binding cleft of apo-hTf. Neither ruthenium nor platinum binding interfered with the recognition of holo-hTf by the transferrin receptor (TfR). The ruthenated/platinated holo-hTf complexes could be internalized via TfR-mediated endocytosis at a similar rate to that of holo-hTf itself. Moreover, the binding to holo-hTf well preserved the bioavailability of the ruthenium complexes, and the hTf-bound 1 and 2 showed a similar cytotoxicity toward the human breast cancer cell line MCF-7 to those of the complexes themselves. However, the conjugation with holo-hTf significantly reduced the cellular uptake of cisplatin and the amount of platinated DNA adducts formed intracellularly, leading to dramatic reduction of cisplatin cytotoxicity toward MCF-7. These findings suggest that hTf can serve as a mediator for the targeting delivery of Ru(arene) anticancer complexes while deactivating cisplatin.

  11. Separation of beta2-transferrin by denaturing gel electrophoresis to detect cerebrospinal fluid in ear and nasal fluids.

    PubMed

    Görögh, Tibor; Rudolph, Pierre; Meyer, Jens Eduard; Werner, Jochen A; Lippert, Burkard M; Maune, Steffen

    2005-09-01

    Cerebrospinal fluid (CSF) leakage is a critical condition with a substantial risk of meningitis. We investigated the use of transferrin isoform analysis as a diagnostic marker for detection of CSF leakage in fluid samples. We analyzed 241 samples from patients with CSF leakage, most commonly presenting as otorrhea or rhinorrhea, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent Western blotting and immunostaining for transferrin. Tears, saliva, nasal fluid, and ear secretions (20 samples each) were analyzed in parallel, and normal human serum served as a control in each experiment. We compared the minimum volume of added CSF that could be detected in secretions by our assay with the minimum volume detected by the prostaglandin-D synthase (beta-trace) test. CSF was admixed with blood in different proportions to determine the influence of blood contamination on the transferrin pattern. In all CSF samples, beta1- and beta2-transferrin were present in nearly equal amounts. In tears and ear secretions, beta2-transferrin migrated in the gel in the same manner as in CSF, but its concentration was noticeably lower than that of beta1-transferrin, a difference that allowed a clear distinction from the transferrin pattern of CSF. In saliva, both transferrin isoforms were also present but could be distinguished from those of other fluids by electrophoretic migration pattern rather than relative concentrations. With the beta-trace test, a minimum of 5 microL of CSF was needed for detection, whereas our beta2-transferrin assay yielded a signal of comparable intensity with a minimum of 2 microL of CSF. Analysis of the transferrin microheterogeneity pattern by SDS-PAGE for the identification of CSF leakage is a highly sensitive and specific method that merits consideration as a routine technique.

  12. Transferrins: iron release from lactoferrin.

    PubMed

    Abdallah, F B; El Hage Chahine, J M

    2000-10-20

    Iron loss in vitro by the iron scavenger bovine lactoferrin was investigated in acidic media in the presence of three different monoanions (NO(3)(-), Cl(-) and Br(-)) and one dianion (SO(4)(2-)). Holo and monoferric C-site lactoferrins lose iron in acidic media (pH< or =3.5) by a four-step mechanism. The first two steps describe modifications in the conformation affecting the whole protein, which occur also with apolactoferrin. These two processes are independent of iron load and are followed by a third step consisting of the gain of two protons. This third step is kinetically controlled by the interaction with two Cl(-), Br(-) and NO(3)(-) or one SO(4)(2-). In the fourth step, iron loss is under the kinetic control of a slow gain of two protons; third-order rate-constants k(2), 4.3(+/-0.2)x10(3), 3.4(+/-0.5)x10(3), 3.3(+/-0.5)x10(3) and 1.5(+/-0.5)x10(3) M(-2) s(-1) when the protein is in interaction with SO(4)(2-), NO(3)(-), Cl(-) or Br(-), respectively. This step is accompanied by the loss of the interaction with the anions; equilibrium constant K(2), 20+/-5 mM, 1.0(+/-0.2)x10(-1), 1.5(+/-0.5)x10(-1) and 1.0(+/-0.3)x10(-1) M(2), for SO(4)(-), NO(3)(-), Cl(-) and Br(-), respectively. This mechanism is very different from that determined in mildly acidic media at low ionic strength (micro<0.5) for the iron transport proteins, serum transferrin and ovotransferrin, with which no prior change in conformation or interaction with anions is required. These differences may result from the fact that in the transport proteins, the interdomain hydrogen bonds that consolidate the closed conformation of the iron-binding cleft occur between amino acid side-chain residues that can protonate in mildly acidic media. With bovine lactoferrin, most of the interdomain hydrogen bonds involved in the C-site and one of those involved in the N-site occur between amino acid side-chain residues that cannot protonate. The breaking of the interdomain H-bond upon protonation can trigger the

  13. Human native kappa opioid receptor functions not predicted by recombinant receptors: Implications for drug design

    PubMed Central

    Broad, John; Maurel, Damien; Kung, Victor W. S.; Hicks, Gareth A.; Schemann, Michael; Barnes, Michael R.; Kenakin, Terrence P.; Granier, Sébastien; Sanger, Gareth J.

    2016-01-01

    If activation of recombinant G protein-coupled receptors in host cells (by drugs or other ligands) has predictive value, similar data must be obtained with native receptors naturally expressed in tissues. Using mouse and human recombinant κ opioid receptors transfected into a host cell, two selectively-acting compounds (ICI204448, asimadoline) equi-effectively activated both receptors, assessed by measuring two different cell signalling pathways which were equally affected without evidence of bias. In mouse intestine, naturally expressing κ receptors within its nervous system, both compounds also equi-effectively activated the receptor, inhibiting nerve-mediated muscle contraction. However, whereas ICI204448 acted similarly in human intestine, where κ receptors are again expressed within its nervous system, asimadoline was inhibitory only at very high concentrations; instead, low concentrations of asimadoline reduced the activity of ICI204448. This demonstration of species-dependence in activation of native, not recombinant κ receptors may be explained by different mouse/human receptor structures affecting receptor expression and/or interactions with intracellular signalling pathways in native environments, to reveal differences in intrinsic efficacy between receptor agonists. These results have profound implications in drug design for κ and perhaps other receptors, in terms of recombinant-to-native receptor translation, species-dependency and possibly, a need to use human, therapeutically-relevant, not surrogate tissues. PMID:27492592

  14. Applying ⁸⁹Zr-Transferrin To Study the Pharmacology of Inhibitors to BET Bromodomain Containing Proteins.

    PubMed

    Doran, Michael G; Carnazza, Kathryn E; Steckler, Jeffrey M; Spratt, Daniel E; Truillet, Charles; Wongvipat, John; Sawyers, Charles L; Lewis, Jason S; Evans, Michael J

    2016-02-01

    Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that (89)Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with (89)Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors.

