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Sample records for plasma transferrin receptor

  1. Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers

    DEFF Research Database (Denmark)

    Hersom, Maria; Helms, Hans Christian; Pretzer, Natasia;

    2016-01-01

    across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated...... in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe......]-transferrin. Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin...

  2. Roles of transferrin receptors in erythropoiesis.

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    Kawabata, Hiroshi; Sakamoto, Soichiro; Masuda, Taro; Uchiyama, Tatsuki; Ohmori, Katsuyuki; Koeffler, H Phillip; Takaori-Kondo, Akifumi

    2016-07-01

    Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands.

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

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    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.

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

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    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.

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

    NARCIS (Netherlands)

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

    2001-01-01

    Background: 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

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

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    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.

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

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    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.

  8. Decreased plasma iron in Alzheimer's disease is due to transferrin desaturation.

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    Hare, Dominic J; Doecke, James D; Faux, Noel G; Rembach, Alan; Volitakis, Irene; Fowler, Christopher J; Grimm, Rudolf; Doble, Philip A; Cherny, Robert A; Masters, Colin L; Bush, Ashley I; Roberts, Blaine R

    2015-03-18

    Plasma iron levels are decreased in Alzheimer's disease (AD) and associated with an idiopathic anemia. We examined iron-binding plasma proteins from AD patients and healthy controls from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Flagship Study of Ageing using size exclusion chromatography-inductively coupled plasma-mass spectrometry. Peak area corresponding to transferrin (Tf) saturation was directly compared to routine pathological testing. We found a significant decrease in transferrin-associated iron in AD that was missed by routine pathological tests of transferrin saturation, and that was able to discriminate between AD and controls. The AD cases showed no significant difference in transferrin concentration, only a decrease in total transferrin-bound iron. These findings support that a previously identified decrease in plasma iron levels in AD patients within the AIBL study is attributable to decreased loading of iron into transferrin, and that this subtle but discriminatory change is not observed through routine pathological testing.

  9. Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant

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    Rangaraj Nandini

    2010-01-01

    Full Text Available Abstract Background Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process. Results We show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking. Conclusions Our results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for

  10. Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium

    Energy Technology Data Exchange (ETDEWEB)

    Chitambar, C.R.; Seligman, P.A.

    1986-12-01

    We have previously shown that human leukemic cells proliferate normally in serum-free media containing various transferrin forms, but the addition of transferrin-gallium leads to inhibition of cellular proliferation. Because gallium has therapeutic potential, the effects of transferrin-gallium on leukemic cell proliferation, transferrin receptor expression, and cellular iron utilization were studied. The cytotoxicity of gallium is considerably enhanced by its binding to transferrin and cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms. Exposure to transferrin-gallium leads to a marked increase in cell surface transferrin binding sites, but despite this, cellular /sup 59/Fe incorporation is inappropriately low. Although shunting of transferrin-gallium to another cellular compartment has not been ruled out, other studies suggest that transferrin-gallium impairs intracellular release of /sup 59/Fe from transferrin by interfering with processes responsible for intracellular acidification. These studies, taken together, demonstrate that inhibition of cellular iron incorporation by transferrin-gallium is a prerequisite for inhibition of cellular proliferation.

  11. Transferrin in ifshes:A review article

    Institute of Scientific and Technical Information of China (English)

    Berhan Asmamaw

    2016-01-01

    Transferrin is a single monomeric glycoprotein of molecular weight of 80 kDa that transports iron involved in many metabolic processes amongst the sites of absorption, storage and utilization, hence considered as the major iron binding protein in the plasma of vertebrate species. In this study, transferrin structure, synthesis, receptor and the mechanism of cellular uptake of iron from transferrin have been reviewed. Besides, the major biological functions of transferrin and the different forms of it (polymorphisms) have been indicated.

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

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    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.

  13. Metabolic Catastrophe in Mice Lacking Transferrin Receptor in Muscle.

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

    2015-11-01

    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.

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

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    Wenjing Xu

    2015-10-01

    Full Text Available 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.

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

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    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.

  16. Serum transferrin receptor, serum ferritin and serum transferrin receptor-ferritin index in adults with iron deficiency anaemia.

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    Nadeem, Seema; Shah, Shahida; Iqbal, Touqueer; Iqbal, Zafar; Hanif, Ejaz

    2011-01-01

    Serum Ferritin (SF) and iron both show acute phase responses to inflammation, so iron may fall and ferritin rise independent of the marrow iron store. Bone marrow iron store has been considered the gold standard, but is invasive, painful and expensive and not suitable for everyone. Serum transferrin receptor (sTfR) which is the concentration of the soluble fragment of transferrin receptor in serum, is an important new haematological parameter. The ratio of sTfR to log SF is known as sTfR-SF index. This study was conducted to evaluate sTfR, Ferritin and sTfR-F Index in diagnosing and differentiating iron deficiency anaemia (IDA) from anaemia of chronic disease (ACD). One hundred and sixteen (116) adult subjects (80 anaemic and 36 controls) who already had their bone marrow examination done for various reasons were included in the study. sTfR, SF, and their index were measured and compared with bone marrow iron stores. Absence of iron stores denoted IDA whereas increased macrophage iron with decreased siderocytes and sideroblasts was diagnostic of ACD. Out of 80 anaemic patients, 47 were diagnosed as IDA while 33 were diagnosed as ACD. In case of IDA the diagnostic accuracy of index was 91.57%, sTfR had accuracy of 85.54% while SF had accuracy of 75.90%. In case of ACD, the diagnostic accuracy of sTfR was 91.30%, index 89.86%, while SF had accuracy of 79.71%. sTfR-SF index is a better parameter than sTfR or ferritin alone but should only be used when the results of these parameters seem altered or a bone marrow aspiration is mandatory for diagnosis of ACD. The estimation of sTfR or index may offer a simple non invasive method that may enable more accurate assessments of iron status in such patients.

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

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    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.

  18. Endocytosis of a functionally enhanced GFP-tagged transferrin receptor in CHO cells.

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    Qi He

    Full Text Available The endocytosis of transferrin receptor (TfR has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81 ± 3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.

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

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    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 serum ferritin concentration 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 malaria-endemic areas.

  20. Evidence for a sequential transfer of iron amongst ferritin,transferrin and transferrin receptor during duodenal absorption of iron in rat and human

    Institute of Scientific and Technical Information of China (English)

    Vasantha L Kolachala; B Sesikeran; K Madhavan Nair

    2007-01-01

    AIM:To elucidate the sequential transfer of iron amongst ferritin,transferrin and transferrin receptor under various iron status conditions.METHODS:Incorporation of 59Fe into mucosal and luminal proteins was carried out in control WKY rats.The sequential transfer of iron amongst ferritin,transferrin and transferrin receptor was carried out in iron deficient,control and iron overloaded rats.The duodenal proteins were subjected to immunoprecipitation and quantitation by specific ELISA and in situ localization by microautoradiography and immunohistochemistry in tandem duodenal sections.Human duodenal biopsy(n = 36)collected from subjects with differing iron status were also stained for these proteins.RESULTS:Ferritin was identified as the major protein that incorporated iron in a time-dependent manner in the duodenal mucosa.The concentration of mucosal ferritin was significantly higher in the iron excess group compared to control,iron deficient groups(731.5 ± 191.96 vs 308.3 ± 123.36,731.5 ± 191.96 vs 256.0 ± 1.19,P < 0.005),while that of luminal transferrin which was significantly higher than the mucosal did not differ among the groups(10.9 ± 7.6 vs 0.87 ± 0.79,11.1 ± 10.3 vs 0.80 ± 1.20,6.8 ± 4.7 vs 0.61 ± 0.63,P < 0.001).In situ grading of proteins and iron,and their superimposition,suggested the occurrence of a sequential transfer of iron.This was demonstrated to occur through the initial binding of iron to luminal transferrin then to absorptive cell surface transferrin receptors.The staining intensity of these proteins varied according to the iron nutrition in humans,with intense staining of transferrin receptor observed in iron deficient subjects.CONCLUSION:It is concluded that the intestine takes up iron through a sequential transfer involving interaction of luminal transferrin,transferrin-transferrin receptor and ferritin.

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

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    Hiroshi Sakane

    Full Text Available 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.

  2. The significance of soluble transferrin receptors in diagnosing iron deficiency anemia

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    Tijanić Ivan

    2015-08-01

    Full Text Available Introduction. In recent years, determination of soluble transferrin receptor levels has been emerging as a test that can reliably indicate iron deficiency in various states, and that is non-invasive and easy to use. The aim of this study was: to determine reference values of soluble transferrin receptor concentrations in serums in our population, to examine the reliability of this method in the diagnosis of anemia due to iron deficiency and associated iron deficiency in anemia accompanying malignant hemopathies, and to identify possible limitations of the test in certain conditions.

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

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    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.

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

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    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

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

    Science.gov (United States)

    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.

  6. Transferrin in fihes: A review article

    Directory of Open Access Journals (Sweden)

    Berhan Asmamaw

    2016-03-01

    Full Text Available Transferrin is a single monomeric glycoprotein of molecular weight of 80 kDa that transports iron involved in many metabolic processes amongst the sites of absorption, storage and utilization, hence considered as the major iron binding protein in the plasma of vertebrate species. In this study, transferrin structure, synthesis, receptor and the mechanism of cellular uptake of iron from transferrin have been reviewed. Besides, the major biological functions of transferrin and the different forms of it (polymorphisms have been indicated.

  7. The role of hepatic transferrin receptor 2 in the regulation of iron homeostasis in the body.

    Directory of Open Access Journals (Sweden)

    Christal A Worthen

    2014-03-01

    Full Text Available Fine tuning of body iron is required to prevent diseases such as iron-overload and anemia. The putative iron-sensor, transferrin receptor 2 (TfR2, is expressed in the liver and mutations in this protein result in the iron-overload disease Type III hereditary hemochromatosis (HH. With the loss of functional TfR2, the liver produces about two-fold less of the peptide hormone hepcidin, which is responsible for negatively regulating iron uptake from the diet. This reduction in hepcidin expression leads to the slow accumulation of iron in the liver, heart, joints, and pancreas and subsequent cirrhosis, heart disease, arthritis, and diabetes. TfR2 can bind iron-loaded transferrin in the bloodstream, and hepatocytes treated with transferrin respond with a two-fold increase in hepcidin expression through stimulation of the BMP-signaling pathway. Loss of functional TfR2 or its binding partner, the original HH protein (HFE, results in a loss of this transferrin-sensitivity. While much is known about the trafficking and regulation of TfR2, the mechanism of its transferrin-sensitivity through the BMP-signaling pathway is still not known.

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

    Science.gov (United States)

    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.

  9. Iron-independent neuronal expression of transferrin receptor mRNA in the rat

    DEFF Research Database (Denmark)

    Moos, Torben; Oates, Phillip S.; Morgan, Evan H.

    1999-01-01

    Hemochromatosis, hippocampus, in situ hybridization, iron deficiency, neurodegeneration, transferrin......Hemochromatosis, hippocampus, in situ hybridization, iron deficiency, neurodegeneration, transferrin...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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 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.

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

    Science.gov (United States)

    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

    2013-03-01

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  16. Ferristatin II promotes degradation of transferrin receptor-1 in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Shaina L Byrne

    Full Text Available Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611 acts by down-regulating transferrin receptor-1 (TfR1 via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679, acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug's activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal (59Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Zhang Deshui

    2012-11-01

    Full Text Available Abstract 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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders

    NARCIS (Netherlands)

    Swart, L. de; Hendriks, J.C.M.; Vorm, L.N. van der; Cabantchik, Z.I.; Evans, P.J.; Hod, E.A.; Brittenham, G.M.; Furman, Y.; Wojczyk, B.; Janssen, M.C.H.; Porter, J.B.; Mattijssen, V.E.; Biemond, B.J.; MacKenzie, M.A.; Origa, R.; Galanello, R.; Hider, R.C.; Swinkels, D.W.

    2016-01-01

    Non-transferrin-bound iron and its labile (redox active) plasma iron component are thought to be potentially toxic forms of iron originally identified in the serum of patients with iron overload. We compared ten worldwide leading assays (6 for non-transferrin-bound iron and 4 for labile plasma iron)

  3. Plasma ferritin and soluble transferrin receptor concentrations and body iron stores identify similar risk factors for iron deficiency but result in different estimates of the national prevalence of iron deficiency and iron-deficiency anemia among women and children in Cameroon.

    Science.gov (United States)

    Engle-Stone, Reina; Nankap, Martin; Ndjebayi, Alex O; Erhardt, Juergen G; Brown, Kenneth H

    2013-03-01

    Available iron status indicators reflect different aspects of metabolism. We compared the prevalence and distribution of iron deficiency (ID) and iron-deficiency anemia (IDA) among Cameroonian women and children, as measured by plasma ferritin, and soluble transferrin receptor concentrations, body iron stores (BIS), and hemoglobin, and evaluated the impact of adjustments for inflammation on these measures. In a nationally representative survey, we randomly selected 30 clusters in each of 3 zones (north, south, and large cities) and 10 households/ cluster, each with a child aged 12-59 mo and a woman 15-49 y. Ferritin and BIS were mathematically adjusted for inflammation, using plasma C-reactive protein and α(1)-acid glycoprotein both as continuous and categorical variables. Inflammation was present in 48.0% of children and 20.8% of women and anemia was diagnosed in 57.6% of children and 38.8% of women. Depending on the iron status indicator applied, the prevalence of ID ranged from 14.2 to 68.4% among children and 11.5 to 31.8% among women, and the prevalence of IDA ranged from 12.0 to 47.4% among children and 9.0 to 19.4% among women; the proportion of anemia associated with ID ranged from 20.8 to 82.3% among children and 23.2 to 50.0% among women. The different iron indicators generally identified similar groups at greatest risk of deficiency, using both conventional and derived cutoffs: younger children, pregnant women, and women and children in the north and rural areas. Research is needed to clarify the relationships between iron status indicators, particularly in the presence of inflammation, to harmonize global data on prevalence of ID.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Seung Min, E-mail: smjeong@catholic.ac.kr [Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Institute for Aging and Metabolic Diseases, College of Medicine, The Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Hwang, Sunsook; Seong, Rho Hyun [School of Biological Sciences and Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742 (Korea, Republic of)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Soichiro Sakamoto

    Full Text Available 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 (TFR1 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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 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 iron deficiency. TfR-F appeared as a useful biomarker that could help physicians to diagnose true iron deficiency in patients with active IBD.

  12. The Transferrin Receptor: A Potential Molecular Imaging Marker for Human Cancer

    Directory of Open Access Journals (Sweden)

    Dagmar Högemann-Savellano

    2003-11-01

    Full Text Available Noninvasive imaging of differences between the molecular properties of cancer and normal tissue has the potential to enhance the detection of tumors. Because overexpression of endogenous transferrin receptor (TfR has been qualitatively described for various cancers and is presumably due to malignant transformation of cells, TfR may represent a suitable target for application of molecular imaging technologies to increase detection of smaller tumors. In the work reported here, investigation into the biology of this receptor using electron microscopy has demonstrated that iron oxide particles targeted to TfR are internalized and accumulate in lysosomal vesicles within cells. Biochemical analysis of the interaction of imaging probes with cells overexpressing the TfR demonstrated that the extent of accumulation, and therefore probe efficacy, is dependent on the nature of the chemical cross-link between transferrin and the iron oxide particle. These data were utilized to design and synthesize an improved imaging probe. Experiments demonstrate that the novel magnetic resonance imaging (MRI probe is sensitive enough to detect small differences in endogenous TfR expression in human cancer cell lines. Quantitative measurement of TfR overexpression in a panel of 27 human breast cancer patients demonstrated that 74% of patient cancer tissues overexpressed the TfR and that the sensitivity of the new imaging agent was suitable to detect TfR overexpression in greater than 40% of these cases. Based on a biochemical and cell biological approach, these studies have resulted in the synthesis and development of an improved MRI probe with the best in vitro and in vivo imaging properties reported to date.

  13. Machupo virus glycoprotein determinants for human transferrin receptor 1 binding and cell entry.

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    Sheli R Radoshitzky

    Full Text Available Machupo virus (MACV is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1. TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors.

  14. Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders.

    Science.gov (United States)

    de Swart, Louise; Hendriks, Jan C M; van der Vorm, Lisa N; Cabantchik, Z Ioav; Evans, Patricia J; Hod, Eldad A; Brittenham, Gary M; Furman, Yael; Wojczyk, Boguslaw; Janssen, Mirian C H; Porter, John B; Mattijssen, Vera E J M; Biemond, Bart J; MacKenzie, Marius A; Origa, Raffaella; Galanello, Renzo; Hider, Robert C; Swinkels, Dorine W

    2016-01-01

    Non-transferrin-bound iron and its labile (redox active) plasma iron component are thought to be potentially toxic forms of iron originally identified in the serum of patients with iron overload. We compared ten worldwide leading assays (6 for non-transferrin-bound iron and 4 for labile plasma iron) as part of an international inter-laboratory study. Serum samples from 60 patients with four different iron-overload disorders in various treatment phases were coded and sent in duplicate for analysis to five different laboratories worldwide. Some laboratories provided multiple assays. Overall, highest assay levels were observed for patients with untreated hereditary hemochromatosis and β-thalassemia intermedia, patients with transfusion-dependent myelodysplastic syndromes and patients with transfusion-dependent and chelated β-thalassemia major. Absolute levels differed considerably between assays and were lower for labile plasma iron than for non-transferrin-bound iron. Four assays also reported negative values. Assays were reproducible with high between-sample and low within-sample variation. Assays correlated and correlations were highest within the group of non-transferrin-bound iron assays and within that of labile plasma iron assays. Increased transferrin saturation, but not ferritin, was a good indicator of the presence of forms of circulating non-transferrin-bound iron. The possibility of using non-transferrin-bound iron and labile plasma iron measures as clinical indicators of overt iron overload and/or of treatment efficacy would largely depend on the rigorous validation and standardization of assays.

  15. A straightforward route to the synthesis of a surface-enhanced Raman scattering probe for targeting transferrin receptor-overexpressed cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang Jing; Wang Zhuyuan; Tan Xuebin; Li Jin; Song Chunyuan; Zhang Ruohu; Cui Yiping, E-mail: cyp@seu.edu.cn [Advanced Photonics Center, Southeast University, Nanjing 210096 (China)

    2010-08-27

    A tumor cell targeting surface-enhanced Raman scattering (SERS) probe has been successfully synthesized by using p-mercaptobenzoic acid (pMBA) as both the SERS reporter and the conjugation agent for attaching transferrin molecules, which shows experimentally the targeting ability for transferrin receptor-overexpressed HeLa cells and exhibits strong SERS signals when being incubated inside cells. To prove that the uptake of such a SERS probe is through a Tf-receptor-mediated endocytosis process, two control experiments: (1) HeLa cells being incubated with the probe at 4 deg. C and (2) HeLa cells being pre-blocked with free transferrin at 37 deg. C, were employed. The difference of SERS intensity between the transferrin-overexpressed HeLa cells and transferrin-pre-blocked HeLa cells indicates that the probe has the potential to selectively target tumor cells.

  16. Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Jiexia Wen

    2013-01-01

    Full Text Available Canine parvovirus (CPV disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo.

  17. Transferrin receptor 1 in the zoonosis and pathogenesis of New World hemorrhagic fever arenaviruses.

    Science.gov (United States)

    Choe, Hyeryun; Jemielity, Stephanie; Abraham, Jonathan; Radoshitzky, Sheli R; Farzan, Michael

    2011-08-01

    At least five New World arenaviruses cause severe human hemorrhagic fevers. These viruses are transmitted to humans through contact with their respective South American rodent hosts. Each uses human transferrin receptor 1 (TfR1) as its obligate receptor. Accidental similarities between human TfR1 and TfR1 orthologs of arenaviral host species enable zoonoses, whereas mice and rats are not infectable because they lack these TfR1 determinants of infection. All pathogenic New World arenaviruses bind to a common region of the apical domain of TfR1. The ability of a New World arenavirus to use human TfR1 is absolutely predictive of its ability to cause hemorrhagic fevers in humans. Nonpathogenic arenaviruses, closely related to hemorrhagic fever arenaviruses, cannot utilize human TfR1 but efficiently enter cells through TfR1 orthologs of their native rodent hosts. Mutagenesis studies suggest that minor changes in the entry glycoproteins of these nonpathogenic viruses may allow human transmission. TfR1 is upregulated as a result of iron sequestration during the acute-phase response to infection, and the severity of disease may result from amplification of viral replication during this response.

  18. Soluble form of canine transferrin receptor inhibits canine parvovirus infection in vitro and in vivo.

    Science.gov (United States)

    Wen, Jiexia; Pan, Sumin; Liang, Shuang; Zhong, Zhenyu; He, Ying; Lin, Hongyu; Li, Wenyan; Wang, Liyue; Li, Xiujin; Zhong, Fei

    2013-01-01

    Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo.

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

    Energy Technology Data Exchange (ETDEWEB)

    Roskams, A.J.; Connor, J.R. (Pennsylvania State Univ., Hershey (United States))

    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.

  20. The Importance of Serum Transferrin Receptor and TfR-F Index in the Diagnosis of Iron Deficiency Accompanied by Acute and Chronic Infections

    National Research Council Canada - National Science Library

    Koşan Çulha, Vildan; Uysal, Zümrüt

    2002-01-01

    This study evaluated the diagnostic superiority of serum transferrin receptor (sTfR) measurement to other laboratory tests performed for the determination of iron deficiency caused by chronic disease...

  1. Transferrin coated nanoparticles: study of the bionano interface in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrzej S Pitek

    Full Text Available It is now well established that the surface of nanoparticles (NPs in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC and after washing (hard corona, HC in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments.

  2. Chromatographic Monoliths for High-Throughput Immunoaffinity Isolation of Transferrin from Human Plasma

    Directory of Open Access Journals (Sweden)

    Irena Trbojević-Akmačić

    2016-06-01

    Full Text Available Changes in protein glycosylation are related to different diseases and have a potential as diagnostic and prognostic disease biomarkers. Transferrin (Tf glycosylation changes are common marker for congenital disorders of glycosylation. However, biological interindividual variability of Tf N-glycosylation and genes involved in glycosylation regulation are not known. Therefore, high-throughput Tf isolation method and large scale glycosylation studies are needed in order to address these questions. Due to their unique chromatographic properties, the use of chromatographic monoliths enables very fast analysis cycle, thus significantly increasing sample preparation throughput. Here, we are describing characterization of novel immunoaffinity-based monolithic columns in a 96-well plate format for specific high-throughput purification of human Tf from blood plasma. We optimized the isolation and glycan preparation procedure for subsequent ultra performance liquid chromatography (UPLC analysis of Tf N-glycosylation and managed to increase the sensitivity for approximately three times compared to initial experimental conditions, with very good reproducibility. This work is licensed under a Creative Commons Attribution 4.0 International License.

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

    Science.gov (United States)

    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 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.

  4. 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.

    Science.gov (United States)

    Byrne, Shaina L; Mason, Anne B

    2009-06-01

    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.

  5. Host-species transferrin receptor 1 orthologs are cellular receptors for nonpathogenic new world clade B arenaviruses.

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    Jonathan Abraham

    2009-04-01

    Full Text Available The ability of a New World (NW clade B arenavirus to enter cells using human transferrin receptor 1 (TfR1 strictly correlates with its ability to cause hemorrhagic fever. Amapari (AMAV and Tacaribe (TCRV, two nonpathogenic NW clade B arenaviruses that do not use human TfR1, are closely related to the NW arenaviruses that cause hemorrhagic fevers. Here we show that pseudotyped viruses bearing the surface glycoprotein (GP of AMAV or TCRV can infect cells using the TfR1 orthologs of several mammalian species, including those of their respective natural hosts, the small rodent Neacomys spinosus and the fruit bat Artibeus jamaicensis. Mutation of one residue in human TfR1 makes it a functional receptor for TCRV, and mutation of four residues makes it a functional receptor for AMAV. Our data support an in vivo role for TfR1 in the replication of most, if not all, NW clade B arenaviruses, and suggest that with modest changes in their GPs the nonpathogenic arenaviruses could use human TfR1 and emerge as human pathogens.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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); pdiabetic and control (p=0.25). Significantly a lower hemoglobin level [10.58±0.67 g/dl vs11.76±1.5 g/dl; pdiabetic 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); porigin. 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 lower level of hemoglobin in diabetic cases is suggestive of iron deficiency anaemia rather than iron overload.

  8. Erythropoietin, ferritin, haptoglobin, hemoglobin and transferrin receptor in metabolic syndrome: a case control study

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    Hämäläinen Päivi

    2012-09-01

    Full Text Available Abstract Background Increased ferritin concentrations are associated with metabolic syndrome (MetS. The association between ferritin as well as hemoglobin level and individual MetS components is unclear. Erythropoietin levels in subjects with MetS have not been determined previously. The aim of this study was to compare serum erythropoietin, ferritin, haptoglobin, hemoglobin, and transferrin receptor (sTFR levels between subjects with and without MetS and subjects with individual MetS components. Methods A population based cross-sectional study of 766 Caucasian, middle-aged subjects (341 men and 425 women from five age groups born in Pieksämäki, Finland who were invited to a health check-up in 2004 with no exclusion criteria. Laboratory analyzes of blood samples collected in 2004 were done during year 2010. MetS was defined by National Cholesterol Education Program criteria. Results 159 (53% men and 170 (40% women of study population met MetS criteria. Hemoglobin and ferritin levels as well as erythropoietin and haptoglobin levels were higher in subjects with MetS (p  Conclusion Subjects with MetS have elevated hemoglobin, ferritin, erythropoietin and haptoglobin concentrations. Higher hemoglobin levels are related to all components of MetS. Higher ferritin levels associate with TG, abdominal obesity, elevated glucose or low high density cholesterol. Haptoglobin levels associate with blood pressure or elevated glucose. However, erythropoietin levels are related only with abdominal obesity. Higher serum erythropoietin concentrations may suggest underlying adipose tissue hypoxemia in MetS.

  9. Targeted Delivery of siRNA to Transferrin Receptor Overexpressing Tumor Cells via Peptide Modified Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Yuran Xie

    2016-10-01

    Full Text Available The use of small interference RNA (siRNA to target oncogenes is a promising treatment approach for cancer. However, siRNA cancer therapies are hindered by poor delivery of siRNA to cancer cells. Transferrin receptor (TfR is overexpressed in many types of tumor cells and therefore is a potential target for the selective delivery of siRNA to cancer cells. Here, we used the TfR binding peptide HAIYPRH (HAI peptide conjugated to cationic polymer branched polyethylenimine (bPEI, optimized the coupling strategy, and the TfR selective delivery of siRNA was evaluated in cells with high (H1299 and low TfR expression (A549 and H460. The HAI-bPEI conjugate exhibited chemico-physical properties in terms of size, zeta-potential, and siRNA condensation efficiency similar to unmodified bPEI. Confocal microscopy and flow cytometry results revealed that HAI-bPEI selectively delivered siRNA to H1299 cells compared with A549 or H460 cells. Moreover, HAI-bPEI achieved more efficient glyceraldehyde 3-phosphate dehydrogenase (GAPDH gene knockdown in H1299 cells compared with bPEI alone. However, despite optimization of the targeting peptide and coupling strategy, HAI-bPEI can only silence reporter gene enhanced green fluorescent protein (eGFP at the protein level when chloroquine is present, indicating that further optimization of the conjugate is required. In conclusion, the HAI peptide may be useful to target TfR overexpressing tumors in targeted gene and siRNA delivery approaches.

  10. Construction of single chain Fv antibody against transferrin receptor and its protein fusion with alkaline phosphatase

    Institute of Scientific and Technical Information of China (English)

    Dao-Feng Yang; Hui-Fen Zhu; Zhi-Hua Wang; Guan-Xin Shen; De-Ying Tian

    2005-01-01

    AIM: To construct fusion protein of a single-chain antibody(scFv) against transferrin receptor (TfR) with alkalinephosphatase (AP).METHODS: The VH-linker-VL, namely scFv gene, wasprepared by amplifying the VH and VL genes from plasmid pGEM-T-VH and pGEM-T-VL with splicing overlap extension polymerase chain reaction (SOE PCR). After the ScFv gene was modified by SfiⅠ and NotⅠ, it was subcloned into the secretory expression vector pUC19/119, and then was transformed into E. coli TG1. The positive colonies were screened by colony PCR and their expressions were induced by IPTG. ScFv gene was gained by digesting ScFv expression vector pUC19/119 with Sfi I and NotⅠ restriction enzymes, then subcloned into expression vector pDAP2, followed by transformation in E. coli TG1. The positive colonies were selected by bacterial colony PCR. The expression of fusion protein (scFv-AP) was induced by IPTG. Its activity was detected by enzyme immunoassay. The molecular weights of scFv and scFv-AP were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).RESULTS: The product of SOE PCR formed a band of 700 bp in agarose gel electrophoresis. SDS-PAGE demonstrated the molecular weight of scFv was 27 ku. Immunofluorescent assay (IFA) demonstrated its reactivity with TfR. The molecular weight of scFv-AP was 75 ku. Enzyme immunoassay showed that scFv-AP could specifically bind to human TfR and play AP activity.CONCLUSION: We have successfully prepared the antihuman TfR scFv and constructed the fusion protein of scFv and AP. It is promising for immunological experiments.

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

    Science.gov (United States)

    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) (Piron 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, PIron 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.

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

    Science.gov (United States)

    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 pchildren 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, pchildren. 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  18. GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN

    DEFF Research Database (Denmark)

    Ralston, E; Ploug, Thorkil

    1996-01-01

    of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex...... (alpha mannosidase II and giantin), of the trans-Golgi network (TGN38), of lysosomes (lgp110), and of early and late endosomes (transferrin receptor and mannose-6-phosphate receptor, respectively), to define the position of their subcellular compartments. By immunofluorescence, GLUT4 appears concentrated...... in the core of the myotubes. It is primarily found around the nuclei, in a pattern suggesting an association with the Golgi complex, which is further supported by colocalization with giantin and by immunogold electron microscopy. GLUT4 appears to be in the trans-most cisternae of the Golgi complex...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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 vegetarians than in omnivores. In children on a vegetarian diet, median hepcidin levels were lower (p 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.

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

    DEFF Research Database (Denmark)

    Schaffert, David Henning; Okholm, Anders Hauge; Sørensen, Rasmus Schøler;

    2016-01-01

    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...... on the origami surface....

  2. Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TU 67).

    Science.gov (United States)

    Herrmann, F; Griffin, J D; Sabbath, K D; Oster, W; Wernet, P; Mertelsmann, R

    1988-04-01

    Anti-TU 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TU 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TU 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TU 67 antibody.

  3. Transferrin iron uptake is stimulated by ascorbate via an intracellular reductive mechanism.

    Science.gov (United States)

    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.

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

    National Research Council Canada - National Science Library

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

    1993-01-01

    .... 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...

  5. The Next Generation Non-competitive Active Polyester Nanosystems for Transferrin Receptor-mediated Peroral Transport Utilizing Gambogic Acid as a Ligand.

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  7. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

    Directory of Open Access Journals (Sweden)

    Benjamin J Tillotson

    Full Text Available The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs against the transferrin receptor (TfR by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes.

  8. Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles

    NARCIS (Netherlands)

    van Dam, EM; Stoorvogel, W

    2002-01-01

    Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the traps-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that

  9. The Use of Soluble Transferrin Receptor in the Detection of rHuEPO Abuse in Sports

    Directory of Open Access Journals (Sweden)

    Lorenzo Gordon

    2010-02-01

    Full Text Available Erythropoietin (EPO increases the number of circulating erythrocytes and muscle oxygenation. The recombinant forms of EPO have indiscriminately been used by athletes, mainly in endurance sports to increase their erythrocytes concentration, thus generating a better delivery of oxygen to the muscle tissue. The administration of recombinant human erythropoietin (rHuEPO except for therapeutic use was prohibited by the International Olympic Committee (IOC and its unauthorized use considered as doping. In the last few years, a number of studies using parameters indicative of accelerated erythropoiesis have investigated a number of indirect methods for the detection of rHuEPO abuse. No single indirect marker has been found that can satisfactorily demonstrated rHuEPO misuse. 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 EPO abuse in sports. Indirect markers of altered erythropoiesis give reliable evidence of current or discontinued rHuEPO usage. This review describes the physical, biological and pharmacokinetic properties of endogenous EPO and its recombinant form. It also discusses the available strategies for the detection of rHuEPO abuse in sports, involving the use of sTfR concentration directly or in mathematical multivariate models.

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

    Science.gov (United States)

    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

  11. 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.

    Science.gov (United States)

    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 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.

  12. A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells

    Directory of Open Access Journals (Sweden)

    Kawamoto Megumi

    2011-08-01

    Full Text Available Abstract Background Transferrin receptor (TfR is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown the elevated expression levels of TfR on cancer cells compared with normal cells. The elevated expression levels of this receptor in malignancies, which is the accessible extracellular protein, can be a fascinating target for the treatment of cancer. We have recently designed novel type of immunotoxin, termed "hybrid peptide", which is chemically synthesized and is composed of target-binding peptide and lytic peptide containing cationic-rich amino acids components that disintegrates the cell membrane for the cancer cell killing. The lytic peptide is newly designed to induce rapid killing of cancer cells due to conformational change. In this study, we designed TfR binding peptide connected with this novel lytic peptide and assessed the cytotoxic activity in vitro and in vivo. Methods In vitro: We assessed the cytotoxicity of TfR-lytic hybrid peptide for 12 cancer and 2 normal cell lines. The specificity for TfR is demonstrated by competitive assay using TfR antibody and siRNA. In addition, we performed analysis of confocal fluorescence microscopy and apoptosis assay by Annexin-V binding, caspase activity, and JC-1 staining to assess the change in mitochondria membrane potential. In vivo: TfR-lytic was administered intravenously in an athymic mice model with MDA-MB-231 cells. After three weeks tumor sections were histologically analyzed. Results The TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC50 values as low as 4.0-9.3 μM. Normal cells were less sensitive to this molecule, with IC50 values > 50 μM. Competition assay using TfR antibody and knockdown of this receptor by siRNA confirmed the specificity of the TfR-lytic hybrid peptide. In addition, it was revealed that this molecule can disintegrate the cell membrane of T47

  13. SOD2 deficient erythroid cells up-regulate transferrin receptor and down-regulate mitochondrial biogenesis and metabolism.

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    Florent M Martin

    Full Text Available BACKGROUND: Mice irradiated and reconstituted with hematopoietic cells lacking manganese superoxide dismutase (SOD2 show a persistent hemolytic anemia similar to human sideroblastic anemia (SA, including characteristic intra-mitochondrial iron deposition. SA is primarily an acquired, clonal marrow disorder occurring in individuals over 60 years of age with uncertain etiology. METHODOLOGY/PRINCIPAL FINDINGS: To define early events in the pathogenesis of this murine model of SA, we compared erythroid differentiation of Sod2⁻/⁻ and normal bone marrow cells using flow cytometry and gene expression profiling of erythroblasts. The predominant transcriptional differences observed include widespread down-regulation of mitochondrial metabolic pathways and mitochondrial biogenesis. Multiple nuclear encoded subunits of complexes I-IV of the electron transport chain, ATP synthase (complex V, TCA cycle and mitochondrial ribosomal proteins were coordinately down-regulated in Sod2⁻/⁻ erythroblasts. Despite iron accumulation within mitochondria, we found increased expression of transferrin receptor, Tfrc, at both the transcript and protein level in SOD2 deficient cells, suggesting deregulation of iron delivery. Interestingly, there was decreased expression of ABCb7, the gene responsible for X-linked hereditary SA with ataxia, a component required for iron-sulfur cluster biogenesis. CONCLUSIONS/SIGNIFICANCE: These results indicate that in erythroblasts, mitochondrial oxidative stress reduces expression of multiple nuclear genes encoding components of the respiratory chain, TCA cycle and mitochondrial protein synthesis. An additional target of particular relevance for SA is iron:sulfur cluster biosynthesis. By decreasing transcription of components of cluster synthesis machinery, both iron utilization and regulation of iron uptake are impacted, contributing to the sideroblastic phenotype.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  16. Targeting immunoliposomes to transferrin receptors on brain capillary endothelial cells as a mean for cargo transport across the blood-brain barrier

    DEFF Research Database (Denmark)

    Johnsen, Kasper Bendix; Larsen, Annette Burkhart; Bruun, Jonas;

    2016-01-01

    transport further into the CNS. We studied transferrin receptor-targeted (OX26) immunoliposomes containing oxaliplatin with the aim of quantifying the uptake of OX26, liposomes and oxaliplatin in BCECs and the remaining CNS. The uptake of the immunoliposomes and their cargo was studied in 18-dayold rats...... higher both in vitro and in vivo when compared to isotypic IgG-conjugated liposomes. Quantitative analyses after capillary depletion revealed cargo transport from BCECs to the remaining CNS, but not differences could be detected between OX26- conjugated and isotype IgG control liposomes in vivo, whereas...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Efrat Harel

    Full Text Available 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.

  1. Radioprotective effect of transferrin targeted citicoline liposomes.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  4. Evolutionary reconstructions of the transferrin receptor of Caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs.

    Directory of Open Access Journals (Sweden)

    Jason T Kaelber

    Full Text Available 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.

  5. Evolutionary reconstructions of the transferrin receptor of Caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs.

    Science.gov (United States)

    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.

  6. Utility of labile plasma iron and transferrin saturation in addition to serum ferritin as iron overload markers in different underlying anemias before and after deferasirox treatment.

    Science.gov (United States)

    Porter, John B; El-Alfy, Mohsen; Viprakasit, Vip; Giraudier, Stephane; Chan, Lee Lee; Lai, Yongrong; El-Ali, Ali; Han, Jackie; Cappellini, Maria D

    2016-01-01

    Plasma markers in addition to serum ferritin (SF) may be useful for the assessment of iron overload; however, predictive utility may differ depending on underlying, transfusion-dependent, anemias. Data were collected before and after 1 year of deferasirox treatment (end of study; EOS) from the large, 1-year EPIC (Evaluation of Patients' Iron Chelation with Exjade(®) ) study. Trends were evaluated between liver iron concentration (LIC), transferrin saturation (TfSat), predose labile plasma iron (LPI) and their relationship to SF categories in 1530 patients: thalassemia major (TM; n = 1114), myelodysplastic syndromes (MDS, n = 336), and sickle-cell disease (SCD, n = 80). Baseline and EOS SF values showed a clear and similar relationship to LIC for all disease groups. TfSat also showed a relationship to SF, most clearly in patients with SCD, where TfSat was lowest in the lowest relative SF category. Unlike SF or LIC, TfSat did not decrease at EOS in any disease group. Baseline LPI was raised in TM and MDS, but not in patients with SCD, decreasing at EOS in both patient groups. After 1 year of chelation therapy, there was a significant trend for greater LPI reduction in patients with TM achieving LIC <7 mg Fe/g dw (P = 0.0137). Despite limitations, SF showed the clearest relationship, of the plasma markers evaluated, to LIC before and after 1 year of deferasirox in patients with TM, MDS, and SCD. In patients with TM, changes in LPI with chelation show a significant relationship to EOS LIC and may provide an additional indicator of chelation response (clinicaltrials.gov identifier: NCT00171821). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  9. Frequency of transferrin receptor positive reticulocytes (TF-Ret) in blood as an indicator of total-body radiation exposure: a pilot study in nuclear medicine patients.

    Science.gov (United States)

    Hempel, Klaus; Haenscheid, Heribert; Biko, Johannes; Hategan, Maria; Kaiser, Franz; Kreissl, Michael; Lorenz, Reinhard; Samnick, Samuel; Schirbel, Andreas; Varazashvili, Lali; Verchenya, Stanislav; Reiners, Christoph

    2012-11-01

    Approximately 3-20% of all reticulocytes in blood of healthy persons are immature and transferrin receptor positive (Tf-Ret). Tf-Ret were measured by flow cytometry in 27 patients treated with three different radiopharmaceuticals labeled with (131)I and in 25 healthy controls. Patients were chronically exposed within 6 days to blood doses from 0.18-1.89 Gy (D6). Typically, two-thirds of D6 was administered within the first day (D1). The study had to be confined to intra-subject investigations due to high biological variability of Tf-Ret counts. A significant radiation-induced decline was found in patients D1 doses that were ≥0.5 Gy. Tf-Ret frequency declined during the first 4 to 5 days of nuclear therapy to about 30-60% of its initial value, and increased in the following 3 days without reaching the initial value. At the time of nadir, the relative frequency of Tf-Ret was more depressed than that of reticulocytes and lymphocytes. The relative Tf-Ret frequency at nadir could be fitted to the equation: %-Tf-Ret=exp-(D1/D(o)). D(o) was found to be 1.0 ± 0.4 Gy (Mean ± SEM). The study shows that Tf-Ret frequency in blood might be a good parameter for estimation of the radiation dose to red marrow.

  10. 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.

    Science.gov (United States)

    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

  11. Targeting urokinase and the transferrin receptor with novel, anti-mitotic N-alkylisatin cytotoxin conjugates causes selective cancer cell death and reduces tumor growth.

    Science.gov (United States)

    Vine, K L; Indira Chandran, V; Locke, J M; Matesic, L; Lee, J; Skropeta, D; Bremner, J B; Ranson, M

    2012-01-01

    Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20(th) and 1/10(th) of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

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

    Science.gov (United States)

    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.

  13. Cross sectional, comparative study of serum erythropoietin, transferrin receptor, ferritin levels and other hematological indices in normal pregnancies and iron deficiency anemia during pregnancy.

    Science.gov (United States)

    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

  14. Generation and functional characterization of the anti-transferrin receptor single-chain antibody-GAL4 (TfRscFv-GAL4 fusion protein

    Directory of Open Access Journals (Sweden)

    Ye Qing

    2012-11-01

    Full Text Available Abstract Background The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Transferrin receptor (TfR is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and TfR binds and intakes of transferrin-iron complex. We have previously generated an anti-TfR single-chain variable fragments of immunoglobulin (scFv which were cloned from hybridoma cell line producing antibody against TfR linked with a 20 aa-long linker sequence (G4S4. In the present study, the anti-TfR single-chain antibody (TfRscFv was fused to DNA-binding domain of the yeast transcription factor GAL4. The recombinant fusion protein, designated as TfRscFv-GAL4, is expected to mediate the entry of DNA-protein complex into targeted tumor cells. Results Fusion protein TfRscFv-GAL4 was expressed in an E. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. The resulting proteins were predominantly monomeric and, upon refolding, became a soluble biologically active bifunctional protein. In biological assays, the antigen-binding activity of the re-natured protein, TfRscFv-GAL4, was confirmed by specific binding to different cancer cells and tumor tissues. The cell binding rates, as indicated by flow cytometry (FCM analysis, ranged from 54.11% to 8.23% in seven different human carcinoma cell lines. It showed similar affinity and binding potency as those of parent full-length mouse anti-TfR antibody. The positive binding rates to tumor tissues by tissue microarrays (TMA assays were 75.32% and 63.25%, but it showed weakly binding with hepatic tissue in 5 cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. In addition, the re-natured fusion protein TfRscFv-GAL4 was used in an ELISA with rabbit anti-GAL4 antibody. The GAL4-DNA functional assay through the GAL4

  15. Enhanced sandwich immunoassay using antibody-functionalized magnetic iron-oxide nanoparticles for extraction and detection of soluble transferrin receptor on a photonic crystal biosensor.

    Science.gov (United States)

    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 (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 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.

  16. Brain tumor-targeted therapy by systemic delivery of siRNA with Transferrin receptor-mediated core-shell nanoparticles.

    Science.gov (United States)

    Wei, Lin; Guo, Xi-Ying; Yang, Ting; Yu, Min-Zhi; Chen, Da-Wei; Wang, Jian-Cheng

    2016-08-20

    Treatment of brain tumor remains a great challenge worldwide. Development of a stable, safe, and effective siRNA delivery system which is able to cross the impermeable blood-brain barrier (BBB) and target glioma cells is necessary. This study aims to investigate the therapeutic effects of intravenous administration of T7 peptide modified core-shell nanoparticles (named T7-LPC/siRNA NPs) on brain tumors. Layer-by-layer assembling of protamine/chondroitin sulfate/siRNA/cationic liposomes followed by T7 peptide modification has been carried out in order to obtain a targeted siRNA delivery system. In vitro cellular uptake experiments demonstrated a higher intracellular fluorescence intensity of siRNA in brain microvascular endothelial cells (BMVECs) and U87 glioma cells when treated with T7-LPC/siRNA NPs compared with PEG-LPC/siRNA NPs. In the co-culture model of BMVECs and U87 cells, a significant down-regulation of EGFR protein expression occurred in the U87 glioma cells after treatment with the T7-LPC/siEGFR NPs. Moreover, the T7-LPC/siRNA NPs had an advantage in penetrating into a deep region of the tumor spheroid compared with PEG-LPC/siRNA NPs. In vivo imaging revealed that T7-LPC/siRNA NPs accumulated more specifically in brain tumor tissues than the non-targeted NPs. Also, in vivo tumor therapy experiments demonstrated that the longest survival period along with the greatest downregulation of EGFR expression in tumor tissues was observed in mice with an intracranial U87 glioma treated with T7-LPC/siEGFR NPs compared with mice receiving other formulations. Therefore, we believe that these transferrin receptor-mediated core-shell nanoparticles are an important potential siRNA delivery system for brain tumor-targeted therapy.

  17. Ultraviolet-B induced expression of hypoxia-inducible factor 1α,transferrin receptor through EGFR/PI3K/AKT/DEC1 pathway

    Institute of Scientific and Technical Information of China (English)

    LI Yanhua; BI Zhigang

    2007-01-01

    The aim of this research was to explore the effects and signaling pathway of ultraviolet-B (UVB) irradiation on the expression of hypoxia-inducible factor 1α (HIF-1α) and transferrin receptor (TfR).HIF-1α protein was measured by Western blot method.Expressions of epidermal growth factor receptor (EGFR),phosphor-EGF-R and TfR after UVB irradiation were determined with flow cytometry.After UVB irradiation,mRNA levels of HIF-1α and TfR were detected by real time-PCR.Results showed that compared with control groups,UVB was able to induce HIF1α and TfR protein expression in a dose- and time-dependent manner in HaCat cells (P < 0.05).TfR mRNA was expressed in a dose-dependent manner and reached a peak at the 8th hour in HaCat cells (P<0.05) whereas HIF-1α mRNA expression was not affected by UVB treatment (P>0.05).The EGFR/PI3K/AKT signaling pathway was required for the induction of HIF-lcx and TfR expression induced by UVB.UVB induced activation of EGFR in HaCat cells and EGFR regulated expression of TfR and HIF-1α.EGFR (-/-) MEF did not increase the HIF 1 expression following UVB irradiation (P>0.05).In contrast,EGFR (+/+) MEF strongly enhanced HIF 1α expression after UVB irradiation (P < 0.05).PD153035,a selective inhibitor of EGFR tyrosine kinase,inhibited the TfR protein expression in UVB-treated cells in a dose-dependent manner (P<0.05).PI3K inhibitors,LY294002 and wortmannin,inhibited HIF-1α and TfR expressions induced by UVB (P < 0.05).The DEC1 (-/-) Ha-Cat cells did not increase their TfR and HIF-1α expressions following UVB irradiation (P>0.05).In contrast,DEC1 (+/+) HaCat cells strongly enhanced TfR and HIF-1α protein expression after UVB irradiation (P<0.05).We conclude that UVB induces TfR and HIF-1α expressions via EGFR/PI3K/AKT/DEC1 signaling pathway.