  15. Reptilian transferrins: evolution of disulphide bridges and conservation of iron-binding center.

    PubMed

    Ciuraszkiewicz, Justyna; Biczycki, Marian; Maluta, Aleksandra; Martin, Samuel; Watorek, Wiesław; Olczak, Mariusz

    2007-07-01

    Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to

  16. Bioadhesive micelles of d-α-tocopherol polyethylene glycol succinate 1000: Synergism of chitosan and transferrin in targeted drug delivery.

    PubMed

    Agrawal, Poornima; Sonali; Singh, Rahul Pratap; Sharma, Gunjan; Mehata, Abhishesh K; Singh, Sanjay; Rajesh, Chellapa V; Pandey, Bajarangprasad L; Koch, Biplob; Muthu, Madaswamy S

    2017-04-01

    The aim of this work was to prepare targeted bioadhesive d-α- tocopheryl glycol succinate 1000 (TPGS) micelles containing docetaxel (DTX) for brain targeted cancer therapy. Considering the unique bioadhesive feature of chitosan, herein, we have developed a synergistic transferrin receptor targeted bioadhesive micelles using TPGS conjugated chitosan (TPGS-chitosan), which target the overexpressed transferrin receptors of glioma cells for brain cancer therapy. The micelles were prepared by the solvent casting method and characterized for their particle size, polydispersity, zeta-potential, surface morphology, drug encapsulation efficiency, and in-vitro release. The IC50 values demonstrated transferrin receptor targeted TPGS-chitosan micelles could be 248 folds more effective than Docel™ after 24h treatment with the C6 glioma cells. Further, time dependent bioadhesive cellular uptake study indicated that a synergistic effect was achieved with the chitosan and transferrin in targeted TPGS-chitosan micelles through the biodhesive property of chitosan as well as transferrin receptor mediated endocytosis. The in-vivo pharmacokinetic results demonstrated that relative bioavailability of non-targeted and targeted micelles were 2.89 and 4.08 times more effective than Docel™ after 48h of treatments, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Prooxidant activity of transferrin and lactoferrin.

    PubMed

    Klebanoff, S J; Waltersdorph, A M

    1990-11-01

    Acceleration of the autoxidation of Fe2+ by apotransferrin or apolactoferrin at acid pH is indicated by the disappearance of Fe2+, the uptake of oxygen, and the binding of iron to transferrin or lactoferrin. The product(s) formed oxidize iodide to an iodinating species and are bactericidal to Escherichia coli. Toxicity to E. coli by FeSO4 (10(-5) M) and human apotransferrin (100 micrograms/ml) or human apolactoferrin (25 micrograms/ml) was optimal at acid pH (4.5-5.0) and with logarithmic phase organisms. Both the iodinating and bactericidal activities were inhibited by catalase and the hydroxyl radical (OH.) scavenger mannitol, whereas superoxide dismutase was ineffective. NaCl at 0.1 M inhibited bactericidal activity, but had little or no effect on iodination. Iodide increased the bactericidal activity of Fe2+ and apotransferrin or apolactoferrin. The formation of OH.was suggested by the formation of the OH.spin-trap adduct (5,5-dimethyl-1-pyroline N-oxide [DMPO]/OH)., with the spin trap DMPO and the formation of the methyl radical adduct on the further addition of dimethyl sulfoxide. (DMPO/OH).formation was inhibited by catalase, whereas superoxide dismutase had little or no effect. These findings suggest that Fe2+ and apotransferrin or apolactoferrin can generate OH.via an H2O2 intermediate with toxicity to microorganisms, and raise the possibility that such a mechanism may contribute to the microbicidal activity of phagocytes.

  18. A novel transferrin/TfR2-mediated mitochondrial iron transport system is disrupted in Parkinson's disease

    PubMed Central

    Mastroberardino, Pier Giorgio; Hoffman, Eric K.; Horowitz, Maxx P.; Betarbet, Ranjita; Taylor, Georgia; Cheng, Dongmei; Na, Hye Mee; Gutekunst, Claire-Anne; Gearing, Marla; Trojanowski, John Q.; Anderson, Marjorie; Chu, Charleen T.; Peng, Junmin; Greenamyre, J. Timothy

    2009-01-01

    More than 80 years after iron accumulation was initially described in the substantia nigra (SN) of Parkinson's disease (PD) patients, the mechanisms responsible for this phenomenon are still unknown. Similarly, how iron is delivered to its major recipients in the cell – mitochondria and the respiratory complexes – has yet to be elucidated. Here, we report a novel transferrin/transferrin receptor 2 (Tf/TfR2)-mediated iron transport pathway in mitochondria of SN dopamine neurons. We found that TfR2 has a previously uncharacterized mitochondrial targeting sequence that is sufficient to import the protein into these organelles. Importantly, the Tf/TfR2 pathway can deliver Tf bound iron to mitochondria and to the respiratory complex I as well. The pathway is redox-sensitive and oxidation of Tf thiols to disulfides induces release from Tf of highly reactive ferrous iron, which contributes to free radical production. In the rotenone model of PD, Tf accumulates in dopamine neurons, with much of it accumulating in the mitochondria. This is associated with iron deposition in SN, similar to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention. PMID:19250966

  19. β1,4-galactosyltransferase 1 is a novel receptor for IgA in human mesangial cells.

    PubMed

    Molyneux, Karen; Wimbury, David; Pawluczyk, Izabella; Muto, Masahiro; Bhachu, Jasraj; Mertens, Peter R; Feehally, John; Barratt, Jonathan

    2017-07-24

    IgA nephropathy is characterized by mesangial deposition of IgA, mesangial cell proliferation, and extracellular matrix production. Mesangial cells bind IgA, but the identity of all potential receptors involved remains incomplete. The transferrin receptor (CD71) acts as a mesangial cell IgA receptor and its expression is upregulated in many forms of glomerulonephritis, including IgA nephropathy. CD71 is not expressed in healthy glomeruli and blocking CD71 does not completely abrogate mesangial cell IgA binding. Previously we showed that mesangial cells express a receptor that binds the Fc portion of IgA and now report that this receptor is an isoform of β-1,4-galactosyltransferase. A human mesangial cell cDNA library was screened for IgA binding proteins and β-1,4-galactosyltransferase identified. Cell surface expression of the long isoform of β-1,4-galactosyltransferase was shown by flow cytometry and confocal microscopy and confirmed by immunoblotting. Glomerular β-1,4-galactosyltransferase expression was increased in IgA nephropathy. IgA binding and IgA-induced mesangial cell phosphorylation of spleen tyrosine kinase and IL-6 synthesis were inhibited by a panel of β-1,4-galactosyltransferase-specific antibodies, suggesting IgA binds to the catalytic domain of β-1,4-galactosyltransferase. Thus, β-1,4-galactosyltransferase is a constitutively expressed mesangial cell IgA receptor with an important role in both mesangial IgA clearance and the initial response to IgA deposition. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  20. Expression of prostacyclin receptor in human megakaryocytes.

    PubMed

    Sasaki, Y; Takahashi, T; Tanaka, I; Nakamura, K; Okuno, Y; Nakagawa, O; Narumiya, S; Nakao, K

    1997-08-01

    Prostacyclin (prostaglandin I2, PGI2) is a potent vasodilator and inhibitor of platelet aggregation. Although it is well known that the specific receptor for prostacyclin (PGI2-R) is abundantly expressed on platelets, PGI2-R expression in megakaryocytes is poorly understood. In this study, we examined its expression in leukemic or normal megakaryocytes. PGI2-R mRNA was expressed in human leukemic cell lines of megakaryocytic nature as evaluated by Northern blot analysis. Phorbol 12-myristate 13-acetate (PMA), interleukin-1 (IL-1), IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin (TPO), and tumor necrosis factor-alpha (TNF-alpha) enhanced PGI2-R mRNA expression. The enhancement of PGI2-R expression by PMA and TPO was associated with the upregulation of platelet factor 4 or glycoprotein IIb mRNA expression. Iloprost, an agonist of prostacyclin, induced significant cyclic (c)AMP synthesis in these leukemic cells indicating that interaction of PGI2-R and its ligand can induce postreceptor signal transduction. Furthermore, iloprost-induced cAMP synthesis was enhanced by the pretreatment with PMA or the cytokines that promoted PGI2-R expression. PMA and TPO also increased the specific binding of [3H]iloprost to these cells. Pooled normal megakaryocytic colonies from TPO-containing semisolid culture of purified human CD34+ cells expressed PGI2-R, which were increased as the megakaryocytes matured with the peak expression before proplatelet formation, as evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). These results indicate that PGI2-R is expressed in human megakaryocytes and is upregulated by cytokines involved in thrombopoiesis or inflammation. Also, it was indicated that megakaryocytic maturation accompanies enhancement of PGI2-R expression.

  1. Characterization of functional urotensin II receptors in human skeletal muscle myoblasts: comparison with angiotensin II receptors.

    PubMed

    Qi, Jian-shen; Minor, Lisa K; Smith, Charles; Hu, Bing; Yang, Jing; Andrade-Gordon, Patricia; Damiano, Bruce

    2005-04-01

    The properties of urotensin II (U-II) receptor (UT receptor) and angiotensin II (ANG II) receptor (AT receptor) in primary human skeletal myoblasts (HSMM) and differentiated skeletal myotubes (HSMMT) were characterized. Radiolabeled U-II and ANG II bound specifically to HSMM with Kd's of 0.31 nM (2311 receptors/cell) and 0.61 nM (18,257 receptors/cell), respectively. The cyclic segment of U-II peptide, CFWKYC, was the minimal sequence required for binding, with the WKY residues essential. Inhibitor studies suggested AT1 is the predominant ANG II receptor. After radioligand binding, under conditions designed to minimize receptor internalization, half the bound U-II was resistant to acid washing suggesting that U-II binds tightly to its receptor in a quasi-irreversible fashion. The AT1 receptor-bound radioligand was completely removed under the same conditions. RT-PCR detected the expression of mRNAs for UT and AT1 receptors. Western blotting showed that U-II and ANG II signaled via ERK1/2 kinase. UT receptor was not lost upon differentiation into myotubes since both mRNA for UT receptor and U-II binding were still present. ANG II receptors were also present as shown by ANG II-induced calcium mobilization.

  2. High-performance liquid affinity chromatography: rapid immunoanalysis of transferrin in serum.

    PubMed

    Ohlson, S; Gudmundsson, B M; Wikström, P; Larsson, P O

    1988-10-01

    We describe a new method for quantitatively measuring substances of clinical interest by high-performance liquid affinity chromatography (HPLAC). As a model system we selected analysis for transferrin in human serum with immobilized antibodies in a high-performance liquid chromatographic system. SelectiSpher-10 Activated Tresyl columns (5 or 10 x 0.5 cm) were used for in situ coupling of polyclonal antibodies to transferrin. The amount of transferrin eluted was determined by integrating the eluted peak at 280 nm. The whole analytical procedure--including injection of sample, washing, elution, and analysis of data--takes only 7 min. We characterized the HPLAC system for analysis of transferrin in several ways: intra-assay CV approximately 3%; inter-assay CV 2-9%; linear response up to 1 mg/mL column volume; detection limit approximately 3 micrograms; analytical recovery 98% +/- 2%; purity of eluted sample greater than 95% (SDS-PAGE). The HPLAC method was compared with "rocket" immunoelectrophoresis, a commonly used method of analysis for transferrin, and there was excellent correlation between the two methods (r = 0.96, n = 60). Benefits of this HPLAC technique include high precision, rapid analysis, and simplified sample handling.