  18. Reference values of serum transferrin receptor (sTfR) and sTfR/log ferritin index in healthy children.

    Science.gov (United States)

    Vázquez-López, María A; López-Ruzafa, Encarnación; Lendinez-Molinos, Francisco; Ortiz-Pérez, María; Ruiz-Tudela, Lucía; Martín-González, Manuel

    2016-03-01

    ABSTARCT The aims of this study were to determine appropriate reference ranges for serum transferrin receptor (sTfR) and sTfR/log ferritin (sTfR-F index) in healthy children and their relationship with iron parameters, erythropoiesis, and other conditions presented by the subject. A total of 902 children with normal iron status, aged 1-11 years, were included in a cross-sectional study. A physical examination was conducted and z-score of body mass index (zBMI) obtained. Complete blood count, iron biomarkers, erythropoietin, C-reactive protein, sTfR, and sTfR/log ferritin were determined. Linear multiple regression was applied to identify the factors that determined sTfR and sTfR-F index variability. Mean values for sTfR and sTfR-F index were 1.22 ± 0.28 mg/L (95% confidence interval [CI]: 1.2-1.23) and 0.87 ± 0.25 (95% CI: 0.85-0.88). The reference intervals (2.5th to 97.5th percentiles [P2.5-P97.5]) were 0.78-1.9 mg/L and 0.49-1.46, respectively. sTfR and sTfR-F values decreased with age (P <.03 and P <.0001, respectively). No changes were observed with sex. Changes in sTfR and sTfR-F index were consistent with ferritin and erythropoietin variations. Iron biomarkers, erythropoietin, and zBMI predicted 19% and 18.1% of the sTfR and sTfR-F index variability. The results provide reference ranges for sTfR and sTfR-F index in healthy children for clinical use in the assessment of body iron status. Both biomarkers are predicted by iron parameters, erythropoietin, and zBMI.

  19. The Homozygous Hemoglobin EE Genotype and Chronic Inflammation Are Associated with High Serum Ferritin and Soluble Transferrin Receptor Concentrations among Women in Rural Cambodia.

    Science.gov (United States)

    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 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 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 meta-analysis were appropriate for use in this population with a high prevalence of Hb disorders and inflammation.

  20. Annotating MYC oncogene status with 89Zr-transferrin imaging

    OpenAIRE

    Holland, Jason P.; Evans, Michael J.; Rice, Samuel L.; Wongvipat, John; Sawyers, Charles L.; Lewis, Jason S.

    2012-01-01

    A non-invasive technology that quantitatively measures the activity of oncogenic signaling pathways could broadly impact cancer diagnosis and treatment using targeted therapies. Here we describe the development of 89Zr-desferrioxamine transferrin (89Zr-Tf), a novel positron emission tomography (PET) radiotracer that binds the transferrin receptor 1 (TFRC, CD71) with high avidity. 89Zr-Tf produces high contrast PET images that quantitatively reflect treatment-induced changes in MYC-regulated T...

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

    Directory of Open Access Journals (Sweden)

    Aya Sugyo

    Full Text Available 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

  2. Utilidad en el post parto de la determinación de protoporfirina eritrocitaria y su relación con el receptor soluble de transferrina Usefulness of erythrocyte protoporphyrin test in the puerperium compared to the soluble transferrin receptor

    Directory of Open Access Journals (Sweden)

    Silvia H. Langini

    2004-08-01

    Full Text Available Se estudió, en 77 puérperas, la relación entre la protoporfirina eritrocitaria (PE, la ferritina sérica (FS, el receptor soluble de transferrina (RsT y los indicadores hematológicos utilizados en la rutina clínica. En sangre venosa se determinó: Hematocrito (Hto, Hemoglobina (Hb, recuento de glóbulos rojos (GR y glóbulos blancos (GB (contador electrónico MEGA; PE (Piomelli; en suero: RsT (ELISA, Orion Diagnostica, FS (ELISA, IMx Ferritina, Abbott y Proteína C-Reactiva (PCR- Látex, Wiener lab. Se analizaron sensibilidad (S, especificidad (E y puntos de corte mediante el modelo ROC (Receiver Operating Characteristics, considerando como gold standard el RsT. Los resultados (media ± DE fueron: Hto (% 35 ± 5; Hb (g/l 113 ± 18; GRx10³/mm³ 3 893 ± 489; VCM (fL 90 ± 6; GB/mm³ 9 543 ± 2.669; PE (µg/dl GR 46 ± 39; RsT (mg/l 4.7 ± 2.8; FS (µg/l 26 ± 31; PCR (Pos/Neg 72/5. La PE no correlacionó con FS, pero sí con el RsT (r=0.323, p=0.007. La S y E de la FS fueron de 83% y 63%, respectivamente, para un punto de corte de 25 µg/l; para la PE la S fue de 38% y la E de 90% para un punto de corte de 53 µg/dl GR. Estos resultados sugieren que ese punto de corte en el puerperio, permitiría detectar con un bajo costo un mayor porcentaje de mujeres (16% en nuestro estudio que presentan deficiencia de Fe pese a sus valores normales de hemoglobina.Erythrocyte protoporphyrin (EP, serum ferritin (SF, soluble transferrin receptor (sTfR and routine hematological laboratory tests were studied in 77 women 24 h post-partum, assisted at Paroissien Hospital (in Buenos Aires Province. Hematocrite (Hct, hemoglobin (Hb, red blood cells (RBC and white blood cells (WBC were determined using an electronic counter (Mega; EP by Piomelli’s; SF by ELISA (IMx Ferritina, Abbott; sTfR by ELISA (Orion Diagnostica and C-Reactive Protein (PCR-Latex, Wiener lab. All determinations were made in fasting blood samples. Statistical analysis (Receiver Operating

  3. Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 (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.

  5. Expression of Hepcidin and Ferroportin in the Placenta, and Ferritin and Transferrin Receptor 1 Levels in Maternal and Umbilical Cord Blood in Pregnant Women with and without Gestational Diabetes

    Science.gov (United States)

    Yang, Anqiang; Zhao, Jun; Lu, Minhua; Gu, Ying; Zhu, Yunlong; Chen, Daozhen; Fu, Jinyan

    2016-01-01

    Background: Regulation of iron transfer from mother to fetus via the placenta is not fully understood and the relationship between stored iron status in the mothers’ serum and gestational diabetes (GDM) in case–control studies is controversial. The present study aimed to detect circulating soluble transferrin receptor (sTfR) and ferritin levels in maternal and umbilical cord blood. We also examined the expression of hepcidin (Hep), transferrin receptor (TfR1), and ferroportin (FPN) in the placenta in pregnant women with and without GDM at full term. Methods: Eighty-two women participated (42 with GDM and 40 without GDM [controls]). Maternal samples were collected at 37–39 weeks’ gestation. Umbilical cord blood was collected at birth. Ferritin and sTfR levels in maternal serum and umbilical cord blood, and Hep, TfR1, and FPN protein expression in plac enta were compared between the GDM and non-GDM groups. Serum ferritin (SF) was measured by electrochemiluminescence assay and sTfR was measured by ELISA. Hep, TfR1, and FPN expression was measured by immunohistochemistry. Results: Maternal serum sTfR levels were significantly elevated in the GDM group compared with the non-GDM group (p = 0.003). SF levels in cord blood in the GDM group were significantly higher than those in the non-GDM group (p = 0.003). However, maternal hemoglobin and SF, and umbilical cord sTfR levels were not different between the groups. In placental tissue, FPN expression was higher and hepcidin expression was lower in the GDM group compared with the non-GDM group (p = 0.000 and p = 0.044, respectively). There was no significant difference in TfR1 between the groups (p = 0.898). Conclusions: Women with GDM transport iron more actively than those without GDM at term pregnancy. Maternal iron metabolism in GDM may play a role in fetal/placental iron demand and in the overall outcome of pregnancy. PMID:27483296

  6. Transferrin-mediated cellular iron delivery.

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Le Guevel, Xavier; Schneider, Marc [Pharmaceutical Nanotechnology, Saarland University, Saarbruecken (Germany); Daum, Nicole, E-mail: Marc.Schneider@mx.uni-saarland.de [Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbruecken (Germany)

    2011-07-08

    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%; {lambda}{sub em} = 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 {mu}g ml{sup -1}-1 mg ml{sup -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. 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)

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Liuxing, E-mail: fenglx@nim.ac.cn; Zhang, Dan; Wang, Jun; Shen, Dairui; Li, Hongmei

    2015-07-16

    Highlights: • Species-unspecific ID-PAGE-LA-ICP-MS was used to quantify Alb and Tf in human serum. • Addition methods of species-unspecific {sup 34}S spike were evaluated. • Isotope change conditions were investigated to reach satisfactory “isotope equilibration”. • Human serum CRM (ERM-DA470k/IFCC) was used to validate the new arrangements. • The developed method offers potential for accurate quantification of protein by ID-PAGE-LA-ICP-MS. - Abstract: 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 {sup 34}S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and {sup 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{sub sp}/m{sub 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

  9. 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.

    Science.gov (United States)

    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 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 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.

  10. 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.

    Science.gov (United States)

    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.

  11. Effects of transferrin on aromatase activity in porcine granulosa cells in vitro.

    Directory of Open Access Journals (Sweden)

    Małgorzata Duda

    2009-01-01

    Full Text Available Proliferating cells have an absolute requirement for iron, which is delivered by transferrin with subsequent intracellular transport via the transferrin receptor. Recent studies have reported that transferrin plays a crucial role in the local regulation of ovarian function, apart from its iron-binding characteristic. Therefore, the present study was undertaken to explore the possible role of transferrin in porcine granulosa cells function by examining its influence on aromatase activity, the most important indicator of follicular cell differentiation. In the first series of studies, pig granulosa cells isolated from small, immature follicles were cultured in the presence of transferrin alone (10 microg/ml or 100 microg/ml or with the addition of FSH (100ng/ml. The second series of studies was undertaken to determine transferrin-stimulated granulosa cells ability to aromatize exogenous testosterone (1x10(-7M. One hour after the establishment of cultures an aromatase inhibitor CGS16949A was added to test its influence on estradiol production. After 48 hours, cultures were terminated and cells were processed for immunocytochemical staining of aromatase. Media were frozen for further estradiol level analysis. Positive immunostaining for aromatase was found in all granulosa cell cultures. The intensity of immunostaining was always stronger in cultures supplemented with FSH whereas the addition of transferrin had no effect. Granulosa cells in vitro synthesized the highest amount of estradiol after the addition of FSH and exogenous testosterone as measured radioimmunologically. Concomitant treatment with FSH and transferrin caused an inhibition of FSH-stimulated aromatase activity. The production of estradiol also declined in the presence of FSH, testosterone and transferrin. This study demonstrates that transferrin had a dose-dependent inhibitory effect on FSH-stimulated aromatase activity, which was confirmed by radioimmunoassay. Our results indicate

  12. Inside job: ligand-receptor pharmacology beneath the plasma membrane.

    Science.gov (United States)

    Babcock, Joseph J; Li, Min

    2013-07-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments. However, these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone. Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell. These additional modes of interaction have been reported for functionally diverse ligands of GPCRs, ion channels, and transporters. Such intracellular drug-target engagements affect cell surface expression. Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation. Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites, and the potential therapeutic opportunities presented by this phenomenon.

  13. Inside job: ligand-receptor pharmacology beneath the plasma membrane

    Institute of Scientific and Technical Information of China (English)

    Joseph J BABCOCK; Min LI

    2013-01-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments.However,these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone.Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell.These additional modes of interaction have been reported for functionally diverse ligands of GPCRs,ion channels,and transporters.Such intracellular drug-target engagements affect cell surface expression.Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation.Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites,and the potential therapeutic opportunities presented by this phenomenon.

  14. Studies on the mitogenic effect of transferrin by membrane signal transduction

    Institute of Scientific and Technical Information of China (English)

    LEUNGTM; PLLIM; 等

    1990-01-01

    One of the earliest events leading to cell activation and growth is the hydrolysis of inositol phospholipids producing various membrane signals induced by an interaction between growth factors or hormones with their respective receptors on the cell membrane [1].To demonstrate the mitogenic action of transferrin,our results show that an addition of transferrin to “serum-deprived” rat hepatoma cells produced a rapid but transient rise in inositol 1,4,5-trisphosphate(IP3) level,and at the same time,an increased intracellular Ca2+ activity and a cytoplasmic alkalinization were observed.These signal transductions further lend support to the mitogenic nature of transferrin.In addition,a possible link between the receptor-mediated endocytosis of transferrin with the generation of intracellular signals is discussed herewith.

  15. 延边黄牛转铁蛋白受体2基因克隆与序列分析%Cloning and Sequence Analysis of the Transferrin Receptor 2 Gene from Yanbian Yellow Cattle

    Institute of Scientific and Technical Information of China (English)

    张立春; 金海国; 李赵志; 任春宇; 曹阳; 周国利; 金鑫

    2012-01-01

    [Objective] The transferrin receptor 2 (TfR2) gene was cloned from Yanbian Yellow cattle, the sequence was analyzed by bioinformatics software and the tissue expression distribution was tested by semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR). [ Method ] The total RNA was extracted from Yanbian Yellow cattle liver. The TfR2 gene was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The TfR2 gene was analyzed by bioinformatic softwares and the TfR2 gene tissue distribution was tested by SqRT-PCR in the end. [Result] The complete CDs and 3' UTR region of TfR2 gene was cloned (GenBank accession: GU553087). The TfR2 gene was 2901 bp in length and contained a 2412 -nucleotides-long open reading frame (ORF) which encoded a protein of 803 amino acid residues. The sequence analysis result showed that Yanbian Yellow cattle TfR2 gene has over 80% homology with mammals in nucleotides, the amino acid sequences of TfR2 gene from different mammals were conserved, especially some amino acid sites which affect its function in human were highly conserved. The protein functional domain analysis showed that TfR2 gene from different mammals were highly conserved. The expression profiles analysis showed that the TfR2 gene was highly expressed in liver, constittitively expressed in spleen, heart, kidney and intestine (duodenum), [conclusion] The TfR2 gene from Yanbian Yellow cattle is highly homologous with other mammals, the functional domain and tissue distribution are highly conserved in different mammals.%[目的]克隆廷边黄牛转铁蛋白受体2 (transferrin receptor 2,TFR2)基因,并分析该基因在不同组织器官中的表达分布.[方法]通过提取肝脏组织总RNA,采用RT-PCR及RACE方法克隆延边黄牛TfR2基因,采用生物信息学软件进行序列分析,用半定量PCR( SqRT-PCR)方法分析TfR2基因在不同组织器官中的表达分布规律.[结果]①成

  16. Probing molecular interaction between transferrin and anti-transferrin by atomic force microscope

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The interaction between transferrin (Tf) and its antibody was investigated by atomic force microscope. Tf-antibody was immobilized on the Au-coated glass slide, and the specific combination between antibody and antigen was also characterized by AFM. The results showed that holo-transferrin was jogged with anti-transferrin, and binded anti-tran- sferrin more tightly than apo-transferrin. The force- distance curves revealed that the affinity of anti-trans- ferrin and holo-transferrin was much stronger than that of apo-transferrin.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. (Chemical Sciences and Engineering Division); ( XSD); (Univ. of Chicago); (Northwestern Univ.)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  20. Applying 89Zr-Transferrin To Study the Pharmacology of Inhibitors to BET Bromodomain Containing Proteins

    Science.gov (United States)

    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

  1. Covalent crosslinking of thyrotropin to thyroid plasma membrane receptors: subunit composition of the thyrotropin receptor.

    Science.gov (United States)

    McQuade, R; Thomas, C G; Nayfeh, S N

    1986-04-01

    The subunit composition of the thyrotropin (TSH) receptor has been characterized using the bifunctional crosslinking agent, disuccinimidyl suberate (DSS), to covalently link [125I]TSH to its receptor. Purified thyroid membranes were labeled with [125I]TSH, and the hormone-receptor complex was crosslinked by incubation with 0.1 mM DSS. Analysis of this crosslinked complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the presence of a specifically labeled hormone-receptor complex, corresponding to a Mr of 68,000 +/- 3000 before correction for the relative molecular mass of TSH. When reducing agents were absent during SDS solubilization, the mobility of the band increased slightly, suggesting the presence of intramolecular disulfide bonds. The labeling of the 68,000 band was specifically inhibited by TSH, but not by other glycoprotein hormones. Specific labeling occurred only in thyroid, and not in liver or muscle plasma membranes. Protease-free immunoglobulin G, isolated from sera of patients with Graves' disease and capable of competing with TSH for binding to its receptor, inhibited the labeling of the 68,000 complex. When the hormone-receptor complex was crosslinked with higher concentrations of DSS (greater than 0.3 mM), a second specifically labeled band was observed, with a Mr of 80,000 +/- 5000. This complex exhibited hormone, tissue, and immunologic specificities similar to those of the 68,000 band. Continuous sucrose density gradient analysis indicated that the intact solubilized receptor possessed a sedimentation coefficient of 10.5 S prior to correction for detergent binding. However, this value increased to 16 S when determined under conditions which took into account the change in hydrodynamic properties attributable to bound Triton X-100. These data suggest that the 80,000 and 68,000 bands represent binding components of the TSH receptor and that the receptor molecule most likely contains

  2. ZRT/IRT-like Protein 14 (ZIP14) Promotes the Cellular Assimilation of Iron from Transferrin*

    OpenAIRE

    Zhao, Ningning; Gao, Junwei; Enns, Caroline A; Knutson, Mitchell D.

    2010-01-01

    ZIP14 is a transmembrane metal ion transporter that is abundantly expressed in the liver, heart, and pancreas. Previous studies of HEK 293 cells and the hepatocyte cell lines AML12 and HepG2 established that ZIP14 mediates the uptake of non-transferrin-bound iron, a form of iron that appears in the plasma during pathologic iron overload. In this study we investigated the role of ZIP14 in the cellular assimilation of iron from transferrin, the circulating plasma protein that normally delivers ...

  3. Differential transferrin expression in placentae from normal and abnormal pregnancies: a pilot study

    Directory of Open Access Journals (Sweden)

    Bukovsky Antonin

    2008-07-01

    Full Text Available Abstract 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 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.

  4. Conjugation of transferrin to azide-modified CdSe/ZnS core-shell quantum dots using cyclooctyne click chemistry.

    Science.gov (United States)

    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.

  5. Carbohydrate deficient transferrin and alcoholism.

    Science.gov (United States)

    Solomons, Hilary Denis

    2012-06-01

    Alcohol abuse is an important public health problem, with major implications in patients with a pre-existing liver pathology of viral origin. Hepatitis C, for example, is one of the diseases in which alcohol consumption can lead to the transition from a fairly benign outline to a potentially life-threatening liver disease. Alcohol abuse is usually identified on the basis of clinical judgment, alcoholism related questionnaires, laboratory tests and, more recently, biomarkers. Also on this list of tests, carbohydrate deficient transferrin (CDT) is widely available and useful for determining recent alcohol consumption, particularly when corroborated with elevation of other liver-associated enzymes. Clinicians should be aware of the indications and limitations of this test in order to better evaluate alcohol consumption in their patients.

  6. Plasma soluble urokinase plasminogen activator receptor in children with urinary tract infection

    DEFF Research Database (Denmark)

    Wittenhagen, Per; Andersen, Jesper Brandt; Hansen, Anita

    2011-01-01

    In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection.......In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection....

  7. 可溶性转铁蛋白受体在慢性病贫血中的应用%The clinical application of soluble transferrin receptor in chronic anemia

    Institute of Scientific and Technical Information of China (English)

    李建花; 裴景亮; 张淑英

    2011-01-01

    目的:探讨可溶性转铁蛋白受体在慢性病贫血中的临床应用.方法:将63例慢性病贫血患者分为慢性病贫血缺铁组(30例)和慢性病贫血非缺铁组(33例),并选择同期23例健康体检者作为健康对照组.三组均采用生物素-亲和素-酶联免疫法定量检测可溶性转铁蛋白受体(sTFR)水平,并结合血清铁(SI)、铁蛋白(SF)、总铁结合力(TIBC)对结果进行分析比较.结果:慢性病贫血缺铁组sTFR为(46.1±29.4)nmol/L,慢性病贫血非缺铁组sTFR为(18.4±5.7)nmol/L,健康对照组sTFR为(15.5±6.2)nmol/L,慢性病贫血缺铁组与其他两组比较,差异有统计学意义(P0.05).结论:sTFR是一个灵敏、可靠的缺铁诊断的定量指标,在慢性病贫血人群的铁状况评估方面具有重要的临床价值.%Objective: To discuss the clinical application of soluble transferrin receptor in chronic anemia. Methods: 63 cases of patients with chronic anemia were divided into chronic anemia with iron deficiency group (30 cases) and chronic anemia without iron deficiency group (33 cases), 23 cases of healthy person were divided into healty group. The concentration of sTFR was detected by the method of biotin-affinity grain enzyme-lined immunosorbent, and the results were comparison analyzed combination the level of TIBC, SI, SF. Results: The sTFR level of chronic anemia with iron deficiency group was (46.1 ±29.4) nmol/L; the sTFR level of chronic anemia without iron deficiency group was (18.4 ±5.7) nmol/L; the sTFR level of healty group was (15.5±6.2) nmol/L; the sTFR level of chronic anemia with iron deficiency group were significantly higher than that of other groups (P0.05). Conclusion: sTFR is an sensitive and reliable quantitative index to diagnose iron deficiency.lt has great clinical value in evaluation iron's situation of chronic anemia population.

  8. Evaluation of Nonferrous Metals as Potential In Vivo Tracers of Transferrin-Based Therapeutics

    Science.gov (United States)

    Zhao, Hanwei; Wang, Shunhai; Nguyen, Son N.; Elci, S. Gokhan; Kaltashov, Igor A.

    2016-02-01

    Transferrin (Tf) is a promising candidate for targeted drug delivery. While development of such products is impossible without the ability to monitor biodistribution of Tf-drug conjugates in tissues and reliable measurements of their levels in blood and other biological fluids, the presence of very abundant endogenous Tf presents a significant impediment to such efforts. Several noncognate metals have been evaluated in this work as possible tracers of exogenous transferrin in complex biological matrices using inductively coupled plasma mass spectrometry (ICP MS) as a detection tool. Placing Ni(II) on a His-tag of recombinant Tf resulted in formation of a marginally stable protein-metal complex, which readily transfers the metal to ubiquitous physiological scavengers, such as serum albumin. An alternative strategy targeted iron-binding pockets of Tf, where cognate Fe(III) was replaced by metal ions known to bind this protein. Both Ga(III) and In(III) were evaluated, with the latter being vastly superior as a tracer (stronger binding to Tf unaffected by the presence of metal scavengers and the retained ability to associate with Tf receptor). Spiking serum with indium-loaded Tf followed by ICP MS detection demonstrated that protein quantities as low as 0.04 nM can be readily detected in animal blood. Combining laser ablation with ICP MS detection allows distribution of exogenous Tf to be mapped within animal tissue cross-sections with spatial resolution exceeding 100 μm. The method can be readily extended to a range of other therapeutics where metalloproteins are used as either carriers or payloads.

  9. Adaptation of transferrin protein and glycan synthesis

    NARCIS (Netherlands)

    G. de Jong (Gerard); W.L. van Noort (W.); R.A. Feelders (Richard); C.M.H. de Jeu-Jaspars (Nel); H.G. van Eijk (Henk)

    1992-01-01

    textabstractWe report the patterns of variability in transferrin structure in pregnancy, iron deficiency anemia, women using oral contraceptives, nonanaemic rheumatoid arthritis, iron deficient rheumatoid arthritis and anemia of the chronic diseases. Changes in microheterogeneity were assessed by cr

  10. Adaptation of transferrin protein and glycan synthesis

    NARCIS (Netherlands)

    G. de Jong (Gerard); W.L. van Noort (W.); R.A. Feelders (Richard); C.M.H. de Jeu-Jaspars (Nel); H.G. van Eijk (Henk)

    1992-01-01

    textabstractWe report the patterns of variability in transferrin structure in pregnancy, iron deficiency anemia, women using oral contraceptives, nonanaemic rheumatoid arthritis, iron deficient rheumatoid arthritis and anemia of the chronic diseases. Changes in microheterogeneity were assessed by

  11. Transferrin Impacts Bacillus thuringiensis Biofilm Levels

    Directory of Open Access Journals (Sweden)

    Bianca Garner

    2016-01-01

    Full Text Available 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.

  12. Activation of μ-opioid receptor and Toll-like receptor 4 by plasma from morphine-treated mice.

    Science.gov (United States)

    Xie, Nan; Gomes, Fabio P; Deora, Vandana; Gregory, Kye; Vithanage, Tharindu; Nassar, Zeyad D; Cabot, Peter J; Sturgess, David; Shaw, Paul N; Parat, Marie-Odile

    2017-03-01

    In this study, we quantified the ability of opioids present in biological samples to activate the μ-opioid receptor and TLR4 using cell-based assays. Each assay was standardised, in the presence of plasma, using morphine, its μ receptor-active metabolite morphine-6 glucuronide (M6G) and its μ receptor-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G). Specificity was verified using antagonists. Morphine- and M6G-spiked plasma samples exhibited μ receptor activation, which M3G-spiked plasma lacked. In contrast, M3G showed moderate but consistent activation of TLR-4. Plasma samples were collected at a number of time points from mice administered morphine (1 or 10mg/kg every 12h for 3days) or saline. Morphine administration led to intermittent μ receptor activation, reversed by μ receptor antagonists, and to TRL4 activation at time points where M3G is measured in plasma. Interestingly, this protocol of morphine administration also led to TLR4-independent NF-κB activation, at time points where M3G was not detected, presumably via elevation of circulating cytokines including, but not limited to, TNFα. Circulating TNFα was increased after three days of morphine administration, and TNFα mRNA elevated in the spleen of morphine-treated mice.

  13. Instrumental comparison of the determination of Cr³+ uptake by human transferrin.

    Science.gov (United States)

    Quarles, C Derrick; Brumaghim, Julia L; Marcus, R Kenneth

    2010-12-01

    UV-VIS absorbance, inductively coupled plasma-optical emission spectroscopy (ICP-OES), and particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) are presented as techniques for determining Cr³+ loading into transferrin (Tf), with and without Fe³+. The methods are compared based on loading percentages (i.e. 100% loading would be equal to 2 M(n+): 1 Tf) determined for Cr³+ loading into apo-transferrin. Spectral interferences and overlapping LMCT bands cause inaccurate chromium (qualitative) and iron (qualitative and quantitative) results for the UV-VIS absorbance method. The ICP-OES and PB/HC-OES methods are in good agreement providing evidence that the PB/HC-OES method is a valid technique for investigating metal-protein complexes. Maximum Cr³+ loading into apo-transferrin over a 24 h period was determined to be 26.8 3.5% by the ICP-OES method and 25.3 2.2% by the PB/HC-OES method. Loading percentages were increased to 49.7 1.9% (ICP-OES) and 55.7 3.2% (PB/HC-OES) when the metal-transferrin solution was allowed to incubate for up to 10 days. Under non-excess carbonate conditions the Cr³+ loading is elevated over a 24 h incubation time, but under physiological conditions the loading is inhibited. Equal loading of Fe³+ and Cr³+ into apo-transferrin was achieved when chromium was at a level more than 5 times in excess of iron. Inhibition of Cr³+ loading was only observed when an excess of Fe³+ was available to bind into apo-transferrin. The ability for Cr³+ to displace Fe3+ from holo-transferrin was observed as small amounts of Cr³+ were loaded into the once occupied metal binding site.

  14. [Improvement of carbohydrate deficient transferrin measurement by capillary zone electrophoresis using immunosubtraction of immunoglobulins and transferrin].

    Science.gov (United States)

    Baraud, J; Schellenberg, F; Pagès, J-C

    2009-01-01

    CDT (Carbohydrate Deficient Transferrin) is considered as the most efficient biomarker of alcohol abuse available for routine use. Among the various methods developed for its measurement, capillary zone electrophoresis (CZE) on the multicapillary analyzer Capillarys2 provides high quality results at high throughput. However, the non CDT specific measurement of protein absorbance at 200 nm may bring abnormal profiles in samples from patients with high polyclonal immunoglobulin level or monoclonal component. We evaluated the automated immunosubtraction procedure developed by the manufacturer in 48 samples with abnormal electrophoretic profiles that potentially could interfere with CZE measurement of CDT. Elimination of the serum immunoglobulins raised the number of interpretable profiles from 19 (40%) to 37 (77%). The immunosubtraction procedure failed in samples with a monoclonal component present at a concentration > 60 g/L and in some samples harbouring a partially degraded C3 fraction. Six samples identified as genetic BC transferrin variants were also included in the study and submitted to an automated transferrin subtraction procedure to ascertain whether the additional peak were actually transferrin glycoforms. After treatment, two samples were classified as homozygote C for transferrin due to the persistence of one of the supposed transferrin peak. In conclusion, immunoglobulin and transferrin subtraction allow a better CDT measurement in most samples with interfering monoclonal components and avoid misclassification of suspected transferrin BC or CD variants.

  15. (111)Indium-transferrin for localization and quantification of gastrointestinal protein loss

    DEFF Research Database (Denmark)

    Simonsen, Jane Angel; Braad, Poul-Erik; Veje, Annegrete;

    2009-01-01

    Objective. To evaluate the indium-111 ((111)In)-transferrin method as a means of localization and quantification of gastrointestinal protein loss. Methods. Fourteen patients and 15 healthy subjects underwent an (111)In-transferrin study consisting of abdominal scintigraphy, whole-body counting...... measurement and determination of plasma activity of (111)In during the course of 5 days. Two of the patients went through a subsequent chromium-51-trichloride test with analysis of radioactivity in faeces in order to compare the results of the two methods. Results. The patients had a mean+/-SEM whole-body...

  16. Structural consequences of binding of UO{sub 2}{sup 2+} to apo-transferrin: Can this protein account for entry of uranium into human cells?

    Energy Technology Data Exchange (ETDEWEB)

    Vidaud, C [CEA Valrho, DSV, DIEP, Service de Biochimie Post Genomique and Toxicologie Nucleaire, F-30207 Bagnols Sur Ceze (France); Vidaud, C.; Gourion-Arsiquaud, S; Rollin-Genetet, F; Torne-Celer, C; Plantevin, S; Pible, O; Quemeneur, E; Berthomieu, C. [CEA Cadarache, Laboratoire de Bioenergetique Cellulaire and Laboratoire des Interactions Proteine-Metal, DSV/DEVM, UMR 6191 CNRS-CEA-Universite Aix-Marseille II, F-13108 Saint-Paul-lez-Durance Cedex (France)

    2007-02-15

    It has been established that transferrin binds a variety of metals. These include toxic uranyl ions which form rather stable uranyl-transferrin derivatives. We determined the extent to which the iron binding sites might accommodate the peculiar topographic profile of the uranyl ion and the consequences of its binding on protein conformation. Indeed, metal intake via endocytosis of the transferrin/transferrin receptor depends on the adequate coordination of the metal in its site, which controls protein conformation and receptor binding. Using UV-vis and Fourier transform infrared difference spectroscopy coupled to a micro-dialysis system, we showed that at both metal binding sites two tyrosines are uranyl ligands, while histidine does not participate with its coordination sphere. Analysis by circular dichroism and differential scanning calorimetry (DSC) showed major differences between structural changes associated with interactions of iron or uranyl with apo-transferrin. Uranyl coordination reduces the level of protein stabilization compared to iron, but this may be simply related to partial lobe closure. The lack of interaction between uranyl-TF and its receptor was shown by flow cytometry using Alexa 488-labeled holo-transferrin. We propose a structural model summarizing our conclusion that the uranyl-TF complex adopts an open conformation that is not appropriate for optimal binding to the transferrin receptor. (authors)

  17. 可溶性转铁蛋白受体在儿童铁缺乏症诊断中的价值%Value of soluble transferrin receptor in the diagnosis of iron deficiency in children

    Institute of Scientific and Technical Information of China (English)

    王亚平; 邵洁; 庄学玲

    2011-01-01

    目的 调查6个月至7岁各年龄段儿童铁缺乏症的发生率;评价可溶性转铁蛋白受体(sTfR)在儿童铁缺乏症筛查中的应用价值.方法 采用诊断性试验方法,检测浙江省杭州市502例来我院体检的6个月至7岁儿童血清STM、铁蛋白(SF)、血清铁(SI)、总铁结合力(TIBC)以及C反应蛋白(CRP)水平,同时检测血常规和血锌原叶F#(ZPP).结果 铁缺乏症的检出率为19.5 %(98/502),其中婴儿组(≦岁)检出率为34.7%,幼儿组(大于1岁、小于或等于3岁)19.4%,学龄前组(大于3岁、小于或等于7岁)14.0% o婴儿组铁缺乏的检出率明显高于其他两个年龄组.婴儿组sTfR均值(2.02±0.73 mg/L)明显高于幼儿组((1.68±0.40 mg/L)和学龄前组(1.67 10.29 mg/L),差异有统计学意义(P < 0.05).sTM诊断铁缺乏的界值在婴儿组为2.02 mg/L(灵敏度70.3%,特异度82.2%),幼儿组为1.85 mg/L(灵敏度71.7%,特异度86.4%),学龄前组为1.85 mg/L(灵敏度77.8%,特异度88.6%).sTf与SF,TIBC,TS,ZPP及MCV具有相关性(r分别为0.107、0.276、-0.139、0.175、-0.140,P均<0.05).结论 7岁以下儿童中,婴儿组是铁缺乏发生率最高的;婴儿组sTfR均值及其诊断铁缺乏的界值均高于其他年龄组;STfR是诊断儿童尤其是婴儿铁缺乏症的一个较敏感指标.%Objective 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. Methods 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. Results 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

  18. Bioadhesive micelles of d-α-tocopherol polyethylene glycol succinate 1000: Synergism of chitosan and transferrin in targeted drug delivery.

    Science.gov (United States)

    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.

  19. Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener's granulomatosis

    NARCIS (Netherlands)

    Boomsma, MM; Stearns-Kurosawa, DJ; Stegeman, CA; Raschi, E; Meroni, PL; Kurosawa, S; Tervaert, JWC

    Elevated soluble thrombomodulin (sTM) levels are an accepted marker of endothelial damage. The physiological significance of plasma endothelial protein C receptor (sEPCR) levels is not known. To assess the relevance of this plasma protein in Wegener's granulomatosis (WG), sEPCR levels were measured

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  2. Maintenance of transferrin polymorphism in pigeons

    Energy Technology Data Exchange (ETDEWEB)

    Frelinger, J.A.

    1972-02-01

    Transferrin, a nonheme iron-binding protein, is polymorphic in most vertebrate species that have been examined. In pigeons, it is controlled by an autosomal gene, with two known codominant alleles, Tf/sup A/ and Tf/sup B/. The two alleles are found in nearly equal frequencies and the three genotypes are at Hardy-Weinberg equilibrium in all populations studied. This report shows that ovotransferrins from heterozygous females inhibit microbial growth, by use of yeast as an assay organism, better than ovetransferrins from either of the homozygous types, or those from a mixture of homozygous types. Heterozygous females hatch a larger percentage of their eggs than homozygous females. This difference is probably accounted for by the transferrin effect. The failure of the mixture of the homozygous types to act like the heterozygous type calls into question the currently accepted structure of transferrin as a monomeric protein. The greater fecundity of heterozygous females can account for the maintenance of transferrin polymorphism in pigeons.

  3. Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin.

    Science.gov (United States)

    Wiejak, J; Surmacz, L; Wyroba, E

    2001-01-01

    The kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY), and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K), and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LY uptake (575 ng LY/mg protein/hr) decreased almost 5-fold within 15 min, reaching plateau at 126 ng/mg protein/hr. Transferrin induced a small increase (7.5%) in the fluid phase uptake rate (after 5 min) followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin-FITC) were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in "mixing" endosomes to which transferrin and LY were targeted. Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC.

  5. Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin

    Directory of Open Access Journals (Sweden)

    J Wiejak

    2009-12-01

    Full Text Available The kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY, and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K, and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LYuptake (575 ng LY/ mg protein /hr decreased almost 5-fold within 15 min, reaching plateau at 126 ng/ mg protein /hr. Transferrin induced a small increase (7.5% in the fluid phase uptake rate (after 5 min followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin– FITC were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in “mixing” endosomes to which transferrin and LY were targeted. Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC.

  6. 益血生胶囊联合蔗糖铁注射液对妊娠贫血患者血清转铁蛋白受体及妊娠结局的影响%Effect of Yi Xuesheng Jiaonang combined with Iron Sucrose Injection on serum transferrin receptor and pregnancy outcome with anemia patients in pregnancy

    Institute of Scientific and Technical Information of China (English)

    章红英; 陈淑群

    2016-01-01

    Objective To explore the effect of Yi Xuesheng Jiaonang combined with Iron Sucrose Injection on anemia in pregnancy on serum transferrin receptor and pregnancy outcome.Methods 78 patients with anemia in pregnancy form December 2014 to December 2015 of the third people’s Hospital of Cixi were collected and randomly divided into control group and treatment group,each had 39 cases.Control group was given 200 mg Iron Sucrose Injection with 100 mL 0.9%sodium chloride injection intravenous infusion,2 times a week; experiment group were treated on the base of control group with 4 pills of Yi Xuesheng Jiaonang,3 times daily.The course was 28d,and two groups all with a course treatment.After the end of treatment,clinical curative effect,serum transferrin receptor level,liver and renal function and the incidence of adverse pregnancy outcome were observed and compared. Results Serum transferrin receptor levels were increased in two groups after treatment (P<0.05),compared with control group,serum transferrin receptor level of treatment group was even higher (P<0.05), alanine aminotransferase (ALT),aspartate aminotransferase (AST),serum creatinine (Cr) and urea nitrogen ( BUN) and other indicators of liver and kidney function of experiment group had no statistical significance.Adverse pregnancy outcome rate betwen two groups was not statistically significant.Conclusion Yi Xuesheng Jiaonang combined with Sucrose Iron Injection therapy for patients with anemia of pregnancy can significantly increased serum transferrin receptor levels,has higher safety and low adverse pregnancy outcomes rate.%目的:探究益血生胶囊联合蔗糖铁注射液对妊娠贫血患者血清转铁蛋白受体及妊娠结局的影响。方法收集2014年12月~2015年12月慈溪市第三人民医院妇产科收治的妊娠贫血患者78例,根据治疗方法不同分为对照组和试验组,每组各39例。2组均实施常规治疗以及对症治疗,对照组给予蔗糖铁注射液200

  7. Drosophila Lipophorin Receptors Recruit the Lipoprotein LTP to the Plasma Membrane to Mediate Lipid Uptake.

    Directory of Open Access Journals (Sweden)

    Míriam Rodríguez-Vázquez

    2015-06-01

    Full Text Available Lipophorin, the main Drosophila lipoprotein, circulates in the hemolymph transporting lipids between organs following routes that must adapt to changing physiological requirements. Lipophorin receptors expressed in developmentally dynamic patterns in tissues such as imaginal discs, oenocytes and ovaries control the timing and tissular distribution of lipid uptake. Using an affinity purification strategy, we identified a novel ligand for the lipophorin receptors, the circulating lipoprotein Lipid Transfer Particle (LTP. We show that specific isoforms of the lipophorin receptors mediate the extracellular accumulation of LTP in imaginal discs and ovaries. The interaction requires the LA-1 module in the lipophorin receptors and is strengthened by a contiguous region of 16 conserved amino acids. Lipophorin receptor variants that do not interact with LTP cannot mediate lipid uptake, revealing an essential role of LTP in the process. In addition, we show that lipophorin associates with the lipophorin receptors and with the extracellular matrix through weak interactions. However, during lipophorin receptor-mediated lipid uptake, LTP is required for a transient stabilization of lipophorin in the basolateral plasma membrane of imaginal disc cells. Together, our data suggests a molecular mechanism by which the lipophorin receptors tether LTP to the plasma membrane in lipid acceptor tissues. LTP would interact with lipophorin particles adsorbed to the extracellular matrix and with the plasma membrane, catalyzing the exchange of lipids between them.

  8. Sigma-1 receptor concentration in plasma of patients with late-life depression: a preliminary study

    Directory of Open Access Journals (Sweden)

    Shimizu H

    2013-12-01

    Full Text Available Hideyuki Shimizu,1 Minoru Takebayashi,2 Masayuki Tani,1 Hiroaki Tanaka,1 Bun Yamagata,1 Kenzo Kurosawa,1 Hiroki Yamada,1 Mitsugu Hachisu,3 Kazue Hisaoka-Nakashima,2 Mami Okada-Tsuchioka,2 Masaru Mimura,4 Akira Iwanami11Department of Neuropsychiatry, Showa University School of Medicine, Tokyo, Japan; 2Department of Psychiatry and Institute for Clinical Research, National Hospital Organization Kure Medical Center, Kure, Japan; 3Department of Clinical Psychopharmacy, Pharmacy School, Showa University, Tokyo, Japan; 4Department of Neuropsychiatry, Keio University School of Medicine, Tokyo, JapanBackground: Recently, the sigma-1 receptor has been shown to play a significant role in the neural transmission of mood by regulating N-methyl-D-aspartate receptors. Additionally, the sigma-1 receptor has been reported to influence cognitive functions including learning and memory. In this study, we measured plasma sigma-1 receptor concentrations before and after antidepressant treatment in patients with late-life major depressive disorder (MDD and explored whether changes in depressive status are related to sigma-1 receptor concentrations.Methods: The study participants were 12 subjects with late-life MDD diagnosed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. All of the participants were over 60 years old. Immediately prior to and 8 weeks after the start of treatment, sigma-1 receptor concentration and mental status, including depressive symptoms (Hamilton Depression Rating Scale; HAM-D, were measured. Treatment for depression was performed according to a developed algorithm based on the choice of treatments. We examined the association between changes in sigma-1 receptor concentration and HAM-D scores during antidepressant treatment. For the measurement of plasma sigma-1 receptor concentration, blood plasma samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Western

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

    Science.gov (United States)

    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.

  10. A balance between B cell receptor and inhibitory receptor signaling controls plasma cell differentiation by maintaining optimal Ets1 levels.

    Science.gov (United States)

    Luo, Wei; Mayeux, Jessica; Gutierrez, Toni; Russell, Lisa; Getahun, Andrew; Müller, Jennifer; Tedder, Thomas; Parnes, Jane; Rickert, Robert; Nitschke, Lars; Cambier, John; Satterthwaite, Anne B; Garrett-Sinha, Lee Ann

    2014-07-15

    Signaling through the BCR can drive B cell activation and contribute to B cell differentiation into Ab-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. In this study, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is downregulated in B cells by BCR or TLR signaling through a pathway dependent on PI3K, Btk, IKK2, and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP1) or membrane receptors CD22 and/or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings indicate that downregulation of Ets1 occurs in response to B cell activation via either BCR or TLR signaling, thereby allowing B cell differentiation and that the maintenance of Ets1 expression is an important function of the inhibitory Lyn → CD22/SiglecG → SHP1 pathway in B cells.

  11. Neural and Oligodendrocyte Progenitor Cells: Transferrin Effects on Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Lucas Silvestroff

    2013-02-01

    Full Text Available 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.

  12. Iron uptake by melanoma cells from the soluble form of the transferrin homologue, melanotransferrin.

    Science.gov (United States)

    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).

  13. Crossing the blood-brain-barrier with transferrin conjugated carbon dots: A zebrafish model study.

    Science.gov (United States)

    Li, Shanghao; Peng, Zhili; Dallman, Julia; Baker, James; Othman, Abdelhameed M; Blackwelder, Patrica L; Leblanc, Roger M

    2016-09-01

    Drug delivery to the central nervous system (CNS) in biological systems remains a major medical challenge due to the tight junctions between endothelial cells known as the blood-brain-barrier (BBB). Here we use a zebrafish model to explore the possibility of using transferrin-conjugated carbon dots (C-Dots) to ferry compounds across the BBB. C-Dots have previously been reported to inhibit protein fibrillation, and they are also used to deliver drugs for disease treatment. In terms of the potential medical application of C-Dots for the treatment of CNS diseases, one of the most formidable challenges is how to deliver them inside the CNS. To achieve this in this study, human transferrin was covalently conjugated to C-Dots. The conjugates were then injected into the vasculature of zebrafish to examine the possibility of crossing the BBB in vivo via transferrin receptor-mediated endocytosis. The experimental observations suggest that the transferrin-C-Dots can enter the CNS while C-Dots alone cannot.

  14. CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Madsen, Mette; Moestrup, Søren K

    2002-01-01

    CD163 is a highly expressed macrophage membrane protein belonging to the scavenger receptor cysteine rich (SRCR) domain family. The CD163 expression is induced by interleukin-6, interleukin-10 and glucocorticoids. Its function has remained unknown until recently when CD163 was identified...... hemolysis. Besides having a detoxificating effect by removing Hb from plasma, the CD163-mediated endocytosis of the Hp-Hb complex may represent a major pathway for uptake of iron in the tissue macrophages. The novel functional linkage of CD163 and Hp, which both are induced during inflammation, also reveal...... as the endocytic receptor binding hemoglobin (Hb) in complex with the plasma protein haptoglobin (Hp). This specific receptor-ligand interaction leading to removal from plasma of the Hp-Hb complex-but not free Hp or Hb-now explains the depletion of circulating Hp in individuals with increased intravascular...

  15. Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling.

    Science.gov (United States)

    You, Changjiang; Marquez-Lago, Tatiana T; Richter, Christian Paolo; Wilmes, Stephan; Moraga, Ignacio; Garcia, K Christopher; Leier, André; Piehler, Jacob

    2016-12-01

    The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane.

  16. Role of the p70 S6 kinase cascade in neutrophilic differentiation and proliferation of HL-60 cells-a study of transferrin receptor-positive and -negative cells obtained from dimethyl sulfoxide- or retinoic acid-treated HL-60 cells.

    Science.gov (United States)

    Kanayasu-Toyoda, Toshie; Yamaguchi, Teruhide; Oshizawa, Tadashi; Kogi, Mieko; Uchida, Eriko; Hayakawa, Takao

    2002-09-01

    Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.

  17. 21 CFR 866.5880 - Transferrin immunological test system.

    Science.gov (United States)

    2010-04-01

    ... transferrin levels aids in the diagnosis of malnutrition, acute inflammation, infection, and red blood cell disorders, such as iron deficiency anemia. (b) Classification. Class II (performance standards)....

  18. Why Do Some T Cell Receptor Cytoplasmic Domains Associate with the Plasma Membrane?

    OpenAIRE

    Philip Anton evan der Merwe; Hao eZhang; Shaun-Paul eCordoba

    2012-01-01

    Based on studies in model systems it has been proposed that the cytoplasmic domains of T cell receptor signaling subunits that have polybasic motifs associate with the plasma membrane, and that this regulates their phosphorylation. Recent experiments in more physiological systems have confirmed membrane association but raised questions as to its function.

  19. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.;

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains...... in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...

  20. Auxiliary Subunits: Shepherding AMPA Receptors to the Plasma Membrane

    Directory of Open Access Journals (Sweden)

    Simon C. Haering

    2014-08-01

    Full Text Available Ionotropic glutamate receptors (iGluRs are tetrameric ligand-gated cation channels that mediate excitatory signal transmission in the central nervous system (CNS of vertebrates. The members of the iGluR subfamily of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptors (AMPARs mediate most of the fast excitatory signal transmission, and their abundance in the postsynaptic membrane is a major determinant of the strength of excitatory synapses. Therefore, regulation of AMPAR trafficking to the postsynaptic membrane is an important constituent of mechanisms involved in learning and memory formation, such as long-term potentiation (LTP and long-term depression (LTD. Auxiliary subunits play a critical role in the facilitation and regulation of AMPAR trafficking and function. The currently identified auxiliary subunits of AMPARs are transmembrane AMPA receptor regulatory proteins (TARPs, suppressor of lurcher (SOL, cornichon homologues (CNIHs, synapse differentiation-induced gene I (SynDIG I, cysteine-knot AMPAR modulating proteins 44 (CKAMP44, and germ cell-specific gene 1-like (GSG1L protein. In this review we summarize our current knowledge of the modulatory influence exerted by these important but still underappreciated proteins.

  1. Effects of PhD examination stress on allopregnanolone and cortisol plasma levels and peripheral benzodiazepine receptor density.

    NARCIS (Netherlands)

    Droogleever Fortuyn, H.A.; Broekhoven, F. van; Span, P.N.; Backstrom, T.; Zitman, F.G.; Verkes, R.J.

    2004-01-01

    Peripheral benzodiazepine receptor (PBR) density in blood platelets and plasma allopregnanolone concentration in humans were determined following acute stress as represented by PhD examination. Fifteen healthy PhD students participated. Heart rate, blood pressure, plasma allopregnanolone, plasma cor

  2. Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

    Science.gov (United States)

    Koldsø, Heidi; Sansom, Mark S P

    2015-11-25

    The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

  3. Elevated transferrin saturation and risk of diabetes

    DEFF Research Database (Denmark)

    Ellervik, Christina; Mandrup-Poulsen, Thomas; Andersen, Henrik Ullits;

    2011-01-01

    OBJECTIVE We tested the hypothesis that elevated transferrin saturation is associated with an increased risk of any form of diabetes, as well as type 1 or type 2 diabetes separately. RESEARCH DESIGN AND METHODS We used two general population studies, The Copenhagen City Heart Study (CCHS, N = 9......,121) and The Copenhagen General Population Study (CGPS, N = 24,195), as well as a 1:1 age- and sex-matched population-based case-control study with 6,129 patients with diabetes from the Steno Diabetes Centre and 6,129 control subjects, totaling 8,535 patients with diabetes and 37,039 control subjects. RESULTS...

  4. Lapatinib Plasma and Tumor Concentrations and Effects on HER Receptor Phosphorylation in Tumor.

    Directory of Open Access Journals (Sweden)

    Neil L Spector

    Full Text Available The paradigm shift in cancer treatment from cytotoxic drugs to tumor targeted therapies poses new challenges, including optimization of dose and schedule based on a biologically effective dose, rather than the historical maximum tolerated dose. Optimal dosing is currently determined using concentrations of tyrosine kinase inhibitors in plasma as a surrogate for tumor concentrations. To examine this plasma-tumor relationship, we explored the association between lapatinib levels in tumor and plasma in mice and humans, and those effects on phosphorylation of human epidermal growth factor receptors (HER in human tumors.Mice bearing BT474 HER2+ human breast cancer xenografts were dosed once or twice daily (BID with lapatinib. Drug concentrations were measured in blood, tumor, liver, and kidney. In a randomized phase I clinical trial, 28 treatment-naïve female patients with early stage HER2+ breast cancer received lapatinib 1000 or 1500 mg once daily (QD or 500 mg BID before evaluating steady-state lapatinib levels in plasma and tumor.In mice, lapatinib levels were 4-fold higher in tumor than blood with a 4-fold longer half-life. Tumor concentrations exceeded the in vitro IC90 (~ 900 nM or 500 ng/mL for inhibition of HER2 phosphorylation throughout the 12-hour dosing interval. In patients, tumor levels were 6- and 10-fold higher with QD and BID dosing, respectively, compared to plasma trough levels. The relationship between tumor and plasma concentration was complex, indicating multiple determinants. HER receptor phosphorylation varied depending upon lapatinib tumor concentrations, suggestive of changes in the repertoire of HER homo- and heterodimers.Plasma lapatinib concentrations underestimated tumor drug levels, suggesting that optimal dosing should be focused on the site of action to avoid to inappropriate dose escalation. Larger clinical trials are required to determine optimal dose and schedule to achieve tumor concentrations that maximally

  5. Sperm-Egg Interaction: Evidence for Boar Sperm Plasma Membrane Receptors for Porcine Zona Pellucida

    Science.gov (United States)

    Peterson, Rudolph N.; Russell, Lonnie; Bundman, Donna; Freund, Matthew

    1980-01-01

    Freshly ejaculated, noncapacitated boar sperm bind rapidly and in large numbers to pig egg zona pellucida in vitro. In the present study, the number of sperm bound decreased sharply when sperm motility was lowered by energy poisons or by reducing the temperature. Highly motile sperm from humans, guinea pigs, and rats, added at concentrations ten times higher than control sperm, did not bind to the porcine zona. At the same high concentration, a small number of hamster and bull sperm bound to the zona. Binding of boar sperm to the zona pellucida was blocked almost completely by diluted whole antiserum to sperm plasma membranes and by univalent (Fab) antibody to these membranes. When antibody to sperm plasma membrane was first absorbed with plasma membrane vesicles, sperm binding was not inhibited. These results provide direct evidence for the existence of sperm plasma membrane receptors for the zona pellucida of the pig.

  6. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

    2015-01-01

    in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...... accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features...

  7. Carbohydrate deficient transferrin (CDT) in alcoholic cirrhosis: a kinetic study

    DEFF Research Database (Denmark)

    Henriksen, Jens Henrik Sahl; Grønbaek, M; Møller, Søren

    1997-01-01

    concentration than controls with a low alcohol intake (detected between carbohydrate deficient transferrin in artery and liver vein or artery and renal vein, either in patients with alcoholic cirrhosis (n = 11) or in controls (n = 8......BACKGROUND/AIMS: Carbohydrate deficient transferrin has been introduced as a marker of excessive alcohol intake. The present study was undertaken in order to measure the circulating level of carbohydrate deficient transferrin in patients with alcoholic cirrhosis and to assess arteriovenous kinetics...... of carbohydrate deficient transferrin in liver and kidney. METHODS/RESULTS: The median value of serum carbohydrate deficient transferrin was 16.0 U/l in patients with alcoholic cirrhosis (n = 41), and this value was not significantly different from that of a normal control group (median 17.4 U/l, n = 55, ns...

  8. Plasma Membrane and Nuclear Localization of G Protein–coupled Receptor Kinase 6A

    OpenAIRE

    Jiang, Xiaoshan; Benovic, Jeffrey L.; Wedegaertner, Philip B.

    2007-01-01

    G protein–coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucl...

  9. Plasma soluble (pro)renin receptor is independent of plasma renin, prorenin, and aldosterone concentrations but is affected by ethnicity.

    Science.gov (United States)

    Nguyen, Geneviève; Blanchard, Anne; Curis, Emmanuel; Bergerot, Damien; Chambon, Yann; Hirose, Takuo; Caumont-Prim, Aurore; Tabard, Sylvie Brailly; Baron, Stéphanie; Frank, Michael; Totsune, Kazuhito; Azizi, Michel

    2014-02-01

    A soluble (pro)renin receptor (sPRR) circulates in plasma and is able to bind renin and prorenin. It is not known whether plasma sPRR concentrations vary with the activity of the renin-angiotensin system. We measured plasma sPRR, renin, prorenin, and aldosterone concentrations in 121 white and 9 black healthy subjects, 40 patients with diabetes mellitus, 41 hypertensive patients with or without renin-angiotensin system blockers, 9 patients with primary aldosteronism, and 10 patients with Gitelman syndrome. Median physiological plasma sPRR concentration was 23.5 ng/mL (interquartile range, 20.9-26.5) under usual uncontrolled sodium diet. sPRR concentration in healthy subjects, unlike renin and prorenin, did not display circadian variation or dependence on age, sex, posture, or hormonal status. sPRR concentrations were ≈25% lower in black than in white subjects, whereas renin concentrations were ≈40% lower. Patients with diabetes mellitus (average renin-high prorenin levels) and with hypertension only (average renin-average prorenin levels) had sPRR concentrations similar to healthy subjects. Renin-angiotensin system blockade was associated with increase of sPRR concentration by ≈12%. sPRR in patients with primary aldosteronism (low renin-low prorenin) and Gitelman syndrome (high renin-high prorenin) were similar and ≈10% higher than in healthy subjects. There was no correlation between sPRR and renin or prorenin. In conclusion, our results show that plasma sPRR concentrations are dependent on ethnicity and independent of renin, prorenin, and aldosterone concentrations in healthy subjects and in patients with contrasted degrees of renin-angiotensin system activity.

  10. Plasma radioiron kinetics in man: explanation for the effect of plasma iron concentration.

    Science.gov (United States)

    Skarberg, K; Eng, M; Huebers, H; Marsaglia, G; Finch, C

    1978-03-01

    The plasma iron turnover was measured in 19 normal subjects. A correlation was found between plasma iron concentration and plasma iron turnover. In addition to the turnover of 55Fe at normal plasma iron concentration (predominantly monoferric transferrin), a second turnover in which the labeled plasma was saturated with iron (to produce predominantly diferric transferrin) was studied with 50Fe. It was demonstrated that diferric transferrin had a greater rate of iron turnover but that the distribution between erythroid and non-erythroid tissues was unchanged. It was concluded that plasma iron turnover is dependent on the monoferric/diferric transferrin ratio in the plasma but that the internal distribution of iron is unaffected.

  11. Hypertension increases urinary excretion of immunoglobulin G, ceruloplasmin and transferrin in normoalbuminuric patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Ohara, Nobumasa; Hanyu, Osamu; Hirayama, Satoshi; Nakagawa, Osamu; Aizawa, Yoshifusa; Ito, Seiki; Sone, Hirohito

    2014-02-01

    Increased urinary excretion of certain plasma proteins, such as immunoglobulin G (IgG), ceruloplasmin and transferrin, with different molecular radii of 55 Å or less and different isoelectric points have been reported to precede development of microalbuminuria in patients who have diabetes mellitus with hypertension. We examined how hypertension affects these urinary proteins in a diabetic state. Excretion of IgG, ceruloplasmin, transferrin, albumin, α2-macroglobulin with a large molecular radius of 88 Å and N-acetylglucosaminidase in first-morning urine samples were measured in normoalbuminuric patients (urinary albumin-to-creatinine ratio hypertension and nondiabetes mellitus (group hypertension, n = 32), type 2 diabetes mellitus and normotension (group diabetes mellitus, n = 52) and type 2 diabetes mellitus and hypertension (group Both, n =45), and in age-matched controls (n = 72). Urinary IgG, ceruloplasmin, transferrin, albumin and N-acetylglucosaminidase and estimated glomerular filtration rate (eGFR) were significantly elevated in groups diabetes mellitus and Both compared with controls. Furthermore, urinary IgG, ceruloplasmin and transferrin in group Both were significantly higher than those in group diabetes mellitus. These exhibited a positive and relatively strong association with eGFR compared with controls. No significant difference in urinary albumin or N-acetylglucosaminidase was found between the two diabetic groups. In contrast, group hypertension had elevated urinary transferrin without any changes in the other compounds. Urinary α2-macroglobulin did not differ among the four groups. These findings suggest that normoalbuminuric diabetic patients without hypertension have both glomerular hemodynamic changes such as increased intraglomerular hydraulic pressure and altered proximal tubules, and that hypertension increases intraglomerular hydraulic pressure. Increased urinary IgG, ceruloplasmin and transferrin may reflect an increase in

  12. A model for the biosynthesis and transport of plasma membrane-associated signaling receptors to the cell surface

    Directory of Open Access Journals (Sweden)

    Sorina Claudia Popescu

    2012-04-01

    Full Text Available Intracellular protein transport is emerging as critical in determining the outcome of receptor-activated signal transduction pathways. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating receptor synthesis and transport to the cell surface. Recent advances in this field indicate that signaling pathways and intracellular transport machinery converge and coordinate to render receptors competent for signaling at their plasma membrane activity sites. The biogenesis and transport to the cell surface of signaling receptors appears to require both general trafficking and receptor-specific factors. Several molecular determinants, residing or associated with compartments of the secretory pathway and known to influence aspects in receptor biogenesis, are discussed and integrated into a predictive cooperative model for the functional expression of signaling receptors at the plasma membrane.

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

    Science.gov (United States)

    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.

  14. Monitoring pulmonary vascular permeability using radiolabeled transferrin

    Energy Technology Data Exchange (ETDEWEB)

    Basran, G.S.; Hardy, J.G.

    1988-07-01

    A simple, noninvasive technique for monitoring pulmonary vascular permeability in patients in critical care units is discussed. High vascular permeability is observed in patients with clinically defined adult respiratory distress syndrome (ARDS) but not in patients with hydrostatic pulmonary edema or in patients with minor pulmonary insults who are considered to be at risk of developing ARDS. The technique has been used in the field of therapeutics and pharmacology to test the effects of the putative antipermeability agents methylprednisolone and terbutaline sulfate. There appears to be a good correlation between the acute inhibitory effect of either drug on transferrin exudation and patient prognosis. Thus, a byproduct of such drug studies may be an index of survival in patients with established ARDS.

  15. Ceruloplasmin/Transferrin Ratio Changes in Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    Rosanna Squitti

    2011-01-01

    Full Text Available The link between iron and Alzheimer's disease (AD has been mainly investigated with a focus on the local accumulation of this metal in specific areas of the brain that are critical for AD. In the present study, we have instead looked at systemic variations of markers of iron metabolism. We measured serum levels of iron, ceruloplasmin, and transferrin and calculated the transferrin saturation and the ceruloplasmin to transferrin ratio (Cp/Tf. Cp/Tf and transferrin saturation increased in AD patients. Cp/Tf ratios also correlated positively with peroxide levels and negatively with serum iron concentrations. Elevated values of ceruloplasmin, peroxides, and Cp/Tf inversely correlated with MMSE scores. Isolated medial temporal lobe atrophy positively correlated with Cp/Tf and negatively with serum iron. All these findings indicate that the local iron accumulation found in brain areas critical for AD should be viewed in the frame of iron systemic alterations.

  16. Binding and endocytosis of monoterbium transferrin by K562 cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Using isotopic labeling of human serum apotransferrin, the binding and the endocytosis of monoterbium transferrin (TbC-apotransferrin, TbC-apotransferrin- FeN) by K562 cells, a human leukemic cell line, have been investigated. There are about (8.58±2.41)×105 binding sites per cell surface at 0℃. The association constant for TbC-apo- transferrin binding is 4.1×107 mol-1@L, for TbC-apo- transferrin-FeN 2.7×107 mol-1@L at 0℃. At pH 7.4, upon warming cells to 37℃, endocytosis starts. The rate constants for the endocytosis are about 0.97 min-1 and 0.31 min-1 and the endocytosis ratio reaches 56% and 80% for TbC-apo- transferrin and TbC-apotransferrin-FeN, respectively.

  17. Assessing Iron Status: Beyond Serum Ferritin and Transferrin Saturation

    National Research Council Canada - National Science Library

    Jay B. Wish

    2006-01-01

    The increasing prevalence of multiple comorbidities among anemic patients with chronic kidney disease has made the use of serum ferritin and transferrin saturation more challenging in diagnosing iron deficiency...

  18. Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia

    OpenAIRE

    Kozyraki, Renata; Fyfe, John; Verroust, Pierre J.; Jacobsen, Christian; Dautry-Varsat, Alice; Gburek, Jakub; Willnow, Thomas E.; Christensen, Erik Ilsø; Søren K. Moestrup

    2001-01-01

    Cubilin is a 460-kDa protein functioning as an endocytic receptor for intrinsic factor vitamin B12 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 stro...

  19. Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

    Science.gov (United States)

    Richter, David; Moraga, Ignacio; Winkelmann, Hauke; Birkholz, Oliver; Wilmes, Stephan; Schulte, Markos; Kraich, Michael; Kenneweg, Hella; Beutel, Oliver; Selenschik, Philipp; Paterok, Dirk; Gavutis, Martynas; Schmidt, Thomas; Garcia, K. Christopher; Müller, Thomas D.; Piehler, Jacob

    2017-07-01

    The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

  20. Impact of Lipid Composition and Receptor Conformation on the Spatio-temporal Organization of μ-Opioid Receptors in a Multi-component Plasma Membrane Model.

    Science.gov (United States)

    Marino, Kristen A; Prada-Gracia, Diego; Provasi, Davide; Filizola, Marta

    2016-12-01

    The lipid composition of cell membranes has increasingly been recognized as playing an important role in the function of various membrane proteins, including G Protein-Coupled Receptors (GPCRs). For instance, experimental and computational evidence has pointed to lipids influencing receptor oligomerization directly, by physically interacting with the receptor, and/or indirectly, by altering the bulk properties of the membrane. While the exact role of oligomerization in the function of class A GPCRs such as the μ-opioid receptor (MOR) is still unclear, insight as to how these receptors oligomerize and the relevance of the lipid environment to this phenomenon is crucial to our understanding of receptor function. To examine the effect of lipids and different MOR conformations on receptor oligomerization we carried out extensive coarse-grained molecular dynamics simulations of crystal structures of inactive and/or activated MOR embedded in an idealized mammalian plasma membrane composed of 63 lipid types asymmetrically distributed across the two leaflets. The results of these simulations point, for the first time, to specific direct and indirect effects of the lipids, as well as the receptor conformation, on the spatio-temporal organization of MOR in the plasma membrane. While sphingomyelin-rich, high-order lipid regions near certain transmembrane (TM) helices of MOR induce an effective long-range attractive force on individual protomers, both long-range lipid order and interface formation are found to be conformation dependent, with a larger number of different interfaces formed by inactive MOR compared to active MOR.

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

    OpenAIRE

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

    2007-01-01

    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. ...

  2. Glucagon and plasma catecholamines during beta-receptor blockade in exercising man

    DEFF Research Database (Denmark)

    Galbo, H; Holst, Janett; Christensen, N J

    1976-01-01

    Seven men ran at 60% of individual maximal oxygen uptake to exhaustion during beta-adrenergic blockade with propranolol (P), during lipolytic blockade with nicotinic acid (N), or without drugs (C). The total work times (83 +/- 9 (P), 122 +/- 8 (N), 166 +/- 10 (C) min, mean and SE) differed...... decrease glucagon concentrations increased progressively in parallel with declining plasma glucose and were at exhaustion always three times preexercise values. Thus beta-adrenergic blockade did not diminish the glucagon response. Nor was this response increased when alpha-receptor stimulation in P...... experiments was intensified. Carbohydrate combustion was smaller and NEFA and glycerol concentrations in serum larger during C experiments. Alanine concentrations were never raised at exhaustion. Accordingly, neither stimulation of adrenergic receptors nor NEFA and alanine concentrations are major...

  3. Three family members with elevated plasma cobalamin, transcobalamin and soluble transcobalamin receptor (sCD320)

    DEFF Research Database (Denmark)

    Hoffmann-Lücke, Elke; Arendt, Johan F B; Nissen, Peter H

    2013-01-01

    BACKGROUND: Plasma cobalamin is requested in order to diagnose cobalamin deficiency and low levels confirm a deficient state. Here, we present three family members with unexpected high levels of cobalamin. METHODS: We included a patient referred for cobalamin measurement due to neurological...... transcobalamin and part of sCD320. CONCLUSIONS: The high cobalamin levels were mainly explained by high levels of holoTC, possibly caused by complex formation with its soluble receptor, sCD320. The family occurrence points to a genetic explanation....

  4. Relationship between alpha-1 receptors and cations in rat liver plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Smart, J.L.

    1986-01-01

    The influence of cations on binding of (/sup 3/H)-prazosin (PRZ), an alpha-1 specific antagonist, to alpha receptor sites in rat liver plasma membranes was examined. All cations tested were able to produce dose-dependent shifts to lower affinity binding sites for PRZ. The maximum number of binding sites was also observed to be altered. Inclusion of cations resulted in a slower observed rate constant for association as well as a delay in the dissociation of specifically bound PRZ following the addition of phentolamine. In contrast, the ability of (-)-norepinephrine to displace PRZ was enhanced by the addition of cations. The influence of alpha-1 receptor stimulation on Na/sup +//K/sup +/-ATPase activity in rat liver was examined by two methods - rat liver plasma membrane Na/sup +//K/sup +/-ATPase activity following liver perfusion in situ and /sup 86/Tb uptake in rat liver slices. The activity of the Na/sup +/ pump was found to be biphasic following exposure to phenylephrine (PE), an alpha-1 agonist. Stimulation (35%) was present over the first two minutes, while activity was inhibited over the interval of 5 to 10 minutes of continued PE exposure. Both phases were blocked by prazosin. The influence of DAG and protein kinase C (PKC) in alpha-1 receptor modulation of the Na/sup +/ pump was studied by employing 4-beta-phorbol (PMA), a phorbol ester which activates PKC. Perfusion of livers with PMA in situ or incubation with slices yielded inhibition of ATPase activity in membranes and /sup 86/Rb uptake in that was qualitatively and quantitatively similar to PE. These results suggest cations may influence receptor function in vivo and in vitro and the inhibitory effects of PE on the sodium pump may be mediated through PKC.

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

    Science.gov (United States)

    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.

  6. TTP specifically regulates the internalization of the transferrin receptor

    DEFF Research Database (Denmark)

    Tosoni, Daniela; Puri, Claudia; Confalonieri, Stefano

    2005-01-01

    with endocytic proteins, including clathrin, dynamin, and the TfR, and localizes selectively to TfR-containing coated-pits (CCP) and -vesicles (CCV). Overexpression of TTP specifically inhibits TfR internalization, and causes the formation of morphologically aberrant CCP, which are probably fission impaired....... This effect is mediated by the SH3 of TTP, which can bind to dynamin, and it is rescued by overexpression of dynamin. Functional ablation of TTP causes a reduction in TfR internalization, and reduced cargo loading and size of TfR-CCV. Tyrosine phosphorylation of either TTP or dynamin prevents...

  7. Association between serum transferrin receptor levels and malaria ...

    African Journals Online (AJOL)

    user

    This study was conducted with financial support from the MRTP under the HRSA (US National. Institutes of ... American Journal of Tropical Medicine and Hygiene 68: 121-127. Hopkins ... Cochrane Database Systematic Review 10:CD006589.

  8. Plasma levels of soluble tumour necrosis factor receptors are increased in coal miners with pneumoconiosis.

    Science.gov (United States)

    Schins, R P; Borm, P J

    1995-10-01

    Among other cytokines, tumour necrosis factor (TNF)-alpha is considered to play a key role in the development of mineral dust related fibrosis. Previously, we showed that ex-vivo release of TNF by peripheral blood monocytes is a marker for progression of coal workers' pneumoconiosis (CWP). Since soluble TNF receptors (sTNF-Rs) are believed to play an important regulatory role in systemic effects of TNF, we measured plasma levels of sTNF-R55 and sTNF-R75 in coal miners with (n = 28) or without (n = 76) CWP and in nonexposed controls (n = 29). sTNF-R75 levels were significantly increased in miners with CWP (2.09 +/- 0.44 ng.mL-1) versus the nonexposed controls (1.86 +/- 0.23 ng.mL-1). Neither sTNF-R55 nor sTNF-R75 were related to exposure, stage of pneumoconiosis, smoking, or (spontaneous or ex-vivo induced) monocyte TNF-release. sTNF-R55 was increased in subjects with medication (especially those using cardiovascular drugs); upon exclusion of these subjects, sTNF-R55 was found also to be significantly increased in CWP. In conclusion, bearing in mind a confounding effect of medication, soluble TNF receptors are elevated in plasma of retired miners with coal workers' pneumoconiosis. These observations further support the important role of TNF-mediated pathways in the pathogenesis of mineral dust related fibrosis.

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

    Science.gov (United States)

    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.

  10. Manganese transport via the transferrin mechanism.

    Science.gov (United States)

    Gunter, Thomas E; Gerstner, Brent; Gunter, Karlene K; Malecki, Jon; Gelein, Robert; Valentine, William M; Aschner, Michael; Yule, David I

    2013-01-01

    Excessive manganese (Mn) uptake by brain cells, particularly in regions like the basal ganglia, can lead to toxicity. Mn(2+) is transported into cells via a number of mechanisms, while Mn(3+) is believed to be transported similarly to iron (Fe) via the transferrin (Tf) mechanism. Cellular Mn uptake is therefore determined by the activity of the mechanisms transporting Mn into each type of cell and by the amounts of Mn(2+), Mn(3+) and their complexes to which these cells are exposed; this complicates understanding the contributions of each transporter to Mn toxicity. While uptake of Fe(3+) via the Tf mechanism is well understood, uptake of Mn(3+) via this mechanism has not been systematically studied. The stability of the Mn(3+)Tf complex allowed us to form and purify this complex and label it with a fluorescent (Alexa green) tag. Using purified and labeled Mn(3+)Tf and biophysical tools, we have developed a novel approach to study Mn(3+)Tf transport independently of other Mn transport mechanisms. This approach was used to compare the uptake of Mn(3+)Tf into neuronal cell lines with published descriptions of Fe(3+) uptake via the Tf mechanism, and to obtain quantitative information on Mn uptake via the Tf mechanism. Results confirm that in these cell lines significant Mn(3+) is transported by the Tf mechanism similarly to Fe(3+)Tf transport; although Mn(3+)Tf transport is markedly slower than other Mn transport mechanisms. This novel approach may prove useful for studying Mn toxicity in other systems and cell types.

  11. Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

    Energy Technology Data Exchange (ETDEWEB)

    Feltner, D.E.; Marasco, W.A.

    1989-06-01

    The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of (3H)FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM (3H)FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. (3H)FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of (3H)FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM (3H)FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.

  12. Plasma extravasation mediated by lipopolysaccharide-induction of kinin B1 receptors in rat tissues

    Directory of Open Access Journals (Sweden)

    Paulo Roberto Wille

    2001-01-01

    Full Text Available The present study was performed to: (a evaluate the effects of kinin B1 (Sar{D-Phe8}-des-Arg9-BK; 10 nmol/kg and B2 (bradykinin (BK; 10 nmol/kg receptor agonists on plasma extravasation in selected rat tissues; (b determine the contribution of a lipopolysaccharide (LPS (100 μ g/kg to the effects triggered by B1 and B2 agonists; and (c characterize the selectivity of B1 ({Leu8}desArg9-BK; 10 nmol/kg and B2 (HOE 140; 10 nmol/kg antagonists as inhibitors of this kinin-induced phenomenon. B1 and B2 agonists were shown to increase plasma extravasation in the duodenum, ileum and also in the urinary bladder of the rat. LPS pretreatment enhanced the plasma extravasation mediated only by the B1 agonist in the duodenum, ileum, trachea, main and segmentar bronchi. These effects were prevented by the B1. but not the B2 antagonist. In normal rats, the B2 antagonist inhibited the effect of B2 agonist in all the tissues analyzed. However, in LPS-treated rats, the B2 antagonist was ineffective in the urinary bladder.

  13. γ-secretase directly sheds the survival receptor BCMA from plasma cells

    Science.gov (United States)

    Laurent, Sarah A.; Hoffmann, Franziska S.; Kuhn, Peer-Hendrik; Cheng, Qingyu; Chu, Yuanyuan; Schmidt-Supprian, Marc; Hauck, Stefanie M.; Schuh, Elisabeth; Krumbholz, Markus; Rübsamen, Heike; Wanngren, Johanna; Khademi, Mohsen; Olsson, Tomas; Alexander, Tobias; Hiepe, Falk; Pfister, Hans-Walter; Weber, Frank; Jenne, Dieter; Wekerle, Hartmut; Hohlfeld, Reinhard; Lichtenthaler, Stefan F.; Meinl, Edgar

    2015-01-01

    Survival of plasma cells is regulated by B-cell maturation antigen (BCMA), a membrane-bound receptor activated by its agonist ligands BAFF and APRIL. Here we report that γ-secretase directly cleaves BCMA, without prior truncation by another protease. This direct shedding is facilitated by the short length of BCMA's extracellular domain. In vitro, γ-secretase reduces BCMA-mediated NF-κB activation. In addition, γ-secretase releases soluble BCMA (sBCMA) that acts as a decoy neutralizing APRIL. In vivo, inhibition of γ-secretase enhances BCMA surface expression in plasma cells and increases their number in the bone marrow. Furthermore, in multiple sclerosis, sBCMA levels in spinal fluid are elevated and associated with intracerebral IgG production; in systemic lupus erythematosus, sBCMA levels in serum are elevated and correlate with disease activity. Together, shedding of BCMA by γ-secretase controls plasma cells in the bone marrow and yields a potential biomarker for B-cell involvement in human autoimmune diseases. PMID:26065893

  14. Receptor-like activity evoked by extracellular ADP in Arabidopsis root epidermal plasma membrane.

    Science.gov (United States)

    Demidchik, Vadim; Shang, Zhonglin; Shin, Ryoung; Colaço, Renato; Laohavisit, Anuphon; Shabala, Sergey; Davies, Julia M

    2011-07-01

    Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca(2+) influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca(2+) in root epidermal protoplasts. An inward Ca(2+)-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca(2+) efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM.

  15. Receptor-Like Activity Evoked by Extracellular ADP in Arabidopsis Root Epidermal Plasma Membrane1

    Science.gov (United States)

    Demidchik, Vadim; Shang, Zhonglin; Shin, Ryoung; Colaço, Renato; Laohavisit, Anuphon; Shabala, Sergey; Davies, Julia M.

    2011-01-01

    Extracellular purine nucleotides are implicated in the control of plant development and stress responses. While extracellular ATP is known to activate transcriptional pathways via plasma membrane (PM) NADPH oxidase and calcium channel activation, very little is known about signal transduction by extracellular ADP. Here, extracellular ADP was found to activate net Ca2+ influx in roots of Arabidopsis (Arabidopsis thaliana) and transiently elevate cytosolic free Ca2+ in root epidermal protoplasts. An inward Ca2+-permeable conductance in root epidermal PM was activated within 1 s of ADP application and repeated application evoked a smaller current. Such response speed and densitization are consistent with operation of equivalents to animal ionotropic purine receptors, although to date no equivalent genes for such receptors have been identified in higher plants. In contrast to ATP, extracellular ADP did not evoke accumulation of intracellular reactive oxygen species. While high concentrations of ATP caused net Ca2+ efflux from roots, equivalent concentrations of ADP caused net influx. Overall the results point to a discrete ADP signaling pathway, reliant on receptor-like activity at the PM. PMID:21562328

  16. Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization

    DEFF Research Database (Denmark)

    Torrisi, M R; Lotti, L V; Belleudi, F;

    1999-01-01

    Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential...... role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments...... of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment...

  17. Soluble urokinase receptor levels in plasma during 5 years of highly active antiretroviral therapy in HIV-1-infected patients

    DEFF Research Database (Denmark)

    Ostrowski, Sisse R; Katzenstein, Terese L; Piironen, Timo;

    2004-01-01

    High blood levels of the soluble urokinase receptor (suPAR) strongly predict increased mortality in human immunodeficiency virus-1 (HIV-1)-infected patients. This study investigated the plasma concentration of suPAR in 29 treatment-naive HIV-1-infected patients during 5 years treatment with highly...... active antiretroviral therapy (HAART). Plasma suPAR decreased after introducing HAART, most pronounced during the first treatment year. The change in plasma suPAR was independent of changes in viral replication and CD4+ cells but it was strongly correlated with plasma levels of the soluble TNF receptor...... is linked to inflammation in untreated as well as HAART-treated HIV-1-infected patients....

  18. Seminal plasma proteomic and gene expression and ngf location and receptors (TRK1 and NGFR) in rabbit genital system

    OpenAIRE

    JÃsy Maria Arruda de Alencar

    2015-01-01

    The aim this study were (i) to map and identify proteins in seminal plasma of New Zealand white rabbits strain, using the techniques of two-dimensional electrophoresis coupled to mass spectrometry and to estimate associations between these proteins with sperm parameters and (ii) characterize expression of the nerve growth factor polypeptide beta ( -NGF) and its cognate neurotrophic receptor tyrosine kinase type 1 (NTRK1), and nerve growth factor receptor (NGFR) at gonads and sex glands in adu...

  19. Plasma adiponectin concentration is associated with skeletal muscle insulin receptor tyrosine phosphorylation, and low plasma concentration precedes a decrease in whole-body insulin sensitivity in humans

    DEFF Research Database (Denmark)

    Stefan, Norbert; Vozarova, Barbora; Funahashi, Tohru;

    2002-01-01

    -induced tyrosine phosphorylation of the insulin receptor (IR) and also increase whole-body insulin sensitivity. To further characterize the relationship between plasma adiponectin concentration and insulin sensitivity in humans, we examined 1) the cross-sectional association between plasma adiponectin......Adiponectin, the most abundant adipose-specific protein, has been found to be negatively associated with degree of adiposity and positively associated with insulin sensitivity in Pima Indians and other populations. Moreover, adiponectin administration to rodents has been shown to increase insulin...... concentration and skeletal muscle IR tyrosine phosphorylation and 2) the prospective effect of plasma adiponectin concentration at baseline on change in insulin sensitivity. Fasting plasma adiponectin concentration, body composition (hydrodensitometry or dual energy X-ray absorptiometry), insulin sensitivity...

  20. Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

    Directory of Open Access Journals (Sweden)

    Sylvie Faucher

    Full Text Available BACKGROUND: IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Ralpha (CD127 and common gamma (CD132 chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127 is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults. METHODOLOGY/PRINCIPAL FINDINGS: We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay's range was 3.2-1000 ng/mL. The concentration of plasma sCD127 was 164+/-104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year. CONCLUSIONS/SIGNIFICANCE: This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.

  1. Transferrin microheterogeneity in rheumatoid arthritis - Relation with disease activity and anemia of chronic disease

    NARCIS (Netherlands)

    R.A. Feelders (Richard); G. Vreugdenhil (Gerard); G. de Jong (G.); A.J.G. Swaak (Antonius); H.G. van Eijk (Henk)

    1992-01-01

    textabstractWe studied the relation between disease activity in rheumatoid arthritis (RA) and the microheterogeneity of transferrin. Using crossed immuno isoelectric focusing, transferrin microheterogeneity patterns were analyzed in sera of healthy individuals, nonanemic RA patients, iron deficient

  2. Partial characterization of transferrins of catfish (Silurus glanis L.) and pike (Esox lucius L.).

    Science.gov (United States)

    Stratil, A; Tomásek, V; Clamp, J R; Williams, J

    1985-01-01

    Basic composition and properties of isolated transferrins of Silurus glanis and Esox lucius have been compared. In transferrin of S. glanis carbohydrate is absent, but it is present in transferrin of E. lucius (2.5%). The N-terminal amino acid is alanine in both species. Mol. wts are 68,400 (S. glanis) and 86,800 (E. lucius). Transferrins of the two species are heterogeneous, but genetic polymorphism was not observed.

  3. Study of the interaction of trivalent actinide and lanthanide ions with human serum transferrin by means of time-resolved laser-fluorescence spectroscopy; Untersuchung der Wechselwirkung trivalenter Actinid- und Lanthanidionen mit humanem Serumtransferrin mittels zeitaufgeloester Laserfluoreszenzspektroskopie

    Energy Technology Data Exchange (ETDEWEB)

    Bauer, Nicole

    2015-04-27

    In the present work the complexation of Cm(III), Eu(III) and Am(III) with human serum transferrin is studied. The aim of this work was the identification and the spectroscopic and thermodynamic characterization of An(III) and Ln(III) transferrin complex species. Different speciation methods, such as time-resolved laser fluorescence spectroscopy (TRLFS), luminescence spectroscopy and EXAFS (Extended X-Ray Absorption Fine Structure) spectroscopy were applied. Using TRLFS two unambiguously different Cm(III) transferrin species were identified for the first time. In the pH range from 3.5 to 9.7 the Cm(III) transferrin species I is formed revealing complexation of the metal ion at a nonspecific site of the protein surface. In case of the Cm(III) transferrin species II Cm(III) is bound at the Fe(III) binding site of the protein resulting in a 4-fold coordination via amino acid groups of the protein (His, Asp, 2 x Tyr) and coordination of two water molecules and three additional ligands, e.g. OH{sup -} or CO{sub 3}{sup 2-}. Due to the kinetic and thermodynamic differences of the binding sites of the N- and C-lobe, the experimental conditions ensure exclusive coordination of Cm(III) at the C-terminal binding site. In addition to the complexation studies of Cm(III) with transferrin, the interaction with the recombinant N-lobe of human serum transferrin (hTf/2N) as a model component for the transferrin N-lobe was investigated. At pH≥7.4 a Cm(III) hTf/2N species with Cm(III) bound at the Fe(III) binding site is formed which is comparable to the Cm(III) transferrin species II. An increase of the temperature from room temperature (T=296 K) to physiological temperature (T=310 K) favors the complexation of Cm(III) with both transferrin and hTf/2N. The complexation of Cm(III) with transferrin was investigated at three different carbonate concentrations (c(carbonate){sub tot}=0 mM, 0,23 mM und 25 mM (physiological carbonate concentration)). An increase of the total carbonate

  4. The plasma level of soluble urokinase receptor is elevated in patients with Streptococcus pneumoniae bacteraemia and predicts mortality

    DEFF Research Database (Denmark)

    Wittenhagen, p; Kronborg, Gitte; Nielsen, H

    2004-01-01

    This multicentre prospective study was conducted to investigate whether the level of the soluble form of urokinase-type plasminogen activator receptor (suPAR) is elevated during pneumococcal bacteraemia and is of predictive value in the early stage of the disease. Plasma levels of suPAR were incr...

  5. Changes in plasma concentrations of interleukin-6 and interleukin-1 receptor antagonists in response to adrenaline infusion in humans

    DEFF Research Database (Denmark)

    Søndergaard, S R; Ostrowski, K.; Ullum, H

    2000-01-01

    To investigate the possible role of adrenaline in the response of interleukin (IL)-6 and IL-1 receptor antagonists (ra) to extreme physiological conditions such as trauma and exercise, we examined the concentrations in the plasma of these cytokines during an adrenaline infusion. Given the fact th...

  6. Constitutive androstane receptor activation decreases plasma apolipoprotein B-containing lipoproteins and atherosclerosis in low-density lipoprotein receptor-deficient mice.

    Science.gov (United States)

    Sberna, Anne-Laure; Assem, Mahfoud; Xiao, Rui; Ayers, Steve; Gautier, Thomas; Guiu, Boris; Deckert, Valérie; Chevriaux, Angélique; Grober, Jacques; Le Guern, Naig; Pais de Barros, Jean-Paul; Moore, David D; Lagrost, Laurent; Masson, David

    2011-10-01

    The goal of this study was to determine the impact of the nuclear receptor constitutive androstane receptor (CAR) on lipoprotein metabolism and atherosclerosis in hyperlipidemic mice. Low-density lipoprotein receptor-deficient (Ldlr(-/-)) and apolipoprotein E-deficient (ApoE(-/-)) mice fed a Western-type diet were treated weekly with the Car agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or the vehicle only for 8 weeks. In Ldlr(-/-) mice, treatment with TCPOBOP induced a decrease in plasma triglyceride and intermediate-density lipoprotein/low-density lipoprotein cholesterol levels (≈30% decrease in both cases after 2 months, Plipoproteins associated with a decrease in hepatic triglyceride content and the repression of several genes involved in lipogenesis. TCPOBOP treatment also induced a marked increase in the very-low-density lipoprotein receptor in the liver, which probably contributed to the decrease in intermediate-density lipoprotein/low-density lipoprotein levels. Atherosclerotic lesions in the aortic valves of TCPOBOP-treated Ldlr(-/-) mice were also reduced (-60%, Plipoprotein receptor, the effect of TCPOBOP on plasma cholesterol levels and the development of atherosclerotic lesions was markedly attenuated. CAR is a potential target in the prevention and treatment of hypercholesterolemia and atherosclerosis.

  7. Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized gold nanoparticles: synthesis, characterization, cytotoxicity and cellular uptake

    Energy Technology Data Exchange (ETDEWEB)

    Parab, Harshala J; Huang, Jing-Hong; Liu, Ru-Shi [Department of Chemistry, National Taiwan University, Taipei 106, Taiwan (China); Lai, Tsung-Ching; Jan, Yi-Hua; Wang, Jui-Ling; Hsiao, Michael; Chen, Chung-Hsuan [Genomics Research Center, Academia Sinica, Taipei 115, Taiwan (China); Hwu, Yeu-Kuang [Institute of Physics, Academia Sinica, Taipei 115, Taiwan (China); Tsai, Din Ping [Department of Physics, National Taiwan University, Taipei 106, Taiwan (China); Chuang, Shih-Yi; Pang, Jong-Hwei S, E-mail: rsliu@ntu.edu.tw, E-mail: mhsiao@gate.sinica.edu.tw [Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan, Taiwan (China)

    2011-09-30

    The feasibility of using gold nanoparticles (AuNPs) for biomedical applications has led to considerable interest in the development of novel synthetic protocols and surface modification strategies for AuNPs to produce biocompatible molecular probes. This investigation is, to our knowledge, the first to elucidate the synthesis and characterization of sodium hexametaphosphate (HMP)-stabilized gold nanoparticles (Au-HMP) in an aqueous medium. The role of HMP, a food additive, as a polymeric stabilizing and protecting agent for AuNPs is elucidated. The surface modification of Au-HMP nanoparticles was carried out using polyethylene glycol and transferrin to produce molecular probes for possible clinical applications. In vitro cell viability studies performed using as-synthesized Au-HMP nanoparticles and their surface-modified counterparts reveal the biocompatibility of the nanoparticles. The transferrin-conjugated nanoparticles have significantly higher cellular uptake in J5 cells (liver cancer cells) than control cells (oral mucosa fibroblast cells), as determined by inductively coupled plasma mass spectrometry. This study demonstrates the possibility of using an inexpensive and non-toxic food additive, HMP, as a stabilizer in the large-scale generation of biocompatible and monodispersed AuNPs, which may have future diagnostic and therapeutic applications.

  8. Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized gold nanoparticles: synthesis, characterization, cytotoxicity and cellular uptake.

    Science.gov (United States)

    Parab, Harshala J; Huang, Jing-Hong; Lai, Tsung-Ching; Jan, Yi-Hua; Liu, Ru-Shi; Wang, Jui-Ling; Hsiao, Michael; Chen, Chung-Hsuan; Hwu, Yeu-Kuang; Tsai, Din Ping; Chuang, Shih-Yi; Pang, Jong-Hwei S

    2011-09-30

    The feasibility of using gold nanoparticles (AuNPs) for biomedical applications has led to considerable interest in the development of novel synthetic protocols and surface modification strategies for AuNPs to produce biocompatible molecular probes. This investigation is, to our knowledge, the first to elucidate the synthesis and characterization of sodium hexametaphosphate (HMP)-stabilized gold nanoparticles (Au-HMP) in an aqueous medium. The role of HMP, a food additive, as a polymeric stabilizing and protecting agent for AuNPs is elucidated. The surface modification of Au-HMP nanoparticles was carried out using polyethylene glycol and transferrin to produce molecular probes for possible clinical applications. In vitro cell viability studies performed using as-synthesized Au-HMP nanoparticles and their surface-modified counterparts reveal the biocompatibility of the nanoparticles. The transferrin-conjugated nanoparticles have significantly higher cellular uptake in J5 cells (liver cancer cells) than control cells (oral mucosa fibroblast cells), as determined by inductively coupled plasma mass spectrometry. This study demonstrates the possibility of using an inexpensive and non-toxic food additive, HMP, as a stabilizer in the large-scale generation of biocompatible and monodispersed AuNPs, which may have future diagnostic and therapeutic applications.

  9. Androgen Receptor Localizes to Plasma Membrane by Binding to Caveolin-1 in Mouse Sertoli Cells.

    Science.gov (United States)

    Deng, Qiong; Wu, Yong; Zhang, Zeng; Wang, Yue; Li, Minghua; Liang, Hui; Gui, Yaoting

    2017-01-01

    The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR) localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens.

  10. Plasma membrane and nuclear localization of G protein coupled receptor kinase 6A.

    Science.gov (United States)

    Jiang, Xiaoshan; Benovic, Jeffrey L; Wedegaertner, Philip B

    2007-08-01

    G protein-coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.

  11. Androgen Receptor Localizes to Plasma Membrane by Binding to Caveolin-1 in Mouse Sertoli Cells

    Directory of Open Access Journals (Sweden)

    Qiong Deng

    2017-01-01

    Full Text Available The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens.

  12. Plasma Membrane and Nuclear Localization of G Protein–coupled Receptor Kinase 6A

    Science.gov (United States)

    Jiang, Xiaoshan; Benovic, Jeffrey L.

    2007-01-01

    G protein–coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization. PMID:17538017

  13. Lectin receptors on the plasma membrane of soybean cells. Binding and lateral diffusion of lectins.

    Science.gov (United States)

    Metcalf, T N; Wang, J L; Schubert, K R; Schindler, M

    1983-08-02

    Protoplasts prepared from suspension cultures of root cells of Glycine max (SB-1 cell line) bound soybean agglutinin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA). Binding studies carried out with 125I-labeled SBA, Con A, and WGA showed that these interactions were saturable and specific. Fluorescence microscopy demonstrated uniform membrane labeling. The mobility of the lectin-receptor complexes was measured by fluorescence redistribution after photobleaching. The diffusion constants (D) for SBA and Con A were 5 X 10(-11) and 7 X 10(-11) cm2/s, respectively. In contrast, WGA yielded a diffusion constant of 3 X 10(-10) cm2/s. Pretreatment of the protoplasts with either SBA or Con A resulted in a 6-fold reduction in the mobility of WGA (D congruent to 5 X 10(-11) cm2/s). The results suggest that the binding of SBA or Con A may lead to alterations of the soybean plasma membrane which, in turn, may restrict the mobility of other receptors.

  14. The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development.

    Science.gov (United States)

    Lin, Wai W; Yi, Zuoan; Stunz, Laura L; Maine, Christian J; Sherman, Linda A; Bishop, Gail A

    2015-09-01

    Tumor necrosis factor receptor-associated factor 3 (TRAF3) is an adaptor protein that inhibits signaling by CD40 and by the receptor for B cell-activating factor (BAFF) and negatively regulates homeostatic B cell survival. Loss-of-function mutations in TRAF3 are associated with human B cell malignancies, in particular multiple myeloma. The cytokine interleukin-6 (IL-6) supports the differentiation and survival of normal and neoplastic plasma cells. We found that mice with a deficiency in TRAF3 specifically in B cells (B-Traf3(-/-) mice) had about twice as many plasma cells as did their littermate controls. TRAF3-deficient B cells had enhanced responsiveness to IL-6, and genetic loss of IL-6 in B-Traf3(-/-) mice restored their plasma cell numbers to normal. TRAF3 inhibited IL-6 receptor (IL-6R)-mediated signaling by facilitating the association of PTPN22 (a nonreceptor protein tyrosine phosphatase) with the kinase Janus-activated kinase 1 (Jak1), which in turn blocked phosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Consistent with these results, the number of plasma cells in the PTPN22-deficient mice was increased compared to that in the wild-type mice. Our findings identify TRAF3 and PTPN22 as inhibitors of IL-6R signaling in B cells and reveal a previously uncharacterized role for TRAF3 in the regulation of plasma cell differentiation.

  15. A prospective study of plasma vitamin D metabolites, vitamin D receptor polymorphisms, and prostate cancer.

    Directory of Open Access Journals (Sweden)

    Haojie Li

    2007-03-01

    Full Text Available BACKGROUND: Vitamin D insufficiency is a common public health problem nationwide. Circulating 25-hydroxyvitamin D3 (25[OH]D, the most commonly used index of vitamin D status, is converted to the active hormone 1,25 dihydroxyvitamin D3 (1,25[OH]2D, which, operating through the vitamin D receptor (VDR, inhibits in vitro cell proliferation, induces differentiation and apoptosis, and may protect against prostate cancer. Despite intriguing results from laboratory studies, previous epidemiological studies showed inconsistent associations of circulating levels of 25(OHD, 1,25(OH2D, and several VDR polymorphisms with prostate cancer risk. Few studies have explored the joint association of circulating vitamin D levels with VDR polymorphisms. METHODS AND FINDINGS: During 18 y of follow-up of 14,916 men initially free of diagnosed cancer, we identified 1,066 men with incident prostate cancer (including 496 with aggressive disease, defined as stage C or D, Gleason 7-10, metastatic, and fatal prostate cancer and 1,618 cancer-free, age- and smoking-matched control participants in the Physicians' Health Study. We examined the associations of prediagnostic plasma levels of 25(OHD and 1,25(OH2D, individually and jointly, with total and aggressive disease, and explored whether relations between vitamin D metabolites and prostate cancer were modified by the functional VDR FokI polymorphism, using conditional logistic regression. Among these US physicians, the median plasma 25(OHD levels were 25 ng/ml in the blood samples collected during the winter or spring and 32 ng/ml in samples collected during the summer or fall. Nearly 13% (summer/fall to 36% (winter/spring of the control participants were deficient in 25(OHD (<20 ng/ml and 51% (summer/fall and 77% (winter/spring had insufficient plasma 25(OHD levels (<32 ng/ml. Plasma levels of 1,25(OH2D did not vary by season. Men whose levels for both 25(OHD and 1,25(OH2D were below (versus above the median had a

  16. Decreased plasma soluble erythropoietin receptor in high-altitude excessive erythrocytosis and Chronic Mountain Sickness.