  3. A microscale protocol for the isolation of transferrin directly from serum.

    PubMed

    Penezić, Ana; Miljuš, Goran; Milutinović, Bojana; Nedić, Olgica

    2017-08-01

    A microscale procedure for the isolation of transferrin directly from human serum (hTf) is described in this study. The protocol is based on three precipitation steps without application of chromatography. It lasts 90min with the initial sample volume of 250μL. The yield of the isolated hTf is 58%, which is considerable in biochemical terms. The purity of the isolated hTf is 97%, as assessed by three methods: electrophoresis followed by protein staining, immunoblotting and HPLC. Immunoblotting with antibodies against other major serum proteins indicated that isolated hTf does not contain albumin, immunoglobulin G or alpha-2-macroglobulin. Lectin dot-blot demonstrated that isolated hTf preserved its glycan moieties. Fluorescent emission spectroscopy of the isolated hTf has shown no changes in tertiary structure. Isolated hTf was approximately 26% saturated with iron ion, which is comparable to physiological value (although a degree of saturation decreases to some extent during isolation procedure). Finally, co-immunoprecipitation experiment confirmed that isolated hTf retained its ligand characteristics crucial for the ligand-receptor type of interaction with the hTf receptor. To conclude, the procedure described in this work, is time and cost-effective, allows multiple sample handling and provides high-purity hTf isolate with preserved structural and functional properties. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Induction of nerve growth factor receptors on cultured human melanocytes

    SciTech Connect

    Peacocke, M.; Yaar, M.; Mansur, C.P.; Chao, M.V.; Gilchrest, B.A. )

    1988-07-01

    Normal differentiation and malignant transformation of human melanocytes involve a complex series of interactions during which both genetic and environmental factors play roles. At present, the regulation of these processes is poorly understood. The authors have induced the expression of nerve growth factor (NGF) receptors on cultured human melanocytes with phorbol 12-tetradecanoate 13-acetate and have correlated this event with the appearance of a more differentiated, dendritic morphology. Criteria for NGF receptor expression included protein accumulation and cell-surface immunofluorescent staining with a monoclonal antibody directed against the human receptor and induction of the messenger RNA species as determined by blot-hybridization studies. The presence of the receptor could also be induced by UV irradiation or growth factor deprivation. The NGF receptor is inducible in cultured human melanocytes, and they suggest that NGF may modulate the behavior of this neural crest-derived cell in the skin.

  5. Glucocorticoid receptor in human respiratory epithelial cells.

    PubMed

    Pujolsa, Laura; Mullol, Joaquim; Picado, Cèsar

    2009-01-01

    Inhaled and intranasal glucocorticoids (GCs) are the most common and effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, and asthma, and the respiratory epithelium is a primary target of GC anti-inflammatory actions. GC effects are mediated through the GC receptor (GR). In humans, one single GR gene gives rise to two main GR products, namely GRalpha and GRbeta, which are subject to translational and posttranslational modifications. GRalpha is expressed in virtually all human cells and tissues, including respiratory epithelial cells, and - at least in vitro - is downregulated by GC. GRalpha mediates the anti-inflammatory actions of GC by activating transcription of anti-inflammatory genes through binding of GRalpha to glucocorticoid response elements (GRE) located in the promoter region of target genes, repressing transcription of proinflammatory genes through direct interaction between GRalpha and proinflammatory transcription factors, such as AP-1 and NF-kappaB (transrepression), and also by destabilizing the mRNA of proinflammatory mediators. GRbeta acts as a dominant negative inhibitor of GRalpha-mediated transactivation and transrepression in certain in vitro studies with transfected cells. The GRbeta message is expressed at low levels in numerous tissues and its protein is mainly expressed in inflammatory cells, although it has also been detected in airway epithelial cells. Increased GRbeta expression has been reported in bronchial asthma and nasal polyposis, and after incubation of cells with certain proinflammatory stimuli. However, the role of GRbeta in modulating GC sensitivity in vivo has been highly debated and is as yet unclear.

  6. Complex of transferrin with ruthenium for medical applications. [Ru 97, Ru 103

    DOEpatents

    Richards, P.; Srivastava, S.C.; Meinken, G.E.

    1980-11-03

    A novel Ruthenium-transferrin complex, prepared by reacting iron-free human transferrin dissolved in a sodium acetate solution at pH 7 with ruthenium by heating at about 40/sup 0/C for about 2 hours, and purifying said complex by means of gel chromatography with pH 7 sodium acetate as eluent. The mono- or di-metal complex produced can be used in nuclear medicine in the diagnosis and/or treatment of tumors and abscesses. Comparitive results with Ga-67-citrate, which is the most widely used tumor-localizing agent in nuclear medicine, indicate increased sensitivity of detection and greater tumor uptake with the Ru-transferrin complex.

  7. Inactivation of transferrin iron binding capacity by the neutrophil myeloperoxidase system

    SciTech Connect

    Clark, R.A.; Pearson, D.W.

    1989-06-05

    Human serum apotransferrin was exposed to the isolated myeloperoxidase-H2O2-halide system or to phorbol ester-activated human neutrophils. Such treatment resulted in a marked loss in transferrin iron binding capacity as well as concomitant iodination of transferrin. Each component of the cell-free system (myeloperoxidase, H2O2, iodide) or neutrophil system (neutrophils, phorbol ester, iodide) was required in order to observe these changes. In the cell-free system, the H2O2 requirement was fulfilled by either reagent H2O2 or the peroxide-generating system glucose oxidase plus glucose. Both loss of iron binding capacity and transferrin iodination by either the myeloperoxidase system or activated neutrophils were blocked by azide or catalase. The isolated peroxidase system had an acidic pH optimum, whereas the intact cell system was more efficient at neutral pH. The kinetics of changes in iron binding capacity and iodination closely paralleled one another, exhibiting t1/2 values of less than 1 min for the myeloperoxidase-H2O2 system, 3-4 min for the myeloperoxidase-glucose oxidase system, and 8 min for the neutrophil system. That the occupied binding site is protected from the myeloperoxidase system was suggested by (1) a failure to mobilize iron from iron-loaded transferrin, (2) an inverse correlation between initial iron saturation and myeloperoxidase-mediated loss of iron binding capacity, and (3) decreased myeloperoxidase-mediated iodination of iron-loaded versus apotransferrin. Since as little as 1 atom of iodide bound per molecule of transferrin was associated with substantial losses in iron binding capacity, there appears to be a high specificity of myeloperoxidase-catalyzed iodination for residues at or near the iron binding sites. Amino acid analysis of iodinated transferrin (approximately 2 atoms/molecule) demonstrated that iodotyrosine was the predominant iodinated species.