    Science.gov (United States)

    Villafuerte, Francisco C; Macarlupú, José Luis; Anza-Ramírez, Cecilia; Corrales-Melgar, Daniela; Vizcardo-Galindo, Gustavo; Corante, Noemí; León-Velarde, Fabiola

    2014-12-01

    Excessive erythrocytosis (EE) is the hallmark of chronic mountain sickness (CMS), a prevalent syndrome in high-altitude Andean populations. Although hypoxemia represents its underlying stimulus, why some individuals develop EE despite having altitude-normal blood erythropoietin (Epo) concentration is still unclear. A soluble form of the Epo receptor (sEpoR) has been identified in human blood and competes directly for Epo with its membrane counterpart (mEpoR). Thus, reduced levels of circulating sEpoR could lead to higher Epo availability and ultimately to EE. We characterized the relationship between Epo and sEpoR, with hematocrit and hemoglobin concentration in healthy highlanders and CMS patients at 4,340 m in Cerro de Pasco, Peru. Our results show that EE patients show decreased plasma sEpoR levels and can be subdivided into two subgroups of normal and high plasma Epo concentration for the altitude of residence, with hemoglobin concentration rising exponentially with an increasing Epo-to-sEpoR ratio (Epo/sEpoR). Also, we showed that the latter varies as an inverse exponential function of arterial pulse O2 saturation. Our findings suggests that EE is strongly associated with higher Epo/sEpoR values, leading to elevated plasma Epo availability to bind mEpoR, and thereby a stronger stimulus for augmented erythropoiesis. Differences in the altitude normal and high Epo CMS patients with a progressively higher Epo/sEpoR supports the hypothesis of the existence of two genetically different subgroups suffering from EE and possibly different degrees of adaptation to chronic high-altitude hypoxia.

  17. Total mortality by elevated transferrin saturation in patients with diabetes

    DEFF Research Database (Denmark)

    Ellervik, Christina; Andersen, Henrik Ullits; Tybjærg-Hansen, Anne;

    2013-01-01

    It is not known to what extent iron overload predicts prognosis in patients with diabetes after diagnosis or whether iron overload is a risk factor independent of the HFE genotype. We investigated total and cause-specific mortality according to increased transferrin saturation (≥ 50 vs....

  18. Isoforms of transferrin in psoriasis patients abusing alcohol

    NARCIS (Netherlands)

    P. Hoefkens (Peter); E.M. Higgins; R.J. Ward (Roberta); H.G. van Eijk (Henk)

    1997-01-01

    textabstractThe different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the 2-sial

  19. Nonrandom distribution of iron in circulating human transferrin.

    Science.gov (United States)

    Zak, O; Aisen, P

    1986-07-01

    By combining the urea gel electrophoresis technique of Makey and Seal with Western immunoblotting, a method has been developed for analyzing the distribution of iron between the two sites of circulating human transferrin. The new method avoids exposure of samples to a nonphysiologic pH that may promote removal or redistribution of iron from the protein; this facilitates examination of multiple samples at one time. Analysis of 21 freshly drawn specimens from normal human subjects confirms previous reports that iron is not randomly distributed in the specific sites of transferrin. Rather, there is a considerable range in the ratio of occupancies of N-terminal and C-terminal sites (N:C ratio), from 0.31 to 6.87 in the present study, with the N-terminal site predominantly occupied in most subjects. The N:C ratio correlates modestly with serum iron concentration (r = .54). Possible flaws in studies indicating a random occupancy of the specific sites of circulating transferrin may lie in the low pH to which samples may be exposed during procedures based on isoelectric focusing or in drawing inferences from data considering only total monoferric transferrin rather than the two distinguishable monoferric species.

  20. Non-transferrin-bound iron (NTBI uptake by T lymphocytes: evidence for the selective acquisition of oligomeric ferric citrate species.

    Directory of Open Access Journals (Sweden)

    Joao Arezes

    Full Text Available Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI. NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.

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

    Science.gov (United States)

    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.

  2. Phosphate inhibits in vitro Fe3+ loading into transferrin by forming a soluble Fe(III)-phosphate complex: a potential non-transferrin bound iron species.

    Science.gov (United States)

    Hilton, Robert J; Seare, Matthew C; Andros, N David; Kenealey, Zachary; Orozco, Catalina Matias; Webb, Michael; Watt, Richard K

    2012-05-01

    In chronic kidney diseases, NTBI can occur even when total iron levels in serum are low and transferrin is not saturated. We postulated that elevated serum phosphate concentrations, present in CKD patients, might disrupt Fe(3+) loading into apo-transferrin by forming Fe(III)-phosphate species. We report that phosphate competes with apo-transferrin for Fe(3+) by forming a soluble Fe(III)-phosphate complex. Once formed, the Fe(III)-phosphate complex is not a substrate for donating Fe(3+) to apo-transferrin. Phosphate (1-10mM) does not chelate Fe(III) from diferric transferrin under the conditions examined. Complexed forms of Fe(3+), such as iron nitrilotriacetic acid (Fe(3+)-NTA), and Fe(III)-citrate are not susceptible to this phosphate complexation reaction and efficiently deliver Fe(3+) to apo-transferrin in the presence of phosphate. This reaction suggests that citrate might play an important role in protecting against Fe(III), phosphate interactions in vivo. In contrast to the reactions of Fe(3+) and phosphate, the addition of Fe(2+) to a solution of apo-transferrin and phosphate lead to rapid oxidation and deposition of Fe(3+) into apo-transferrin. These in vitro data suggest that, in principle, elevated phosphate concentrations can influence the ability of apo-transferrin to bind iron, depending on the oxidation state of the iron.

  3. The interleukin-6 receptor alpha-chain (CD126) is expressed by neoplastic but not normal plasma cells.

    Science.gov (United States)

    Rawstron, A C; Fenton, J A; Ashcroft, J; English, A; Jones, R A; Richards, S J; Pratt, G; Owen, R; Davies, F E; Child, J A; Jack, A S; Morgan, G

    2000-12-01

    Interleukin-6 (IL-6) is reported to be central to the pathogenesis of myeloma, inducing proliferation and inhibiting apoptosis in neoplastic plasma cells. Therefore, abrogating IL-6 signaling is of therapeutic interest, particularly with the development of humanized anti-IL-6 receptor (IL-6R) antibodies. The use of such antibodies clinically requires an understanding of IL-6R expression on neoplastic cells, particularly in the cycling fraction. IL-6R expression levels were determined on plasma cells from patients with myeloma (n = 93) and with monoclonal gammopathy of undetermined significance (MGUS) or plasmacytoma (n = 66) and compared with the levels found on normal plasma cells (n = 11). In addition, 4-color flow cytometry was used to assess the differential expression by stage of differentiation and cell cycle status of the neoplastic plasma cells. IL-6R alpha chain (CD126) was not detectable in normal plasma cells, but was expressed in approximately 90% of patients with myeloma. In all groups, the expression levels showed a normal distribution. In patients with MGUS or plasmacytoma, neoplastic plasma cells expressed significantly higher levels of CD126 compared with phenotypically normal plasma cells from the same marrow. VLA-5(-) "immature" plasma cells showed the highest levels of CD126 expression, but "mature" VLA-5(+) myeloma plasma cells also overexpressed CD126 when compared with normal subjects. This study demonstrates that CD126 expression is restricted to neoplastic plasma cells, with little or no detectable expression by normal cells. Stromal cells in the bone marrow microenvironment do not induce the overexpression because neoplastic cells express higher levels of CD126 than normal plasma cells from the same bone marrow in individuals with MGUS. (Blood. 2000;96:3880-3886)

  4. Transferrin variation and genetic structure of reindeer populations in Scandinavia

    Directory of Open Access Journals (Sweden)

    Knut H. Røed

    1987-06-01

    Full Text Available Polyacrylamide gel electrophoresis was used to analyse transferrin variation in herds of semi-domestic reindeer from Scandinavia. The results are compared with previously reported values for other populations of both semi-domestic and wild reindeer using the same techniques as in the present study. In all populations the number of alleles was high, ranging from seven to eleven, and the heterozygosity was correspondingly high, with a mean of 0.749. This high genetic variation in all populations suggests that inbreeding is not widespread among Scandinavian reindeer. The pattern of allele frequency distribution indicates a high degree of genetic heterogeneity in the transferrin locus, both between the different semi-domestic herds and between the different wild populations. The mean value of genetic distance was 0.069 between semi-domestic herds and 0.091 between wild populations. Between semi-domestic and wild populations the genetic distance was particularly high, with a mean of 0.188. This high value was mainly due to a different pattern in the distribution of the two most common transferrin alleles: Tfu was most common among semi-domestic herds, while TfEI was most common among wild populations. These differences in transferrin allele distribution are discussed in relation to possible different origins of semi-domestic and wild reindeer in Scandinavia, or alternatively, to different selection forces acting on transferrin genotypes in semi-domestic and wild populations.Transferrin-variasjon og genetisk struktur hos rein i Skandinavia.Abstact in Norwegian / Sammendrag: Transferrin-variasjon i tamreinflokker ble analysert ved hjelp av polyacrylamid gel elektroforese. Resultatene er sammenlignet med verdier som tidligere er beskrevet for både tamrein og villrein hvor det ble benyttet samme metode som i denne undersøkelsen. I alle populasjonene ble det registrert et høyt antall alleler (7-11 og heterozygositeten var tilsvarende høy med en

  5. Monocyte and plasma expression of TAM ligand and receptor in renal failure: Links to unregulated immunity and chronic inflammation.

    Science.gov (United States)

    Lee, Iris J; Hilliard, Brendan A; Ulas, Mehriban; Yu, Daohai; Vangala, Chandan; Rao, Swati; Lee, Jean; Gadegbeku, Crystal A; Cohen, Philip L

    2015-06-01

    Chronic inflammation is increased in patients with chronic kidney disease (CKD) and contributes to cardiovascular morbidity and mortality. Specific immune mechanisms and pathways that drive and maintain chronic inflammation in CKD are not well described. The TAM ligands (Gas6 and protein S) and receptors (Axl and Mer) have been recently recognized as playing a prominent role in immune regulation. The receptors exist in both soluble and cell-bound forms; the soluble receptors (sAxl and sMer) are believed to compete with the bound receptors and thus inhibit their function. In this study, we determined the expression of cell-bound and soluble TAM proteins in patients with CKD. CKD patients had significantly lower expression of Mer in monocytes, yet increased expression of soluble TAM receptors sAxl and sMer in plasma compared to controls. The metalloproteinase ADAM 17, responsible for cleavage of Mer to its soluble form, was increased in patient monocytes. Elevated levels of soluble TAM receptors were more evident in patients with progressive renal failure. These observations suggest that functional deficiency of TAM receptor-mediated regulation of inflammation may contribute to chronic inflammation in patients with CKD.

  6. Enhanced blood-brain barrier transmigration using a novel transferrin embedded fluorescent magneto-liposome nanoformulation

    Science.gov (United States)

    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.

  7. MicroRNA-144 regulates hepatic ATP binding cassette transporter A1 and plasma high-density lipoprotein after activation of the nuclear receptor farnesoid X receptor.

    Science.gov (United States)

    de Aguiar Vallim, Thomas Q; Tarling, Elizabeth J; Kim, Tammy; Civelek, Mete; Baldán, Ángel; Esau, Christine; Edwards, Peter A

    2013-06-07

    The bile acid receptor farnesoid X receptor (FXR) regulates many aspects of lipid metabolism by variouscomplex and incompletely understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism. To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism. ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma high-density lipoprotein (HDL)-cholesterol levels. Here, we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lowers hepatic ABCA1 and plasma HDL levels. We identified 2 complementary sequences to miR-144 in the 3' untranslated region of ABCA1 mRNA that are necessary for miR-144-dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I protein, whereas overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL-cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL-cholesterol. In addition, we used tissue-specific FXR-deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal, FXR. Finally, we identified functional FXR response elements upstream of the miR-144 locus, consistent with direct FXR regulation. We have identified a novel pathway involving FXR, miR-144, and ABCA1 that together regulate plasma HDL-cholesterol.

  8. Gallbladder motor function, plasma cholecystokinin and cholecystokinin receptor of gallbladder in cholesterol stone patients

    Institute of Scientific and Technical Information of China (English)

    Jian Zhu; Tian-Quan Han; Sheng Chen; Yu Jiang; Sheng-Dao Zhang

    2005-01-01

    AIM: To study the interactive relationship of gallbladder motor function, plasma cholecystokinin (CCK) and cholecystokinin A receptor (CCK-R) of gallbladder in patients with cholesterol stone disease.METHODS: Gallbladder motility was studied by ultrasonography in 33 patients with gallbladder stone and 10 health subjects as controls. Plasma CCK concentration was measured by radioimmunoassay in fasting status (CCK-f) and in 30 min after lipid test meal (CCK-30).Radioligand method was employed to analyze the amount and activity of CCK-R from 33 gallstone patients having cholecystectomy and 8 persons without gallstone died of severe trauma as controls.RESULTS: The percentage of cholesterol in the gallstone composition was more than 70%. The cholesterol stone type was indicated for the patients with gallbladder stone in this study. Based on the criterion of gallbladder residual fraction of the control group, 33 gallstone patients were divided into two subgroups, contractor group (14 cases)and non-contractor group (19 cases), The concentration of CCK-30 was significantly higher in non-contractor group than that in both contractor group and control group (55.86±3.86 pmol/l vs 37.85±0.88 pmol/l and 37.95±0.74 pmol/L, P<0.01), but there was no difference between contractor group and control group. Meanwhile no significant difference of the concentration of CCK-f could be observed among three groups. The amount of CCK-R was lower in non-contractor group than those in both control group and contractor group (10.27±0.94 fmol/mg vs24.59±2.39 fmol/mg and 22.66±0.55 fmol/mg, P<0.01).The activity of CCK-R shown as KD in non-contractor group decreased compared to that in control group and contractor group. Only was the activity of CCK-R lower in contractor group than that in control group. The ejection fraction correlated closely with the amount of CCK-R (r = 0.9683,P<0.01), and the concentration of CCK-30 correlated negatively with the amount of CCK-R closely (r = -0

  9. Vacuolar Sorting Receptor (VSR) Proteins Reach the Plasma Membrane in Germinating Pollen Tubes

    Institute of Scientific and Technical Information of China (English)

    Hao Wang; Xiao-Hong Zhuang; Stefan Hillmer; David G. Robinson; Li-Wen Jiang

    2011-01-01

    Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants.Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-Golgi network (TGN) in various plant cell types,including suspension culture cells,root cells,developing and germinating seeds.Here,we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes.Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that,in addition to the previously demonstrated PVC/MVB localization,VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution.Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes.Using a high-speed Spinning Disc Confocal Microscope,the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR.In contrast,the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.

  10. Changes in levels of serum soluble transferrin receptor before and after high-flux hemodialysis in patients with uremia and its related factors%尿毒症患者高通量血液透析前后血清可溶性转铁蛋白受体水平变化及其影响因素分析

    Institute of Scientific and Technical Information of China (English)

    殷培; 李冀军; 宋岩; 陈凤锟; 张壹言; 姚凤华; 赵长征

    2015-01-01

    Objective To investigate the changes in levels of serum soluble transferrin receptor ( sTfR) after switc-hing from maintenance hemodialysis to high-flux hemodialysis ( HFD) in patients with uremia, and to analyze its related factors.Methods Forty patients undergoing low-flux maintenance hemodialysis were treated by high-flux maintenance he-modialysis for 12 months.The levels of sTfR, iron metabolism indices, anemia index and inflammatory index were meas-ured before HFD treatment ( T0 ) and after HFD treatment for 2 months ( T1 ) , 6 months ( T2 ) and 12 months ( T3 ) .Pear-son correlation and multiple regression method were used to analyze the relationship between serum sTfR and other indexes. At the same time, we observed the changing trend of erythropoietin and the amount of iron therapy.Results Serum sTfR level was significantly decreased at T1 , T2 and T3 as compared with that at T0 , and significant difference was found ( all P<0.01).Pearson correlation analysis showed that sTfR was positively correlated with Ret%, SF (r=0.154, 0.152;all P<0.05) and was negatively correlated with Hb, HCT, β2-MG and TBC ( r =-0.251, -0.299, -0.384 and-0.166;P<0.01 or P<0.05).Multiple regression showed that HCT, TSAT andβ2-MG levels were closed related with serum sTfR level in HFD patients (all P<0.01).The erythropoietin and iron dosage in this group reduced with prolonged HFD treatment.Conclusions HFD can effectively improve renal anemia in patients with maintenance hemodialysis.The detection of sTfR level reflects the effects of iron reserves and erythropoietic stimulation on patients.%目的:探讨尿毒症患者由常规血液透析转换为高通量血液透析治疗后血清中可溶性转铁蛋白受体( sTfR)水平的变化及相关影响因素。方法选择维持性血液透析患者40例,转换为高通量血液透析治疗12个月。分别于高通量血液透析治疗前( T0)及治疗后2个月( T1)、6个月( T2)、12个月( T3)测定血

  11. Control of transferrin expression by β-amyloid through the CP2 transcription factor.

    Science.gov (United States)

    Jang, Sang-Min; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Kang, Eun-Jin; Kim, Chul Geun; Kim, Hyun-Jung; Choi, Kyung-Hee

    2010-10-01

    Accumulation of β-amyloid protein (Aβ) is one of the most important pathological features of Alzheimer's disease. Although Aβ induces neurodegeneration in the cortex and hippocampus through several molecular mechanisms, few studies have evaluated the modulation of transcription factors during Aβ-induced neurotoxicity. Therefore, in this study, we investigated the transcriptional activity of transcription factor CP2 in neuronal damage mediated by Aβ (Aβ(1-42) and Aβ(25-35) ). An unbiased motif search of the transferrin promoter region showed that CP2 binds to the transferrin promoter, an iron-regulating protein, and regulates transferrin transcription. Ectopic expression of CP2 led to increased transferrin expression at both the mRNA and protein levels, whereas knockdown of CP2 down-regulated transferrin mRNA and protein expression. Moreover, CP2 trans-activated transcription of a transferrin reporter gene. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay showed that CP2 binds to the transferrin promoter region. Furthermore, the binding affinity of CP2 to the transferrin promoter was regulated by Aβ, as Aβ (Aβ(1-42) and Aβ(25-35) ) markedly increased the binding affinity of CP2 for the transferrin promoter. Taken together, these results suggest that CP2 contributes to the pathogenesis of Alzheimer's disease by inducing transferrin expression via up-regulating its transcription.

  12. Transferrin gene frequencies in Cádiz (southern Spain).

    Science.gov (United States)

    Gamero, J J; Romero, J L; Vizcaya, M A; Arufe, I

    1990-12-01

    The genetic polymorphism of transferrin (Tf) was studied in a sample of 385 healthy unrelated subjects of both sexes resident in the province of Cádiz (southern Spain). Isoelectric focusing was carried out in polyacrylamide gels, followed by staining with Coomassie Blue R250. The gene frequencies obtained were as follows: Tf C1, 0.7922; Tf C2, 0.1883; Tf C3, 0.0195.

  13. Transferrin and Haemoglobin types in the African Elephant (Loxodonta Africana

    Directory of Open Access Journals (Sweden)

    D. R Osterhoff

    1972-01-01

    Full Text Available In a random sample of 84 elephants from the Kruger National Park and five elephants from the Addo Elephant National Park, biochemical polymorphism in the serum transferrins could be established. It seems that elephants in the Kruger and Addo Parks are genetically similar but further studies are indicated to confirm these preliminary findings. For the haemo- globin investigations 109 blood samples were available, all originating from the Kruger National Park and all revealing only one type of haemoglobin.

  14. Radioreceptor assay to study the affinity of benzodiazepines and their receptor binding activity in human plasma including their active metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Dorow, R.G.; Seidler, J.; Schneider, H.H. (Schering A.G., Berlin (Germany, F.R.))

    1982-04-01

    A radioreceptor assay has been established to measure the receptor affinities of numerous benzodiazepines in clinical use. The time course of receptor binding activity was studied by this method in the plasma of eight healthy subjects randomly treated with 1mg lormetazepam (Noctamid(R)), 2mg flunitrazepam (Rohypnol(R)), and 10mg diazepam (Valium(R)), and placebo on a cross-over basis. Blood samples were collected up to 154h after treatment. Receptor affinities of numerous benzodiazepines in vitro show good correlation with therapeutic human doses (r=0.96) and may be predictive of drug potency in man. Mean peak plasma levels of lormetazepam binding equivalents were 4.8+-1 ng/ml at 2h after lormetazepam, 7.2+-1.8 ng/ml at 8h after flunitrazepam, and 17.9+-2.7 ng/ml at 15h after diazepam. Plasma elimination halflives of benzodiazepine binding equivalents were 9.3, 23 and 63h, respectively. Slow elimination of benzodiazepine binding equivalents following flunitrazepam and diazepam may be due to persistent active metabolites.

  15. Plasma adiponectin concentration is associated with skeletal muscle insulin receptor tyrosine phosphorylation, and low plasma concentration precedes a decrease in whole-body insulin sensitivity in humans.

    Science.gov (United States)

    Stefan, Norbert; Vozarova, Barbora; Funahashi, Tohru; Matsuzawa, Yuji; Weyer, Christian; Lindsay, Robert S; Youngren, Jack F; Havel, Peter J; Pratley, Richard E; Bogardus, Clifton; Tataranni, P Antonio

    2002-06-01

    Adiponectin, the most abundant adipose-specific protein, has been found to be negatively associated with degree of adiposity and positively associated with insulin sensitivity in Pima Indians and other populations. Moreover, adiponectin administration to rodents has been shown to increase insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and also increase whole-body insulin sensitivity. To further characterize the relationship between plasma adiponectin concentration and insulin sensitivity in humans, we examined 1) the cross-sectional association between plasma adiponectin concentration and skeletal muscle IR tyrosine phosphorylation and 2) the prospective effect of plasma adiponectin concentration at baseline on change in insulin sensitivity. Fasting plasma adiponectin concentration, body composition (hydrodensitometry or dual energy X-ray absorptiometry), insulin sensitivity (insulin-stimulated glucose disposal, hyperinsulinemic clamp), and glucose tolerance (75-g oral glucose tolerance test) were measured in 55 Pima Indians (47 men and 8 women, aged 31 +/- 8 years, body fat 29 +/- 8% [mean +/- SD]; 50 with normal glucose tolerance, 3 with impaired glucose tolerance, and 2 with diabetes). Group 1 (19 subjects) underwent skeletal muscle biopsies for the measurement of basal and insulin-stimulated tyrosine phosphorylation of the IR (stimulated by 100 nmol/l insulin). The fold increase after insulin stimulation was calculated as the ratio between maximal and basal phosphorylation. Group 2 (38 subjects) had follow-up measurements of insulin-stimulated glucose disposal. Cross-sectionally, plasma adiponectin concentration was positively associated with insulin-stimulated glucose disposal (r = 0.58, P < 0.0001) and negatively associated with percent body fat (r = -0.62, P < 0.0001) in the whole group. In group 1 plasma adiponectin was negatively associated with the basal (r = -0.65, P = 0.003) and positively associated with the fold increase in IR

  16. Endogenous plasma estradiol in healthy men is positively correlated with cerebral cortical serotonin 2A receptor binding

    DEFF Research Database (Denmark)

    Frokjaer, Vibe G.; Erritzoe, David; Juul, Anders;

    2010-01-01

    Background: Sex-hormones influence brain function and are likely to play a role in the gender predisposition to mood and anxiety disorders. Acute fluctuations of sex-hormone levels including hormonal replacement therapy appear to affect serotonergic neurotransmission, but it is unknown if baseline...... = 0.0001), whereas no independent effects of testosterone could be demonstrated. Correction for other factors of importance for 5-HT2A receptor binding did not change the result. A voxel-based analysis suggested that there were no regional differences in the estradiol effect on cortical 5-HT2A...... receptor binding. Conclusions: Our data show a positive correlation between endogenous plasma estradiol levels and cortical 5-HT2A receptor binding in healthy men, whereas, no independent effect of testosterone was demonstrated. We speculate that this association could be mediated through effects on gene...

  17. Protein Disulfide Isomerase Chaperone ERP-57 Decreases Plasma Membrane Expression of the Human GnRH Receptor

    Science.gov (United States)

    Yánez, Rodrigo Ayala; Conn, P. Michael

    2012-01-01

    Retention of misfolded proteins by the endoplasmic reticulum (ER) is a quality control mechanism involving the participation of endogenous chaperones such as calnexin (CANX) which interact and restrict plasma membrane expression of gonadotropin releasing hormone receptor (GnRHR), a G protein coupled receptor. CANX also interacts with ERP-57, a thiol oxidoreductase chaperone present in the ER. CANX along with ERP-57, promotes the formation of disulfide bond bridges in nascent proteins. The human GnRH receptor (hGnRHR) is stabilized by two disulfide bond bridges (Cys14-Cys200 and Cys114-Cys196), that, when broken, its expression at plasma membrane decreases. To determine if the presence of chaperones CANX and ERP-57 exert an influence over membrane routing and second messenger activation, we assessed the effect of various mutants including those with broken bridges (Cys→Ala) along with the wild type hGnRHR. The effect of chaperones on mutants was insignificant, whereas the overexpression of ERP-57 led to a wild type hGnRHR retention which was further enhanced by cotransfection with CANX cDNA disclosing receptor retention by ERP-57 augmented by CANX, suggesting a quality control mechanism. PMID:20029959

  18. BF-1--a novel selective 5-HT2B receptor antagonist blocking neurogenic dural plasma protein extravasation in guinea pigs.

    Science.gov (United States)

    Schmitz, Beate; Ullmer, Christoph; Segelcke, Daniel; Gwarek, Mirella; Zhu, Xin-Ran; Lübbert, Hermann

    2015-03-15

    Serotonin 5-HT2B receptor antagonists have been proposed as migraine prophylactic drugs, but previously available 5-HT2B receptor antagonists displayed multiple monoaminergic side effects and had to be withdrawn from the market. Here, we set out to identify a novel antagonist with high affinity and selectivity towards 5-HT2B receptors. To test the affinity of new compounds towards various receptors, we generated a broad series of cells functionally coupling human monoaminergic receptors to luciferase. Using the cell lines we revealed pimethixene (1-methyl-4-(9H-thioxanthen-9-ylidene)piperidine) as highly potent, albeit non-selective 5-HT2B receptor antagonist and optimized its chemical structure to create highly potent and selective 5-HT2B receptor antagonists. We selected the methoxythioxanthene BF-1 for further analysis. In comparison to pimethixene, it lacked high affinities to 5-HT1A, 5-HT2A, 5-HT2C, histamine H1, dopamine D1 and D2 as well as muscarinic M1 and M2 receptors. BF-1 was tested as potential migraine prophylactic drug by blocking meta-chlorophenylpiperazine, (mCPP) or BW723C86 (5-((thiophen-2-yl)methoxy)-α-methyltryptamine) induced neurogenic dural plasma protein extravasation in a guinea pig model that may resemble a migraine attack. BF-1 was significantly more potent in this assay compared to the well know non-selective 5-HT2B antagonists, methysergide ((6aR,9R)-N-[(2S)-1-Hydroxybutan-2-yl]-4,7-dimethyl-6,6a,8,9-tetrahydroindolo[4,3-fg]quinoline-9-carboxamide) or pizotifen (4-(1-methyl-4-piperidylidine)-9,10-dihydro-4H-benzo-[4,5]cyclohepta[1,2]-thiophene). Therefore, we propose BF-1 as a new compound that may be developed for prophylactic migraine treatment without the typical monoaminergic side effects.

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

    Science.gov (United States)

    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, phTf, 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 docking experiments. In conclusion, these results suggest that the seven Cp2M(IV)Cl2 used for this study can interact with apo-hTf, and their affinity was directly and inversely

  20. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.

  1. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  2. MicroRNA-144 Regulates Hepatic ABCA1 and Plasma HDL Following Activation of the Nuclear Receptor FXR

    Science.gov (United States)

    de Aguiar Vallim, Thomas Q.; Tarling, Elizabeth J.; Kim, Tammy; Civelek, Mete; Baldán, Ángel; Esau, Christine; Edwards, Peter A.

    2014-01-01

    Rationale The bile acid receptor Farnesoid-X-Receptor (FXR) regulates many aspects of lipid metabolism by various complex and not fully understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism. Objective To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism. Methods and Results ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma High Density Lipoprotein (HDL)-cholesterol levels. Here we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lower hepatic ABCA1 and plasma HDL levels. We identified two complementary sequences to miR-144 in the 3′ untranslated region (UTR) of ABCA1 mRNA that are necessary for miR-144-dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I (ApoA-I) protein, whilst overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL-cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL-cholesterol. In addition, we utilized tissue-specific FXR deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal FXR. Finally, we identified functional FXR response elements (FXREs) upstream of the miR-144 locus, consistent with direct FXR regulation. Conclusion We have identified a novel pathway involving FXR, miR-144 and ABCA1 that together regulate plasma HDL cholesterol. PMID:23519696

  3. Transferrin serves as a mediator to deliver organometallic ruthenium(II) anticancer complexes into cells.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  6. Plasma membrane nucleolin is a receptor for the anticancer aptamer AS1411 in MV4-11 leukemia cells.

    Science.gov (United States)

    Soundararajan, Sridharan; Wang, Li; Sridharan, Vijayalakshmi; Chen, Weiwei; Courtenay-Luck, Nigel; Jones, David; Spicer, Eleanor K; Fernandes, Daniel J

    2009-11-01

    AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunoblotting of the biotinylated proteins showed that full-length (106 kDa) and truncated forms of nucleolin were present on the cell surface. In contrast, K-562 cells, which are 4-fold less sensitive than MV4-11 cells to AS1411, showed no full-length nucleolin and lesser amounts of the truncated forms of nucleolin on the cell surface. Incubation of MV4-11 cells with [(32)P]AS1411 and immunoprecipitation of the plasma membrane fraction with anti-nucleolin antibody demonstrated the presence of [(32)P]AS1411-nucleolin complexes. Anti-nucleolin antibody inhibited binding of fluorescein isothiocyanate (FITC)-AS1411 to plasma membrane nucleolin 56 +/- 10% SE (P AS1411 only. Cellular uptake of [(32)P]AS1411 into MV4-11 cells was blocked by a 20-fold excess of unlabeled AS1411 but not by a 20-fold excess of the biologically inactive oligonucleotide CRO-26. Uptake was approximately 3-fold faster into MV4-11 cells than into K-562 cells. Partial knockdown of plasma membrane and cytosolic nucleolin in MCF-7 cells resulted in a 3-fold decrease in AS1411 uptake. These results provide evidence that plasma membrane nucleolin is a functional receptor for AS1411 in MV4-11 cells.

  7. [Epidermal growth factor receptor expression and epidermal growth factor blood plasma content in simple and complex endometrial hyperplasia].

    Science.gov (United States)

    Dznelashvili, N; Kasradze, D; Tavartkiladze, A; Mariamidze, A

    2014-01-01

    The goal of our study was to concurrently determine the prognostic significance of Epidermal Growth Factor Receptor (EGFR) expression in endometrium and Epidermal Growth Factor (EGF) blood content in simple and complex hyperplasia. In order to detect EGFR expression, immunohistochemical examination of endometrial scarp from 35 patients was done along with HPLC (High performance liquid chromatography) method, for measuring EGF blood plasma content. The numerical data obtained were processed statistically using computer program SPSS-12. According to the results: 1. A significant/marked increase in EGF blood plasma level together with pronounced EGFR expression in simple endometrial hyperplasia (without atypia) suggests that simple hyperplasia is likely to transform into complex form, while unchanged level of EGF against the background of mild EGFR expression is probably indicative of not very bad prognosis. 2. Normal indices of EGF blood plasma level in simple endometrial hyperplasia (without atypia), accompanied by mild EGFR expression is suggestive of good prognosis. 3. A sharp or extremely sharp increase in EGF blood plasma level with pronounced EGFR expression in complex endometrial hyperplasia (without atypia) is likely to indicate poor prognosis that may lead to the transformation into atypical form. However, unchanged EGF blood plasma level against the background of mild EGFR expression in complex endometrial hyperplasia (without atypia) is likely to point to not very bad prognosis. 4. A marked increase in EGF blood plasma level with a pronounced EGFR expression in complex endometrial hyperplasia (without atypia) is likely to indicate poor prognosis that may lead to the transformation into atypical form. Because it is evident that drastic increase in EGF blood plasma level is not necessary, other factor should be suspected to play the major role, i.e the substance that will (or will not) withstand neoplasia.

  8. Plasma Levels of Soluble Receptor for Advanced Glycation End Products and Coronary Atherosclerosis: Possible Correlation with Clinical Presentation

    Directory of Open Access Journals (Sweden)

    Colomba Falcone

    2013-01-01

    Full Text Available Receptor for Advanced Glycation End-products (RAGE is a multi-ligand receptor ubiquitous present on epithelial, neuronal, vascular and inflammatory cells, usually expressed at low levels in homeostasis and to increased degrees at sites of stress or injury. The aim of the present study was to evaluate sRAGE plasma levels in patients with Acute Coronary Syndrome (ACS and to assess its diagnostic efficacy in identification of patients with acute events. Plasma levels of sRAGE were determined in 860 patients with Coronary Artery Disease (CAD: 530 patients presented stable angina and 330 were observed during acute ischemic event (147 with unstable angina and 183 with myocardial infarction. sRAGE plasma levels were significantly lower in patients with ACS than in patients with stable angina: [median 584 pg/mL (IQR: 266–851 pg/mL in MI patients, median 769 pg/mL (IQR: 394–987 pg/mL in patients with unstable angina, median 834 pg/mL (IQR 630–1005 pg/mL in patients with stable angina; P<0.001]. sRAGE levels did not differ among ACS patients stratified by the extent of coronary artery disease. In conclusion, this study confirm the role of sRAGE in activation and progression of inflammatory process and suggests the possibility that sRAGE can be considered an indicator of destabilization of vulnerable plaque.

  9. The effect of glycosylation on the transferrin structure: A molecular dynamic simulation analysis.

    Science.gov (United States)

    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.

  10. The plasma concentration of HDL-associated apoM is influenced by LDL receptor-mediated clearance of apoB-containing particles.

    Science.gov (United States)

    Christoffersen, Christina; Benn, Marianne; Christensen, Pernille M; Gordts, Philip L S M; Roebroek, Anton J M; Frikke-Schmidt, Ruth; Tybjaerg-Hansen, Anne; Dahlbäck, Björn; Nielsen, Lars B

    2012-10-01

    ApoM is mainly associated with HDL. Nevertheless, we have consistently observed positive correlations of apoM with plasma LDL cholesterol in humans. Moreover, LDL receptor deficiency is associated with increased plasma apoM in mice. Here, we tested the idea that plasma apoM concentrations are affected by the rate of LDL receptor-mediated clearance of apoB-containing particles. We measured apoM in humans each carrying one of three different LDL receptor mutations (n = 9) or the apoB3500 mutation (n = 12). These carriers had increased plasma apoM (1.34 ± 0.13 µM, P = 0.003, and 1.23 ± 0.10 µM, P = 0.02, respectively) as compared with noncarriers (0.93 ± 0.04 µM). When we injected human apoM-containing HDL into Wt (n = 6) or LDL receptor-deficient mice (n = 6), the removal of HDL-associated human apoM was delayed in the LDL receptor-deficient mice. After 2 h, 54 ± 5% versus 90 ± 8% (P LDL receptor-deficient mice, respectively. Finally, we compared the turnover of radio-iodinated LDL and plasma apoM concentrations in 45 normocholesterolemic humans. There was a negative correlation between plasma apoM and the fractional catabolic rate of LDL (r = -0.38, P = 0.009). These data suggest that the plasma clearance of apoM, despite apoM primarily being associated with HDL, is influenced by LDL receptor-mediated clearance of apoB-containing particles.

  11. A microscale protocol for the isolation of transferrin directly from serum.

    Science.gov (United States)

    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.

  12. Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

    Science.gov (United States)

    Conway, B R; Minor, L K; Xu, J Z; D'Andrea, M R; Ghosh, R N; Demarest, K T

    2001-12-01

    Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding

  13. The plasma level of soluble urokinase receptor is elevated in patients with Streptococcus pneumoniae bacteraemia and predicts mortality

    DEFF Research Database (Denmark)

    Wittenhagen, P; Kronborg, G; Weis, N;

    2004-01-01

    This multicentre prospective study was conducted to investigate whether the level of the soluble form of urokinase-type plasminogen activator receptor (suPAR) is elevated during pneumococcal bacteraemia and is of predictive value in the early stage of the disease. Plasma levels of suPAR were...... (n = 117; p cerebral symptoms and high serum concentrations of protein YKL-40 and suPAR were associated significantly with mortality (p ....05). In multivariate analysis, only suPAR remained a significant predictor of death (mortality rate of 13 for suPAR levels of > 10 ng/mL; 95% CI: 1.1-158). The increase in suPAR levels may reflect increased expression by vascular or inflammatory cells in the setting of pneumococcal sepsis. This plasma protein may...

  14. Functional variation in the arginine vasopressin 2 receptor as a modifier of human plasma von Willebrand factor levels

    DEFF Research Database (Denmark)

    Nossent, Anne Yaël; Robben, J H; Deen, P M T

    2010-01-01

    SUMMARY OBJECTIVES: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain-of-function variations in the V2R gene (AVPR2) would lead to higher plasma levels of VWF....... The functionality of the G12E variant was studied in stably transfected MDCKII cells, expressing constructs of either 12G-V2R or 12E-V2R. Both V2R variants were fully glycosylated and expressed on the basolateral membrane. The binding affinity of V2R for AVP was increased three-fold in 12E-V2R-green fluorescent...

  15. Early and exudative age-related macular degeneration is associated with increased plasma levels of soluble TNF receptor II

    DEFF Research Database (Denmark)

    Faber, Carsten; Jehs, Tina Maria Ludowika; Juel, Helene Baek;

    2015-01-01

    PURPOSE: We have recently identified homeostatic alterations in the circulating T cells of patients with age-related macular degeneration (AMD). In cultures of retinal pigment epithelial cells, we have demonstrated that T-cell-derived cytokines induced the upregulation of complement, chemokines...... and other proteins implicated in AMD pathogenesis. The purpose of this study was to test whether increased plasma levels of cytokines were present in patients with AMD. METHODS: We conducted a case-control study. Age-related macular degeneration status was assessed using standardized multimodal imaging...... techniques. Plasma was isolated from freshly drawn peripheral venous blood samples and analysed for interleukin (IL)15, IL18, interferon (IFN)γ, soluble tumour necrosis factor (TNF) receptor II (sTNFRII) and complement factor H (CFH) Y402H genotype. RESULTS: We included 136 individuals with early or late...

  16. Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Belhage, B; Schousboe, A

    1991-01-01

    , the density of the GABAA receptors was significantly increased in the THIP-treated cultures as compared to the control cultures and this effect of THIP was particularly pronounced in the processes. GABAA receptors were occasionally observed to form 'hot spots' in process-like structures and again......The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level...... by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However...

  17. Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study

    DEFF Research Database (Denmark)

    Hansen, G H; Belhage, B; Schousboe, A

    1991-01-01

    The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level...... by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However...... at the EM level using the preembedding immunogold technique. It is likely that low-affinity GABAA receptors are preferentially located in the cell processes and to a considerable extent in the form of 'hot spots'. However, these 'hot spots' also contain high-affinity receptors....

  18. Plasma Membrane Nucleolin Is a Receptor for the Anticancer Aptamer AS1411 in MV4-11 Leukemia Cells

    OpenAIRE

    Soundararajan, Sridharan; Wang, Li; Sridharan, Vijayalakshmi; Chen, Weiwei; Courtenay-Luck, Nigel; Jones, David; Spicer, Eleanor K.; Fernandes, Daniel J.

    2009-01-01

    AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunobl...

  19. Heat shock protein 27 is required for sex steroid receptor trafficking to and functioning at the plasma membrane.

    Science.gov (United States)

    Razandi, Mahnaz; Pedram, Ali; Levin, Ellis R

    2010-07-01

    Classical sex steroid receptors (SRs) localize at the plasma membranes (PMs) of cells, initiating signal transduction through kinase cascades that contribute to steroid hormone action. Palmitoylation of the SRs is required for membrane localization and function, but the proteins that facilitate this modification and subsequent receptor trafficking are unknown. Initially using a proteomic approach, we identified that heat shock protein 27 (Hsp27) binds to a motif in estrogen receptor alpha (ERalpha) and promotes palmitoylation of the SR. Hsp27-induced acylation occurred on the ERalpha monomer and augmented caveolin-1 interactions with ERalpha, resulting in membrane localization, kinase activation, and DNA synthesis in breast cancer cells. Oligomerization of Hsp27 was required, and similar results were found for the trafficking of endogenous progesterone and androgen receptors to the PMs of breast and prostate cancer cells, respectively. Small interfering RNA (siRNA) knockdown of Hsp27 prevented sex SR trafficking to and signaling from the membrane. These results identify a conserved and novel function for Hsp27 with potential as a target for interrupting signaling from membrane sex SRs to tumor biology in hormone-responsive cancers.

  20. Effect of non-surgical periodontal treatment on transferrin serum levels in patients with chronic periodontitis.

    Science.gov (United States)

    Shirmohamadi, Adileh; Chitsazi, Mohamad Taghi; Faramarzi, Masoumeh; Salari, Ashkan; Naser Alavi, Fereshteh; Pashazadeh, Nazila

    2016-01-01

    Background. Transferrin is a negative acute phase protein, which decreases during inflammation and infection. The aim of the present investigation was to evaluate changes in the transferrin serum levels subsequent to non-surgical treatment of chronic periodontal disease. Methods. Twenty patients with chronic periodontitis and 20 systemically healthy subjects without periodontal disease, who had referred to Tabriz Faculty of Dentistry, were selected. Transferrin serum levels and clinical periodontal parameters (pocket depth, clinical attachment level, gingival index, bleeding index and plaque index) were measured at baseline and 3 months after non-surgical periodontal treatment. Data were analyzed with descriptive statistical methods (means ± standard deviations). Independent samples t-test was used to compare transferrin serum levels and clinical variables between the test and control groups. Paired samples t-test was used in the test group for comparisons before and after treatment. Statistical significance was set at P < 0.05. Results. The mean transferrin serum level in patients with chronic periodontitis (213.1 ± 9.2 mg/dL) was significantly less than that in periodontally healthy subjects (307.8 ± 11.7 mg/dL). Three months after periodontal treatment, the transferrin serum level increased significantly (298.3 ± 7.6 mg/dL) and approached the levels in periodontally healthy subjects (P < 0.05). Conclusion. The decrease and increase in transferrin serum levels with periodontal disease and periodontal treatment, respectively, indicated an inverse relationship between transferrin serum levels and chronic periodontitis.

  1. Changes in transferrin are related to changes in insulin resistance: the SLIM study

    NARCIS (Netherlands)

    Roumen, C.; Feskens, E.J.M.; Jansen, E.H.; Saris, W.H.; Blaak, E.E.

    2008-01-01

    Aims To determine the effect of a lifestyle intervention on serum transferrin and ferritin levels and the relationship between changes in transferrin and ferritin and changes in glucose tolerance and insulin resistance. Methods Randomized controlled lifestyle intervention directed at a healthy diet

  2. Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line

    Directory of Open Access Journals (Sweden)

    R. Bertazolli-Filho

    2007-10-01

    Full Text Available The trabecular meshwork (TM is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.

  3. Changes in transferrin are related to changes in insulin resistance: the SLIM study

    NARCIS (Netherlands)

    Roumen, C.; Feskens, E.J.M.; Jansen, E.H.; Saris, W.H.; Blaak, E.E.

    2008-01-01

    Aims To determine the effect of a lifestyle intervention on serum transferrin and ferritin levels and the relationship between changes in transferrin and ferritin and changes in glucose tolerance and insulin resistance. Methods Randomized controlled lifestyle intervention directed at a healthy diet

  4. Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma

    DEFF Research Database (Denmark)

    Møller, Holger Jon; Peterslund, Niels Anker; Graversen, Jonas Heilskov

    2002-01-01

    The hemoglobin scavenger receptor (HbSR/CD163) is an interleukin-6- and glucocorticoid-regulated macrophage/monocyte receptor for uptake of haptoglobin-hemoglobin complexes. Moreover, there are strong indications that HbSR serves an anti-inflammatory function. Immunoprecipitation and immunoblotting...

  5. Use of chromatofocusing to detect a transferrin variant in serum of alcoholic subjects.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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.

  7. Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane

    DEFF Research Database (Denmark)

    Geisler, C; Dietrich, J; Nielsen, B L;

    1998-01-01

    amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane...... and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic...

  8. Reducing elevated plasma LDL cholesterol: the central role of the LDL receptor.

    Science.gov (United States)

    Vincent, J

    2014-07-01

    Elevated low-density lipoprotein cholesterol (LDL-C) is an established risk factor for cardiovascular disease (CVD), and reduction of elevated LDL-C reduces mortality in patients at risk. This benefit has evolved from the use of statins and knowledge of the LDL receptor (LDLR). The most potent drugs used for dyslipidemias act by mechanisms that involve this receptor. Advances in molecular genetics and understanding of the regulation of this receptor have revealed several pharmacological targets that are being explored to develop more targeted therapies for dyslipidemias.

  9. Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle

    DEFF Research Database (Denmark)

    Lundby, Carsten; Hellsten, Ylva; Jensen, Mie B. F.;

    2008-01-01

    The presence and potential physiological role of the erythropoietin receptor (Epo-R) were examined in human skeletal muscle. In this study we demonstrate that Epo-R is present in the endothelium, smooth muscle cells, and in fractions of the sarcolemma of skeletal muscle fibers. To study...... the potential effects of Epo in human skeletal muscle, two separate studies were conducted: one to study the acute effects of a single Epo injection on skeletal muscle gene expression and plasma hormones and another to study the effects of long-term (14 wk) Epo treatment on skeletal muscle structure. Subjects...... (n = 11) received a single Epo injection of 15,000 IU (double blinded, cross over, placebo). A single Epo injection reduced myoglobin and increased transferrin receptor and MRF-4 mRNA content within 10 h after injection. Plasma hormones remained unaltered. Capillarization and fiber hypertrophy...

  10. IN SILICO EVALUATION OF ANGIOTENSIN II RECEPTOR ANTAGONIST’S PLASMA PROTEIN BINDING USING COMPUTED MOLECULAR DESCRIPTORS

    Directory of Open Access Journals (Sweden)

    Jadranka Odović

    2014-03-01

    Full Text Available The discovery of new pharmacologically active substances and drugs modeling led to necessity of predicting drugs properties and its ADME data. Angiotensin II receptor antagonists are a group of pharmaceuticals which modulate the renin-angiotensin-aldosterone system and today represent the most commonly prescribed anti-hypertensive drugs. The aim of this study was to compare different molecular properties of seven angiotensin II receptor antagonists / blockers (ARBs, (eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and their plasma protein binding (PPB data. Several ARBs molecular descriptors were calculated using software package Molinspiration Depiction Software as well as Virtual Computational Chemistry Laboratory (electronic descriptor – PSA, constitutional parameter – Mw, geometric descriptor – Vol, lipophilicity descriptors - logP values, aqueous solubility data – logS. The correlations between all collected descriptors and plasma protein binding data obtained from relevant literature were established. In the simple linear regression poor correlations were obtained in relationships between PPB data and all calculated molecular descriptors. In the next stage of the study multiple linear regression (MLR was used for correlation of PPB data with two different descriptors as independent variables. The best correlation (R2=0.70 with P<0.05 was established between PPB data and molecular weight with addition of volume values as independent variables. The possible application of computed molecular descriptors in drugs protein binding evaluation can be of great importance in drug research.

  11. Self-assembled targeted nanoparticles based on transferrin-modified eight-arm-polyethylene glycol–dihydroartemisinin conjugate

    Science.gov (United States)

    Liu, Kefeng; Dai, Lin; Li, Chunxiao; Liu, Jing; Wang, Luying; Lei, Jiandu

    2016-07-01

    Poor delivery of insoluble anticancer drugs has so far precluded their clinical application. In this study, an efficient tumor targeted-nanoparticle delivery system, transferrin-eight-arm-polyethylene glycol–dihydroartemisinin nanoparticles (TF-8arm-PEG-DHA NPs) for the vehiculation of dihydroartemisinin (DHA) was first prepared and evaluated for its targeting efficiency and cytotoxicity in vitro and in vivo to Lewis lung carcinoma (LLC) cells, which overexpress transferrin receptors (TFRs). The synthesized TF-8arm-PEG–DHA NPs had high solubility (~102 fold of free DHA), relatively high drug loading (~10 wt% DHA), long circulating half-life and moderate particle size (~147 nm). The in vitro cytotoxicity and in vivo tumor growth inhibition studies in LLC-tumor bearing mice confirmed the enhanced efficacy of TF-modified 8arm-PEG-DHA NPs compared to free DHA and non-modified 8arm-PEG-DHA NPs. All these results together supported that the formulation developed in this work exhibited great potential as an effective tumor targeting delivery system for insoluble anticancer drugs.

  12. Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron.

    Science.gov (United States)

    van der Ende, A; du Maine, A; Simmons, C F; Schwartz, A L; Strous, G J

    1987-06-25

    Growing human choriocarcinoma BeWo b24 cells contain 1.5 X 10(6) functional cell surface transferrin binding sites and 2.0 X 10(6) intracellular binding sites. These cells rapidly accumulate iron at a rate of 360,000 iron atoms/min/cell. During iron uptake the transferrin and its receptor recycle at least each 19 min. The accumulated iron is released from the BeWo cells at a considerable rate. The time required to release 50% of previously accumulated iron into the extracellular medium is 30 h. This release process is cell line-specific as HeLa cells release very little if any iron. The release of iron by BeWo cells is stimulated by exogenous chelators such as apotransferrin, diethylenetriaminepenta-acetic acid, desferral, and apolactoferrin. The time required to release 50% of the previously accumulated iron into medium supplemented with chelator is 15 h. In the absence of added chelators iron is released as a low molecular weight complex, whereas in the presence of chelator the iron is found complexed to the chelator. Uptake of iron is inhibited by 250 microM primaquine or 2.5 microM monensin. However, the release of iron is not inhibited by these drugs. Intracellular iron is stored bound to ferritin. A model for the release of iron by BeWo cells and its implication for transplacental iron transport is discussed.

  13. Determination of the functional size of oxytocin receptors in plasma membranes from mammary gland and uterine myometrium of the rat by radiation inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Soloff, M.S.; Beauregard, G.; Potier, M.

    1988-05-01

    Gel filtration of detergent-solubilized oxytocin (OT) receptors in plasma membrane fractions from both regressed mammary gland and labor myometrium of the rat, showed that specific (/sup 3/H)OT binding was associated with a heterogeneously sized population of macromolecules. As radiation inactivation is the only method available to measure the apparent molecular weights of membrane proteins in situ, we used this approach to define the functional sizes of OT receptors. The results indicate that both mammary and myometrial receptors are uniform in size and of similar molecular mass. Mammary and myometrial receptors were estimated to be 57.5 +/- 3.8 (SD) and 58.8 +/- 1.6 kilodaltons, respectively. Knowledge of the functional size of OT receptors will be useful in studies involving the purification and characterization of the receptor and associated membrane components.

  14. Endosome-to-Plasma Membrane Recycling of VEGFR2 Receptor Tyrosine Kinase Regulates Endothelial Function and Blood Vessel Formation.

    Science.gov (United States)

    Jopling, Helen M; Odell, Adam F; Pellet-Many, Caroline; Latham, Antony M; Frankel, Paul; Sivaprasadarao, Asipu; Walker, John H; Zachary, Ian C; Ponnambalam, Sreenivasan

    2014-04-29

    Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis.

  15. Study of the Effect of Leeching on Plasma Endothelin and Soluble Interleukin-2 Receptor in Patients with Systemic Lupus Erythematosus

    Institute of Scientific and Technical Information of China (English)

    CHENG Shi-ping; LIU Jia-lin; YUAN Jing

    2005-01-01

    Objective:to explore the mechanism of leeching in treating systemic lupus erythematosus (SLE). Methods: Forty-four patients with SLE were randomly divided into conventional corticosteroid treated group (control group, n =20) and conventional treatment group with leeching intervention added (leeching group, n= 24). Before and after treatment the concentration of plasma endothelin (ET) and soluble interleukin-2 receptor (sIL-2R) were determined. Results: Before treatment the level of plasma ET and sIL-2R in the SLE patients were all higher than those in the normal healthy group, (P<0.01). But after treatment the level of these in both groups were significantly improved than those of before treatment (P<0.05), and comparison between these two treated groups showed that the difference between them was significant (P<0.05).Conclusion: Leeching added to conventional treatment of SLE could be more effective in improving the level of plasma ET and sIL-2R, and ameliorating the impairment of renal tissues.

  16. Spectroscopic studies of the interaction mechanisms between mono-caffeoylquinic acids and transferrin

    Science.gov (United States)

    Guan, Yanqing; Dong, Jing; Chen, Shizhong; Liu, Meixian; Wang, Daidong; Zhang, Xiaotian; Wang, Hong; Lin, Zongtao

    2017-06-01

    Transferrin (Tf) is an important protein responsible for circulating and transporting iron into cytoplasm. Tf can be taken into cells through endocytosis mediated by Tf receptor, which usually overexpresses in cancer cells. The Tf-Tf receptor pathway opens a possible avenue for novel targeted cancer therapy by utilizing Tf-binding active compounds. Among which, anti-cancer active caffeoylquinic acids (CQAs) were recently found to be promising Tf-binders by our group. For better understanding the anti-cancer activities of CQAs, it is important to unveil the binding mechanisms between CQAs and Tf. In this study, the fluorescence quenching, surface plasmon resonance (SPR), circular dichroism (CD) and molecular docking were used to investigate the interactions between CQA and Tf. The results showed that the calculated apparent association constants of interactions between 1-, 3-, 4- and 5-CQA and Tf at 298 K were 7.97 × 105 M- 1, 4.36 × 107 M- 1, 6.58 × 105 M- 1 and 4.42 × 106 M- 1, respectively. The thermodynamic parameters indicated that the interaction between 1-, 3-, 5-CQA and Tf is due to H-bonding, and electrostatic interactions were likely involved in the binding of 4-CQA and Tf. The CD results indicated that bindings of 1-CQA, 4-CQA and 5-CQA with Tf resulted in more stretched β-turn and random coil translated from β-sheet. In contrast, 3-CQA led to more stable a-helix conformation. Molecular docking studies of CQAs with Tf further displayed that CQAs were able to interact with residues near Fe3 + binding site. The spectroscopic studies revealed the action mechanisms, thermodynamics and interacting forces between CQAs and Tf, and thus are helpful for future design and discovery of Tf-binders for targeted cancer therapy applying Tf-Tf receptor pathway.

  17. Higher plasma soluble Receptor for Advanced Glycation End Products (sRAGE) levels are associated with incident cardiovascular disease and all-cause mortality in type 1 diabetes

    DEFF Research Database (Denmark)

    Nin, Johanna W M; Jorsal, Anders; Ferreira, Isabel;

    2010-01-01

    To investigate the associations of plasma levels of soluble receptor for advanced glycation end products (sRAGE) with incident cardiovascular disease (CVD) and all-cause mortality in type 1 diabetes and the extent to which any such associations could be explained by endothelial and renal dysfunct......To investigate the associations of plasma levels of soluble receptor for advanced glycation end products (sRAGE) with incident cardiovascular disease (CVD) and all-cause mortality in type 1 diabetes and the extent to which any such associations could be explained by endothelial and renal...... dysfunction, low-grade inflammation, arterial stiffness, and advanced glycation end products (AGEs)....

  18. A facile drug delivery system preparation through the interaction between drug and iron ion of transferrin

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Lin [Nanjing Normal University, Jiangsu Key Laboratory Biofunctional Materials, Key Laboratory of Applied Photochemistry, Analysis and Testing Center, College of Chemistry and Materials Science (China); Liu, Jihua [China Pharmaceutical University, Department of Complex Prescription of TCM (China); Wei, Shaohua; Ge, Xuefeng; Zhou, Jiahong, E-mail: zhoujiahong@njnu.edu.cn [Nanjing Normal University, Jiangsu Key Laboratory Biofunctional Materials, Key Laboratory of Applied Photochemistry, Analysis and Testing Center, College of Chemistry and Materials Science (China); Yu, Boyang, E-mail: boyangyu59@163.com [China Pharmaceutical University, Department of Complex Prescription of TCM (China); Shen, Jian [Nanjing Normal University, Jiangsu Key Laboratory Biofunctional Materials, Key Laboratory of Applied Photochemistry, Analysis and Testing Center, College of Chemistry and Materials Science (China)

    2013-09-15

    Many anticancer drugs have the capability to form stable complex with metal ions. Based on such property, a simple method to combine these drugs with transferrin, through the interaction between drug and Fe ion of transferrin, to improve their anticancer activity, is proposed. To demonstrate this technique, the complex of photosensitive anticancer drug hypocrellin A and transferrin was prepared by such facile method. The results indicated that the complex of hypocrellin A and transferrin can stabilize in aqueous solution. In vitro studies have demonstrated the superior cancer cell uptake ability of hypocrellin A-transferrin complex to the free hypocrellin A. Significant damage to such drug-impregnated tumor cells was observed upon irradiation and the cancer cells killing ability of hypocrellin A-transferrin was stronger than the free hypocrellin A within a certain range of concentrations. The above results demonstrated the validity and potential of our proposed strategy to prepare the drug delivery system of this type of anti-cancer drugs and transferrin.

  19. Immunological measurement of transferrin compared with chemical measurement of total iron-binding capacity.

    Science.gov (United States)

    Tsung, S H; Rosenthal, W A; Milewski, K A

    1975-07-01

    Because of uncertainty as to the molecular weight of transferrin, a previous comparison [Von der Heul et al., Clin. Chim. Acta 38, 347 (1972)] between transferrin content of serum and total iron-binding capacity cannot be definitive. We found a conversion factor for expressing the maximum amount of iron bound by 1 mg of transferrin. We compared the resulting calculated value with values obtained by three other methods for measuring total iron-binding capacity. We agree with the previous observation that the latter, as measured radioisotopically, give higher results than would be judged from the transferrin content but the same as those for two chemical methods. The diffusion rate of transferrin in agar was the same irrespective of the degree of iron saturation. Serum transferrin concentrations were low in patients with anemia resulting from malignancy, chronic disorders, and cirrhosis of the liver, and high or normal in patients with iron deficiency anemia and in pregnant women or women who were taking birth-control pills. Measurement of transferrin concentration can be used to distinguish iron deficiency anemia from anemia resulting from chronic disorders, but offers no advantages over existing methods for estimating total iron-binding capacity.

  20. Differential mass spectrometry of rat plasma reveals proteins that are responsive to 17beta-estradiol and a selective estrogen receptor modulator PPT.

    Science.gov (United States)

    Zhao, Xuemei; Deyanova, Ekaterina G; Lubbers, Laura S; Zafian, Pete; Li, Jenny J; Liaw, Andy; Song, Qinghua; Du, Yi; Settlage, Robert E; Hickey, Gerry J; Yates, Nathan A; Hendrickson, Ronald C

    2008-10-01

    Estrogens are a class of steroid hormones that interact with two related but distinct nuclear receptors, estrogen receptor (ER) alpha and beta. To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17beta-estradiol (E2) or an ERalpha-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPT-responsive proteins, such as serine protease inhibitor family members.

  1. Cell-permeable and plasma-stable peptidomimetic inhibitors of the postsynaptic density-95/N-methyl-D-aspartate receptor interaction

    DEFF Research Database (Denmark)

    Bach, Anders*; Eildal, Jonas Nii Nortey*; Stuhr-Hansen, Nicolai

    2011-01-01

    The protein--protein interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treating ischemic brain diseases, neuropathic pain, and Alzheimer's disease. We have previously demonstrated that N-alkylated tetrapeptides are potent inhibitors...... to identification of N-cyclohexylethyl-ETA(S)V (54) as the most potent, plasma-stable and cell-permeable inhibitor, which is a promising tool in unraveling the therapeutic potential of the PSD-95/NMDA receptor interaction....

  2. Identification of the Oxidized Low-Density Lipoprotein Scavenger Receptor CD36 in Plasma

    DEFF Research Database (Denmark)

    Handberg, Aase; Levin, Klaus; Højlund, Kurt

    2006-01-01

    BACKGROUND: Macrophage CD36 scavenges oxidized low-density lipoprotein, leading to foam cell formation, and appears to be a key proatherogenic molecule. Increased expression of CD36 has been attributed to hyperglycemia and to defective macrophage insulin signaling in insulin resistance. Premature...... atherosclerosis is the major cause of morbidity and mortality in type 2 diabetes. Here, we report the identification of a soluble form of CD36 (sCD36) in plasma and hypothesize that sCD36 would be elevated in patients with type 2 diabetes and insulin resistance. METHODS AND RESULTS: sCD36 in plasma...... was demonstrated by immunopurification and Western blotting. We established ELISA assays to determine sCD36 in plasma and measured sCD36 in obese type 2 diabetic patients, obese nondiabetic relatives, and obese and lean control subjects. sCD36 was markedly elevated in type 2 diabetic patients compared with both...

  3. Adhesion and receptor clustering stabilizes lateral heterogeneity in cell plasma membranes

    Science.gov (United States)

    Veatch, Sarah

    2013-03-01

    The thermodynamic properties of plasma membrane lipids play a vital role in many functions that initiate at the mammalian cell surface. Some functions are thought to occur, at least in part, because plasma membrane lipids have a tendency to separate into two distinct liquid phases, called liquid-ordered and liquid-disordered. We find that isolated cell plasma membranes are poised near a miscibility critical point separating these two liquid phases, and postulate that critical composition fluctuations provide the physical basis of functional membrane heterogeneity in intact cells. In this talk I will describe several possible mechanisms through which dynamic fluctuations can be stabilized in super-critical membranes, and will present some preliminary evidence suggesting that these structures can be visualized in intact cells using quantitative super-resolution fluorescence localization imaging.

  4. Plasma membrane Toll-like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon-β.

    Science.gov (United States)

    Boye, Louise; Welsby, Iain; Lund, Lisbeth Drozd; Goriely, Stanislas; Frøkiaer, Hanne

    2016-11-01

    Lactobacillus acidophilus induces a potent interferon-β (IFN-β) response in dendritic cells (DCs) by a Toll-like receptor 2 (TLR2) -dependent mechanism, in turn leading to strong interleukin-12 (IL-12) production. In the present study, we investigated the involvement of different types of endocytosis in the L. acidophilus-induced IFN-β and IL-12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN-β and IL-12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place in the immature cells as well as by spleen tyrosine kinase (Syk) -dependent phagocytosis but without involvement of plasma membrane TLR2. Stimulation with TLR2 or TLR4 ligands increased macropinocytosis in a Syk-independent manner. As a consequence, incubation of DCs with TLR ligands before incubation with L. acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN-β and IL-12 was strongly inhibited. As L. acidophilus-induced IFN-β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading to increased macropinocytosis abrogates IFN-β induction we conclude that plasma membrane TLR stimulation leading to increased macropinocytosis decreases endosomal induction of IFN-β and speculate that this is due to competition between compartments for molecules involved in the signal pathways. In summary, endosomal signalling by L. acidophilus that leads to IFN-β and IL-12 production is inhibited by TLR stimulation from the plasma membrane.

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

    Science.gov (United States)

    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.

  6. INCREASED PLASMA-CONCENTRATIONS OF INTERLEUKIN-1 RECEPTOR ANTAGONIST IN NEONATAL SEPSIS

    NARCIS (Netherlands)

    DEBONT, ESJM; DELEIJ, LHFM; OKKEN, A; BAARSMA, R; KIMPEN, JLL

    Newborns are prone to severe infections and sepsis. Cytokines such as tumor necrosis factor-alpha and IL-1 beta play a major role in the initiation of the host response to infections. IL-1 receptor antagonist (IL-1ra) is a naturally occurring antagonist of IL-1 beta. we hypothesized that low IL-1ra

  7. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    Science.gov (United States)

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  8. Genome-wide association study of NMDA receptor coagonists in human cerebrospinal fluid and plasma

    NARCIS (Netherlands)

    Luykx, J. J.; Bakker, S. C.; Visser, W. F.; Verhoeven-Duif, N.; Buizer-Voskamp, J. E.; Den Heijer, J. M.; Boks, M. P. M.; Sul, J. H.; Eskin, E.; Ori, A. P.; Cantor, R. M.; Vorstman, J.; Strengman, E.; DeYoung, J.; Kappen, T. H.; Pariama, E.; van Dongen, E. P. A.; Borgdorff, P.; Bruins, P.; de Koning, T. J.; Kahn, R. S.; Ophoff, R. A.

    2015-01-01

    The N-methyl-D-aspartate receptor (NMDAR) coagonists glycine, D-serine and L-proline play crucial roles in NMDAR-dependent neurotransmission and are associated with a range of neuropsychiatric disorders. We conducted the first genome-wide association study of concentrations of these coagonists and t

  9. Genome-wide association study of NMDA receptor coagonists in human cerebrospinal fluid and plasma

    NARCIS (Netherlands)

    Luykx, J. J.; Bakker, S. C.; Visser, W. F.; Verhoeven-Duif, N.; Buizer-Voskamp, J. E.; den Heijer, J. M.; Boks, M. P M; Sul, J. H.; Eskin, E.; Ori, A. P.; Cantor, R. M.; Vorstman, J.; Strengman, E.; DeYoung, J.; Kappen, T. H.; Pariama, E.; van Dongen, E. P A; Borgdorff, P.; Bruins, P.; de Koning, T. J.; Kahn, R. S.; Ophoff, R. A.

    2015-01-01

    The N-methyl-d-aspartate receptor (NMDAR) coagonists glycine, d-serine and l-proline play crucial roles in NMDAR-dependent neurotransmission and are associated with a range of neuropsychiatric disorders. We conducted the first genome-wide association study of concentrations of these coagonists and t

  10. Elevated plasma urokinase receptor predicts low birth weight in maternal malaria

    DEFF Research Database (Denmark)

    Ostrowski, S R; Shulman, C E; Peshu, N

    2007-01-01

    The blood level of soluble urokinase receptor (suPAR) is increased and associated with a poor clinical or fatal outcome in children with acute malaria. This study hypothesized that the suPAR level would be associated with foetal outcome in maternal malaria. suPAR was measured by ELISA in maternal...

  11. Mosapride, a selective serotonin 5-HT4 receptor agonist, and alogliptin, a selective dipeptidyl peptidase-4 inhibitor, exert synergic effects on plasma active GLP-1 levels and glucose tolerance in mice.

    Science.gov (United States)

    Nonogaki, Katsunori; Kaji, Takao

    2015-12-01

    Pharmacologic stimulation of serotonin 5-HT4 receptors increased plasma active glucagon-like-peptide-1 (GLP-1) levels independent of feeding, and that pharmacologic stimulation of 5-HT4 receptors and pharmacologic inhibition of dipeptidyl peptidase-4 exerted synergic effects on plasma active GLP-1 levels and glucose tolerance in mice.

  12. One-Step Self-Assembling Method to Prepare Dual-Functional Transferrin Nanoparticles for Antitumor Drug Delivery.

    Science.gov (United States)

    Wang, Kaikai; Yuan, Ahu; Yu, Jiaqian; Wu, Jinhui; Hu, Yiqiao

    2016-03-01

    Protein-based nanoparticles hold great promise in both preclinical and clinical practices due to their high biocompatibility and biodegradability. However, the complicated preparations often denature proteins, which subsequently diminish their bioactivity. To overcome these drawbacks, we developed a one-step self-assembling method for preparing protein-based nanoparticles. Transferrin (Tf), a targeting protein, was mixed with 2-mercaptoethanol to break disulfide bonds. Using this method, Tf-PTX-NPs (paclitaxel-loaded Tf nanoparticles) could be readily obtained. Tf-PTX-NPs were round and their diameter could be controlled in the range of 5-200 nm. The bioactivity of Tf to its receptor after forming nanoparticles was also confirmed in vitro. Tf-PTX-NPs also could inhibit the tumor growth to some extent in a mice tumor xenograft model. Therefore, using this self-assembling method, we fabricated this antitumor Tf-based nanoparticle, in which Tf acted as both the targeting moiety and drug carrier.

  13. The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

    Science.gov (United States)

    Cho, DI; Min, C; Jung, KS; Cheong, SY; Zheng, M; Cheong, SJ; Oak, MH; Cheong, JH; Lee, BK; Kim, KM

    2012-01-01

    BACKGROUND AND PURPOSE Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH The N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS Shortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONS Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties. PMID:22117524

  14. Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia.

    Science.gov (United States)

    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.

  15. Prognostic value of plasma soluble urokinase plasminogen activator receptor (suPAR) in Danish patients with recurrent epithelial ovarian cancer (REOC)

    DEFF Research Database (Denmark)

    Begum, Farah Diba; Høgdall, Estrid V S; Riisbo, Rikke

    2006-01-01

    The level of the soluble urokinase plasminogen activator receptor (suPAR) is elevated in tumour tissue from several types of cancer. This is the first study aiming to predict the prognosis for survival by the use of a pre-chemotherapeutic plasma suPAR value in 71 patients with recurrent epithelial...

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

    Science.gov (United States)

    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.

  17. Transferrin saturation ratio and risk of total and cardiovascular mortality in the general population.

    LENUS (Irish Health Repository)

    Stack, A G

    2014-08-01

    The transferrin saturation (TSAT) ratio is a commonly used indicator of iron deficiency and iron overload in clinical practice but precise relationships with total and cardiovascular mortality are unclear.

  18. TRANSFERRIN POLYMORPHISM IN FOUR LOCAL BREEDS OF GOAT IN CENTRAL JAVA, INDONESIA

    Directory of Open Access Journals (Sweden)

    E. Kurnianto

    2012-12-01

    Full Text Available The objectives of this study were to determine the gene frequency and individual heterozygosity of transferrin in four local breeds of goat in Central Java-Indonesia. The number of blood samples were taken from 96 heads of goat, in which each of breeds were 24 samples, those were Kejobong (Purbalingga regency, Ettawa Grade (Purworejo regency, Kacang (Grobogan regency and Jawarandu (Pemalang regency. Polyacrilamide Gel Electrophoresis was performed to detect the bands of blood plasm protein. Gen frequency was calculated using general formula of population genetics. Estimated heterozygosity and individual heterosizygosity were calculated to analysis the equilibrium condition of transferrin. Result showed there was two allele of transferrin, namely TfA and TfB. Gene frequency of TfA was higher than that of TfB. Transferrin gene and genotypes were in disequilibrium of Hardy-Weinberg Law.

  19. Decreased plasma soluble erythropoietin receptor in high-altitude excessive erythrocytosis and Chronic Mountain Sickness

    OpenAIRE

    Villafuerte, Francisco C.; Macarlupú, José Luis; Anza-Ramírez, Cecilia; Corrales-Melgar, Daniela; Vizcardo-Galindo, Gustavo; Corante, Noemí; León-Velarde, Fabiola

    2014-01-01

    Excessive erythrocytosis (EE) is the hallmark of chronic mountain sickness (CMS), a prevalent syndrome in high-altitude Andean populations. Although hypoxemia represents its underlying stimulus, why some individuals develop EE despite having altitude-normal blood erythropoietin (Epo) concentration is still unclear. A soluble form of the Epo receptor (sEpoR) has been identified in human blood and competes directly for Epo with its membrane counterpart (mEpoR). Thus, reduced levels of circulati...

  20. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  1. Remodeling of plasma lipoproteins in patients with rheumatoid arthritis: Interleukin-6 receptor-alpha inhibition with tocilizumab.

    Science.gov (United States)

    Lee, Janet S; Chapman, M John; Piraino, Paolo; Lamerz, Jens; Schindler, Thomas; Cutler, Paul; Dernick, Gregor

    2016-02-01

    Rheumatoid arthritis (RA) is associated with increased cardiovascular risk, mediated in part by elevated circulating interleukin-6 levels and proinflammatory changes in plasma lipoproteins. We hypothesized that RA patients acquire inflammation-induced modifications to the protein cargo of circulating lipoproteins that may be reversed by tocilizumab, an interleukin-6 receptor-alpha inhibitor. Size-exclusion chromatography and reverse-phase protein arrays using 29 antibodies against 26 proteins were applied at baseline and after tocilizumab treatment to analyze the distributions of apolipoproteins, enzymes, lipid transfer proteins, and other associated proteins in plasma lipoprotein fractions from 20 women with RA. A 30% reduction in high-density lipoprotein (HDL)-associated serum amyloid A4 and complement C4 occurred with tocilizumab. Levels of C-reactive protein, associated or comigrating with HDL and low-density lipoprotein (LDL) peaks, were reduced on treatment by approximately 80% and 24%, respectively. Reductions in lipoprotein-associated phospholipase A2, lipoprotein (a), and cholesteryl ester transfer protein in the LDL fraction suggest reductions in LDL-associated proatherogenic factors. Elevations in very low-density lipoprotein (VLDL) enriched with apolipoprotein E were equally observed. Tocilizumab treatment led to reductions in proinflammatory components and proatherogenic proteins associated with HDL. Whether changes in the proteome of VLDL, LDL, and HDL induced by anti-inflammatory tocilizumab treatment in RA patients modify cardiovascular disease risk requires further investigation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. The α7 nicotinic acetylcholine receptor function in hippocampal neurons is regulated by the lipid composition of the plasma membrane.

    Science.gov (United States)

    Colón-Sáez, José O; Yakel, Jerrel L

    2011-07-01

    The α7 nicotinic acetylcholine receptors (nAChRs) play an important role in cellular events such as neurotransmitter release, second messenger cascades, cell survival and apoptosis. In addition, they are a therapeutic target for the treatment of neurological disorders such as Alzheimer's disease and schizophrenia, and drugs that potentiate α7 nAChRs through the regulation of desensitization are currently being developed. Recently, these channels were found to be localized into lipid rafts. Here we show that the disruption of lipid rafts in rat primary hippocampal neurons, through cholesterol-scavenging drugs (methyl-β-cyclodextrin) and the enzymatic breakdown of sphingomyelin (sphingomyelinase), results in significant changes in the desensitization kinetics of native and expressed α7 nAChRs. These effects can be prevented by cotreatment with cholesterol and sphingomyelin, and can be mimicked by treatment with cholesterol and sphingomyelin synthesis inhibitors (mevastatin and myriocin, respectively), suggesting that the effects on desensitization kinetics are indeed due to changes in the levels of cholesterol and sphingomyelin in the plasma membrane. These data provide new insights into themechanism of desensitization of α7 nAChRs by providing evidence that the lipid composition of the plasma membrane can modulate the activity of the α7 nAChRs.

  3. Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

    Science.gov (United States)

    Cho, Gota; Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Skowronski, Mariusz T; Shono, Masayuki; Nielsen, Søren; Ishikawa, Yasuko

    2015-04-01

    The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Effects of transferrin conjugated multi-walled carbon nanotubes in lung cancer delivery

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Rahul Pratap [Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005 (India); Sharma, Gunjan [Genotoxicology and Cancer Biology Lab, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi 221005 (India); Sonali [Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005 (India); Singh, Sanjay [Department of Pharmaceutics, Indian Institute of Technology (BHU), Varanasi 221005 (India); Patne, Shashikant C.U. [Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005 (India); Pandey, Bajarangprasad L. [Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005 (India); Koch, Biplob, E-mail: kochbiplob@gmail.com [Genotoxicology and Cancer Biology Lab, Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi 221005 (India); Muthu, Madaswamy S., E-mail: muthubits@rediffmail.com [Department of Pharmaceutics, Indian Institute of Technology (BHU), Varanasi 221005 (India); Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005 (India)

    2016-10-01

    The aim of this study was to develop multi-walled carbon nanotubes (MWCNT) which were covalently conjugated with transferrin by carbodiimide chemistry and loaded with docetaxel as a model drug for effective treatment of lung cancer in comparison with the commercial docetaxel injection (Docel™). D-Alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) was used as amphiphilic surfactant to improve the aqueous dispersity and biocompatibility of MWCNT. Human lung cancer cells (A549 cells) were employed as an in-vitro model to access cellular uptake, cytotoxicity, cellular apoptosis, cell cycle analysis, and reactive oxygen species (ROS) of the docetaxel/coumarin-6 loaded MWCNT. The cellular uptake results of transferrin conjugated MWCNT showed higher efficiency in comparison with free C6. The IC{sub 50} values demonstrated that the transferrin conjugated MWCNT could be 136-fold more efficient than Docel™ after 24 h treatment with the A549 cells. Flow cytometry analysis confirmed that cancerous cells appeared significantly (P < 0.05) in the sub-G1 phase for transferrin conjugated MWCNT in comparison with Docel™. Results of transferrin conjugated MWCNT have showed better efficacy with safety than Docel™. - Highlights: • It shows the development of transferrin conjugated MWCNT formulation of DTX for the effective treatment of lung cancer. • Evaluated the cellular uptake, cytotoxicity, cellular apoptosis, cell cycle, and ROS level of the DTX/C6 loaded MWCNT. • The IC{sub 50} values demonstrated that the transferrin conjugated MWCNT could be 136-fold more effective than Docel™. • Safety of the DTX formulations were studied by the measurements of ALP, LDH and total protein count levels in BAL fluid. • Results of transferrin conjugated MWCNT have showed better efficacy with safety than Docel™ in lung cancer delivery.

  5. Evolutionary relationships of a "primitive" shark (Heterodontus) assessed by micro-complement fixation of serum transferrin.

    Science.gov (United States)

    Davies, D H; Lawson, R; Burch, S J; Hanson, J E

    1987-01-01

    The evolutionary relationships of six sharks were investigated by comparing their transferrins using the micro-complement fixation method. The immunological distances observed were used to build a tree that confirms that the squaloid and galeoid species examined belong to two separate groups and that Heterodontus, a genus of hitherto uncertain position, belongs with the galeoids. The divergence time estimated from the transferrin comparisons is roughly 240 +/- 65 million years between Heterodontus and galeoids.

  6. Total mortality by transferrin saturation levels: two general population studies and a metaanalysis

    DEFF Research Database (Denmark)

    Ellervik, Christina; Tybjærg-Hansen, Anne; Nordestgaard, Børge G

    2011-01-01

    There is evidence for increased mortality in patients with clinically overt hereditary hemochromatosis. Whether increased transferrin saturation (TS), as a proxy for iron overload is associated with increased mortality in the general population is largely unknown.......There is evidence for increased mortality in patients with clinically overt hereditary hemochromatosis. Whether increased transferrin saturation (TS), as a proxy for iron overload is associated with increased mortality in the general population is largely unknown....

  7. Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Jagadeesh, G.; Deth, R.C.

    1987-11-01

    Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of (/sup 3/H)prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate (Gpp(NH)p). A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. (/sup 3/H)Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with (/sup 3/H)prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.

  8. The plasma concentration of HDL-associated apoM is influenced by LDL receptor-mediated clearance of apoB-containing particles

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Benn, Marianne; Christensen, Pernille Møller;

    2012-01-01

    are affected by the rate of LDL receptor-mediated clearance of apoB-containing particles. We measured apoM in humans each carrying one of three different LDL receptor mutations (n = 9) or the apoB3500 mutation (n = 12). These carriers had increased plasma apoM (1.34 ± 0.13 µM, P = 0.003, and 1.23 ± 0.10 µM, P...... catabolic rate of LDL (r = -0.38, P = 0.009). These data suggest that the plasma clearance of apoM, despite apoM primarily being associated with HDL, is influenced by LDL receptor-mediated clearance of apoB-containing particles....

  9. Purification of transferrins and lactoferrin using DEAE affi-gel blue.

    Science.gov (United States)

    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.

  10. Rapid screening of transferrin-binders in the flowers of Bauhinia blakeana Dunn by on-line high-performance liquid chromatography-diode-array detector-electrospray ionization-ion-trap-time-of-flight-mass spectrometry-transferrin-fluorescence detection system.

    Science.gov (United States)

    Liu, Meixian; Dong, Jing; Lin, Zongtao; Niu, Yanyan; Zhang, Xiaotian; Jiang, Haixiu; Guo, Ning; Li, Wei; Wang, Hong; Chen, Shizhong

    2016-06-10

    Transferrin (Transferrin, TRF, TF) has drawn increasing attention in cancer therapy due to its potential applications in drug delivery. TF receptor, highly expressed in tumor cells, recognizes and transports Fe(3+)-TF into cells to release iron into cytoplasm. Thus, discovering TF-binding compounds has become an active research area and is of great importance for target therapy. In this study, an on-line analysis method was established for screening TF-binding compounds from the flowers of Bauhinia blakeana Dunn using a high-performance liquid chromatography-diode-array detector-multi-stage mass spectrometry-transferrin-fluorescence detector (HPLC-DAD-MS(n)-TF-FLD) method. As a result, 33 of 80 identified or tentatively characterized compounds in the sample were TF-binding active. Twenty-five flavonol glycosides and eight phenolic acids were identified as TF-binders. Twelve of these active compounds together with six standard compounds were used to study the dose-response effects and structure-activity relationships of flavonoids and phenolic acids. The method was validated by vitexin with a good linearity in the range of concentrations used in the study. The limit of detection for vitexin was 0.1596 nmol. Our study indicated that the established method is simple, rapid and sensitive for screening TF-binding active compounds in the extract of Bauhinia blakeana Dunn, and therefore is important for discovering potential anti-cancer ingredients from complex samples for TF related drug delivery.

  11. Plasma soluble erythropoietin receptor is decreased during sleep in Andean highlanders with Chronic Mountain Sickness

    OpenAIRE

    Villafuerte, Francisco C.; Corante, Noemí; Anza-Ramírez, Cecilia; Figueroa-Mujíca, Rómulo; Vizcardo-Galindo, Gustavo; Mercado, Andy; Macarlupú, José Luis; León-Velarde, Fabiola

    2016-01-01

    Excessive erythrocytosis (EE) is the main sign of Chronic Mountain Sickness (CMS), a highly prevalent syndrome in Andean highlanders. Low pulse O2 saturation (SpO2) during sleep and serum androgens have been suggested to contribute to EE in CMS patients. However, whether these factors have a significant impact on the erythropoietin (Epo) system leading to EE is still unclear. We have recently shown that morning soluble Epo receptor (sEpoR), an endogenous Epo antagonist, is decreased in CMS pa...

  12. [Carbohydrate deficient transferrin and ethyl glucuronide: markers for alcohol use].

    Science.gov (United States)

    Paling, Erik P; Mostert, Leendert J

    2013-01-01

    In this article, we report on the usefulness of physicians testing for carbohydrate deficient transferrin (CDT) and ethyl glucuronide (EtG) when there are doubts about alcohol use by their patients. A 44-year-old male consulted his general practitioner with depressive symptoms and denied using alcohol. Laboratory examination revealed an elevated CDT value. The latter was caused by chronic alcohol use. The second patient, a 32-year-old female with known alcohol dependence and receiving inpatient treatment at an addiction clinic, came back from leave. She denied having consumed alcohol and her blood alcohol concentration was zero. Examination of her urine showed an elevated EtG/creatinine ratio. This was caused by having had a few drinks during her leave and could not have been caused by using mouthwash or disinfection soap. We describe how to use the results of CDT and EtG testing in the therapeutic process and give recommendations for patient communication before performing these two tests.

  13. Evolutionary diversification of the vertebrate transferrin multi-gene family.

    Science.gov (United States)

    Hughes, Austin L; Friedman, Robert

    2014-11-01

    In a phylogenetic analysis of vertebrate transferrins (TFs), six major clades (subfamilies) were identified: (a) S, the mammalian serotransferrins; (b) ICA, the mammalian inhibitor of carbonic anhydrase (ICA) homologs; (c) L, the mammalian lactoferrins; (d) O, the ovotransferrins of birds and reptiles; (e) M, the melanotransferrins of bony fishes, amphibians, reptiles, birds, and mammals; and (f) M-like, a newly identified TF subfamily found in bony fishes, amphibians, reptiles, and birds. A phylogenetic tree based on the joint alignment of N-lobes and C-lobes supported the hypothesis that three separate events of internal duplication occurred in vertebrate TFs: (a) in the common ancestor of the M subfamily, (b) in the common ancestor of the M-like subfamily, and (c) in the common ancestor of other vertebrate TFs. The S, ICA, and L subfamilies were found only in placental mammals, and the phylogenetic analysis supported the hypothesis that these three subfamilies arose by gene duplication after the divergence of placental mammals from marsupials. The M-like subfamily was unusual in several respects, including the presence of a uniquely high proportion of clade-specific conserved residues, including distinctive but conserved residues in the sites homologous to those functioning in carbonate binding of human serotransferrin. The M-like family also showed an unusually high proportion of cationic residues in the positively charged region corresponding to human lactoferrampin, suggesting a distinctive role of this region in the M-like subfamily, perhaps in antimicrobial defense.

  14. The association among cytochrome P450 3A, progesterone receptor polymorphisms, plasma 17-alpha hydroxyprogesterone caproate concentrations, and spontaneous preterm birth.

    Science.gov (United States)

    Bustos, Martha L; Caritis, Steve N; Jablonski, Kathleen A; Reddy, Uma M; Sorokin, Yoram; Manuck, Tracy; Varner, Michael W; Wapner, Ronald J; Iams, Jay D; Carpenter, Marshall W; Peaceman, Alan M; Mercer, Brian M; Sciscione, Anthony; Rouse, Dwight J; Ramin, Susan M

    2017-09-01

    Infants born preterm birth are the leading cause of mortality in children preterm birth by 33% in women with history of spontaneous preterm birth. We demonstrated previously that plasma concentrations of 17-alpha hydroxyprogesterone caproate vary widely among pregnant women and that women with 17-alpha hydroxyprogesterone caproate plasma concentrations in the lowest quartile had spontaneous preterm birth rates of 40% vs rates of 25% in those women with higher concentrations. Thus, plasma concentrations are an important factor in determining drug efficacy but the reason 17-alpha hydroxyprogesterone caproate plasma concentrations vary so much is unclear. Predominantly, 17-alpha hydroxyprogesterone caproate is metabolized by CYP3A4 and CYP3A5 enzymes. We sought to: (1) determine the relation between 17-alpha hydroxyprogesterone caproate plasma concentrations and single nucleotide polymorphisms in CYP3A4 and CYP3A5; (2) test the association between progesterone receptor single nucleotide polymorphisms and spontaneous preterm birth; and (3) test whether the association between plasma concentrations of 17-alpha hydroxyprogesterone caproate and spontaneous preterm birth varied by progesterone receptor single nucleotide polymorphisms. In this secondary analysis, we evaluated genetic polymorphism in 268 pregnant women treated with 17-alpha hydroxyprogesterone caproate, who participated in a placebo-controlled trial to evaluate the benefit of omega-3 supplementation in women with history of spontaneous preterm birth. Trough plasma concentrations of 17-alpha hydroxyprogesterone caproate were measured between 25-28 weeks of gestation after a minimum of 5 injections of 17-alpha hydroxyprogesterone caproate. We extracted DNA from maternal blood samples and genotyped the samples using TaqMan (Applied Biosystems, Foster City, CA) single nucleotide polymorphism genotyping assays for the following single nucleotide polymorphisms: CYP3A4*1B, CYP3A4*1G, CYP3A4*22, and CYP3A5*3; and rs

  15. Soluble Urokinase-Type Plasminogen Activator Receptor Plasma Concentration May Predict Susceptibility to High Altitude Pulmonary Edema

    Science.gov (United States)

    Zügel, Stefanie; Schoeb, Michele; Auinger, Katja; Dehnert, Christoph; Maggiorini, Marco

    2016-01-01

    Introduction. Acute exposure to high altitude induces inflammation. However, the relationship between inflammation and high altitude related illness such as high altitude pulmonary edema (HAPE) and acute mountain sickness (AMS) is poorly understood. We tested if soluble urokinase-type plasminogen activator receptor (suPAR) plasma concentration, a prognostic factor for cardiovascular disease and marker for low grade activation of leukocytes, will predict susceptibility to HAPE and AMS. Methods. 41 healthy mountaineers were examined at sea level (SL, 446 m) and 24 h after rapid ascent to 4559 m (HA). 24/41 subjects had a history of HAPE and were thus considered HAPE-susceptible (HAPE-s). Out of the latter, 10/24 HAPE-s subjects were randomly chosen to suppress the inflammatory cascade with dexamethasone 8 mg bid 24 h prior to ascent. Results. Acute hypoxic exposure led to an acute inflammatory reaction represented by an increase in suPAR (1.9 ± 0.4 at SL versus 2.3 ± 0.5 at HA, p < 0.01), CRP (0.7 ± 0.5 at SL versus 3.6 ± 4.6 at HA, p < 0.01), and IL-6 (0.8 ± 0.4 at SL versus 3.3 ± 4.9 at HA, p < 0.01) in all subjects except those receiving dexamethasone. The ascent associated decrease in PaO2 correlated with the increase in IL-6 (r = 0.46, p < 0.001), but not suPAR (r = 0.27, p = 0.08); the increase in IL-6 was not correlated with suPAR (r = 0.16, p = 0.24). Baseline suPAR plasma concentration was higher in the HAPE-s group (2.0 ± 0.4 versus 1.8 ± 0.4, p = 0.04); no difference was found for CRP and IL-6 and for subjects developing AMS. Conclusion. High altitude exposure leads to an increase in suPAR plasma concentration, with the missing correlation between suPAR and IL-6 suggesting a cytokine independent, leukocyte mediated mechanism of low grade inflammation. The correlation between IL-6 and PaO2 suggests a direct effect of hypoxia, which is not the case for suPAR. However, suPAR plasma concentration measured before hypoxic exposure may predict

  16. Genetic variation in estrogen receptor, C-reactive protein and fibrinogen does not predict the plasma levels of inflammation markers after longterm hormone replacement therapy

    DEFF Research Database (Denmark)

    de Maat, Moniek P M; Madsen, Jonna Skov; Langdahl, Bente Lomholt;

    2007-01-01

    Markers of inflammation, such as C-reactive protein (CRP) and fibrinogen, are associated with the risk of atherothrombosis. Plasma levels of these markers of inflammation are affected by hormone replacement therapy (HRT) and modulated by smoking. We studied whether genetic variation in the estrogen...... receptor- 1 (ESR1), CRP and fibrinogen-beta genes influences the plasma levels of inflammation markers after HRT. Plasma CRP and fibrinogen were measured after five years follow-up in healthy postmenopausal women (per-protocol group) who were randomised to hormone therapy (n=187) or no treatment (n=249......). The effect of HRT, smoking and genetic variations in ESR1 (PvuII and XbaI), CRP (1444C/T) and fibrinogen-beta (FGB, -455G/A) were determined. The plasma concentration of CRP was higher in the HRT group than in the control group (2.03 mg/l and 1.41 mg/l, respectively; p

  17. Protein Kinase D and Gβγ Subunits Mediate Agonist-evoked Translocation of Protease-activated Receptor-2 from the Golgi Apparatus to the Plasma Membrane.

    Science.gov (United States)

    Jensen, Dane D; Zhao, Peishen; Jimenez-Vargas, Nestor N; Lieu, TinaMarie; Gerges, Marina; Yeatman, Holly R; Canals, Meritxell; Vanner, Stephen J; Poole, Daniel P; Bunnett, Nigel W

    2016-05-20

    Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca(2+) signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases.

  18. A membrane-proximal, C-terminal α-helix is required for plasma membrane localization and function of the G Protein-coupled receptor (GPCR) TGR5.

    Science.gov (United States)

    Spomer, Lina; Gertzen, Christoph G W; Schmitz, Birte; Häussinger, Dieter; Gohlke, Holger; Keitel, Verena

    2014-02-07

    The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of β-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the β2 adrenergic receptor (β2AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5β2AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the β2AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.

  19. Improvement of Plasma Biomarkers after Switching Stroke Patients from Other Angiotensin II Type I Receptor Blockers to Olmesartan.

    Science.gov (United States)

    Tada, Yoshiteru; Yagi, Kenji; Uno, Masaaki; Matsushita, Nobuhisa; Kanematsu, Yasuhisa; Kuwayama, Kazuyuki; Shimada, Kenji; Nishi, Kyoko; Hirasawa, Motohiro; Satomi, Junichiro; Kitazato, Keiko T; Kageji, Teruyoshi; Matsuura, Eiji; Nagahiro, Shinji

    2015-07-01

    Managing hypertension is crucial for preventing stroke recurrence. Some stroke patients experience resistant hypertension. In our experimental stroke model, olmesartan increased the expression of angiotensin (Ang) II converting enzyme-2. We hypothesized that switching to olmesartan affects biomarkers and the blood pressure (BP) in stroke patients whose BP is insufficiently controlled by standard doses of Ang II type I receptor blockers (ARBs) other than olmesartan. We recruited 25 patients to study our hypothesis. All had a history of stroke or silent cerebral infarction. We switched them to olmesartan (10-40 mg per day) for 12 weeks and determined their plasma level of Ang-(1-7), peroxiredoxin, oxidized low-density lipoprotein (oxLDL)/β-2-glycoprotein I (β2GPI) complex, adiponectin, high mobility group box 1 (HMGB1), and tumor necrosis factor-α (TNFα) and recorded their BP before and after olmesartan treatment. After switching the patients to olmesartan, their plasma level of Ang-(1-7) as a vasoprotective indicator and adiponectin regulating metabolic syndrome was increased, and peroxiredoxin and the oxLDL/β2GPI complex indicating its antioxidative stress and its proatherogenicity were lower than their baseline. This suggests that olmesartan may be more effective than other ARBs to improve these conditions. Neither HMGB1 nor TNFα reflecting an inflammatory response was affected, suggesting that the anti-inflammatory effects of olmesartan are similar to those of other ARBs. The recommended BP (olmesartan. No adverse events occurred. Switching from other ARBs to olmesartan may be a promising therapeutic option in patients with resistant hypertension. Copyright © 2015 National Stroke Association. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  1. Exosomes: Tunable Nano Vehicles for Macromolecular Delivery of Transferrin and Lactoferrin to Specific Intracellular Compartment.

    Science.gov (United States)

    Malhotra, Himanshu; Sheokand, Navdeep; Kumar, Santosh; Chauhan, Anoop S; Kumar, Manoj; Jakhar, Priyanka; Boradia, Vishant M; Raje, Chaaya I; Raje, Manoj

    2016-05-01

    Due to their abundant ubiquitous presence, rapid uptake and increased requirement in neoplastic tissue, the delivery of the iron carrier macromolecules transferrin (Tf) and lactoferrin (Lf) into mammalian cells is the subject of intense interest for delivery of drugs and other target molecules into cells. Utilizing exosomes obtained from cells of diverse origin we confirmed the presence of the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which has recently been characterized as a Tf and Lf receptor. Using a combination of biochemical, biophysical and imaging based methodologies, we demonstrate that GAPDH present in exosomes captures Tf and Lf and subsequently effectively delivers these proteins into mammalian cells. Exosome vesicles prepared had a size of 51.2 ± 23.7 nm. They were found to be stable in suspension with a zeta potential (ζ-potential) of -28.16 ± 1.15 mV. Loading of Tf/Lf did not significantly affect ζ-potential of the exosomes. The carrier protein loaded exosomes were able to enhance the delivery of Tf/Lf by 2 to 3 fold in a diverse panel of cell types. Ninety percent of the internalized cargo via this route was found to be specifically delivered into late endosome and lysosomes. We also found exosomes to be tunable nano vehicles for cargo delivery by varying the amount of GAPDH associated with exosome. The current study opens a new avenue of research for efficient delivery of these vital iron carriers into cells employing exosomes as a nano delivery vehicle.