  8. Engineered Polymer-Transferrin Conjugates as Self-Assembling Targeted Drug Delivery Systems.

    PubMed

    Makwana, Hiteshri; Mastrotto, Francesca; Magnusson, Johannes Pall; Sleep, Darrell; Hay, Joanna; Nicholls, Karl J; Allen, Stephanie; Alexander, Cameron

    2017-03-28

    Polymer-protein conjugates can be engineered to self-assemble into discrete and well-defined drug delivery systems which combine the advantages of receptor targeting and controlled drug release. We designed specific conjugates of the iron-binding and transport protein, transferrin (Tf), to combine the advantages of this serum-stable protein as a targeting agent for cancer cells with self-assembling polymers to act as carriers of cytotoxic drugs. Tf variants were expressed with cysteine residues at sites spanning different regions of the protein surface and the polymer conjugates grown from these variants were compared with polymer conjugates grown from non-selectively derivatised sites on native Tf. The resulting synthetic biopolymer hybrids were evaluated for self-assembly properties, size and topology, ability to carry an anti-cancer drug (paclitaxel) and cytotoxicity with and without a drug payload in a representative human colon cancer cell line. The results demonstrated that the engineered Tf variant polymer conjugates formed better-defined self-assembled nanoparticles than the non-selectively derivatised conjugates and showed greater efficacy in paclitaxel delivery. A polymer conjugate grown from a specific Tf variant, S415C was found to be taken up rapidly into cancer cells expressing the Tf-receptor, and, while tolerated well by cells in the absence of drugs, was as cytotoxic as free paclitaxel when loaded with the drug. Importantly, the S415C conjugate polymer was not the most active variant in Tf-receptor binding, suggesting that the nanoscale self-assembly of the polymer-protein hybrid is also a key factor in delivery efficacy. The data overall suggest new design rules for polymer-biopolymer hybrids and therapeutic delivery systems which include engineering specific residues for conjugation which mediate nanoscale assembly as well as control of ligand-receptor interactions to target specific cell types.

  9. Simultaneous Profiling of 194 Distinct Receptor Transcripts in Human Cells

    PubMed Central

    Kang, Byong H.; Jensen, Karin J.; Hatch, Jaime A.; Janes, Kevin A.

    2013-01-01

    Many signal transduction cascades are initiated by transmembrane receptors with the presence or absence and abundance of receptors dictating cellular responsiveness. Here, we provide a validated array of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) reagents for high-throughput profiling of the presence and relative abundance of transcripts for 194 transmembrane receptors in the human genome. We found that the qRT-PCR array had greater sensitivity and specificity for the detected receptor transcript profiles compared to conventional oligonucleotide microarrays or exon microarrays. The qRT-PCR array also distinguished functional receptor presence versus absence more accurately than deep sequencing of adenylated RNA species, RNA-seq. By applying qRT-PCR-based receptor transcript profiling to 40 human cell lines representing four main tissues (pancreas, skin, breast, and colon), we identified clusters of cell lines with enhanced signaling capabilities and revealed a role for receptor silencing in defining tissue lineage. Ectopic expression of the interleukin 10 (IL-10) receptor encoding gene IL10RA in melanoma cells engaged an IL-10 autocrine loop not otherwise present in this cell type, which altered signaling, gene expression, and cellular responses to proinflammatory stimuli. Our array provides a rapid, inexpensive, and convenient means for assigning a receptor signature to any human cell or tissue type. PMID:23921087

  10. Transmembrane signaling by a chimera of the Escherichia coli aspartate receptor and the human insulin receptor.

    PubMed Central

    Moe, G R; Bollag, G E; Koshland, D E

    1989-01-01

    Since many receptors apparently contain only one or two membrane-spanning segments, their transmembrane topology should be similar. This feature suggests that these receptors share common mechanisms of transmembrane signaling. To test the degree of conservation of signaling properties, a chimeric receptor containing the ligand-binding extracellular domain of the Escherichia coli aspartate chemoreceptor and the cytosolic portion of the human insulin receptor was constructed. This chimeric receptor is active as a tyrosine kinase, and aspartate stimulates its activity. Some interesting differences are noted in the target proteins phosphorylated by the chimera compared to the wild-type insulin receptor. These results indicate that features of the signaling mechanisms used by these diverse receptors are conserved, but that interesting changes in the protein properties are caused by differences in the neighboring domains. Images PMID:2548185

  11. Theranostic vitamin E TPGS micelles of transferrin conjugation for targeted co-delivery of docetaxel and ultra bright gold nanoclusters.

    PubMed

    Muthu, Madaswamy S; Kutty, Rajaletchumy Veloo; Luo, Zhentao; Xie, Jianping; Feng, Si-Shen

    2015-01-01

    The aim of this work was to develop an advanced theranostic micelles of D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), which are conjugated with transferrin for targeted co-delivery of docetaxel (DTX) as a model drug and ultra bright gold clusters (AuNC) as a model imaging agent for simultaneous cancer imaging and therapy. The theranostic micelles with and without transferrin conjugation were prepared by the solvent casting method and characterized for their particle size, polydispersity, surface chemistry, drug encapsulation efficiency, drug loading and cellular uptake efficiency. Transferrin receptors expressing MDA-MB-231-luc breast cancer cells and NIH-3T3 fibroblast cells (control cells without transferrin receptor expression) were employed as an in vitro model to access cytotoxicity of the formulations. The overexpression of transferrin receptor on the surface of MDA-MB-231-luc cells was confirmed by flow cytometry. The biodistribution study and theranostic efficacy of the micelles were investigated by using the Xenogen IVIS(®) Spectrum imaging system, which includes AuNC based fluorescence imaging and luciferase induced bioluminescence imaging on MDA-MB-231-luc tumor bearing SCID mice. The IC50 values demonstrated that the non-targeted and targeted micelles could be 15.31 and 71.73 folds more effective than Taxotere(®) after 24 h treatment with the MDA-MB-231-luc cells. Transferrin receptor targeted delivery of such micelles was imaged in xenograft model and showed their great advantages for real-time tumor imaging and inhibition of tumor growth.

  12. Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia.

    PubMed

    Kozyraki, R; Fyfe, J; Verroust, P J; Jacobsen, C; Dautry-Varsat, A; Gburek, J; Willnow, T E; Christensen, E I; Moestrup, S K

    2001-10-23

    Cubilin is a 460-kDa protein functioning as an endocytic receptor for intrinsic factor vitamin B(12) complex in the intestine and as a receptor for apolipoprotein A1 and albumin reabsorption in the kidney proximal tubules and the yolk sac. In the present study, we report the identification of cubilin as a novel transferrin (Tf) receptor involved in catabolism of Tf. Consistent with a cubilin-mediated endocytosis of Tf in the kidney, lysosomes of human, dog, and mouse renal proximal tubules strongly accumulate Tf, whereas no Tf is detectable in the endocytic apparatus of the renal tubule epithelium of dogs with deficient surface expression of cubilin. As a consequence, these dogs excrete increased amounts of Tf in the urine. Mice with deficient synthesis of megalin, the putative coreceptor colocalizing with cubilin, also excrete high amounts of Tf and fail to internalize Tf in their proximal tubules. However, in contrast to the dogs with the defective cubilin expression, the megalin-deficient mice accumulate Tf on the luminal cubilin-expressing surface of the proximal tubule epithelium. This observation indicates that megalin deficiency causes failure in internalization of the cubilin-ligand complex. The megalin-dependent, cubilin-mediated endocytosis of Tf and the potential of the receptors thereby to facilitate iron uptake were further confirmed by analyzing the uptake of (125)I- and (59)Fe-labeled Tf in cultured yolk sac cells.