  2. Targeting etoposide to acute myelogenous leukaemia cells using nanostructured lipid carriers coated with transferrin

    Science.gov (United States)

    Khajavinia, Amir; Varshosaz, Jaleh; Jafarian Dehkordi, Abbas

    2012-10-01

    The aim of the present study was to evaluate the diverse properties of transferrin (Tf)-conjugated nanostructured lipid carriers (NLCs) prepared using three different fatty amines, including stearylamine (SA), dodecylamine (DA) and spermine (SP), and two different methods for Tf coupling. Etoposide-loaded NLCs were prepared by an emulsion-solvent evaporation method followed by probe sonication. Chemical coupling of NLCs with Tf was mediated by an amide linkage between the surface-exposed amino group of the fatty amine and the carboxyl group of the protein. The physical coating was performed in a Ringer-Hepes buffer medium. NLCs were characterized by their particle size, zeta potential, polydispersity index, drug entrapment percentage, drug release profiles and Tf-coupling efficiency. The cytotoxicity of NLCs on K562 acute myelogenous leukaemia cells was studied by MTT assay, and their cellular uptake was studied by a flow cytometry method. SA-containing NLCs showed the lowest particle size, the highest zeta potential and the largest coupling efficiency values. The drug entrapment percentage and the zeta potential decreased after Tf coupling, but the average particle size increased. SP-containing formulations released their drug contents comparatively slower than SA- or DA-containing NLCs. Unconjugated NLCs released moderately more drug than Tf-NLCs. Flow cytometry studies revealed enhanced cellular uptake of Tf-NLCs compared to unconjugated ones. Blocking Tf receptors resulted in a significantly higher cell survival rate for Tf-NLCs. The highest cytotoxic activity was observed in the chemically coupled SA-containing nanoparticles, with an IC50 value of 15-fold lower than free etoposide.

  3. The Vasopressin Type-2 Receptor and Prostaglandin Receptors EP2 and EP4 can Increase Aquaporin-2 Plasma Membrane Targeting Through a cAMP Independent Pathway

    DEFF Research Database (Denmark)

    Olesen, Emma Tina Bisgaard; Moeller, Hanne Bjerregaard; Assentoft, Mette

    2016-01-01

    Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be cA...

  4. Temporal responses of cutaneous blood flow and plasma catecholamine concentrations to histamine H1- or H2-receptor stimulation in man

    DEFF Research Database (Denmark)

    Knigge, U; Alsbjørn, B; Thuesen, B;

    1988-01-01

    continuously with a laser Doppler flowmeter, and noradrenaline and adrenaline concentrations were determined in blood samples drawn every 15 min. The infusion of histamine caused an immediate and sustained vasodilatation. The Concomitant infusion of mepyramine prevented the immediate vasodilatation, but had...... noradrenaline, while the increase during concomitant H1-receptor blockade was delayed but achieved the level observed during the histamine infusion. The response to histamine during H2-receptor blockade was small and transient. The rise in plasma adrenaline was not significant. These findings suggest...

  5. Comparative neuroprotective properties of stilbene and catechin analogs: action via a plasma membrane receptor site?

    Science.gov (United States)

    Bastianetto, Stéphane; Dumont, Yvan; Han, Yingshan; Quirion, Rémi

    2009-01-01

    Various studies have reported on the neuroprotective effects of polyphenols, widely present in food, beverages, and natural products. For example, we have shown that resveratrol, a polyphenol enriched in red wine and other foods such as peanuts, protects hippocampal cells against beta-amyloid (Abeta)-induced toxicity, a key protein involved in the neuropathology of Alzheimer disease. This effect involves, at least in part, the capacity of resveratrol to activate the phosphorylation of delta isoform of protein kinase C (PKC-delta). The neuroprotective action of resveratrol is shared by piceatannol, a stilbene derivative, as well as by tea-derived catechin gallate esters. The thioflavin T assay indicated that all these polyphenols inhibited the formation of Abeta fibrils, suggesting that this action likely also contributes to their neuroprotective effects. Binding and autoradiographic studies revealed that the effects of polyphenols might involve specific binding sites that are particularly enriched in the choroid plexus in the rat brain. Interestingly, the choroid plexus secretes transthyretin, a protein that has been shown to modulate Abeta aggregation and that may be critical to the maintenance of normal learning capacities in aging. Taken together, these data suggest that polyphenols target multiple enzymes/proteins, leading to their neuroprotective actions, possibly through action via specific plasma membrane binding sites.

  6. Plasma soluble erythropoietin receptor is decreased during sleep in Andean highlanders with Chronic Mountain Sickness.

    Science.gov (United States)

    Villafuerte, Francisco C; Corante, Noemí; Anza-Ramírez, Cecilia; Figueroa-Mujíca, Rómulo; Vizcardo-Galindo, Gustavo; Mercado, Andy; Macarlupú, José Luis; León-Velarde, Fabiola

    2016-07-01

    Excessive erythrocytosis (EE) is the main sign of Chronic Mountain Sickness (CMS), a highly prevalent syndrome in Andean highlanders. Low pulse O2 saturation (SpO2) during sleep and serum androgens have been suggested to contribute to EE in CMS patients. However, whether these factors have a significant impact on the erythropoietin (Epo) system leading to EE is still unclear. We have recently shown that morning soluble Epo receptor (sEpoR), an endogenous Epo antagonist, is decreased in CMS patients suggesting increased Epo availability (increased Epo/sEpoR). The present study aimed to characterize the nocturnal concentration profile of sEpoR and Epo and their relationship with SpO2, Hct, and serum testosterone in healthy highlanders (HH) and CMS patients. Epo and sEpoR concentrations were evaluated every 4 h (6 PM to 6 AM) and nighttime SpO2 was continuously monitored (10 PM to 6 AM) in 39 male participants (CMS, n = 23; HH, n = 16) aged 21-65 yr from Cerro de Pasco, Peru (4,340 m). CMS patients showed higher serum Epo concentrations throughout the night and lower sEpoR from 10 PM to 6 AM. Consequently, Epo/sEpoR was significantly higher in the CMS group at every time point. Mean sleep-time SpO2 was lower in CMS patients compared with HH, while the percentage of sleep time spent with SpO2 < 80% was higher. Multiple-regression analysis showed mean sleep-time SpO2 and Epo/sEpoR as significant predictors of hematocrit corrected for potential confounders (age, body mass index, and testosterone). Testosterone levels were associated neither with Hct nor with erythropoietic factors. In conclusion, our results show sustained erythropoietic stimulus driven by the Epo system in CMS patients, further enhanced by a continuous exposure to accentuated nocturnal hypoxemia.

  7. Changes in the plasma membrane in metabolic disease: impact of the membrane environment on G protein-coupled receptor structure and function.

    Science.gov (United States)

    Desai, Aditya J; Miller, Laurence J

    2017-07-10

    Drug development targeting GPCRs often utilizes model heterologous cell expression systems, reflecting an implicit assumption that the membrane environment has little functional impact on these receptors or on their responsiveness to drugs. However, much recent data have illustrated that membrane components can have an important functional impact on intrinsic membrane proteins. This review is directed toward gaining a better understanding of the structure of the plasma membrane in health and disease, and how this organelle can influence GPCR structure, function and regulation. It is important to recognize that the membrane provides a potential mode of lateral allosteric regulation of GPCRs and can affect the effectiveness of drugs and their biological responses in various disease states, which can even vary among individuals across the population. The type 1 cholecystokinin receptor is reviewed as an exemplar of a class A GPCR that is affected in this way by changes in the plasma membrane. © 2017 The British Pharmacological Society.

  8. The glycation site specificity of human serum transferrin is a determinant for transferrin's functional impairment under elevated glycaemic conditions.

    Science.gov (United States)

    Silva, André M N; Sousa, Paulo R H; Coimbra, João T S; Brás, Natércia F; Vitorino, Rui; Fernandes, Pedro A; Ramos, Maria J; Rangel, Maria; Domingues, Pedro

    2014-07-01

    The mechanisms involving iron toxicity in diabetes mellitus are not completely understood. However, the spontaneous reaction of reducing sugars with protein amino groups, known as glycation, has been shown to compromise the action of Tf (transferrin), the systemic iron transporter. In order to understand the structural alterations that impair its function, Tf was glycated in vitro and the modification sites were determined by MS. Iron binding to glycated Tf was assessed and a computational approach was conducted to study how glycation influences the iron-binding capacity of this protein. Glycated Tf samples were found to bind iron less avidly than non-modified Tf and MS results revealed 12 glycation sites, allowing the establishment of Lys534 and Lys206 as the most vulnerable residues to this modification. Their increased susceptibility to glycation was found to relate to their low side-chain pKa values. Lys534 and Lys206 participate in hydrogen bonding crucial for iron stabilization in the C- and N-lobes of the protein respectively, and their modification is bound to influence iron binding. Furthermore, the orientation of the glucose residues at these sites blocks the entrance to the iron-binding pocket. Molecular dynamics simulations also suggested that additional loss of iron binding capacity may result from the stereochemical effects induced by the glycation of lysine residues that prevent the conformational changes (from open to closed Tf forms) required for metal binding. Altogether, the results indicate that Tf is particularly vulnerable to glycation and that this modification targets spots that are particularly relevant to its function.

  9. TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP.

    Science.gov (United States)

    Brejchová, Jana; Sýkora, Jan; Ostašov, Pavel; Merta, Ladislav; Roubalová, Lenka; Janáček, Jiří; Hof, Martin; Svoboda, Petr

    2015-03-01

    Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a "membrane fence" model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more "fluid" and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R-Gq/G11α protein coupling. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Mating behavior induces changes of expression of Fos protein, plasma testosterone and androgen receptors in the accessory olfactory bulb (AOB) of the male mandarin vole Microtus mandarinus

    OpenAIRE

    Fengqin HE, Fadao TAI

    2009-01-01

    In order to investigate the neuroendocrine mechanism of the mating behavior in the adult male mandarin voles Microtus mandarinus, the radioimmunoassay (RIA) and immunohistochemistry methods were used to investigate the differences in plasma testosterone (T) concentrations and distribution of T immunoreactive neurons (T-IRs), androgen receptor immunoreactive neurons (AR-IRs) and Fos protein immunoreactive neurons (Fos-IRs) in the accessory olfactory bulb (AOB) and the main olfactory bulb (MOB)...

  11. Effects of Acute Pinitol Supplementation on Plasma Pinitol Concentration, Whole Body Glucose Tolerance, and Activation of the Skeletal Muscle Insulin Receptor in Older Humans

    OpenAIRE

    Stull, A. J.; Wood, K V; Thyfault, J. P.; Campbell, W.W.

    2009-01-01

    Limited research with rodents and humans suggests that oral ingestion of pinitol (3-O-methyl-d-chiro-inositol) might positively influence glucose tolerance. This double-blinded, placebo-controlled, and cross-over study assessed the effects of acute pinitol supplementation on plasma pinitol concentration, glucose tolerance, insulin sensitivity, and activation of the skeletal muscle insulin receptor. Fifteen older, nondiabetic subjects (62 ± 1 years, mean ± SEM) completed four, 1-day trials. Su...

  12. Soluble TNF-alpha receptor 1 and IL-6 plasma levels in humans subjected to the sleep deprivation model of spaceflight

    Science.gov (United States)

    Shearer, W. T.; Reuben, J. M.; Mullington, J. M.; Price, N. J.; Lee, B. N.; Smith, E. O.; Szuba, M. P.; Van Dongen, H. P.; Dinges, D. F.

    2001-01-01

    BACKGROUND: The extent to which sleep loss may predispose astronauts to a state of altered immunity during extended space travel prompts evaluation with ground-based models. OBJECTIVE: We sought to measure plasma levels of selected cytokines and their receptors, including the putative sleep-regulation proteins soluble TNF-alpha receptor (sTNF-alpha R) I and IL-6, in human subjects undergoing 2 types of sleep deprivation during environmental confinement with performance demands. METHODS: Healthy adult men (n = 42) were randomized to schedules that varied in severity of sleep loss: 4 days (88 hours) of partial sleep deprivation (PSD) involving two 2-hour naps per day or 4 days of total sleep deprivation (TSD). Plasma samples were obtained every 6 hours across 5 days and analyzed by using enzyme-linked immunoassays for sTNF-alpha RI, sTNF-alpha RII, IL-6, soluble IL-2 receptor, IL-10, and TNF-alpha. RESULTS: Interactions between the effects of time and sleep deprivation level were detected for sTNF-alpha RI and IL-6 but not for sTNF-alpha RII, soluble IL-2 receptor, IL-10, and TNF-alpha. Relative to the PSD condition, subjects in the TSD condition had elevated plasma levels of sTNF-alpha RI on day 2 (P =.04), day 3 (P =.01), and across days 2 to 4 of sleep loss (P =.01) and elevated levels of IL-6 on day 4 (P =.04). CONCLUSIONS: Total sleep loss produced significant increases in plasma levels of sTNF-alpha RI and IL-6, messengers that connect the nervous, endocrine, and immune systems. These changes appeared to reflect elevations of the homeostatic drive for sleep because they occurred in TSD but not PSD, suggesting that naps may serve as the basis for a countermeasures approach to prolonged spaceflight.

  13. Endocytosis of adiponectin receptor 1 through a clathrin-and Rab5-dependent pathway

    Institute of Scientific and Technical Information of China (English)

    Qiurong Ding; Zhenzhen Wang; Yan Chen

    2009-01-01

    In eukaryotic cells, receptor endocytosis is a key event regulating signaling transduction. Adiponectin receptors belong to a new receptor family that is distinct from G-protein-coupled receptors and has critical roles in the pathogen-esis of diabetes and metabolic syndrome. Here, we analyzed the endocytosis of adiponectin and adiponectin receptor 1 (AdipoR1) and found that they are both internalized into transferrin-positive compartments that follow similar traffic routes. Blocking clathrin-mediated endocytosis by expressing Epsl5 mutants or depleting K+ trapped AdipoRl at the plasma membrane, and K+ depletion abolished adiponectin internalization, indicating that the endocytosis of AdipoRl and adiponectin is clathrin-dependent. Depletion of K+ and overexpression of Eps15 mutants enhance adiponectin-stimulated AMP-activated protein kinase phosphorylation, suggesting that the endocytosis of AdipoR1 might down-regulate adiponectin signaling. In addition, AdipoR1 colocalizes with the small GTPase Rab5, and a dominant negative Rab5 abrogates AdipoR1 endocytosis. These data indicate that AdipoRl is internalized through a clathrin- and Rab5-dependent pathway and that endocytosis may play a role in the regulation of adiponectin signaling.

  14. Inhibitory Effect of the Punica granatum Fruit Extract on Angiotensin-II Type I Receptor and Thromboxane B2 in Endothelial Cells Induced by Plasma from Preeclamptic Patients.

    Science.gov (United States)

    Kusumawati, Widya; Keman, Kusnarman; Soeharto, Setyawati

    2016-01-01

    This study aims to evaluate whether the Punica granatum fruit extract modulates the Angiotensin-II Type I receptor (AT1-R) and thromboxane B2 level in endothelial cells induced by plasma from preeclamptic patients. Endothelial cells were obtained from human umbilical vascular endothelial cells. At confluence, endothelial cells were divided into five groups, which included endothelial cells exposed to 2% plasma from normal pregnancy (NP), endothelial cells exposed to 2% plasma from preeclamptic patients (PP), and endothelial cells exposed to PP in the presence of ethanolic extract of Punica granatum (PP + PG) at the following three doses: 14; 28; and 56 ppm. The expression of AT1-R was observed by immunohistochemistry technique, and thromboxane B2 level was done by immunoassay technique. Plasma from PP significantly increased AT1-R expression and thromboxane B2 levels compared to cells treated by normal pregnancy plasma. The increasing of AT1-R expression significantly (P Punica granatum extract. Moreover, the increasing of thromboxane B2 levels significantly (P Punica granatum extract. We further concluded that Punica granatum fruit protects and inhibits the sensitivity of endothelial cells to plasma from preeclamptic patients due to inhibition of AT1-R expression (56 ppm) and reduced thromboxane B2 levels (14 ppm).

  15. Genetic variation in estrogen receptor, C-reactive protein and fibrinogen does not predict the plasma levels of inflammation markers after longterm hormone replacement therapy

    DEFF Research Database (Denmark)

    de Maat, Moniek P M; Madsen, Jonna Skov; Langdahl, Bente;

    2007-01-01

    ). The effect of HRT, smoking and genetic variations in ESR1 (PvuII and XbaI), CRP (1444C/T) and fibrinogen-beta (FGB, -455G/A) were determined. The plasma concentration of CRP was higher in the HRT group than in the control group (2.03 mg/l and 1.41 mg/l, respectively; p ... receptor- 1 (ESR1), CRP and fibrinogen-beta genes influences the plasma levels of inflammation markers after HRT. Plasma CRP and fibrinogen were measured after five years follow-up in healthy postmenopausal women (per-protocol group) who were randomised to hormone therapy (n=187) or no treatment (n=249...... concentration (n.s.). Genetic polymorphisms in ESR1, CRP and fibrinogen were not associated with an effect of HRT on the CRP and fibrinogen plasma levels, and no significant interaction with smoking was observed. In conclusion, higher plasma levels of CRP and lower plasma levels of fibrinogen were observed...

  16. Plasma soluble urokinase-type plasminogen activator receptor level is independently associated with coronary microvascular function in patients with non-obstructive coronary artery disease

    DEFF Research Database (Denmark)

    Mekonnen, Girum; Corban, Michel T; Hung, Olivia Y;

    2015-01-01

    , medications profiles and hs-CRP, suPAR remained an independent predictor of CFR (B = -0.30, p = 0.04), indicating an independent association between suPAR level and coronary microvascular function. CONCLUSIONS: In this cross-sectional study, plasma suPAR level was an independent predictor of coronary......BACKGROUND: Soluble urokinase-type plasminogen activator receptor (suPAR) is a novel biomarker released from leukocytes and endothelial cells that has been associated with atherosclerotic cardiovascular disease. We hypothesized that plasma suPAR level is an independent predictor of coronary...... microvascular function. METHODS: Coronary blood flow velocity and plasma suPAR levels were evaluated in patients with non-obstructive coronary artery disease. Coronary flow reserve (CFR) was calculated as the ratio of hyperemic to basal average peak blood flow velocity and coronary microvascular dysfunction...

  17. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  18. The TNF-alpha system in heart failure and after heart transplantation : plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    NARCIS (Netherlands)

    van Riemsdijk-van Overbeeke, I C; Baan, C C; Niesters, H G; Hesse, C J; Loonen, E H; Balk, A H; Maat, A P; Weimar, W

    BACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To

  19. The TNF-alpha system in heart failure and after heart transplantation : plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    NARCIS (Netherlands)

    van Riemsdijk-van Overbeeke, I C; Baan, C C; Niesters, H G; Hesse, C J; Loonen, E H; Balk, A H; Maat, A P; Weimar, W

    1999-01-01

    BACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To asse

  20. A computational analysis of non-genomic plasma membrane progestin binding proteins: signaling through ion channel-linked cell surface receptors.

    Science.gov (United States)

    Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K

    2013-12-11

    A number of plasma membrane progestin receptors linked to non-genomic events have been identified. These include: (1) α1-subunit of the Na(+)/K(+)-ATPase (ATP1A1), (2) progestin binding PAQR proteins, (3) membrane progestin receptor alpha (mPRα), (4) progesterone receptor MAPR proteins and (5) the association of nuclear receptor (PRB) with the plasma membrane. This study compares: the pore-lining regions (ion channels), transmembrane (TM) helices, caveolin binding (CB) motifs and leucine-rich repeats (LRRs) of putative progesterone receptors. ATP1A1 contains 10 TM helices (TM-2, 4, 5, 6 and 8 are pores) and 4 CB motifs; whereas PAQR5, PAQR6, PAQR7, PAQRB8 and fish mPRα each contain 8 TM helices (TM-3 is a pore) and 2-4 CB motifs. MAPR proteins contain a single TM helix but lack pore-lining regions and CB motifs. PRB contains one or more TM helices in the steroid binding region, one of which is a pore. ATP1A1, PAQR5/7/8, mPRα, and MAPR-1 contain highly conserved leucine-rich repeats (LRR, common to plant membrane proteins) that are ligand binding sites for ouabain-like steroids associated with LRR kinases. LRR domains are within or overlap TM helices predicted to be ion channels (pore-lining regions), with the variable LRR sequence either at the C-terminus (PAQR and MAPR-1) or within an external loop (ATP1A1). Since ouabain-like steroids are produced by animal cells, our findings suggest that ATP1A1, PAQR5/7/8 and mPRα represent ion channel-linked receptors that respond physiologically to ouabain-like steroids (not progestin) similar to those known to regulate developmental and defense-related processes in plants.

  1. The BBSome Controls Energy Homeostasis by Mediating the Transport of the Leptin Receptor to the Plasma Membrane.

    Directory of Open Access Journals (Sweden)

    Deng-Fu Guo

    2016-02-01

    Full Text Available Bardet-Biedl syndrome (BBS is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb. Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity.

  2. The BBSome Controls Energy Homeostasis by Mediating the Transport of the Leptin Receptor to the Plasma Membrane.

    Science.gov (United States)

    Guo, Deng-Fu; Cui, Huxing; Zhang, Qihong; Morgan, Donald A; Thedens, Daniel R; Nishimura, Darryl; Grobe, Justin L; Sheffield, Val C; Rahmouni, Kamal

    2016-02-01

    Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity.

  3. Measurement of NMDA Receptor Antagonist, CPP, in Mouse Plasma and Brain Tissue Following Systematic Administration Using Ion-Pair LCMS/MS.

    Science.gov (United States)

    Gemperline, Erin; Laha, Kurt; Scarlett, Cameron O; Pearce, Robert A; Li, Lingjun

    2014-08-21

    (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) is a competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and is routinely used with rodent models to investigate the role of NMDA receptors in brain function. This highly polar compound is difficult to separate from biological matrices. A reliable and sensitive assay was developed for the determination of CPP in plasma and tissue. In order to overcome the challenges relating to the physicochemical properties of CPP we employed an initial separation using solid phase extraction harnessing mixed-mode anion exchange. Then an ion-pair UPLC C18 separation was performed followed by MS/MS with a Waters Acquity UPLC interfaced to an AB Sciex QTrap 5500 mass spectrometer, which was operated in positive ion ESI mode. Multiple reaction monitoring (MRM) mode was utilized to detect the analyte and internal standard. The precursor to product ions used for quantitation for CPP and internal standard were m/z 252.958 → 207.100 and 334.955 → 136.033, respectively. This method was applied to a pharmacokinetic study and examined brain tissue and plasma concentrations following intravenous and intraperitoneal injections of CPP. The elimination half-life (t1/2) of CPP was 8.8 minutes in plasma and 14.3 minutes in brain tissue, and the plasma to brain concentration ratio was about 18:1. This pharmacokinetic data will aid the interpretation of the vast number of studies using CPP to investigate NMDA receptor function in rodents and the method itself can be used to study many other highly polar analytes of interest.

  4. Plasma fibrinogen levels are associated with epidermal growth factor receptor gene mutation in Chinese patients with non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    Jianfei Zhu; Ling Cai; Haoxian Yang; Yinsheng Wen; Junye Wang; Tiehua Rong; Lanjun Zhang

    2013-01-01

    Objective: The plasma fibrinogen levels had not only been used as an independent prognostic parameter for thepatients with non-small cell lung cancer (NSCLC), but also as a promising biomarker for evaluating the efficacy of chemotherapy. This study aimed to investigate the correlation between the plasma fibrinogen levels and epidermal growth factor receptor (EGFR) gene mutation and clinical-pathological characteristics of Chinese patients with NSCLC. Methods: In this retrospective study, NSCLC specimens collected from 352 patients between November 2009 and November 2011 were selected to detect EGFR gene mutation with real-time polymerase chain reaction (RT-PCR). In these specimens, 308 ones were also detected EGFR gene copy number with fluorescence in situ hybridization (FISH). Coagulation makers were examined prior to the operations. The association between the plasma fibrinogen levels and EGFR gene mutation and clinical-pathological characteristics were analyzed using SPSS 16.0 software. Results: The median pre-operation plasma fibrinogen level was 3.55 g/L (109/352) patients with higher plasma fibrinogen level (> 4.0 g/L). The lower plasma fibrinogen levels correlated significantly with EGFR gene mutations (P < 0.001), the similar result was seen in platelet counts (P = 0.026). A linear correlation was found between the plasma fibrinogen levels and the platelet counts in NSCLC patients (R2 = 0.209, P < 0.001). Pre-operationplasma fibrinogen levels correlated with gender (P < 0.001), smoking status (P < 0.001), and histology (P < 0.001). There weresignificant link between the above clinical-pathological characteristics and EGFR gene mutations. In addition, EGFR gene mutationwas correlated with FISH-positive status (P < 0.001). Moreover, both plasma fibrinogen level (P = 0.024) and the EGFRgene copy number (P = 0.040) had significant relationships with the pathological TNM stage. Conclusion: This study showedthat a significant relevance between plasma fibrinogen

  5. Apolipoprotein E mediates enhanced plasma high-density lipoprotein cholesterol clearance by low-dose streptococcal serum opacity factor via hepatic low-density lipoprotein receptors in vivo.

    Science.gov (United States)

    Rosales, Corina; Tang, Daming; Gillard, Baiba K; Courtney, Harry S; Pownall, Henry J

    2011-08-01

    Recombinant streptococcal serum opacity factor (rSOF) mediates the in vitro disassembly of human plasma high-density lipoprotein (HDL) into lipid-free apolipoprotein (apo) A-I, a neo-HDL that is cholesterol poor, and a cholesteryl ester-rich microemulsion (CERM) containing apoE. Given the occurrence of apoE on the CERM, we tested the hypothesis that rSOF injection into mice would reduce total plasma cholesterol clearance via apoE-dependent hepatic low-density lipoprotein receptors (LDLR). rSOF (4 μg) injection into wild-type C57BL/6J mice formed neo-HDL, CERM, and lipid-free apoA-I, as observed in vitro, and reduced plasma total cholesterol (-43%, t(1/2)=44±18 minutes) whereas control saline injections had a negligible effect. Similar experiments with apoE(-/-) and LDLR(-/-) mice reduced plasma total cholesterol ≈0% and 20%, respectively. rSOF was potent; injection of 0.18 μg of rSOF produced 50% of maximum reduction of plasma cholesterol 3 hours postinjection, corresponding to a ≈0.5-mg human dose. Most cholesterol was cleared hepatically (>99%), with rSOF treatment increasing clearance by 65%. rSOF injection into mice formed a CERM that was cleared via hepatic LDLR that recognize apoE. This reaction could provide an alternative mechanism for reverse cholesterol transport.

  6. Integrative proteome and transcriptome analysis of extramedullary erythropoiesis and its reversal by transferrin treatment in a mouse model of beta-thalassemia.

    Science.gov (United States)

    Vallelian, Florence; Gelderman-Fuhrmann, Monique P; Schaer, Christian A; Puglia, Michele; Opitz, Lennart; Baek, Jin Hyen; Vostal, Jaroslav; Buehler, Paul W; Schaer, Dominik J

    2015-02-06

    Beta-thalassemia results from mutations of the β-hemoglobin (Hbb) gene and reduced functional Hbb synthesis. Excess α-Hb causes globin chain aggregation, oxidation, cytoskeletal damage, and increased red blood cell clearance. These events result in anemia, altered iron homeostasis, and expansion of extramedullary erythropoiesis. Serum transferrin (Tf) is suggested to be an important regulator of erythropoiesis in murine models of thalassemia. The present study was conducted to establish a quantitative proteomic and transcriptomic analysis of transferrin-modulated extramedullary erythropoiesis in the spleen of wild type and thalassemic Hbb(th3/+) mice. Our LC-MS/MS protein analysis and mRNA sequencing data provide quantitative expression estimates of 1590 proteins and 24,581 transcripts of the murine spleen and characterize key processes of erythropoiesis and RBC homeostasis such as the whole heme synthesis pathway as well as critical components of the red blood cell antioxidant systems and the proliferative cell cycling pathway. The data confirm that Tf treatment of nontransfused Hbb(th3/+) mice induces a systematic correction of these processes at a molecular level. Tf treatment of Hbb(th3/+) mice for 60 days leads to a complete molecular restoration of the normal murine spleen phenotype. These findings support further investigation of plasma-derived Tf as a treatment for thalassemia.

  7. Encapsulation of paclitaxel into lauric acid-O-carboxymethyl chitosan-transferrin micelles for hydrophobic drug delivery and site-specific targeted delivery.

    Science.gov (United States)

    Nam, Joung-Pyo; Park, Seong-Cheol; Kim, Tae-Hun; Jang, Jae-Yeang; Choi, Changyong; Jang, Mi-Kyeong; Nah, Jae-Woon

    2013-11-30

    Transferrin/PEG/O-carboxymethyl chitosan/fatty acid/paclitaxel (TPOCFP) micelles were tested for suitability as a drug carrier characterized by low cytotoxicity, sustained release, high cellular uptake, and site-specific targeted delivery of hydrophobic drugs. Characterization, drug content, encapsulation efficiency, and in vitro drug release were investigated. When the feeding amount of paclitaxel (PTX) was increased, the drug content increased, but loading efficiency decreased. TPOCFP micelles had a spherical shape, with a particle size of approximately 140-649 nm. In vitro cell cytotoxicity and hemolysis assays were conducted to confirm the safety of the micelles. Anticancer activity and confocal laser scanning microscopy (CLSM) were used to confirm the targeting efficiency of target ligand-modified TPOCFP micelles. Anticancer activity and CLSM results clearly demonstrated that transferrin-modified TPOCFP micelles were quickly taken up by the cell. The endocytic pathway of TPOCFP micelles was analyzed by flow cytometry, revealing transfection via receptor-mediated endocytosis. These results suggest that PTX-encapsulated TPOCFP micelles may be used as an effective cancer-targeting drug delivery system for chemotherapy.

  8. A comparative, cross-species investigation of the properties and roles of transferrin- and lactoferrin-binding protein B from pathogenic bacteria.

    Science.gov (United States)

    Ostan, N; Morgenthau, A; Yu, R H; Gray-Owen, S D; Schryvers, A B

    2017-02-01

    Pathogenic bacteria from the families Neisseriaeceae and Moraxellaceae acquire iron from their host using surface receptors that have the ability to hijack iron from the iron-sequestering host proteins transferrin (Tf) and lactoferrin (Lf). The process of acquiring iron from Tf has been well-characterized, including the role of the surface lipoprotein transferrin-binding protein B (TbpB). In contrast, the only well-defined role for the homologue, LbpB, is in its protection against cationic antimicrobial peptides, which is mediated by regions present in some LbpBs that are highly enriched in glutamic or aspartic acid. In this study we compare the Tf-TbpB and the Lf-LbpB interactions and examine the protective effect of LbpB against extracts from human and transgenic mouse neutrophils to gains insights into the physiological roles of LbpB. The results indicate that in contrast to the Tf-TbpB interaction, Lf-LbpB interaction is sensitive to pH and varies between species. In addition, the results with transgenic mouse neutrophils raise the question of whether there is species specificity in the cleavage of Lf to generate cationic antimicrobial peptides or differences in the potency of peptides derived from mouse and human Lf.

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

    Science.gov (United States)

    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.

  10. Transferrin conjugated poly (γ-glutamic acid-maleimide-co-L-lactide)-1,2-dipalmitoylsn-glycero-3-phosphoethanolamine copolymer nanoparticles for targeting drug delivery.

    Science.gov (United States)

    Zhao, Caiyan; Liu, Xiaoguang; Liu, Junxing; Yang, Zhiwei; Rong, Xianghui; Li, Mingjun; Liang, Xingjie; Wu, Yan

    2014-11-01

    Targeted drug delivery strategies have shown great potential in solving some problems of chemotherapy, such as non-selectivity and severe side effects, thus enhancing the anti-tumor efficiency of chemotherapeutic agents. In this work, we have prepared a novel nanoparticle consisted of amphiphilic poly(γ-glutamic acid-maleimide-co-L-lactide)-1,2-dipalmitoylsn-glycero-3-phosphoethanolamine (γ-PGA-MAL-PLA-DPPE) copolymer decorated with transferrin (Tf), which can specifically deliver anti-cancer drug paclitaxel (PTX) to the tumor cells for targeting chemotherapy. These nanoparticles (NPs) have preferable particle size, high encapsulation efficiency and a pH-dependent release profile. As expected, The Tf modification mediate specific targeting to nasopharyngeal carcinoma (C666-1) cells and human cervical carcinoma (Hela) cells with the transferrin receptor (TfR) overexpressed and enhance cellular uptake of the NPs, as demonstrated by flow cytometry and confocal microscopy assays. In vitro cytotoxicity studies reveal that the NPs have excellent biocompatibility, and the presence of Tf enhance the activity of PTX to the targeted cells. All these results prove that Tf modified γ-PGA-MAL-PLA-DPPE NPs could facilitate the tumor-specific therapy. Therefore, such a targeting drug delivery system provides significant advances toward cancer therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Bovine prolactin elevates hTF expression directed by a tissue-specific goat β-casein promoter through prolactin receptor-mediated STAT5a activation.

    Science.gov (United States)

    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.

  12. High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

    Directory of Open Access Journals (Sweden)

    Lenka Roubalova

    Full Text Available HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v, Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%, the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy is increased; affinity of response (potency is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between

  13. Impact of Oral Iron Challenges on Circulating Non-Transferrin-Bound Iron in Healthy Guatemalan Males

    NARCIS (Netherlands)

    Schuemann, Klaus; Kroll, Sylvia; Romero-Abal, Maria-Eugenia; Georgiou, Niki A.; Marx, Jo J. M.; Weiss, Guenter; Solomons, Noel W.

    2012-01-01

    Introduction:Oral iron as a supplement has been associated with adverse health consequences, especially in the context of young children with active malaria. A potential aggravating role of non-transferrin-bound iron (NTBI) has been proposed. Material and Methods: NTBI responses in both a fasting an

  14. Impact of Oral Iron Challenges on Circulating Non-Transferrin-Bound Iron in Healthy Guatemalan Males

    NARCIS (Netherlands)

    Schuemann, Klaus; Kroll, Sylvia; Romero-Abal, Maria-Eugenia; Georgiou, Niki A.; Marx, Jo J. M.; Weiss, Guenter; Solomons, Noel W.

    2012-01-01

    Introduction:Oral iron as a supplement has been associated with adverse health consequences, especially in the context of young children with active malaria. A potential aggravating role of non-transferrin-bound iron (NTBI) has been proposed. Material and Methods: NTBI responses in both a fasting an

  15. Genetic resistance of carp (Cyprinus carpio L.) to Trypanoplasma borreli: influence of transferrin polymorphisms

    NARCIS (Netherlands)

    Jurecka, P.M.; Wiegertjes, G.F.; Rakus, K.L.; Pilarczyk, A.; Irnazarow, I.

    2009-01-01

    In serum most of the iron molecules are bound to transferrin (Tf), which is a highly polymorphic protein in fish. Tf is an essential growth factor for mammalian trypanosomes. We performed a series of experiments with Trypanoplasma borreli to detect putative correlations between different Tf genotype

  16. Expression of SLAM (CD150) cell-surface receptors on human B-cell subsets: from pro-B to plasma cells.

    Science.gov (United States)

    De Salort, Jose; Sintes, Jordi; Llinàs, Laia; Matesanz-Isabel, Jessica; Engel, Pablo

    2011-01-30

    The SLAM (CD150) family receptors are leukocyte cell-surface glycoproteins involved in leukocyte activation. These molecules and their adaptor protein SAP contribute to the effective germinal center formation, generation of high-affinity antibody-secreting plasma cells, and memory B cells, thereby facilitating long-term humoral immune response. Multi-color flow cytometric analysis was performed to determine the expression of CD48 (SLAMF2), CD84 (SLAMF5), CD150 (SLAM or SLAMF1), CD229 (Ly9 or SLAMF3), CD244 (2B4 or SLAMF4), CD319 (CRACC, CS1, or SLAMF7), and CD352 (NTB-A or SLAMF6) on human cell lines and B-cell subsets. The following subsets were assessed: pro-B, pre-B, immature-B, and mature-B cells from bone marrow; transitional and B1/B2 subsets from peripheral blood; and naïve, pre-germinal center, germinal center, memory, plasmablasts, and plasma cells from tonsil and spleen. All receptors were expressed on B cells, with the exception of CD244. SLAM family molecules were widely distributed during B-cell development, maturation and terminal differentiation into plasmablasts and plasma cells, but their expression among various B-cell subsets differed significantly. Such heterogeneous expression patterns suggest that SLAM molecules play an essential and non-redundant role in the control of humoral immune responses. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Effects of AT1 Receptor Blockade on Plasma Thromboxane A2 (TXA2 Level and Skin Microcirculation in Young Healthy Women on Low Salt Diet

    Directory of Open Access Journals (Sweden)

    Ana Cavka

    2013-10-01

    Full Text Available Objective: To determine the effect of AT1 receptor antagonism on skin microcirculation and plasma level of thromboxane A2 (TXA2. Methods: Healthy women (n=20 maintained 7 days low salt (LS diet (intake 2 metabolite thromboxane B2 (TXB2 and plasma renin activity (PRA aldosterone concentration, electrolytes (Na+, K+, as well as blood pressure and heart rate were determined before and after study protocols. Results: PRA and aldosterone increased significantly after 7 days of both LS diet and LS diet+losartan. LS diet or LS diet+losartan administrations had no significant effect on post-occlusion hyperemia While there was no change in TXB2 after LS diet TXB2 significantly increased after one week of LS+losartan compared to control levels (cTXB2 pg/mL control 101±80 vs. LS diet+losartan 190±116, pConclusion: These data suggest that inhibition of AT1 receptors could lead to activation of AT2 receptors, which maintain hyperemia, despite the increased level of vasoconstrictor TXA2. These findings also suggest an important role of crosstalk between renin-angiotensin system (RAS and arachidonic acid metabolites in the regulation of microcirculation under physiological conditions.

  18. The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2

    OpenAIRE

    2011-01-01

    Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inh...

  19. Effects of acute pinitol supplementation on plasma pinitol concentration, whole body glucose tolerance, and activation of the skeletal muscle insulin receptor in older humans.

    Science.gov (United States)

    Stull, A J; Wood, K V; Thyfault, J P; Campbell, W W

    2009-05-01

    Limited research with rodents and humans suggests that oral ingestion of pinitol (3- O-methyl- D- CHIRO-inositol) might positively influence glucose tolerance. This double-blinded, placebo-controlled, and cross-over study assessed the effects of acute pinitol supplementation on plasma pinitol concentration, glucose tolerance, insulin sensitivity, and activation of the skeletal muscle insulin receptor. Fifteen older, nondiabetic subjects (62+/-1 years, mean+/-SEM) completed four, 1-day trials. Subjects consumed a non-nutritive beverage with nothing (placebo) or 1,000 mg pinitol. Sixty minutes later, the subjects consumed beverages that were either energy- and carbohydrate-free (Sham) or contained 75 g glucose (OGTT). Blood samples were collected frequently over the 240-min testing period. For the OGTT trials only, vastus lateralis samples were obtained before the placebo and pinitol supplementation and 60 min after consuming the 75 g glucose beverage. Plasma pinitol concentration increased and was maintained for 240 min. Pinitol did not influence the fasting state and 180-min area under the curves for plasma glucose and insulin during the Sham and OGTT trials or hepatic (placebo 0.83+/-0.08; pinitol 0.80+/-0.08) and whole-body (placebo 6.10+/-0.54; pinitol 6.22+/-0.52) insulin sensitivities. Activation of the muscle insulin receptor was increased by 140% with glucose ingestion (Pre 0.62+/-0.12; Post 1.49+/-0.35), but pinitol did not influence this response. These results show that the pinitol supplement was quickly absorbed, but did not acutely influence indices of whole-body glucose tolerance and insulin sensitivity, or the activation of the skeletal muscle insulin receptor in older, nondiabetic humans.

  20. The TNF-alpha system in heart failure and after heart transplantation: plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    OpenAIRE

    Riemsdijk-van Overbeeke, Iza; Baan, Carla; Niesters, Bert; Hesse, Cees; Loonen, E.H.M.; Weimar, Willem; Balk, Aggie; Maat, Alex

    1999-01-01

    textabstractBACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To assess the activity of the TNF-alpha system in patients with heart failure and after heart transplantation. METHODS: We measured TNF-alpha mRNA expression of peripheral blood mononuclear ce...

  1. Mosapride citrate, a 5-HT₄ receptor agonist, increased the plasma active and total glucagon-like peptide-1 levels in non-diabetic men.

    Science.gov (United States)

    Aoki, Kazutaka; Kamiyama, Hiroshi; Masuda, Kiyomi; Togashi, Yu; Terauchi, Yasuo

    2013-01-01

    Mosapride citrate, a selective agonist of the 5-hydroxytryptaine (5-HT)₄ receptor, is typically used to treat heartburn, nausea, and vomiting associated with chronic gastritis or to prepare for a barium enema X-ray examination. Mosapride citrate reportedly improves insulin sensitivity in patients with type 2 diabetes. As mosapride citrate activates the motility of the gastrointestinal tract, we hypothesized that mosapride citrate affects incretin secretion. We examined the effect of the administration of mosapride citrate on the plasma glucose, serum insulin, plasma glucagon, and plasma incretin levels before breakfast and at 60, 120, and 180 min after breakfast in men with normal glucose tolerance (NGT) or impaired glucose tolerance (IGT) to exclude gastropathy. Mosapride citrate was administered according to two different intake schedules (C: control (no drug), M: mosapride citrate 20 mg) in each of the subject groups. The area under the curve (AUC) of the plasma glucose levels was smaller in the M group than in the C group. The time profiles for the serum insulin levels at 60 and 120 min after treatment with mosapride citrate tended to be higher, although the difference was not statistically significant. The AUCs of the plasma active and total glucagon-like peptide-1 (GLP-1) levels were significantly larger in the M group than in the C group. No significant difference in the AUC of the plasma glucose-dependent insulinotropic polypeptide (GIP) level was observed between the two groups. Our results suggest that mosapride citrate may have an antidiabetic effect by increasing GLP-1 secretion.

  2. Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Jung-Hwan Lee

    Full Text Available The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP-induced radicals on the epidermal growth factor receptor (EGFR, which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals.

  3. Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Lee, Jung-Hwan; Om, Ji-Yeon; Kim, Yong-Hee; Kim, Kwang-Mahn; Choi, Eun-Ha; Kim, Kyoung-Nam

    2016-01-01

    The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP)-induced radicals on the epidermal growth factor receptor (EGFR), which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals.

  4. Transferrin-PEG-PE modified dexamethasone conjugated cationic lipid carrier mediated gene delivery system for tumor-targeted transfection

    Science.gov (United States)

    Wang, Wei; Zhou, Fang; Ge, Linfu; Liu, Ximin; Kong, Fansheng

    2012-01-01

    Background The main barriers to non-viral gene delivery include cellular and nuclear membranes. As such, the aim of this study was to develop a type of vector that can target cells through receptor-mediated pathways and by using nuclear localization signal (NLS) to increase the nuclear uptake of genetic materials. Methods A dexamethasone (Dexa)-conjugated lipid was synthesized as the material of the solid lipid nanoparticles (SLNs), and transferrin (Tf) was linked onto polyethylene glycol-phosphatidylethanolamine (PEG-PE) to obtain Tf-PEG-PE ligands for the surface modification of the carriers. The in vitro transfection efficiency of the novel modified vectors was evaluated in human hepatoma carcinoma cell lines, and in vivo effects were observed in an animal model. Results Tf-PEG-PE modified SLNs/enhanced green fluorescence protein plasmid (pEGFP) had a particle size of 222 nm and a gene loading quantity of 90%. Tf-PEG-PE-modified SLNs/pEGFP (Tf-SLNs/pEGFP) displayed remarkably higher transfection efficiency than non-modified SLNs/pEGFP and the vectors not containing Dexa, both in vitro and in vivo. Conclusion It can be concluded that Tf and Dexa could function as an excellent active targeting ligand to improve the cell targeting and nuclear targeting ability of the carriers, and the resulting nanomedicine could be a promising active targeting drug/gene delivery system. PMID:22679364

  5. Sex and species differences in plasma testosterone and in counts of androgen receptor-positive cells in key brain regions of Sceloporus lizard species that differ in aggression.

    Science.gov (United States)

    Hews, Diana K; Hara, Erina; Anderson, Maurice C

    2012-05-01

    We studied neuroendocrine correlates of aggression differences in adults of two Sceloporus lizard species. These species differ in the degree of sex difference in aggressive color signals (belly patches) and in aggression: Sceloporus undulatus (males blue, high aggression; females white, low aggression) and Sceloporus virgatus (both sexes white, lower aggression). We measured plasma testosterone and counted cells expressing androgen receptor-like immunoreactivity to the affinity-purified polyclonal AR antibody, PG-21, in three brain regions of breeding season adults. Male S. undulatus had the highest mean plasma testosterone and differed significantly from conspecific females. In contrast, there was no sex difference in plasma testosterone concentrations in S. virgatus. Male S. undulatus also had the highest mean number of AR-positive cells in the preoptic area: the sexes differed in S. undulatus but not in S. virgatus, and females of the two species did not differ. In the ventral medial hypothalamus, S. undulatus males had higher mean AR cell counts compared to females, but again there was no sex difference in S. virgatus. In the habenula, a control brain region, the sexes did not differ, and although the sex by species interaction significant was not significant, there was a trend (p=0.050) for S. virgatus to have higher mean AR cell counts than S. undulatus. Thus hypothalamic AR cell counts paralleled sex and species differences in aggression, as did mean plasma testosterone levels in these breeding-season animals.

  6. Peroxisome Proliferator-Activated Receptor Activation is Associated with Altered Plasma One-Carbon Metabolites and B-Vitamin Status in Rats.

    Science.gov (United States)

    Lysne, Vegard; Strand, Elin; Svingen, Gard F T; Bjørndal, Bodil; Pedersen, Eva R; Midttun, Øivind; Olsen, Thomas; Ueland, Per M; Berge, Rolf K; Nygård, Ottar

    2016-01-05

    Plasma concentrations of metabolites along the choline oxidation pathway have been linked to increased risk of major lifestyle diseases, and peroxisome proliferator-activated receptors (PPARs) have been suggested to be involved in the regulation of key enzymes along this pathway. In this study, we investigated the effect of PPAR activation on circulating and urinary one-carbon metabolites as well as markers of B-vitamin status. Male Wistar rats (n = 20) received for 50 weeks either a high-fat control diet or a high-fat diet with tetradecylthioacetic acid (TTA), a modified fatty acid and pan-PPAR agonist with high affinity towards PPARα. Hepatic gene expression of PPARα, PPARβ/δ and the enzymes involved in the choline oxidation pathway were analyzed and concentrations of metabolites were analyzed in plasma and urine. TTA treatment altered most biomarkers, and the largest effect sizes were observed for plasma concentrations of dimethylglycine, nicotinamide, methylnicotinamide, methylmalonic acid and pyridoxal, which were all higher in the TTA group (all p Pparα mRNA was increased after TTA treatment, but genes of the choline oxidation pathway were not affected. Long-term TTA treatment was associated with pronounced alterations on the plasma and urinary concentrations of metabolites related to one-carbon metabolism and B-vitamin status in rats.

  7. The Transferrin Receptor at the Blood-Brain Barrier - exploring the possibilities for brain drug delivery

    NARCIS (Netherlands)

    Visser, Corine

    2005-01-01

    There are many diseases of the central nervous system (CNS), like Parkinson's disease, Alzheimer's disease, depression, schizophrenia, epilepsy, migraine headache, and HIV infection in the brain. However, treatment is difficult since many drugs cannot reach the brain in sufficient quantities due to

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

    Science.gov (United States)

    2011-07-01

    and form enveloped virions [1]. Seven arenaviruses cause viral hemorrhagic fever in humans: the Old World arenaviruses Lassa and ‘Lujo,’ and the New...hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to... fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular

  9. Expression of the neuronal transferrin receptor is age dependent and susceptible to iron deficiency

    DEFF Research Database (Denmark)

    Moos, Torben; Oates, Phillip S.; Morgan, Evan H.

    1998-01-01

    Blood-brain barrier, choroid plexus, developing brain, metabolism, nutrition, neurodegenerative disorders......Blood-brain barrier, choroid plexus, developing brain, metabolism, nutrition, neurodegenerative disorders...

  10. The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2.

    Directory of Open Access Journals (Sweden)

    Mark N Adams

    Full Text Available Protease-activated receptor-2 (PAR2 is a G protein coupled receptor (GPCR activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca²⁺ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.

  11. The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2

    Science.gov (United States)

    Adams, Mark N.; Christensen, Melinda E.; He, Yaowu; Waterhouse, Nigel J.; Hooper, John D.

    2011-01-01

    Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. PMID:22140500

  12. The effects of antifreeze peptide III (AFP) and insulin transferrin selenium (ITS) on cryopreservation of chimpanzee (Pan troglodytes) spermatozoa.

    Science.gov (United States)

    Younis, A I; Rooks, B; Khan, S; Gould, K G

    1998-01-01

    We investigated the effects of antifreeze peptides (AFP) and insulin transferrin selenium (ITS) on the motility and membrane integrity of chimpanzee (Pan troglodytes) spermatozoa after chilling (0-5 degrees C) and thawing. The effects of three thawing procedures, in the presence or absence of AFP and ITS, on sperm motility and on the status of the plasma membrane and acrosome were also examined. During chilling, AFP and ITS seem mildly cytotoxic, as the progressive motility and velocity (curvilinear and straight line) declined significantly at AFP concentrations of 1, 10, and 100 microg/ml and at ITS concentrations of 1 and 10 microg/ml. However, at a concentration of 100 microg/ml, ITS was able to protect sperm during short-term hypothermic storage. Addition of AFP or ITS at 100 microg/ml to test egg yolk-glycerol extender during freezing significantly (P < 0.05) increased postthaw motility, plasma membrane integrity, and acrosome integrity. The mean (+/-SE) motility recovery rate increased from 28.9 +/- 3.9%, for the untreated control, to 59.2 +/- 5.8% and 67.8 +/- 7.4%, for ITS and AFP, respectively. The effects of the thawing procedure were influenced by the presence of AFP during the freezing cycle. An improved motility recovery rate of 67 +/- 4.2% was obtained when chimpanzee sperm frozen in test egg yolk-glycerol extender supplemented with AFP were thawed rapidly at 37 degrees C, compared to 47 +/- 5.2% and 44 +/- 8.2% for slow (23 degrees C) and ultrarapid (75 degrees C) thawing, respectively. The motility recovery after thawing of ITS-treated semen at 23 degrees C, 37 degrees C, or 75 degrees C was not significantly different. Semen frozen without AFP or ITS and thawed at 75 degrees C was seriously (P < 0.05) damaged. This study provides evidence that AFP- or ITS-supplemented semen extender improves postthaw sperm motility in the chimpanzee.

  13. Investigation of the selective androgen receptor modulators S1, S4 and S22 and their metabolites in equine plasma using high-resolution mass spectrometry.

    Science.gov (United States)

    Hansson, Annelie; Knych, Heather; Stanley, Scott; Thevis, Mario; Bondesson, Ulf; Hedeland, Mikael

    2016-04-15

    Selective androgen receptor modulators (SARMs) are prohibited in sports due to their performance enhancing ability. It is important to investigate the metabolism to determine appropriate targets for doping control. This is the first study where the equine metabolites of SARMs S1, S4 (Andarine) and S22 (Ostarine) have been studied in plasma. Each SARM was administered to three horses as an intravenous bolus dose and plasma samples were collected. The samples were pretreated with protein precipitation using cold acetonitrile before separation by liquid chromatography. The mass spectrometric analysis was performed using negative electrospray, quadrupole time-of-flight mass spectrometry operated in MS(E) mode and triple-quadrupole mass spectrometry operated in selected reaction monitoring mode. For the quantification of SARM S1, a deuterated analogue was used as internal standard. The numbers of observed metabolites were eight, nine and four for the SARMs S1, S4 and S22, respectively. The major metabolite was formed by the same metabolic reactions for all three SARMs, namely amide hydrolysis, hydroxylation and sulfonation. The values of the determined maximum plasma concentrations were in the range of 97-170 ng/mL for SARM S1, 95-115 ng/mL for SARM S4 and 92-147 ng/mL for SARM S22 and the compounds could be detected for 96 h, 12 h and 18 h, respectively. The maximum plasma concentration of SARMs S1, S4 and S22 was measured in the first sample (5 min) after administration and they were eliminated fast from plasma. The proposed targets to be used in equine doping control are the parent compounds for all three SARMs, but with the metabolite yielding the highest response as a complementary target. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Peroxisome proliferator-activated receptor (PPAR) delta genetic polymorphism and its association with insulin resistance index and fasting plasma glucose concentrations in Chinese subjects.

    Science.gov (United States)

    Hu, C; Jia, W; Fang, Q; Zhang, R; Wang, C; Lu, J; Xiang, K

    2006-12-01

    Previous studies have shown that the peroxisome proliferator-activated receptor delta (PPARD) genetic polymorphism affects cholesterol metabolism in Whites. This association was not observed in a Korean population in a separate study, but this study showed a link between the PPARD polymorphism and body weight and fasting plasma glucose. The purpose of this study was to determine whether polymorphisms of PPARD influence glucose and cholesterol metabolism in Chinese subjects. We investigated the association between the polymorphism (-87T/C) of the human PPARD gene and phenotypes related to body weight, insulin sensitivity, glucose and lipid metabolism in Chinese subjects. Unrelated Chinese subjects (n = 663) in Shanghai were studied; 287 had newly diagnosed Type 2 diabetes mellitus and 376 were non-diabetic control subjects over 40 years old. Clinical parameters were collected and genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. In normal glucose tolerant (NGT) subjects, the C allele carriers had higher fasting plasma glucose concentrations (P = 0.0078) and a lower insulin sensitivity index (ISI) (P = 0.0365). The C allele carriers also showed higher concentrations of low-density lipoprotein cholesterol (P = 0.0261) and percentage of body fat (P = 0.0357). There was a trend towards higher visceral adiposity in C allele carriers, but the difference was not significant (P = 0.0830). In diabetes patients, similar results were detected for plasma glucose concentrations (fasting plasma glucose P polymorphism is associated with higher fasting plasma glucose concentrations in both NGT and diabetic subjects, largely due to impaired insulin sensitivity.

  15. Liquid chromatography-mass spectrometric method for determination of the non-imidazole H3-receptor antagonist UPR1056 in rat plasma.

    Science.gov (United States)

    Vacondio, Federica; Silva, Claudia; Morini, Giovanni; Bordi, Fabrizio; Flammini, Lisa; Barocelli, Elisabetta; Mor, Marco

    2011-07-01

    The non-imidazole H3 receptor antagonist UPR1056 was dosed in plasma samples from rats individually administered with a single i.p. dose of 1.25 mg/kg by means of a newly validated HPLC-MS method. UPR1056 was extracted from rat plasma by protein precipitation with acetonitrile and was separated by linear gradient elution, employing water and methanol both additioned with 0.05% trifluoroacetic acid as mobile phases. UPR1056 was detected in MS using an electrospray ion source operating in positive ion mode. Acquisition was performed in single ion monitoring mode at m/z=349.3. The method was validated over the concentration range of 17.43-1743 ng/mL (50-5000 pmol/mL). Within- and between-run precision for the low, mid and high quality controls (QC) levels were 6.75% or less and accuracy ranged from 95.8 to 107.6%. The lower limit of quantification was 17.43 ng/mL. The analysis of the time course of UPR1056 concentrations over the 24-h period revealed a C(max) of 1147 ng/mL after 2 h from peripheral administration of the non-imidazole H(3)-receptor antagonist, with a prolonged elimination half-life of over 9 h.

  16. 2D-ELDOR study of heterogeneity and domain structure changes in plasma membrane vesicles upon cross-linking of receptors.

    Science.gov (United States)

    Chiang, Yun-Wei; Costa-Filho, Antonio J; Baird, Barbara; Freed, Jack H

    2011-09-08

    2D electron-electron double resonance (2D-ELDOR) with the "full Sc-" method of analysis is applied to the study of plasma membrane vesicles. Membrane structural changes upon antigen cross-linking of IgE receptors (IgE-FcεRI) in plasma membrane vesicles (PMVs) isolated from RBL-2H3 mast cells are investigated, for the first time, by means of these 2D-ELDOR techniques. Spectra of 1-palmitoyl-2-(16-doxyl stearoyl) phosphatidylcholine (16-PC) from PMVs before and after this stimulation at several temperatures are reported. The results demonstrate a coexistence of liquid-ordered (L(o)) and liquid-disordered (L(d)) components. We find that upon cross-linking, the membrane environment is remodeled to become more disordered, as shown by a moderate increase in the population of the L(d) component. This change in the relative amount of the L(o) versus L(d) components upon cross-linking is consistent with a model wherein the IgE receptors, which when clustered by antigen to cause cell stimulation, lead to more disordered lipids, and their dynamic and structural properties are slightly altered. This study demonstrates that 2D-ELDOR, analyzed by the full Sc- method, is a powerful approach for capturing the molecular dynamics in biological membranes. This is a particular case showing how 2D-ELDOR can be applied to study physical processes in complex systems that yield subtle changes.

  17. Treatment with a GLP-1 receptor agonist diminishes the decrease in free plasma leptin during maintenance of weight loss

    DEFF Research Database (Denmark)

    Iepsen, E W; Lundgren, J; Dirksen, C

    2015-01-01

    BACKGROUND: Recent studies indicate that glucagon-like peptide (GLP)-1 inhibits appetite in part through regulation of soluble leptin receptors. Thus, during weight loss maintenance, GLP-1 receptor agonist (GLP-1RA) administration may inhibit weight loss-induced increases in soluble leptin...... receptors thereby preserving free leptin levels and preventing weight regain. METHODS: In a randomized controlled trial, 52 healthy obese individuals were, after a diet-induced 12% body weight loss, randomized to treatment with or without administration of the GLP-1RA liraglutide (1.2 mg per day). In case...... of weight gain, low-calorie diet products were allowed to replace up to two meals per day to achieve equal weight maintenance. Glucose tolerance and hormone responses were investigated before and after weight loss and after 52 weeks weight maintenance. Primary end points: increase in soluble leptin receptor...

  18. Experimental treatment of breast cancer-bearing BALB/c mice by artemisinin and transferrin-loaded magnetic nanoliposomes.

    Science.gov (United States)

    Gharib, Amir; Faezizadeh, Zohreh; Mesbah-Namin, Seyed Ali Reza; Saravani, Ramin

    2015-05-01

    The combination of artemisinin and transferrin exhibits versatile anticancer activities. In previous, we successfully prepared artemisinin and transferrin-loaded magnetic nanoliposomes and evaluated their anti-proliferative activity against MCF-7 and MDA-MB-231 cell lines in vitro. In this study, we investigate the in vivo anti-breast cancer activity of artemisinin and transferrin-loaded magnetic nanoliposome against breast transplanted tumors in BALB/c mice model. Artemisinin and transferrin-loaded magnetic nanoliposomes were prepared and characterized for some physiochemical properties. Pieces of tumor tissue from the breast cancer-bearing BALB/c mice were transplanted subcutaneously to the syngeneic female BALB/c mice. In the presence of the external magnet that placed at the breast tumor site, the tissue distribution and tumor-suppressing effects of prepared nanoliposomes on tumor growth was evaluated. The prepared nanoliposomes have fine spherical shape, rough surface, nano-sized diameter and magnetic properties. At 2 h after treatment, the intravenous administration of artemisinin and transferrin-loaded magnetic nanoliposomes followed using the magnetic field approximately produced 10- and 5.5-fold higher levels of artemisinin and transferrin in the tumors, respectively, compared with free artemisinin and transferrin. Moreover, in the presence of an external magnetic field, the prepared nanoliposomes could significantly induce apoptosis in the mice breast cancer cells as well as could reduce tumor volume in tumorized mice at 15 days after treatment. The data suggested that the artemisinin and transferrin-loaded magnetic nanoliposomes would be a good choice for the breast tumor-targeted therapy, due to its high targeting efficiency.

  19. Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

    Science.gov (United States)

    Chen, Huiyong; Choesang, Tenzin; Li, Huihui; Sun, Shuming; Pham, Petra; Bao, Weili; Feola, Maria; Westerman, Mark; Li, Guiyuan; Follenzi, Antonia; Blanc, Lionel; Rivella, Stefano; Fleming, Robert E; Ginzburg, Yelena Z

    2016-03-01

    Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

  20. Intact and cleaved plasma soluble urokinase receptor in patients with metastatic colorectal cancer treated with oxaliplatin with or without cetuximab

    DEFF Research Database (Denmark)

    Tarpgaard, Line Schmidt; Christensen, Ib Jarle; Høyer-Hansen, Gunilla

    2015-01-01

    Circulating forms of the urokinase plasminogen activator receptor (uPAR) are associated with prognosis in patients with colorectal cancer. Preclinical studies have shown that uPAR can influence the state of phosphorylation and signalling activity of the epidermal growth factor receptor (EGFR...... with FLOX + cetuximab as compared to patients with KRAS wild-type and high levels of suPAR. These results thus support the preclinical findings and should be further tested in an independent clinical data set....

  1. Enhanced optical immunosensor based on surface plasmon resonance for determination of transferrin.

    Science.gov (United States)

    Liu, Xia; Sun, Ying; Song, Daqian; Zhang, Qinglin; Tian, Yuan; Zhang, Hanqi

    2006-01-15

    Wavelength modulation surface plasmon resonance biosensors (SPR) using colloidal Au nanoparticles and double-linker sensing membrane enhancement are reported for determination of transferrin. The 2-mercaptoethylamine (MEA) was immobilized on the biosensor surface with traditional amine coupling method. The interaction between colloidal Au nanoparticles and MEA was investigated. The anti-transferrin was immobilized on the biosensor surface prepared with staphylococcal protein A (SPA). The interaction of the antibody and antigen was monitored in real time. The good response was obtained in the concentration range 1-20, 0.1-20 and 0.05-20 microg/mL for directly immune assay, double-linker assay and colloidal Au-amplified assay. The result clearly demonstrates that these methods may obtain significantly enhancement of sensitivity for the wavelength modulation SPR biosensor.

  2. Loss of genetic variability at the transferrin locus in five hatchery stocks of tambaqui (Colossoma macropomum

    Directory of Open Access Journals (Sweden)

    Calcagnotto Daniela

    2000-01-01

    Full Text Available Knowledge and conservation of the genetic variability in stocks maintained as live gene banks have become a high priority task for Brazilian fish culture. The aim of the present survey was to assess the transferrin allelic diversity of five hatchery stocks of tambaqui (Colossoma macropomum. The tambaqui stock from Pentecoste, the oldest maintained in Brazilian hatchery stations, retained three of the six alleles detected in wild populations of tambaqui from the Amazon River. Other hatchery stocks, directly or indirectly derived from the Pentecoste stock, did not show transferrin allelic variability. Insufficient number of founders and genetic drift due to sampling errors seem to be the main causes leading to loss of genetic diversity in tambaqui hatchery stocks. Appropriate management strategies are required in order to improve the genetic potential of tambaqui stocks in Brazil.

  3. Beta-2-transferrin to detect cerebrospinal fluid pleural effusion: a case report

    Directory of Open Access Journals (Sweden)

    Smith Jennifer C

    2009-03-01

    Full Text Available Abstract Introduction Pleural effusion secondary to ventriculoperitoneal shunt insertion is a rare and potentially life-threatening occurrence. Case presentation We describe a 14-month-old Caucasian boy who had a ventriculoperitoneal shunt inserted for progressive hydrocephalus of unknown etiology. Two and a half months post-shunt insertion, the patient presented with mild respiratory distress. A chest radiograph revealed a large right pleural effusion and a shunt series demonstrated an appropriately placed distal catheter tip. A subsequent abdominal ultrasound revealed marked ascites. Fluid drained via tube thoracostomy was sent for beta-2-transferrin electrophoresis. A positive test was highly suggestive of cerebral spinal fluid hydrothorax. Post-externalization of the ventriculoperitoneal shunt, the ascites and pleural effusion resolved. Conclusion Testing for beta-2-transferrin protein in pleural fluid may serve as a useful technique for diagnosing cerebrospinal fluid hydrothorax in patients with ventriculoperitoneal shunts.

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

    Science.gov (United States)

    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.

  5. Transferrin hypoglycosylation in hereditary fructose intolerance: using the clues and avoiding the pitfalls.

    Science.gov (United States)

    Adamowicz, M; Płoski, R; Rokicki, D; Morava, E; Gizewska, M; Mierzewska, H; Pollak, A; Lefeber, D J; Wevers, R A; Pronicka, E

    2007-06-01

    Hereditary fructose intolerance (HFI) is caused by a deficiency of aldolase B due to mutations of the ALDOB gene. The disease poses diagnostic problems because of unspecific clinical manifestations. We report three cases of HFI all of whom had a chronic disease with neurological, nephrological or gastroenterological symptoms, whereas nutritional fructose intolerance, the pathognomonic sign of HFI, was apparent only in retrospect. In all patients a hypoglycosylated pattern of transferrin isoforms was found but was misinterpreted as a sign of CDG Ix. The correct diagnosis was achieved with marked delay (26, 36 and 24 months, respectively) by sequencing of the ALDOB gene two common mutations were identified on both alleles or on one (A150P/A175D, A150P/-, and A150P/A175D). The diagnosis was further supported by normalization of transferrin isoforms on a fructose-free diet. Data available in two patients showed that following the fructose restriction the type I pattern of carbohydrate-deficient transferrin detectable on fructose-containing diet disappeared after 3-4 weeks. These cases illustrate that in the first years of life HFI may show misleading variability in clinical presentation and that protein glycosylation analysis such as transferrin isofocusing may give important diagnostic clues. However, care should be taken not to misinterpret the abnormal results as CDG Ix as well as to remember that a normal profile does not exclude HFI due to the possibility of spontaneous fructose restriction in the diet. The presented data also emphasize the usefulness of ALDOB mutation screening for diagnosis of HFI.

  6. Insulin-like growth factor-I and transferrin mediate growth and survival of Chinese hamster ovary cells.

    Science.gov (United States)

    Sunstrom, N A; Gay, R D; Wong, D C; Kitchen, N A; DeBoer, L; Gray, P P

    2000-01-01

    The aim of this investigation was to elucidate the roles of insulin-like growth factor-I (IGF-I) and transferrin in the survival and proliferation of Chinese hamster ovary (CHO) cells upon withdrawal of serum. For this purpose, we employed DNA analysis and flow cytometry to compare CHO cell lines expressing either IGF-I alone or IGF-I and transferrin. The ability of cells to cycle and the occurrence of apoptosis were monitored in these cells in serum-free medium. These results indicate that IGF-I alone is able to maintain the viability of CHO cells for an extended length of time in the absence of serum. Transferrin alone does not promote survival or proliferation. Only in the presence of both IGF-I and transferrin do cells survive and proliferate. Therefore, in attached CHO cultures, IGF-I alone does not stimulate cell proliferation but is a requirement for growth in serum-free medium in cooperation with transferrin. We report on the dual role of IGF-I as a survival factor in CHO cells and its interlocking role with transferrin to stimulate cell growth.

  7. Fructose rich diet differently affects angiotensin II receptors content in the nucleus and a plasma membrane fraction of visceral adipose tissue.

    Science.gov (United States)

    Bundalo, Maja; Djordjevic, Ana; Bursac, Biljana; Zivkovic, Maja; Koricanac, Goran; Stankovic, Aleksandra

    2017-08-03

    The adipose tissue renin-angiotensin system (RAS) is proposed to be a pathophysiological link between adipose tissue dysregulation and metabolic disorders induced by fructose rich diet (FRD). RAS can act intracellularly. We hypothesized that adipocytes nuclear membranes possess angiotensin receptors type 1 and type 2 (AT1R and AT2R) that couple to nuclear signaling pathways and regulate oxidative gene expression under the FRD. We analyzed the effect of nine-week consumption of 10% fructose solution on biochemical parameters, adipocyte morphology and expression of AT1R, AT2R, AT1R-associated protein (ATRAP), NOX4, MMP-9, and MnSOD in adipose tissue of Wistar rats. We detected AT1R and AT2R in nuclear fraction. FRD decreased protein level of nuclear angiotensin receptors, while increased AT1R and decreased AT2R levels were observed at plasma membrane. FRD increased mRNA level of ATRAP, while MnSOD mRNA and protein levels were decreased. No significant differences were observed for MMP9 and NOX4 mRNA levels. These findings coincide with hyperleptinemia, elevated blood pressure and triglyceride level, as well as with unchanged visceral adipose tissue mass and morphology upon FRD. Besides providing evidence for nuclear localization of angiotensin receptors in visceral adipose tissue, this study also points to the different effect of FRD on AT1R expression in different cellular compartments. Elevated blood pressure and decreased antioxidative capacity in visceral fat of fructose fed rats were accompanied by an increased AT1R level in plasma membrane while upregulation of ATRAP and decrease of nuclear membrane AT1R suggest an increase in capacity for attenuation of excessive AT1R signaling and visceral adiposity.

  8. Genetic variants in glucocorticoid and mineralocorticoid receptors are associated with concentrations of plasma cortisol, muscle glycogen content, and meat quality traits in male Nellore cattle.

    Science.gov (United States)

    Poleti, M D; DeRijk, R H; Rosa, A F; Moncau, C T; Oliveira, P S; Coutinho, L L; Eler, J P; Balieiro, J C C

    2015-04-01

    The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are key components in the regulation of the hypothalamic-pituitary-adrenal neuroendocrine axis and coordinate the physiological response to stress agents to reestablish homeostasis. Genetic variations of GR (NR3C1) and MR (NR3C2) genes could explain the alterations in animals to adapt to challenges, and therefore, their influence on production traits. The present study aimed to identify single-nucleotide polymorphisms (SNPs) in the bovine NR3C1 and NR3C2 genes and explore their associations to relevant traits of beef cattle production. Genotypes and phenotypes were collected from 241 male Nellore cattle (119 noncastrated and 122 castrated surgically) with an average of 24 ± 1.2 mo of age and live weight of 508 ± 39 kg. The traits evaluated were concentrations of plasma adrenocorticotropic hormone (ACTH) and cortisol, muscle glycogen and lactate content, and pH, color, cooking loss, and shear force of longissimus thoracis measured on the 1st, 7th, and 14th days postmortem. Five SNPs were identified, 2 in the NR3C1 gene and 3 in the NR3C2 gene. There was an associative relationship between the SNP NR3C1_1 g.3293A>G and postmortem plasma concentration of cortisol (P = 0.0008). The SNPs NR3C2_1 g.115T>C and NR3C2_2 g.570T>C were associated with muscle glycogen content (P = 0.0306 and P = 0.0158), postmortem plasma concentration of ACTH (P = 0.0118 and P = 0.0095), and cooking loss of the steak aged 1 d (P = 0.0398 and P = 0.0423). Haplotype analysis showed associations of GR haplotypes with postmortem plasma concentrations of cortisol and MR haplotypes with meat color, cooking losses, muscle glycogen content, and plasma concentrations of ACTH. The associations observed in the present study show that SNPs in GR and MR genes are related with changes of hypothalamic-pituitary-adrenal axis activity and metabolic profile in cattle, leading to individual variation in meat quality traits.

  9. The 5-HT1D/1B receptor agonist sumatriptan enhances fear of simulated speaking and reduces plasma levels of prolactin.

    Science.gov (United States)

    de Rezende, Marcos Gonçalves; Garcia-Leal, Cybele; Graeff, Frederico Guilherme; Del-Ben, Cristina Marta

    2013-12-01

    This study measured the effects of the preferential 5-HT1D/1B receptor agonist sumatriptan in healthy volunteers who performed the Simulated Public Speaking Test (SPST), which recruits the neural network involved in panic disorder and social anxiety disorder. In a double-blind, randomised experiment, 36 males received placebo (12), 50 mg (12) or 100 mg (12) of sumatriptan 2 h before the SPST. Subjective, physiological and hormonal measures were taken before, during and after the test. The dose of 100 mg of sumatriptan increased speech-induced fear more than either a 50mg dose of the drug or placebo. The largest dose of sumatriptan also enhanced vigilance more than placebo, without any change in blood pressure, heart rate or electrical skin conductance. Sumatriptan decreased plasma levels of prolactin. A significant but moderate increase in plasma cortisol after SPST occurred, independent of treatment. Because sumatriptan decreases 5-HT release into the extracellular space, the potentiation of SPST-induced fear caused by the drug supports the hypothesis that 5-HT attenuates this emotional state. As acute administration of antidepressants has also been shown to enhance speaking fear and increase plasma prolactin, in contrast to sumatriptan, the 5-HT regulation of stress-hormone release is likely to be different from that of emotion.

  10. Sterol regulatory element-binding protein-1 determines plasma remnant lipoproteins and accelerates atherosclerosis in low-density lipoprotein receptor-deficient mice.

    Science.gov (United States)

    Karasawa, Tadayoshi; Takahashi, Akimitsu; Saito, Ryo; Sekiya, Motohiro; Igarashi, Masaki; Iwasaki, Hitoshi; Miyahara, Shoko; Koyasu, Saori; Nakagawa, Yoshimi; Ishii, Kiyoaki; Matsuzaka, Takashi; Kobayashi, Kazuto; Yahagi, Naoya; Takekoshi, Kazuhiro; Sone, Hirohito; Yatoh, Shigeru; Suzuki, Hiroaki; Yamada, Nobuhiro; Shimano, Hitoshi

    2011-08-01

    Sterol regulatory element-binding protein-1 (SREBP-1) is nutritionally regulated and is known to be a key transcription factor regulating lipogenic enzymes. The goal of this study was to evaluate the roles of SREBP-1 in dyslipidemia and atherosclerosis. Transgenic mice that overexpress SREBP-1c in the liver and SREBP-1-deficient mice were crossed with low-density lipoprotein receptor (LDLR)-deficient mice, and the plasma lipids and atherosclerosis were analyzed. Hepatic SREBP-1c overexpression in LDLR-deficient mice caused postprandial hypertriglyceridemia, increased very-low-density lipoprotein (VLDL) cholesterol, and decreased high-density lipoprotein cholesterol in plasma, which resulted in accelerated aortic atheroma formation. Conversely, absence of SREBP-1 suppressed Western diet-induced hyperlipidemia in LDLR-deficient mice and ameliorated atherosclerosis. In contrast, bone marrow-specific SREBP-1 deficiency did not alter the development of atherosclerosis. The size of nascent VLDL particles secreted from the liver was increased in SREBP-1c transgenic mice and reduced in SREBP-1-deficient mice, accompanied by upregulation and downregulation of phospholipid transfer protein expression, respectively. Hepatic SREBP-1c determines plasma triglycerides and remnant cholesterol and contributes to atherosclerosis in hyperlipidemic states. Hepatic SREBP-1c also regulates the size of nascent VLDL particles.

  11. The phospholipid scramblases 1 and 4 are cellular receptors for the secretory leukocyte protease inhibitor and interact with CD4 at the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Bénédicte Py

    Full Text Available Secretory leukocyte protease inhibitor (SLPI is secreted by epithelial cells in all the mucosal fluids such as saliva, cervical mucus, as well in the seminal liquid. At the physiological concentrations found in saliva, SLPI has a specific antiviral activity against HIV-1 that is related to the perturbation of the virus entry process at a stage posterior to the interaction of the viral surface glycoprotein with the CD4 receptor. Here, we confirm that recombinant SLPI is able to inhibit HIV-1 infection of primary T lymphocytes, and show that SLPI can also inhibit the transfer of HIV-1 virions from primary monocyte-derived dendritic cells to autologous T lymphocytes. At the molecular level, we show that SLPI is a ligand for the phospholipid scramblase 1 (PLSCR1 and PLSCR4, membrane proteins that are involved in the regulation of the movements of phospholipids between the inner and outer leaflets of the plasma membrane. Interestingly, we reveal that PLSCR1 and PLSCR4 also interact directly with the CD4 receptor at the cell surface of T lymphocytes. We find that the same region of the cytoplasmic domain of PLSCR1 is involved in the binding to CD4 and SLPI. Since SLPI was able to disrupt the association between PLSCR1 and CD4, our data suggest that SLPI inhibits HIV-1 infection by modulating the interaction of the CD4 receptor with PLSCRs. These interactions may constitute new targets for antiviral intervention.

  12. Metabolism of glycosylated human salivary amylase: in vivo plasma clearance by rat hepatic endothelial cells and in vitro receptor mediated pinocytosis by rat macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Niesen, T.E.; Alpers, D.H.; Stahl, P.D.; Rosenblum, J.L.

    1984-09-01

    Salivary-type amylase normally comprises about 60% of the amylase activity in human serum, but only a small fraction is a glycosylated isoenzyme (amylase A). In contrast, 1/3 of amylase in human saliva is glycosylated. Since glycosylation can affect circulatory clearance, we studied the clearance of amylase A in rats and its uptake by rat alveolar macrophages. Following intravenous injection, /sup 125/I-labeled amylase A disappeared rapidly from plasma (t 1/2 . 9 min) and accumulated in the liver. Simultaneous injection of mannose-albumin slowed its clearance to a rate comparable to that of /sup 125/I-labeled nonglycosylated salivary amylase (t 1/2 . 45 min). In contrast, galactose-albumin had no effect. Electron microscope autoradiography of the liver following injection of /sup 125/I-labeled amylase A revealed a localization of grains over the hepatic endothelial cells. In vitro studies indicated that amylase A is taken up by alveolar macrophages via receptor-mediated pinocytosis. Uptake was linear over time, saturable, and inhibited by mannan and mannose-albumin, but not by galactose-albumin. We conclude that amylase A, which is a naturally occurring human glycoprotein with at most three terminal L-fucose residues per molecule, is recognized in rats by a mannose receptor located on hepatic endothelial cells. We speculate that this receptor, by rapidly clearing circulating amylase A, may be responsible for the low level of amylase A in human serum.

  13. Acceleration of Lung Regeneration by Platelet-Rich Plasma Extract through the Low-Density Lipoprotein Receptor-Related Protein 5-Tie2 Pathway.

    Science.gov (United States)

    Mammoto, Tadanori; Chen, Zhao; Jiang, Amanda; Jiang, Elisabeth; Ingber, Donald E; Mammoto, Akiko

    2016-01-01

    Angiogenesis, the growth of new blood vessels, plays a key role in organ development, homeostasis, and regeneration. The cooperation of multiple angiogenic factors, rather than a single factor, is required for physiological angiogenesis. Recently, we have reported that soluble platelet-rich plasma (PRP) extract, which contains abundant angiopoietin-1 and multiple other angiogenic factors, stimulates angiogenesis and maintains vascular integrity in vitro and in vivo. In this report, we have demonstrated that mouse PRP extract increases phosphorylation levels of the Wnt coreceptor low-density lipoprotein receptor-related protein 5 (LRP5) and thereby activates angiogenic factor receptor Tie2 in endothelial cells (ECs) and accelerates EC sprouting and lung epithelial cell budding in vitro. PRP extract also increases phosphorylation levels of Tie2 in the mouse lungs and accelerates compensatory lung growth and recovery of exercise capacity after unilateral pneumonectomy in mice, whereas soluble Tie2 receptor or Lrp5 knockdown attenuates the effects of PRP extract. Because human PRP extract is generated from autologous peripheral blood and can be stored at -80°C, our findings may lead to the development of novel therapeutic interventions for various angiogenesis-related lung diseases and to the improvement of strategies for lung regeneration.

  14. The Effects of Temperature, Viscosity, and Molecular Size on the Aluminum-27 QCT NMR of Transferrins

    Science.gov (United States)

    Aramini, James M.; Vogel, Hans J.

    1996-02-01

    A number of reports in recent years have demonstrated the feasibility of detecting quadrupolar metal ions bound tightly to rather large proteins via the quadrupolar central transition (QCT) NMR approach. In this article, an in-depth investigation of several interesting properties of transferrin-bound27Al NMR signals, namely, their dependence on temperature, viscosity, and molecular size is presented. It is shown that (1) decreasing temperature and (2) increasing viscosity by adding reagents such as glycerol and ethylene glycol perturb only the linewidths of transferrin-bound27Al signals, and, in fact, produce a decrease in signal linewidth. These effects are in accord with quadrupolar relaxation theory, which predicts that the linewidth of the central transition of a half-integer quadrupolar nucleus should decrease with increasing correlation time of the protein under nonextreme narrowing conditions. Furthermore, it is demonstrated that these trends, which are completely opposite to those generally observed in NMR spectroscopy, can be exploited to monitor ovotransferrin half-molecule reassociation reactions. In combination with the peculiar properties of transferrin-bound quadrupolar nuclei reported in the literature to date, the phenomena described here provide the basis for understanding the conditions and experimental parameters which may facilitate the application of the QCT NMR technique to the study of other quadrupolar nuclei and proteins.

  15. Transferrin gene polymorphisms and population genetic studies of Atlantic cod (Gadus morhua)

    Institute of Scientific and Technical Information of China (English)

    Berhan Asmamaw

    2016-01-01

    Objective:To detect single nucleotide polymorphisms (SNPs) in the cod transferrin gene by comparing the sequences from Norwegian (North East Atlantic Ocean) and Canadian (North West Atlantic Ocean) specimen, and to quantify the genetic variation and differentiation in East and West Atlantic cod populations. Methods:cDNA sequences between individuals of Canadian (North West Atlantic Ocean) and Norwegian (North East Atlantic Ocean) origin were aligned. Allele frequencies of theSNPs were used to discriminate the different Atlantic cod populations in West/East Atlantic Ocean, and the Baltic Sea. Results: The sequence alignment detected19SNPs, of which 18 of them resulted in amino acid changes in the transferrin protein. Nonsynonymous to synonymous site substitution ratio (dn/ds) was by far greater than 1 providing an evidence for the existence of positive selection. The West Atlantic cod populations showed high values of heterozygosity and the Baltic populations were found to be inbred. Conclusions: This study identified and indicated transferrin gene polymorphisms that can be used for population differentiations.

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

    Science.gov (United States)

    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. Higher plasma soluble Receptor for Advanced Glycation End Products (sRAGE) levels are associated with incident cardiovascular disease and all-cause mortality in type 1 diabetes: a 12-year follow-up study

    DEFF Research Database (Denmark)

    Nin, Johanna W M; Jorsal, Anders; Merces Ferreira, Isabel Maria;

    2010-01-01

    To investigate the associations of plasma levels of soluble receptor for advanced glycation end products (sRAGE) with incident cardiovascular disease (CVD) and all-cause mortality in type 1 diabetes and the extent to which any such associations could be explained by endothelial and renal dysfunct......To investigate the associations of plasma levels of soluble receptor for advanced glycation end products (sRAGE) with incident cardiovascular disease (CVD) and all-cause mortality in type 1 diabetes and the extent to which any such associations could be explained by endothelial and renal...... dysfunction, low-grade inflammation, arterial stiffness, and advanced glycation end products (AGEs)....

  18. Expression of tranferrin receptors in the pineal gland of postnatal and adult rats and its alteration in hypoxia and melatonin treatment.

    Science.gov (United States)

    Kaur, C; Sivakumar, V; Ling, E A

    2007-02-01

    Transferrin receptors (Tfrc) are membrane bound glycoproteins which function to mediate cellular uptake of iron from transferrin. We examined expression of Tfrc in the pineal gland of rats of different ages from 1 day to 12 weeks. The mRNA and protein expression of Tfrc increased up to 6 weeks of age and decreased in 12 week rats. Tfrc immunoreactivity was observed on pinealocytes and macrophages/microglia. By immunoelectron microscopy, the immunoreaction in pinealocytes was observed in the cytosol, on mitochondria and plasma membrane whereas in macrophages/microglia it was localized on the plasma membrane in 1-day to 2-week old rats. In older rats, the immunoreaction product in pinealocytes was associated with the plasma membrane and mitochondria only. Iron localization was observed in pinealocytes as well as macrophages/microglia. It is suggested that Tfrc are required for uptake of iron for cell proliferation and maturation in the pineal gland upto 6 weeks of age. The significance of Tfrc expression on mitochondria is speculative. They may be involved in iron transport to the mitochondria or for regulation of the secretory activity of pinealocytes. The TfrcmRNA and protein expression increased significantly in response to hypoxia in 12-week rats and this coincided with intense iron staining of the pinealocytes and macrophages/microglia. It is concluded that increased expression of Tfrc in response to hypoxia leads to excess cellular uptake of iron which may be damaging to the cells. Melatonin administration in hypoxic rats may prove to be beneficial as it reduced the Tfrc expression.

  19. [Can mannose-binding lectin and plasma level of soluble urokinase receptor be used in diagnosis and treatment monitorization of brucellosis patients?].

    Science.gov (United States)

    Karsen, Hasan; Cesur, Salih; Karaağaç, Leman; Binici, Irfan; Fidan, Yasemin; Oğüş, Elmas; Demiröz, Ali Pekcan

    2012-07-01

    The aim of this study was to evaluate the diagnostic value of serum mannose-binding lectin (MBL) and plasma soluble urokinase plasminogen activator receptor (SuPAR) levels in monitoring the treatment in patients with brucellosis, by comparing their levels before and after treatment with the values obtained from healthy control group. Thirty brucellosis patients (mean age: 25.8 ± 12.2 years; 15 were male) and 28 healthy controls (mean age: 29.3 ± 12.3 years; 15 were male) were included in the study. Patients were diagnosed with brucellosis according to the characteristic clinical findings and by brucella standard tube agglutination test (SAT) titer ≥ 1/160 and/or blood culture positivity. Serum MBL (Antibodyshop, Denmark) and plasma SuPAR (Virogates, Denmark) levels were investigated with commercial ELISA kits. In our study, no statistical significance was observed between the pre-treatment (13.8 ± 13.4 ng/ml) and post-treatment (12.4 ± 13.1 ng/ml) MBL levels of the patient group and MBL levels of the control group (16.5 ± 14.8 ng/ml) (p> 0.05). Moreover, the mean SuPAR levels measured in pre-treatment and post-treatment plasma samples of the brucellosis patients was 5.1 ± 1.9 ng/ml and 2.9 ± 1.3 ng/ml, respectively, while the mean SuPAR level was 1.8 ± 0.5 ng/ml in the control group. The difference between mean SuPAR levels of patients in pre- and post-treatment samples was found statistically significant (p 0.001). In conclusion, plasma SuPAR level would be a useful marker for the diagnosis and treatment follow up of the patients with brucellosis.

  20. Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

    Energy Technology Data Exchange (ETDEWEB)

    Shukla, S.D.; Morrison, W.J.; Klachko, D.M.

    1989-07-01

    Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and (3H)PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of (3H)PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).

  1. Synthesis and characterization of tumor-targeted copolymer nanocarrier modified by transferrin

    Directory of Open Access Journals (Sweden)

    Liu R

    2015-05-01

    Full Text Available Ran Liu,1,2 Yonglu Wang,1,3 Xueming Li,3 Wen Bao,1,2 Guohua Xia,1,2 Wei Chen,3 Jian Cheng,1,2 Yuanlong Xu,3 Liting Guo,1,2 Baoan Chen1,21Department of Hematology (Key Department of Jiangsu Medicine, Zhongda Hospital, Medical School, 2Faculty of Oncology, Medical School, Southeast University, 3College of Pharmacy, Nanjing University of Technology, Nanjing, People’s Republic of China Abstract: To increase the encapsulation of hydrophilic antitumor agent daunorubicin (DNR and multidrug resistance reversal agent tetrandrine (Tet in the drug delivery system of nanoparticles (NPs, a functional copolymer NP composed of poly(lactic-co-glycolic acid (PLGA, poly-l-lysine (PLL, and polyethylene glycol (PEG was synthesized and then loaded with DNR and Tet simultaneously to construct DNR/Tet–PLGA–PLL–PEG-NPs using a modified double-emulsion solvent evaporation/diffusion method. And to increase the targeted antitumor effect, DNR/Tet–PLGA–PLL–PEG-NPs were further modified with transferrin (Tf due to its specific binding to Tf receptors (TfR, which is highly expressed on the surface of tumor cells. In this study, the influence of the diversity of formulation parameters was investigated systematically, such as drug loading, mean particle size, molecular weight, the concentration of PLGA–PLL–PEG–Tf, volume ratio of acetone to dichloromethane, the concentration of polyvinyl alcohol (PVA in the external aqueous phase, the volume ratio of the internal aqueous phase to the external aqueous phase, and the type of surfactants in the internal aqueous phase. Meanwhile, its possible effect on cell viability was evaluated. Our results showed that the regular spherical DNR/Tet–PLGA–PLL–PEG–Tf-NPs with a smooth surface, a relatively low polydispersity index, and a diameter of 213.0±12.0 nm could be produced. The encapsulation efficiency was 70.23%±1.91% for DNR and 86.5%±0.70% for Tet, the moderate drug loading was 3.63%±0.15% for DNR and 4

  2. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

    NARCIS (Netherlands)

    Gouget, A.; Senchou, V.; Govers, F.; Sanson, A.; Barre, A.; Rougé, P.; Pont-Lezica, R.; Canut, H.

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsi

  3. D-2-like receptor stimulation decreases effective renal plasma flow and glomerular filtration rate in spontaneously hypertensive rats

    NARCIS (Netherlands)

    de Vries, PAM; de Jong, PE; de Zeeuw, D; Navis, GJ

    2002-01-01

    In spontaneously hypertensive rats (SHRs) the dopaminergic D-1-like renal vasodilator response is impaired. The renal vascular response to D-2-like receptor stimulation in vivo is incompletely known. Therefore, renal hemodynamics were studied in conscious SHRs during continuous infusion of D-2-like

  4. Peroxisome Proliferator-Activated Receptor ß/ (PPARß/) but Not PPAR Serves as a Plasma Free Fatty Acid Sensor in Liver

    NARCIS (Netherlands)

    Sanderson, L.; Degerhardt, T.; Desvergne, B.; Koppen, A.; Kalkhoven, E.; Müller, M.R.; Kersten, A.H.

    2009-01-01

    Peroxisome proliferator-activated receptor alpha (PPAR alpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction

  5. [Concentration of ferritin, transferrin and iron as a markers of iron deficiency in healthy women in reproductive age].

    Science.gov (United States)

    Chełchowska, Magdalena; Laskowska-Klita, Teresa; Leibschang, Jerzy

    2007-01-01

    Iron deficiency anemia in pregnancy continues to be a clinical problem, which contributes to maternal and fetal morbidity. Iron store deficiency leads to iron deficient erythropoiesis and to negative iron balance when the iron supply is insufficient to maintain normal concentration of hemoglobin. The aim of this study was aimed to establish concentration of ferritin, transferrin and iron as a markers of iron deficiency in healthy women in reproductive age came for control examination to Institute of Mother and Child in Warsaw. In serum of 108 healthy, multiparas in age up to 40 years from urban agglomerations, middle-class non-pregnant women concentration of iron, ferritin, transferrin and transferrin saturation were determined by commercially available kits (Hoffman-La Roche, Switzerland). Mean concentration of iron, ferritin and transferrin were among normal values. Low level of iron (below 49 microg/dl) was observed in serum of 12%, this of ferritin (below 20 ng/ml) in 22% and of transferrin (below 252 mg/dl) in 15% of studied women. Transferrin saturation lower than 15% was observed in 9 patients. The obtained values were age dependent. The lowest values of total iron were observed in the youngest group I (below 25 years old) and were accompanied with ferritin level below 20 ng/ml in 26% of women. Low ferritin values were also observed in serum of 22% patients of group II (25-35 years old) and only in 14% of women older than 35 years (group III). Saturation of transferrin lower than 15%, which indicated deficiency of iron for erythropoiesis, was observed in 26%, 13% and 19% patients of group I, II and III respectively. Obtained results indicated that in population of studied women in reproductive age, subclinical iron deficiency in 20% and negative iron balance in 10% could be observed. Therefore, iron status, especially store ferritin, should be assessed very carefully as a component of medical care.

  6. Cross-sectional study of expression of divalent metal transporter-1, transferrin, and hepcidin in blood of smelters who are occupationally exposed to manganese

    Directory of Open Access Journals (Sweden)

    Qiyuan Fan

    2016-09-01

    Full Text Available Background Manganese (Mn is widely used in industries including the manufacture of Mn-iron (Fe alloy. Occupational Mn overexposure causes manganism. Mn is known to affect Fe metabolism; this study was designed to test the hypothesis that workers exposed to Mn may have an altered expression of mRNAs encoding proteins in Fe metabolism. Methods Workers occupationally exposed to Mn (n = 71 from a Mn–Fe alloy factory and control workers without Mn-exposure (n = 48 from a pig-iron plant from Zunyi, China, were recruited for this study. Blood samples were collected into Trizol-containing tubes. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. Metal concentrations were quantified by atomic absorption spectrophotometry. Results Working environment and genetic background of both groups were similar except for marked differences in airborne Mn concentrations (0.18 mg/m3 in Mn–Fe alloy factory vs. 0.0022 mg/m3 in pig-Fe plant, and in blood Mn levels (34.3 µg/L vs. 10.4 µg/L. Mn exposure caused a significant decrease in the expression of divalent metal transporter-1 (DMT1, transferrin (Tf and hepcidin by 58.2%, 68.5% and 61.5%, respectively, as compared to controls, while the expression of transferrin receptor (TfR was unaltered. Linear regression analysis revealed that expressions of DMT1, Tf and hepcidin were inversely correlated with the accumulative Mn exposure; the correlation coefficients (r are −0.47, −0.54, and −0.49, respectively (p < 0.01. Conclusion The data suggest that occupational Mn exposure causes decreased expressions of DMT1, Tf and hepcidin in blood cells; the finding will help understand the mechanism underlying Mn exposure-associated alteration in Fe homeostasis among workers.

  7. Plasma membrane Toll-like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon-β

    DEFF Research Database (Denmark)

    Boye, Louise; Welsby, Iain; Lund, Lisbeth Drozd

    2016-01-01

    Lactobacillus acidophilus induces a potent interferon-β (IFN-β) response in dendritic cells (DCs) by a Toll-like receptor 2 (TLR2) -dependent mechanism, in turn leading to strong interleukin-12 (IL-12) production. In the present study, we investigated the involvement of different types...... of endocytosis in the L. acidophilus-induced IFN-β and IL-12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN-β and IL-12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place....... acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN-β and IL-12 was strongly inhibited. As L. acidophilus-induced IFN-β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading...