  13. Iron uptake by melanoma cells from the soluble form of the transferrin homologue, melanotransferrin.

    PubMed

    Food, Michael R; Des Richardson, R

    2002-01-01

    Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue that can also exist in a soluble form (sMTf). Considering the high homology of MTf to Tf, it is possible to suggest that sMTf could bind to the high affinity transferrin receptor 1 (TfR1) or lower affinity TfR2. We have used sMTf labelled with 59Fe to examine its ability to donate Fe to cells. Our experiments demonstrate that sMTf is far less effective than Tf at donating Fe to cells and this does not occur via specific receptors. Indeed, the uptake of sMTf by cells occurred via a non-specific process (e.g. adsorptive pinocytosis).

  14. Cell uptake of a biosensor detected by hyperpolarized 129Xe NMR: the transferrin case.

    PubMed

    Boutin, Céline; Stopin, Antoine; Lenda, Fatimazohra; Brotin, Thierry; Dutasta, Jean-Pierre; Jamin, Nadège; Sanson, Alain; Boulard, Yves; Leteurtre, François; Huber, Gaspard; Bogaert-Buchmann, Aurore; Tassali, Nawal; Desvaux, Hervé; Carrière, Marie; Berthault, Patrick

    2011-07-01

    For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructs based on the non-selective grafting of cryptophane precursors on holo-transferrin. This biological system was chosen because there are many receptors on the cell surface, and endocytosis further increases this density. The study of these biosensors with K562 cell suspensions via fluorescence microscopy and (129)Xe NMR indicates a strong interaction, as well as interesting features such as the capacity of xenon to enter the cryptophane even when the biosensor is endocytosed, while keeping a high level of polarization. Despite a lack of specificity for transferrin receptors, undoubtedly due to the hydrophobic character of the cryptophane moiety that attracts the biosensor into the cell membrane, these biosensors allow the first in-cell probing of biological events using hyperpolarized xenon.

  15. Transferrin liposomes of docetaxel for brain-targeted cancer applications: formulation and brain theranostics.

    PubMed

    Sonali; Singh, Rahul Pratap; Singh, Nitesh; Sharma, Gunjan; Vijayakumar, Mahalingam R; Koch, Biplob; Singh, Sanjay; Singh, Usha; Dash, Debabrata; Pandey, Bajarangprasad L; Muthu, Madaswamy S

    2016-05-01

    Diagnosis and therapy of brain cancer was often limited due to low permeability of delivery materials across the blood-brain barrier (BBB) and their poor penetration into the brain tissue. This study explored the possibility of utilizing theranostic d-alpha-tocopheryl polyethylene glycol 1000 succinate mono-ester (TPGS) liposomes as nanocarriers for minimally invasive brain-targeted imaging and therapy (brain theranostics). The aim of this work was to formulate transferrin conjugated TPGS coated theranostic liposomes, which contain both docetaxel and quantum dots (QDs) for imaging and therapy of brain cancer. The theranostic liposomes with and without transferrin decoration were prepared and characterized for their particle size, polydispersity, morphology, drug encapsulation efficiency, in-vitro release study and brain theranostics. The particle sizes of the non-targeted and targeted theranostic liposomes were found below 200 nm. Nearly, 71% of drug encapsulation efficiency was achieved with liposomes. The drug release from transferrin conjugated theranostic liposomes was sustained for more than 72 h with 70% of drug release. The in-vivo results indicated that transferrin receptor-targeted theranostic liposomes could be a promising carrier for brain theranostics due to nano-sized delivery and its permeability which provided an improved and prolonged brain targeting of docetaxel and QDs in comparison to the non-targeted preparations.

  16. Oligodendrocyte differentiation and signaling after transferrin internalization: a mechanism of action.

    PubMed

    Pérez, María Julia; Fernandez, Natalia; Pasquini, Juana María

    2013-10-01

    Oligodendrocytes are the cells producing the myelin membrane around the axons in the central nervous system and, although apotransferrin (aTf) is required for oligodendrocyte differentiation, the underlying mechanisms are not fully understood. Fyn tyrosine kinase, a member of the Src family of proteins, has been shown to play an important role in myelination by up-regulating the expression of myelin basic protein; however, a molecular link between aTf and Fyn kinase signaling pathway during oligodendrocytes differentiation has not been established yet. Our aim was to investigate whether Fyn kinase, MEK/ERK and PI3K/Akt signaling pathways are required for aTf-stimulation of oligodendrocyte differentiation and also to determine if the transferrin receptor is involved in these mechanisms. Treatment of primary cultures of oligodendroglial precursor cells with aTf leads to Fyn kinase activation by a mechanism that involves transferrin receptor. In turn, Fyn kinase activation promotes MEK-mediated transient phosphorylation of ERK1/2. On the other hand, transferrin receptor internalization also produces rapid and sustained activation of Akt, which involves phosphatidylinositol 3-kinase (PI3K) activation. Finally, aTf incorporated through clathrin-mediated endocytosis increases myelin basic protein, F3-contactin and β-tubulin through Fyn/MEK/ERK pathways, as well as an activation of the PI3K/Akt pathway. Our results also demonstrate that the activation of the pathways necessary for oligodendroglial precursor cell maturation is dependent on AP2 recruitment onto the plasma membrane for clathrin-mediated endocytosis of transferrin receptor.

  17. Accelerated degradation of 160 kDa epidermal growth factor (EGF) receptor precursor by the tyrosine kinase inhibitor herbimycin A in the endoplasmic reticulum of A431 human epidermoid carcinoma cells.

    PubMed Central

    Murakami, Y; Mizuno, S; Uehara, Y

    1994-01-01

    The effect of herbimycin A on the biosynthesis of epidermal growth factor (EGF) receptor was examined in human epidermoid carcinoma A431 cells. Cells were pulse-labelled with [35S]methionine, and EGF receptor biosynthesis was quantified by immunoprecipitation using a monoclonal anti-(EGF receptor) antibody. In the presence of herbimycin A, an immature 160 kDa EGF receptor precursor accumulated in 1 h and disappeared completely in 4 h. Pulse-labelled 160 kDa receptor precursor in the absence of herbimycin A, however, was converted normally into a 170 kDa one by chase with herbimycin A. Herbimycin A affected neither the synthesis of the secreted form of EGF receptor devoid of cytoplasmic domain, nor that of the transferrin receptor in A431 cells. The herbimycin A-induced degradation of 160 kDa EGF receptor precursor was not inhibited by an inhibitor of lysosomal enzymes, NH4Cl. Endoglycosidase H digestion of the 160 kDa precursor converted it into the deglycosylated 130 kDa precursor peptide. These results suggested that herbimycin A selectively acted on the EGF receptor precursor during the synthesis of the 160 kDa form, probably on the cytoplasmic domain, to form an aberrant molecule which was subjected to rapid degradation in the endoplasmic reticulum. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8037692

  18. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    PubMed Central

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  19. Expression of the Endocannabinoid Receptors in Human Fascial Tissue

    PubMed Central

    Fede, C.; Albertin, G.; Petrelli, L.; Sfriso, M.M.; Biz, C.; Caro, R. De; Stecco, C.

    2016-01-01

    Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2) in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation. PMID:27349320

  20. Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis

    SciTech Connect

    Craven, C.M.; Alexander, J.; Eldridge, M.; Kushner, J.P.; Bernstein, S.; Kaplan, J.

    1987-05-01

    Genetically hypotransferrinemic mice accumulate iron in the liver and pancreas. A similar pattern of tissue iron accumulation occurs in humans with hereditary hemochromatosis. In both disorders, there is a decrease plasma concentration of apotransferrin. To test the hypothesis that nontransferrin-bound iron exists and is clear by the parenchymal tissues, the tissue distribution of /sup 59/Fe was studied in animals lacking apotransferrin. Two groups of animals were used: normal rats and mice whose transferrin had been saturated by an intravenous injection of nonradiolabeled iron, and mice with congential hypotransferrinemia. In control animals, injected /sup 59/Fe was found primarily in the bone marrow and spleen. In the transferrin iron-saturated animals, injected /sup 59/Fe accumulated in the liver and pancreas. Gastrointestinally absorbed iron in hypotransferrinemic or transferrin iron-saturated mice was deposited in the liver. This indicates that newly absorbed iron is released from mucosal cells not bound to transferrin. Clearance studies demonstrated that transferrin-bound /sup 59/Fe was removed from the circulation of rats with a half-time of 50 min. In transferrin iron-saturated animals, injected /sup 59/Fe was removed with a half-time of <30 s. Analysis of the distribution of /sup 59/Fe in serum samples by polyacrylamide gel electrophoresis demonstrated the presence of /sup 59/Fe not bound to transferrin. These results demonstrate the existence of and uptake system for non-transferrin-bound iron. These observations support the hypothesis that parenchymal iron overload is consequence of reduced concentrations of apotransferrin.

  1. Characterization of muscarinic receptor subtypes in human tissues

    SciTech Connect

    Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

    1988-01-01

    The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with (/sup 3/H)Pirenzepine and (/sup 3/H)N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M/sub 1/ neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M/sub 1/, the cardiac M/sub 2/ and the glandular M/sub 3/.

  2. Melanocortin MC₁ receptor in human genetics and model systems.

    PubMed

    Beaumont, Kimberley A; Wong, Shu S; Ainger, Stephen A; Liu, Yan Yan; Patel, Mira P; Millhauser, Glenn L; Smith, Jennifer J; Alewood, Paul F; Leonard, J Helen; Sturm, Richard A

    2011-06-11

    The melanocortin MC(1) receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC(1) receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC(1) receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC(1) receptor and the molecular mechanisms underlying the association between melanocortin MC(1) receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC(1) receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC(1) receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC(1) receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC(1) receptor polymorphism.

  3. Human Receptor Activation by Aroclor 1260, a Polychlorinated Biphenyl Mixture

    PubMed Central

    Wahlang, Banrida; Falkner, K. Cameron; Clair, Heather B.; Al-Eryani, Laila; Prough, Russell A.; States, J. Christopher; Coslo, Denise M.; Omiecinski, Curtis J.; Cave, Matthew C.

    2014-01-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  4. Expression of glutamate receptor subunits in human cancers.

    PubMed

    Stepulak, Andrzej; Luksch, Hella; Gebhardt, Christine; Uckermann, Ortrud; Marzahn, Jenny; Sifringer, Marco; Rzeski, Wojciech; Staufner, Christian; Brocke, Katja S; Turski, Lechoslaw; Ikonomidou, Chrysanthy

    2009-10-01

    Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

  5. Evidence for Alpha Receptors in the Human Ureter

    NASA Astrophysics Data System (ADS)

    Madeb, Ralph; Knopf, Joy; Golijanin, Dragan; Bourne, Patricia; Erturk, Erdal

    2007-04-01

    An immunohistochemical and western blot expression analysis of human ureters was performed in order to characterize the alpha-1-adrenergic receptor distribution along the length of the human ureteral wall. Mapping the distribution will assist in understanding the potential role alpha -1-adrenergic receptors and their subtype density might have in the pathophysiology of ureteral colic and stone passage. Patients diagnosed with renal cancer or bladder cancer undergoing nephrectomy, nephroureterectomy, or cystectomy had ureteral specimens taken from the proximal, mid, distal and tunneled ureter. Tissues were processed for fresh frozen examination and fixed in formalin. None of the ureteral specimens were involved with cancer. Serial histologic sections and immunohistochemical studies were performed using antibodies specific for alpha-1-adrenergic receptor subtypes (alpha 1a, alpha 1b, alpha 1d). The sections were examined under a light microscope and scored as positive or negative. In order to validate and quantify the alpha receptor subtypes along the human ureter. Western blotting techniques were applied. Human ureter stained positively for alpha -1-adrenergic receptors. Immunostaining appeared red, with intense reaction in the smooth muscle of the ureter and endothelium of the neighboring blood vessels. There was differential expression between all the receptors with the highest staining for alpha-1D subtype. The highest protein expression for all three subtypes was in the renal pelvis and decreased with advancement along the ureter to the distal ureter. At the distal ureter, there was marked increase in expression as one progressed towards the ureteral orifice. The same pattern of protein expression was exhibited for all three alpha -1-adrenergic receptor subtypes. We provide preliminary evidence for the ability to detect and quantify the alpha-1-receptor subtypes along the human ureter which to the best of our knowledge has never been done with

  6. Targeting xenobiotic receptors PXR and CAR in human diseases

    PubMed Central

    Banerjee, Monimoy; Robbins, Delira; Chen, Taosheng

    2014-01-01

    Nuclear receptors such as the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are xenobiotic receptors regulating not only drug metabolism and disposition but also various human diseases such as cancer, diabetes, inflammatory disease, metabolic disease and liver diseases, suggesting that PXR and CAR are promising targets for drug discovery. Consequently, there is an urgent need to discover and develop small molecules that target these PXR- and/or CAR-mediated human-disease-related pathways for relevant therapeutic applications. This review proposes approaches to target PXR and CAR, either individually or simultaneously, in the context of various human diseases, taking into consideration the structural differences between PXR and CAR. PMID:25463033

  7. Cloning, functional expression and characterization of a human olfactory receptor.

    PubMed

    Hatt, H; Gisselmann, G; Wetzel, C H

    1999-05-01

    The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed and characterized the first human olfactory receptor (OR 17-40). Application of a mixture of hundred different odorants elicited a transient increase in intracellular calcium at HEK 293-cells which were transfected with a plasmid containing the receptor encoding DNA and a membrane import sequence. By subdividing the odorant mixture in smaller groups we could identify a single component which represented the only effective substance: helional. Testing some structurally closely related molecules we found only one other compound which also could activate the receptor: heliotropyl acetone. All other compounds tested were completely ineffective. These findings represent the beginning of molecular understanding of odorant recognition in humans.

  8. The Insect Ortholog of the Human Orphan Cytokine Receptor CRLF3 Is a Neuroprotective Erythropoietin Receptor

    PubMed Central

    Hahn, Nina; Knorr, Debbra Y.; Liebig, Johannes; Wüstefeld, Liane; Peters, Karsten; Büscher, Marita; Bucher, Gregor; Ehrenreich, Hannelore; Heinrich, Ralf

    2017-01-01

    The cytokine erythropoietin (Epo) mediates various cell homeostatic responses to environmental challenges and pathological insults. While stimulation of vertebrate erythrocyte production is mediated by homodimeric “classical” Epo receptors, alternative receptors are involved in neuroprotection. However, their identity remains enigmatic due to complex cytokine ligand and receptor interactions and conflicting experimental results. Besides the classical Epo receptor, the family of type I cytokine receptors also includes the poorly characterized orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including human and various insect species. By making use of the more simple genetic makeup of insect model systems, we studied whether CRLF3 is a neuroprotective Epo receptor in animals. We identified a single ortholog of CRLF3 in the beetle Tribolium castaneum, and established protocols for primary neuronal cell cultures from Tribolium brains and efficient in vitro RNA interference. Recombinant human Epo as well as the non-erythropoietic Epo splice variant EV-3 increased the survival of serum-deprived brain neurons, confirming the previously described neuroprotective effect of Epo in insects. Moreover, Epo completely prevented hypoxia-induced apoptotic cell death of primary neuronal cultures. Knockdown of CRLF3 expression by RNA interference with two different double stranded RNA (dsRNA) fragments abolished the neuroprotective effect of Epo, indicating that CRLF3 is a crucial component of the insect Epo-responsive receptor. This suggests that a common urbilaterian ancestor of the orphan human and insect cytokine receptor CRLF3 served as a neuroprotective receptor for an Epo-like cytokine. Our work also suggests that vertebrate CRLF3, like its insect ortholog, might represent a tissue protection-mediating receptor. PMID:28769759

  9. The Insect Ortholog of the Human Orphan Cytokine Receptor CRLF3 Is a Neuroprotective Erythropoietin Receptor.

    PubMed

    Hahn, Nina; Knorr, Debbra Y; Liebig, Johannes; Wüstefeld, Liane; Peters, Karsten; Büscher, Marita; Bucher, Gregor; Ehrenreich, Hannelore; Heinrich, Ralf

    2017-01-01

    The cytokine erythropoietin (Epo) mediates various cell homeostatic responses to environmental challenges and pathological insults. While stimulation of vertebrate erythrocyte production is mediated by homodimeric "classical" Epo receptors, alternative receptors are involved in neuroprotection. However, their identity remains enigmatic due to complex cytokine ligand and receptor interactions and conflicting experimental results. Besides the classical Epo receptor, the family of type I cytokine receptors also includes the poorly characterized orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including human and various insect species. By making use of the more simple genetic makeup of insect model systems, we studied whether CRLF3 is a neuroprotective Epo receptor in animals. We identified a single ortholog of CRLF3 in the beetle Tribolium castaneum, and established protocols for primary neuronal cell cultures from Tribolium brains and efficient in vitro RNA interference. Recombinant human Epo as well as the non-erythropoietic Epo splice variant EV-3 increased the survival of serum-deprived brain neurons, confirming the previously described neuroprotective effect of Epo in insects. Moreover, Epo completely prevented hypoxia-induced apoptotic cell death of primary neuronal cultures. Knockdown of CRLF3 expression by RNA interference with two different double stranded RNA (dsRNA) fragments abolished the neuroprotective effect of Epo, indicating that CRLF3 is a crucial component of the insect Epo-responsive receptor. This suggests that a common urbilaterian ancestor of the orphan human and insect cytokine receptor CRLF3 served as a neuroprotective receptor for an Epo-like cytokine. Our work also suggests that vertebrate CRLF3, like its insect ortholog, might represent a tissue protection-mediating receptor.

  10. Cloning of a parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) cDNA from a rat osteosarcoma (UMR 106) cell line: Chromosomal assignment of the gene in the human, mouse, and rat genomes

    SciTech Connect

    Pausova, Z.; Bourdon, J.; Clayton, D.; Janicic, N.; Goltzman, D.; Hendy, G.N. ); Mattei, M.G. ); Seldin, M.F. ); Riviere, M.; Szpirer, J. )

    1994-03-01

    Complementary DNAs spanning the entire coding region of the rat parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) were isolated from a rat osteosarcoma (UMR 106) cell-line cDNA library. The longest of these clones (rPTHrec4) was used to chromosomally assign the PTHR gene in the human, rat, and mouse genomes. By somatic cell hybrid analysis, the gene was localized to