  8. Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

    Science.gov (United States)

    Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

    2016-08-01

    Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

  9. Enhanced plasma ghrelin levels in Helicobacter pylori-colonized,interleukin-1-receptor type 1-homozygous knockout (IL-1R1-/-) mice

    Institute of Scientific and Technical Information of China (English)

    Yuka Abiko; Hidekazu Suzuki; Tatsuhiro Masaoka; Sachiko Nomura; Kumiko Kurabayashi; Hiroshi Hosoda; Kenji Kangawa; Toshifumi Hibi

    2005-01-01

    AIM: Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor, and it plays a role in stimulating the growth hormone secretion, food intake,body weight gain and gastric motility. Eradication of Helicobacter pylori(H pylori) was shown to be associated with increase of the body weight. On the other hand, H pylori infection evokes the release of gastric IL-1β. The present study was designed to investigate the involvement of the gastric IL-1 signal in the ghrelin dynamics in H pyloricolonized mice.METHODS: Twelve-week-old female IL-1-receptor type 1-homozygous-knockout mice (IL-1R1-/-) and their wild-type littermates (WT) were orally inoculated with H pylori (Hp group), while other cohorts received oral inoculation of culture medium (Cont group). Thirteen weeks after the inoculation, the mice were examined. The plasma and stomach ghrelin levels and the gastric preproghrelin mRNA were measured.RESULTS: Although the WT mice with H pylori infection showed a significantly decreased body weight as compared with that of the animals without H pylori infection,H pylori infection did not influence the body weight of the IL-1R1-knockout (IL-1R1-/-) mice. In the H pylori-infected IL-1R1-/-mice, the total and active ghrelin levels in the plasma were significantly increased, and the gastric ghrelin level was decreased. No significant differences were noted in the gastric preproghrelin mRNA expression.CONCLUSION: Ghrelin secretion triggered by H pylori infection might be suppressed by IL-1β, the release of which is also induced by the infection, resulting in the body weight loss of mice with H pylori infection.

  10. Protein deficiency in pregnant rats causes decreased levels of plasma somatomedin and its carrier protein associated with reduced plasma levels of placental lactogen and hepatic lactogenic receptor number.

    Science.gov (United States)

    Pilistine, S J; Munro, H N

    1984-03-01

    Rats were fed either a 20% lactalbumin (control) or a 5% lactalbumin (low protein) diet for the last 2 weeks of pregnancy. At day 20 of gestation, rat serum placental lactogen levels, measured by radioreceptor assay, were significantly decreased by the low protein diet, thus confirming our earlier findings. The number of microsomal membrane lactogenic receptors, measured on the maternal livers at the end of pregnancy, was severely reduced in the livers of the low protein group, whereas protein deficiency did not affect binding affinity. Serum concentrations of somatomedin, measured by a competitive binding assay after acid treatment of the serum to remove endogenous carrier protein, were extensively reduced in the low protein group. The amounts of the somatomedin carrier proteins in the serum were assayed by separation on Sephacryl-S300 columns into higher- and lower-molecular-weight fractions peak 2 and peak 3, respectively. For the low protein diet group, both fractions showed a reduction in binding capacity, more marked in the case of peak 2. Since placental lactogen is known to influence output of somatomedin by the liver, we hypothesize that protein deficiency during pregnancy causes a fall in serum somatomedin level by reducing secretion of placental lactogen, which regulates its production by the liver.

  11. In vitro isotopic determination of diffusion volumes by transferrin labelled with indium 111. study of the correlation with SARI 125; Determination isotopique in vitro de volumes de diffusion par la transferrine marquee a l'indium 111. Etude de la correlation avec la SARI 125

    Energy Technology Data Exchange (ETDEWEB)

    Porot, C.L. [CHU Jean-Minjoz, Service de medecine nucleaire, 25 - Besancon (France); Angoue, O.R. [CHU Jean-Minjoz, laboratoire de biophysique et statistiques, 25 - Besancon (France); Berthetc, L.O. [CHU Jean-Minjoz, 25 - Besancon (France); Ungureanu, C.O.; Boulahdour, H.A.

    2010-07-01

    Serum albumin labeled with iodine 125 (S.A.R.I. 125) is the reference tracer used in measuring isotopic plasma volume. It has been causing a suspension of manufacturing leading to a supply disruption and resulting in the search for an alternative to measure plasma volume under consideration for measuring blood volume. Plasma transferrin labeled with indium-111 (Tf-{sup 111}In) is a potentially useful marker. To this end, we assessed the level of activity to be administered to determine a volume of distribution. The study of the correlation between the volume of distribution values obtained with S.A.R.I. 125 and Tf-{sup 111}In was then performed. Tf is an autologous protein which the labelling is easy and stable. Tf-{sup 111}In is a valid alternative to the S.A.R.I. 125 for measuring the plasma volume. The activity required for this examination shall not exceed 100 micro curies. (N.C.)

  12. Plasma levels of leptin and soluble leptin receptor and polymorphisms of leptin gene -18G > A and leptin receptor genes K109R and Q223R, in survivors of childhood acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Gozdzik Jolanta

    2011-06-01

    Full Text Available Abstract Background Approximately 20% of children and adolescents in Europe are overweight. Survivors of pediatric acute lymphoblastic leukemia (ALL are at increased risk of overweight and obesity. The purpose of this study was to assess leptin and leptin soluble receptor levels, as well as polymorphisms of selected genes in survivors of pediatric ALL, and the influence of chemo- and radiotherapy on development of overweight in the context of leptin regulation. Methods Eighty two patients (55% males, of median age 13.2 years (m: 4.8 years; M: 26.2 years were included in the study. The ALL therapy was conducted according to modified Berlin-Frankfurt-Munster (BFM; n = 69 regimen or New York (n = 13 regimen. In 38% of patients cranial radiotherapy (CRT was used in median dose of 18.2Gy (m: 14Gy; M: 24Gy. Median age at diagnosis was 4.5 (m: 1 year; M: 16.9 years and median time from completion of ALL treatment was 3.2 years (m: 0.5 year; M: 4.3 years. Patients with BMI ≥85 percentile were classified as overweight. Correlation of plasma levels of leptin and leptin soluble receptor, and polymorphisms of leptin gene -18G > A, leptin receptor genes K109R and Q223R, and the overweight status were analyzed in relation to gender, intensity of chemotherapy (high intensity vs. standard intensity regimens and to the use of CRT. Results Significant differences of leptin levels in patients treated with and without CRT, both in the entire study group (22.2+/- 3.13 ng/ml vs. 14.9+/-1.6 ng/ml; p Conclusions The prevalence of overweight in our cohort was higher than in general European population (31% vs 20% and increased regardless of the use of CRT. Leptin and leptin receptor levels may be used as useful markers of high risk of becoming overweight in ALL survivors, particularly in females treated with CRT. Polymorphisms of leptin gene -18G > A and leptin receptor genes K109R and Q223R were not associated with overweight status in ALL survivors.

  13. Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development

    DEFF Research Database (Denmark)

    Petersen, Yvette M; Hartmann, Bolette; Holst, Jens Juul

    2003-01-01

    transcription polymerase chain reaction) during pre- and postnatal development and the relationship between these variables and small intestinal growth in enterally and parenterally fed fetal and newborn pigs (premature and term-delivered, 92 and 100% gestation, respectively). Plasma GLP-2 concentrations...... of colostrum in fetal pigs at 92% gestation compared with untreated controls (59 +/- 11 vs. 7 +/- 2 pmol/L, P parenteral infusion of elemental nutrients, but the time course...... in these pigs. We conclude that the introduction of enteral feeding transiently increases plasma GLP-2 concentrations and decreases small intestinal GLP-2R mRNA levels during pig development. GLP-2 may play a role in the growth of the small intestine around birth and weaning via a response to enteral nutrition....

  14. The Receptor for Advanced Glycation End Products (Rage) and Its Ligands in Plasma and Infrainguinal Bypass Vein.

    Science.gov (United States)

    Malmstedt, J; Frebelius, S; Lengquist, M; Jörneskog, G; Wang, J; Swedenborg, J

    2016-04-01

    The aim was to investigate whether RAGE and its ligands are associated with infrainguinal bypass outcome in patients with and without diabetes. This was a prospective observational cohort. Patients (n = 68) with (n = 38) and without (n = 30) diabetes undergoing infrainguinal vein bypass for peripheral arterial disease were followed for 3 years. Endosecretory RAGE (esRAGE), S100A12, advanced glycation end products, and carboxymethyl-lysine (CML) were determined in plasma using ELISA. The influence of plasma levels on the main outcome (amputation free survival) was evaluated using Cox proportional hazard analysis. Plasma esRAGE, CML, and S100A12 in healthy controls (n = 30) without cardiovascular disease matched for sex and age were compared with patients, using the Mann-Whitney U test. Veins from bypass surgery procedures were stained and S100A12, RAGE, AGE, and CML were determined using immunohistochemistry. Forty-six patients survived with an intact leg during follow up. Seventeen died (median survival time 702 days, IQR 188-899 day), and six had amputations. High plasma S100A12 was associated with reduced amputation free survival (hazard ratio [HR] 2.99; 95% CI 1.24-7.24) when comparing levels above the 75th percentile with levels below. The increased risk was unchanged adjusting for age, sex, and diabetes. Diabetic patients had higher plasma S100A12 (11.75 ng/mL; 95% CI 8.12-15.38 ng/mL) than non-diabetic patients (5.0141 ng/mL; 95% CI 3.62-6.41 ng/mL), whereas plasma CML, esRAGE, and AGE were similar. Plasma CML and S100A12 were higher in patients than in controls (1.25 μg/mL, 95% CI 1.18-1.32 μg/mL vs. 0.8925 μg/mL, 95% CI 0.82-0.96 μg/mL; and 8.7 μg/mL, 95% CI 6.52-10.95 μg/mL vs. 3.47 μg/mL, 95% CI 2.95-3.99 μg/mL, respectively). The proportion of vein tissue stained for AGE (21%), RAGE (5%), CML (9%) and S100A12 (3%), were similar in patients with and without diabetes. Plasma S100A12 and CML are elevated in peripheral arterial disease and markers of

  15. Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Kannan, Vivekanandan; Gadamsetty, Deepak; Rose, Madhankumar; Maria, Stella; Mustafa, Imran; Khedkar, Anand; Dave, Nitesh; Arumugam, Muruganandam; Iyer, Harish

    2010-05-01

    A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 microm 50 mm x 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 microg/ml when 100 microl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C(max)) 0.40, 0.57, 1.95 microg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31microg/ml on Day 28 for low, mid and high dose treated animals.

  16. Clinical Value of Plasma Soluble Urokinase-Type Plasminogen Activator Receptor Levels in Term Neonates with Infection or Sepsis: A Prospective Study

    Directory of Open Access Journals (Sweden)

    Tania Siahanidou

    2014-01-01

    Full Text Available Background. suPAR, the soluble form of the urokinase-type plasminogen activator receptor, has been identified as a biomarker of infection in adults but its properties in neonatal infection are not known. Methods. Plasma suPAR levels were determined by ELISA in 47 term neonates with infection (19 bacterial and 28 viral and in 18 healthy neonates as controls. Thirteen out of 47 infected neonates were septic. In all infected neonates, suPAR levels were repeated at 24 hours, 48 hours, 3–5 days, and 7–10 days following admission. Results. Plasma suPAR levels were significantly increased in infected neonates upon admission, whereas they were highest in septic neonates, in comparison with controls P<0.001 and correlated positively with serum CRP levels (P=0.001. At infection subsidence, suPAR concentrations decreased significantly in comparison with baseline (P<0.001 but remained higher than in controls (P=0.01. Receiver operating characteristic analysis resulted in significant areas under the curve for detecting either infected or septic neonates, but not for discriminating between bacterial and viral cause of infection. Conclusions. suPAR is a diagnostic biomarker of infection or sepsis in term neonates; however, it cannot discriminate bacterial from viral infections and also its utility for monitoring the response to treatment is questioned.

  17. Association of suboptimal health status with psychosocial stress, plasma cortisol and mRNA expression of glucocorticoid receptor α/β in lymphocyte.

    Science.gov (United States)

    Yan, Yu-Xiang; Dong, Jing; Liu, You-Qin; Zhang, Jie; Song, Man-Shu; He, Yan; Wang, Wei

    2015-01-01

    Suboptimal health status (SHS) has become a new public health challenge in China. This study investigated whether high SHS is associated with psychosocial stress, changes in cortisol level and/or glucocorticoid receptor (GR) isoform expression. Three-hundred eighty-six workers employed in three companies in Beijing were recruited. The SHS score was derived from data collection in the SHS questionnaire (SHSQ-25). The short standard version of the Copenhagen Psychosocial Questionnaire (COPSOQ) was used to assess job-related psychosocial stress. The mean value of the five scales of COPSOQ and distribution of plasma cortisol and mRNA expression of GRα/GRβ between the high level of SHS group and the low level of SHS group were compared using a general linear model procedure. Multiple linear regression analysis was used to analyze the effect of psychosocial stress on SHS. We identified three factors that were predictive of SHS, including "demands at work", "interpersonal relations and leadership" and "insecurity at work". Significantly higher levels of plasma cortisol and GRβ/GRα mRNA ratio were observed among the high SHS group. High level of SHS is associated with decreased mRNA expression of GRα. This study confirmed the association between chronic psychosocial stress and SHS, indicating that improving the psychosocial work environment may reduce SHS and then prevent chronic diseases effectively.

  18. Plasma FGF21 concentrations, adipose fibroblast growth factor receptor-1 and β-klotho expression decrease with fasting in northern elephant seals.

    Science.gov (United States)

    Suzuki, Miwa; Lee, Andrew Y; Vázquez-Medina, José Pablo; Viscarra, Jose A; Crocker, Daniel E; Ortiz, Rudy M

    2015-05-15

    Fibroblast growth factor (FGF)-21 is secreted from the liver, pancreas, and adipose in response to prolonged fasting/starvation to facilitate lipid and glucose metabolism. Northern elephant seals naturally fast for several months, maintaining a relatively elevated metabolic rate to satisfy their energetic requirements. Thus, to better understand the impact of prolonged food deprivation on FGF21-associated changes, we analyzed the expression of FGF21, FGF receptor-1 (FGFR1), β-klotho (KLB; a co-activator of FGFR) in adipose, and plasma FGF21, glucose and 3-hydroxybutyrate in fasted elephant seal pups. Expression of FGFR1 and KLB mRNA decreased 98% and 43%, respectively, with fasting duration. While the 80% decrease in mean adipose FGF21 mRNA expression with fasting did not reach statistical significance, it paralleled the 39% decrease in plasma FGF21 concentrations suggesting that FGF21 is suppressed with fasting in elephant seals. Data demonstrate an atypical response of FGF21 to prolonged fasting in a mammal suggesting that FGF21-mediated mechanisms have evolved differentially in elephant seals. Furthermore, the typical fasting-induced, FGF21-mediated actions such as the inhibition of lipolysis in adipose may not be required in elephant seals as part of a naturally adapted mechanism to support their unique metabolic demands during prolonged fasting. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. High plasma levels of intact and cleaved soluble urokinase receptor reflect immune activation and are independent predictors of mortality in HIV-1-infected patients

    DEFF Research Database (Denmark)

    Ostrowski, Sisse Rye; Piironen, Timo; Høyer-Hansen, Gunilla

    2005-01-01

    ). In multivariate Cox analysis adjusting for CD4+ count, HIV RNA, beta2-microglobulin, hemoglobin and clinical stage, higher levels of suPAR(I-III) and suPAR(II-III) were independent predictors of increased mortality risk (P ...BACKGROUND: High blood levels of soluble urokinase receptor (suPAR) measured by enzyme-linked immunoassay (ELISA) (bulk measurement of 3-domain and 2-domain suPAR [suPAR(I-III), suPAR(II-III)], and suPAR(I-III) ligand complexes) strongly predict mortality in HIV-1-infected patients. This study...... investigated plasma levels of suPAR(I-III), suPAR(II-III), and 1-domain suPAR [suPAR(I)] and their predictive value for survival in HIV patients. METHODS: Plasma suPAR was measured by ELISA and 3 different time-resolved fluorescence immunoassays detecting suPAR(I-III), suPAR(I-III) plus suPAR(II-III), and su...

  20. Comparative effects of pioglitazone and rosiglitazone on plasma levels of soluble receptor for advanced glycation end products in type 2 diabetes mellitus patients.

    Science.gov (United States)

    Oz Gul, Ozen; Tuncel, Ercan; Yilmaz, Yusuf; Ulukaya, Engin; Gul, Cuma Bulent; Kiyici, Sinem; Oral, Arzu Yilmaztepe; Guclu, Metin; Ersoy, Canan; Imamoglu, Sazi

    2010-01-01

    Low levels of soluble receptor for advanced glycation end products (sRAGE) have been associated with the occurrence of vascular complications in patients with type 2 diabetes mellitus. Preliminary evidence has suggested that thiazolidinediones have the ability to modulate circulating levels of this molecule in the hyperglycemic milieu. The aim of this pilot study was to assess the differential effect of 2 different thiazolidinediones-pioglitazone and rosiglitazone-on plasma levels of sRAGE in type 2 diabetes mellitus patients. Sixty type 2 diabetes mellitus subjects were randomly assigned to receive pioglitazone (30 mg/d, n = 19), rosiglitazone (4 mg/d, n = 20), or placebo (medical nutrition therapy, n = 21) for 12 weeks. Changes in plasma glucose, glycosylated hemoglobin, insulin resistance (homeostasis model assessment), total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, and sRAGE were evaluated at baseline and after 12 weeks. At 12 weeks, the pioglitazone (P diabetes mellitus patients, pioglitazone-but not rosiglitazone-significantly raised sRAGE, which may contribute to its antiatherogenic effects.

  1. Correlation of Ferritin and Transferrin Serum with hsCRP and F2-Isoprostane in Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Waode Nurfina

    2010-12-01

    Full Text Available BACKGROUND: The low inflammatory state that accompanies the Metabolic Syndrome (MetS associates with the overexpression of oxidative stress. Ferritin and Transferrin serum are often used to measure iron status and their concentrations are altered in several metabolic conditions. We hypothesized that concentration of Ferritin and Transferrin serum increase in Metabolic Syndrome (MetS and correlate with the inflammation and oxidative stress. METHODS: We studied 65 male MetS patients, aged 43.26±7.16 years. Iron metabolism was measured by concentration of Ferritin and Transferrin serums, while inflammatory and oxidative stress by high sensitivity C-reactive Protein (hsCRP and F2-Isoprostane. RESULTS: Concentration of Ferritin 315.70±188.63 ng/L and Transferrin 2.36±0.31 g/L increased along with increasing components of MetS. Concentration of Ferritin serum had a positive correlation with hsCRP (r=0.220 and F2-Isoprostane (r=0.023. CONCLUSIONS: Serum concentration of Ferritin increased in the MetS and correlates with hsCRP and F2-Isoprostane. KEYWORDS: metabolic syndrome, ferritin, transferrin, hsCRP, F2-isoprostane.

  2. Epidermal growth factor receptor genotype in plasma DNA and outcome of chemotherapy in the Chinese patients with advanced non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    ZHUO Ming-lei; DUAN Jian-chun; WANG Yu-yan; GUO Qing-zhi; LIU Xu-yi; LIU Ning-hong; WANG Jie; WU Mei-na; ZHAO Jun; Sonya Wei Song; BAI Hua; WANG Shu-hang; YANG Lu; AN Tong-tong; WANG Xin

    2011-01-01

    Background The genotype of epidermal growth factor receptor (EGFR) is associated with tyrosine kinase inhibitor and effectiveness of therapy,but its role in cytotoxic chemotherapy is still unknown.Previous studies indicated that certain EGFR mutations were associated with response and progression free survival following platinum based chemotherapy.Our recent studies have identified that EGFR genotypes in the tumour tissues were not associated with response to the first-line chemotherapy in Chinese patients with advanced non-small cell lung cancer (NSCLC).In this study,we investigated associations of EGFR genotypes from plasma of patients with advanced NSCLC and response to first-line chemotherapy and prognosis.Methods We enrolled 145 advanced NSCLC patients who had received first-line chemotherapy in our department.We examined plasma EGFR genotypes for these patients and associations of EGFR mutations with response to chemotherapy and clinical outcomes.Results There were 54 patients with known EGFR mutations and 91 cases of wild types.No significant difference was detected in the response rate to first-line chemotherapy between mutation carriers and wild-type patients (37.0% vs.31.9%).The median survival time and 1-,2-year survival rates were higher in mutation carriers than wild-types (24months vs.18 months,85.7% vs.65.7% and 43.7% vs.25.9%,P=0.047).Clinical stage (IV vs.Ⅲb),response to the first-line chemotherapy (partial vs.no) and EGFR genotype were independent prognostic factors.Conclusion Plasma EGFR mutations in the Chinese patients with advanced NSCLC is not a predictor for the response to first-line chemotherapy,but an independent prognostic factor indicating longer survival.

  3. Mating behavior induces changes of expression of Fos protein, plasma testosterone and androgen receptors in the accessory olfactory bulb (AOB of the male mandarin vole Microtus mandarinus

    Directory of Open Access Journals (Sweden)

    Fengqin HE, Fadao TAI

    2009-08-01

    Full Text Available In order to investigate the neuroendocrine mechanism of the mating behavior in the adult male mandarin voles Microtus mandarinus, the radioimmunoassay (RIA and immunohistochemistry methods were used to investigate the differences in plasma testosterone (T concentrations and distribution of T immunoreactive neurons (T-IRs, androgen receptor immunoreactive neurons (AR-IRs and Fos protein immunoreactive neurons (Fos-IRs in the accessory olfactory bulb (AOB and the main olfactory bulb (MOB following exposure to clean hard-wood shavings (control group, soiled bedding (exposure group or contact with an estrous female (mating group. Results showed that plasma T concentration was significantly higher in the mating group than that in the exposure group, and both the mating group and the exposure group displayed significantly higher plasma T concentration than the control group. T-IRs, AR-IRs and Fos-IRs were investigated with the immunohistochemistry method in granule cell (GC and mitral cell (MC of the MOB and the AOB in the three groups. There were significantly more T-IRs, AR-IRs and Fos-IRs in MC and GC of the AOB in the mating group than that in the exposure group or the control group. T-IRs, AR-IRs and Fos-IRs did not show significant differences between the exposure group and the control group. Furthermore, obvious differences in MC and GC of the MOB were not found among the three groups. The results confirm that both changes of T and AR in the AOB might be underlying mating behavior in the adult male mandarin voles [Current Zoology 55 (4: 288–295, 2009].

  4. Plasma epidermal growth factor receptor mutation testing with a chip-based digital PCR system in patients with advanced non-small cell lung cancer.

    Science.gov (United States)

    Kasahara, Norimitsu; Kenmotsu, Hirotsugu; Serizawa, Masakuni; Umehara, Rina; Ono, Akira; Hisamatsu, Yasushi; Wakuda, Kazushige; Omori, Shota; Nakashima, Kazuhisa; Taira, Tetsuhiko; Naito, Tateaki; Murakami, Haruyasu; Koh, Yasuhiro; Mori, Keita; Endo, Masahiro; Nakajima, Takashi; Yamada, Masanobu; Kusuhara, Masatoshi; Takahashi, Toshiaki

    2017-04-01

    Epidermal growth factor receptor (EGFR) mutation testing is a companion diagnostic to determine eligibility for treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). Recently, plasma-based EGFR testing by digital polymerase chain reaction (dPCR), which enables accurate quantification of target DNA, has shown promise as a minimally invasive diagnostic. Here, we aimed to evaluate the accuracy of a plasma-based EGFR mutation test developed using chip-based dPCR-based detection of 3 EGFR mutations (exon 19 deletions, L858R in exon 21, and T790M in exon 20). Forty-nine patients with NSCLC harboring EGFR-activating mutations were enrolled, and circulating free DNAs (cfDNAs) were extracted from the plasma of 21 and 28 patients before treatment and after progression following EGFR-TKI treatment, respectively. Using reference genomic DNA containing each mutation, the detection limit of each assay was determined to be 0.1%. The sensitivity and specificity of detecting exon 19 deletions and L858R mutations, calculated by comparing the mutation status in the corresponding tumors, were 70.6% and 93.3%, and 66.7% and 100%, respectively, showing similar results compared with previous studies. T790M was detected in 43% of 28 cfDNAs after progression with EGFR-TKI treatment, but in no cfDNAs before the start of the treatment. This chip-based dPCR assay can facilitate detection of EGFR mutations in cfDNA as a minimally invasive method in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. The protective role of transferrin in Müller glial cells after iron-induced toxicity.

    Science.gov (United States)

    Picard, Emilie; Fontaine, Isabelle; Jonet, Laurent; Guillou, Florian; Behar-Cohen, Francine; Courtois, Yves; Jeanny, Jean-Claude

    2008-05-20

    Transferrin (Tf) expression is enhanced by aging and inflammation in humans. We investigated the role of transferrin in glial protection. We generated transgenic mice (Tg) carrying the complete human transferrin gene on a C57Bl/6J genetic background. We studied human (hTf) and mouse (mTf) transferrin localization in Tg and wild-type (WT) C57Bl/6J mice using immunochemistry with specific antibodies. Müller glial (MG) cells were cultured from explants and characterized using cellular retinaldehyde binding protein (CRALBP) and vimentin antibodies. They were further subcultured for study. We incubated cells with FeCl(3)-nitrilotriacetate to test for the iron-induced stress response; viability was determined by direct counting and measurement of lactate dehydrogenase (LDH) activity. Tf expression was determined by reverse transcriptase-quantitative PCR with human- or mouse-specific probes. hTf and mTf in the medium were assayed by ELISA or radioimmunoassay (RIA), respectively. mTf was mainly localized in retinal pigment epithelium and ganglion cell layers in retina sections of both mouse lines. hTf was abundant in MG cells. The distribution of mTf and hTf mRNA was consistent with these findings. mTf and hTf were secreted into the medium of MG cell primary cultures. Cells from Tg mice secreted hTf at a particularly high level. However, both WT and Tg cell cultures lose their ability to secrete Tf after a few passages. Tg MG cells secreting hTf were more resistant to iron-induced stress toxicity than those no longer secreted hTf. Similarly, exogenous human apo-Tf, but not human holo-Tf, conferred resistance to iron-induced stress on MG cells from WT mice. hTf localization in MG cells from Tg mice was reminiscent of that reported for aged human retina and age-related macular degeneration, both conditions associated with iron deposition. The role of hTf in protection against toxicity in Tg MG cells probably involves an adaptive mechanism developed in neural retina to

  6. Transferrin-modified nanostructured lipid carriers as multifunctional nanomedicine for codelivery of DNA and doxorubicin

    Directory of Open Access Journals (Sweden)

    Han Y

    2014-08-01

    Full Text Available Yiqun Han,1,† Ying Zhang,2,† Danni Li,3 Yuanyuan Chen,1 Jiping Sun,1 Fansheng Kong4 1Department of Respiratory Medicine, General Hospital of Ji’nan Command, PLA, 2Center of Interventional Therapy, Ji’nan Infectious Disease Hospital, 3Department of Internal Neurology, Ji’nan Central Hospital Affiliated to Shandong University, 4Department of Hematology, General Hospital of Ji’nan Command, PLA, Ji’nan, People’s Republic of China †These two authors contributed equally to this work Background: Nanostructured lipid carriers (NLC, composed of solid and liquid lipids, and surfactants are potentially good colloidal drug carriers. The aim of this study was to develop surface-modified NLC as multifunctional nanomedicine for codelivery of enhanced green fluorescence protein plasmid (pEGFP and doxorubicin (DOX. Methods: Two different nanocarriers: pEGFP- and DOX-loaded NLC, and solid lipid nanoparticles (SLN were prepared. Transferrin-containing ligands were used for the surface coating of the vectors. Their average size, zeta potential, and drug encapsulation capacity were evaluated. In vitro transfection efficiency of the modified vectors was evaluated in human alveolar adenocarcinoma cell line (A549 cells, and in vivo transfection efficiency of the modified vectors was evaluated in a mouse bearing A549 cells model. Results: Transferrin-modified DOX and pEGFP coencapsulated NLC (T-NLC has a particle size of 198 nm and a +19 mV surface charge. The in vitro cell viabilities of the T-NLC formulations were over 80% compared with the control. T-NLC displayed remarkably greater gene transfection efficiency and enhanced antitumor activity than DOX- and pEGFP-coencapsulated SLN in vivo.Conclusion: The results demonstrate that T-NLC noticeably enhanced antitumor activity through the combination of gene therapy with chemotherapy. Also coating of active transferrin improved the lung cancer cell-targeting of the carriers. In summary, the novel gene

  7. Vascular endothelial growth factor receptor inhibitor SU5416 suppresses lymphocyte generation and immune responses in mice by increasing plasma corticosterone.

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    Jamison J Grailer

    Full Text Available Inhibitors of vascular endothelial growth factor and its receptors (VEGFRs are attractive therapeutic candidates for cancer treatment. One such small molecule VEGFR inhibitor, SU5416, limits angiogenesis in vivo and is widely used for investigating VEGFR signaling in tumor pathophysiology. Herein, we describe novel actions of SU5416 on the immune system. Treatment of mice with SU5416 for 3 days induced significant reductions in size and cellularity of peripheral lymph nodes. Interestingly, SU5416 did not affect initial lymphocyte localization to peripheral lymph nodes but did reduce lymphocyte accumulation during long-term migration assays. Treatment with SU5416 also induced severe loss of double-positive thymocytes resulting in thymic atrophy and a reduction in peripheral B cells. Furthermore, immune responses following immunization were reduced in mice treated with SU5416. Findings of thymic atrophy and reduced weight gain during SU5416 treatment suggested elevated corticosterone levels. Indeed, a significant 5-fold increase in serum corticosterone was found 4 hours after treatment with SU5416. Importantly, adrenalectomy negated the effects of SU5416 treatment on primary immune tissues, and partial reversal of SU5416-induced changes was observed following blockade of glucocorticoid receptors. SU5416 has been reported to inhibit the activation of latent transforming growth factor (TGF-β, a cytokine involved in the regulation of glucocorticoid release by the adrenal glands. Interestingly, treatment with a TGF-β receptor inhibitor, showed a similar phenotype as SU5416 treatment, including elevated serum corticosterone levels and thymic atrophy. Therefore, these results suggest that SU5416 induces glucocorticoid release directly from the adrenal glands, possibly by inhibition of TGF-β activation.

  8. Effect of fish oil on lateral mobility of prostaglandin F2α (FP) receptors and spatial distribution of lipid microdomains in bovine luteal cell plasma membrane in vitro.

    Science.gov (United States)

    Plewes, M R; Burns, P D; Graham, P E; Hyslop, R M; Barisas, B G

    2017-01-01

    Lipid microdomains are ordered regions on the plasma membrane of cells, rich in cholesterol and sphingolipids, ranging in size from 10 to 200 nm in diameter. These lipid-ordered domains may serve as platforms to facilitate colocalization of intracellular signaling proteins during agonist-induced signal transduction. It is hypothesized that fish oil will disrupt the lipid microdomains, increasing spatial distribution of these lipid-ordered domains and lateral mobility of the prostaglandin (PG) F2α (FP) receptors in bovine luteal cells. The objectives of this study were to examine the effects of fish oil on (1) the spatial distribution of lipid microdomains, (2) lateral mobility of FP receptors, and (3) lateral mobility of FP receptors in the presence of PGF2α on the plasma membrane of bovine luteal cells in vitro. Bovine ovaries were obtained from a local abattoir and corpora lutea were digested using collagenase. In experiment 1, lipid microdomains were labeled using cholera toxin subunit B Alexa Fluor 555. Domains were detected as distinct patches on the plasma membrane of mixed luteal cells. Fish oil treatment decreased fluorescent intensity in a dose-dependent manner (P oil treatment on lateral mobility of FP receptors. Fish oil treatment increased microdiffusion and macrodiffusion coefficients of FP receptors as compared to control cells (P oil-treated cells (P oil treatment. Lateral mobility of receptors was decreased within 5 min following the addition of ligand for control cells (P oil-treated cells (P > 0.10). The data presented provide strong evidence that fish oil causes a disruption in lipid microdomains and affects lateral mobility of FP receptors in the absence and presence of PGF2α.

  9. DMSO Enhances TGF-β Activity by Recruiting the Type II TGF-β Receptor From Intracellular Vesicles to the Plasma Membrane.

    Science.gov (United States)

    Huang, Shuan Shian; Chen, Chun-Lin; Huang, Franklin W; Hou, Wei-Hsien; Huang, Jung San

    2016-07-01

    Dimethyl sulfoxide (DMSO) is used to treat many diseases/symptoms. The molecular basis of the pharmacological actions of DMSO has been unclear. We hypothesized that DMSO exerts some of these actions by enhancing TGF-β activity. Here we show that DMSO enhances TGF-β activity by ∼3-4-fold in Mv1Lu and NMuMG cells expressing Smad-dependent luciferase reporters. In Mv1Lu cells, DMSO enhances TGF-β-stimulated expression of P-Smad2 and PAI-1. It increases cell-surface expression of TGF-β receptors (TβR-I and/or TβR-II) by ∼3-4-fold without altering their cellular levels as determined by (125) I-labeled TGF-β-cross-linking/Western blot analysis, suggesting the presence of large intracellular pools in these cells. Sucrose density gradient ultracentrifugation/Western blot analysis reveals that DMSO induces recruitment of TβR-II (but not TβR-I) from its intracellular pool to plasma-membrane microdomains. It induces more recruitment of TβR-II to non-lipid raft microdomains than to lipid rafts/caveolae. Mv1Lu cells transiently transfected with TβR-II-HA plasmid were treated with DMSO and analyzed by indirect immunofluoresence staining using anti-HA antibody. In these cells, TβR-II-HA is present as a vesicle-like network in the cytoplasm as well as in the plasma membrane. DMSO causes depletion of TβR-II-HA-containing vesicles from the cytoplasm and co-localization of TβR-II-HA and cveolin-1 at the plasma membrane. These results suggest that DMSO, a fusogenic substance, enhances TGF-β activity presumably by inducing fusion of cytoplasmic vesicles (containing TβR-II) and the plasma membrane, resulting in increased localization of TβR-II to non-lipid raft microdomains where canonical signaling occurs. Fusogenic activity of DMSO may play a pivotal role in its pharmacological actions involving membrane proteins with large cytoplasmic pools. J. Cell. Biochem. 117: 1568-1579, 2016. © 2015 Wiley Periodicals, Inc.

  10. Comparison of three methods for detecting epidermal growth factor receptor mutations in plasma DNA samples of Chinese patients with advanced non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    QIN Ling; ZHONG Wei; ZHANG Li; LI Long-yun; WANG Meng-zhao

    2011-01-01

    Background Epidermal growth factor receptor (EGFR) mutations can predict tumor response to tyrosine kinase inhibitors (TKIs). Detecting EGFR mutations in plasma DNA samples in patients with advanced non-small cell lung cancer is challenging and promising. We compared three methods for detecting plasma EGFR mutations, including direct DNA sequencing, denaturing high-performance liquid chromatography (DHPLC) and Scorpions Amplification Refractory Mutation System (Scorpions ARMS).Methods Plasma DNA samples from 73 patients with stage ⅢB to Ⅳ adenocarcinoma were analyzed for EGFR mutations in exons 19 (deletion mutation) and 21(L858R mutation) using direct DNA sequencing, DHPLC and Scorpions ARMS. Sensitivities of the three methods were compared and the relationship between EGFR mutations and patients'survival was analyzed.Results In 73 patients, we detected EGFR mutations in 5 samples (6.9%) by direct DNA sequencing, in 22 samples (30.1%) by DHPLC, and in 28 samples (38.4%) by Scorpions ARMS. EGFR mutations were found in 13 samples in exon 19 and in 9 samples in exon 21 by DHPLC, while we found mutations in 15 samples in exon 19 and in 13 samples in exon 21 by Scorpions ARMS. Among the 73 patients, there was 90.4% concordance between DHPLC and Scorpions ARMS (66/73, K=0.79, P=0.07). Of the 73 patients, 46 patients were treated with gefitinib, including 18 patients with mutations and 28 patients without mutations as determined by Scorpions ARMS. The 18 patients with mutations had a significantly longer progression-free survival (PFS) time (median PFS was 21.0 months) than the 28 patients without mutations (median PFS was 7.0 months) (P=0.022).Conclusions Among the three methods for detecting EGFR mutations in plasma DNA samples of patients with advanced lung adenocarcinoma, direct gene sequencing had the lowest sensitivity, while Scorpion ARMS showed the highest mutation detecting capability. DHPLC is slightly less sensitive than Scorpion ARMS. EGFR

  11. Predicting C282Y Homozygote Genotype for Hemochromatosis Using Serum Ferritin and Transferrin Saturation Values from 44,809 Participants of the HEIRS Study

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    Andrew Lim

    2014-01-01

    Full Text Available INTRODUCTION: The simultaneous interpretation of serum ferritin level and transferrin saturation has been used as a clinical guide to diagnose genetic hemochromatosis. The Hemochromatosis and Iron Overload Screening (HEIRS Study screened 101,168 North American participants for serum ferritin level and transferrin saturation, and C282Y genotyping for the HFE gene.

  12. Interrelationship between Manganese and Iron for Binding to Apo-Transferrin

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    T. Hassanzadeh Ghasabeh

    2003-07-01

    Full Text Available Manganese is an essential trace elenent. There is a little evidence for deficiency of manganese in human , while as its toxicity has been reported in several cases. Manganese toxicity occurrs in humans exposed to high enviromental concentrations (for example workers in the dry battery production and may be particularly important in the neonatal period. The chemical similarities between manganese and iron and their binding to apo-transferrin (apo-tf may lead to the disturbances of iron metabolism. In the present project the interrelationship of anemia and manganese toxicity by techniques such as Affinity chromatography ; Equilibrium dialysis ; Urea polyacrylamide gel electrophoresis ; Spectrophotometric titration was investigated. The charactristics of Mn and Fe binding to apo-tf have been investigated and compared in this article . Using Equilibrium dialysis technique the binding of Fe and Mn to apo-tf was also studied. The binding constant of Mn to apo-tf was calculated using scatchared plot analysis. Addition of Mn (1.5 g/ml to reaction mixture containing Fe and apo-tf reduced Fe binding to apo-transferrin in comparison to control. Using Urea polyacrylamide gel electrophoresis (Urea-PAGE technique conforming the binding of Mn to apo-tf. The data that has been presented in this article elucidated the probable mechanism by which Mn interference with Fe metabolism; which result in the apperance of anemia.

  13. Impact of oral iron challenges on circulating non-transferrin-bound iron in healthy Guatemalan males.

    Science.gov (United States)

    Schümann, Klaus; Kroll, Sylvia; Romero-Abal, Maria-Eugenia; Georgiou, Niki A; Marx, Jo J M; Weiss, Günter; Solomons, Noel W

    2012-01-01

    Oral iron as a supplement has been associated with adverse health consequences, especially in the context of young children with active malaria. A potential aggravating role of non-transferrin-bound iron (NTBI) has been proposed. NTBI responses in both a fasting and post-oral iron dosing situation were related to serum iron concentration and ferritin status. Fasting and 1, 2, and 3 h postdose serum samples were obtained in conjunction with oral ferrous sulfate supplementation in aqueous solution of 0, 15, 30, 60, 120 and 240 mg Fe in a cohort of 8 healthy Guatemalan men over a 9-week metabolic protocol. Hemoglobin, serum ferritin, percent transferrin saturation, serum iron and NTBI were all measured. Circulating levels of serum iron and NTBI increased in a graded fashion in response to oral iron, with the relative increment for NTBI slightly greater than that of iron. Detectable NTBI was occasionally measured in fasting specimens, more frequently in subjects with high ferritin status. Post-iron NTBI responses, by contrast, were higher in normal-ferritin subjects in absolute terms, and rose with increasing postabsorptive serum iron responses. The appearance and response of circulating NTBI were consistent with recognized principles of iron regulation. Copyright © 2012 S. Karger AG, Basel.

  14. Plasma Concentrations of Hepcidin in Anemic Zimbabwean Infants.

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    Tatenda G Mupfudze

    Full Text Available Anemia in infancy is a global public health problem. We evaluated the relative contributions of iron deficiency and inflammation to infant anemia.We measured plasma hepcidin, ferritin, soluble transferrin receptor (sTfR, alpha-1-acid glycoprotein and C-reactive protein (CRP by ELISA on archived plasma from 289 HIV-unexposed anemic or non-anemic Zimbabwean infants at ages 3 mo, 6 mo and 12 mo. Among anemic infants, we determined the proportion with iron-deficiency anemia (IDA and anemia of inflammation (AI. We undertook regression analyses of plasma hepcidin and anemia status, adjusting for sex, age and birthweight.Anemic infants at 3 mo were more stunted and had higher CRP (median 0.45 vs 0.21 mg/L; P = 0.037 and hepcidin (median 14.7 vs 9.7 ng/mL; P = 0.022 than non-anemic infants, but similar levels of ferritin and sTfR; 11% infants had IDA and 15% had AI. Anemic infants at 6 mo had higher hepcidin (median 7.9 vs 4.5 ng/mL; P = 0.016 and CRP (median 2.33 vs 0.32 mg/L; P<0.001, but lower ferritin (median 13.2 vs 25.1 μg/L; P<0.001 than non-anemic infants; 56% infants had IDA and 12% had AI. Anemic infants at 12 mo had lower ferritin (median 3.2 vs 22.2 μg/L; P<0.001 and hepcidin (median 0.9 vs 1.9 ng/mL; P = 0.019, but similar CRP levels; 48% infants had IDA and 8% had AI. Comparing anemic with non-anemic infants, plasma hepcidin was 568% higher, 405% higher and 64% lower at 3 mo, 6 mo and 12 mo, respectively, after adjusting for sex and birthweight (all p<0.01. Plasma hepcidin declined significantly with age among anemic but not non-anemic infants. Girls had 61% higher hepcidin than boys, after adjusting for age, anemia and birthweight (p<0.001.Anemia is driven partly by inflammation early in infancy, and by iron deficiency later in infancy, with plasma hepcidin concentrations reflecting the relative contribution of each. However, there is need to better characterize the drivers of hepcidin during infancy in developing countries.

  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.

    Science.gov (United States)

    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 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 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 F-FDG, as shown through PET imaging and biodistribution studies. (89)Zr-Tf is a useful tool to interrogate MYC via TfR-targeted PET imaging in TNBC. This data could lead to investigations of

  16. Independent associations of polymorphisms in vitamin D binding protein (GC) and vitamin D receptor (VDR) genes with obesity and plasma 25OHD3 levels demonstrate sex dimorphism.

    Science.gov (United States)

    Almesri, Norah; Das, Nagalla S; Ali, Muhallab E; Gumaa, Khalid; Giha, Hayder Ahmed

    2016-04-01

    We investigated a possible association between polymorphisms in vitamin D binding protein (GC) and vitamin D receptor (VDR) genes and obesity in Bahraini adults. For this purpose, 406 subjects with varying body mass indexes (BMIs) were selected. Plasma levels of 25-hydroxyvitamin D3 (25OHD3) were measured by chemiluminescence immunoassay. Six single nucleotide polymorphisms, 2 in the VDR gene (rs731236 TC and rs12721377 AG) and 4 in the GC gene (rs2282679 AC, rs4588 CA, rs7041 GT, and rs2298849 TC), were genotyped by real-time polymerase chain reaction. We found that the rs7041 minor allele (G) and rare genotype (GG) were associated with higher BMI (p = 0.007 and p = 0.012, respectively), but they did not influence 25OHD3 levels. However, the minor alleles of rs2282679 (A) and rs4588 (C) were associated with low 25OHD3 plasma levels (p = 0.039 and p = 0.021, respectively), but not with BMI. Having categorized the subjects based on their sex, we found that (i) rs7041 GG associated with high BMI in females (p = 0.003), (ii) rs4588 CC associated with high BMI in females (p = 0.034) and low 25OHD3 levels in males (p = 0.009), and (iii) rs12721377 AA associated with low 25OHD3 levels in females (p = 0.039). Notably, none of the common haplotypes (6 in the GC gene and 3 in the VDR gene) were associated with BMI. Therefore, polymorphisms in the GC (rs2282679, rs4588, rs7041) and VDR (rs12721377) genes were independently associated with obesity and 25OHD3 levels with a clear sex dimorphism.

  17. The regulator of G protein signaling (RGS) domain of G protein-coupled receptor kinase 5 (GRK5) regulates plasma membrane localization and function.

    Science.gov (United States)

    Xu, Hua; Jiang, Xiaoshan; Shen, Ke; Fischer, Christopher C; Wedegaertner, Philip B

    2014-07-01

    The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of the RGS domain, displays greatly decreased coIP interactions. Both forcing dimerization of GRK5-EE, via fusion to leucine zipper motifs, and appending an extra C-terminal membrane-binding region to GRK5-EE (GRK5-EE-CT) recover PM localization. In addition, GRK5-EE displays a decreased ability to inhibit PAR1-induced calcium release compared with GRK5 wild type (wt). In contrast, PM-localized GRK5-EE-CaaX (appending a C-terminal prenylation and polybasic motif from K-ras) or GRK5-EE-CT shows comparable ability to GRK5 wt to inhibit PAR1-induced calcium release. The results suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function. © 2014 Xu, Jiang, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. Trace Amine-Associated Receptor 1 Localization at the Apical Plasma Membrane Domain of Fisher Rat Thyroid Epithelial Cells Is Confined to Cilia

    Science.gov (United States)

    Szumska, Joanna; Qatato, Maria; Rehders, Maren; Führer, Dagmar; Biebermann, Heike; Grandy, David K.; Köhrle, Josef; Brix, Klaudia

    2015-01-01

    Background The trace amine-associated receptor 1 (Taar1) is one member of the Taar family of G-protein-coupled receptors (GPCR) accepting various biogenic amines as ligands. It has been proposed that Taar1 mediates rapid, membrane-initiated effects of thyronamines, the endogenous decarboxylated and deiodinated relatives of the classical thyroid hormones T4 and T3. Objectives Although the physiological actions of thyronamines in general and 3-iodothyronamine (T1AM) in particular are incompletely understood, studies published to date suggest that synthetic T1AM-activated Taar1 signaling antagonizes thyromimetic effects exerted by T3. However, the location of Taar1 is currently unknown. Methods To fill this gap in our knowledge we employed immunofluorescence microscopy and a polyclonal antibody to detect Taar1 protein expression in thyroid tissue from Fisher rats, wild-type and taar1-deficient mice, and in the polarized FRT cells. Results With this approach we found that Taar1 is expressed in the membranes of subcellular compartments of the secretory pathway and on the apical plasma membrane of FRT cells. Three-dimensional analyses further revealed Taar1 immunoreactivity in cilial extensions of postconfluent FRT cell cultures that had formed follicle-like structures. Conclusions The results suggest Taar1 transport along the secretory pathway and its accumulation in the primary cilium of thyrocytes. These findings are of significance considering the increasing interest in the role of cilia in harboring functional GPCR. We hypothesize that thyronamines can reach and activate Taar1 in thyroid follicular epithelia by acting from within the thyroid follicle lumen, their potential site of synthesis, as part of a nonclassical mechanism of thyroid autoregulation. PMID:26601071

  19. Lipid domain structure of the plasma membrane revealed by patching of membrane components.

    Science.gov (United States)

    Harder, T; Scheiffele, P; Verkade, P; Simons, K

    1998-05-18

    Lateral assemblies of glycolipids and cholesterol, "rafts," have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.

  20. Aberrant plasma IL-7 and soluble IL-7 receptor levels indicate impaired T-cell response to IL-7 in human tuberculosis.

    Directory of Open Access Journals (Sweden)

    Christian Lundtoft

    2017-06-01

    Full Text Available T-cell proliferation and generation of protective memory during chronic infections depend on Interleukin-7 (IL-7 availability and receptivity. Regulation of IL-7 receptor (IL-7R expression and signalling are key for IL-7-modulated T-cell functions. Aberrant expression of soluble (s and membrane-associated (m IL-7R molecules is associated with development of autoimmunity and immune failure in acquired immune deficiency syndrome (AIDS patients. Here we investigated the role of IL-7/IL-7R on T-cell immunity in human tuberculosis. We performed two independent case-control studies comparing tuberculosis patients and healthy contacts. This was combined with follow-up examinations for a subgroup of tuberculosis patients under therapy and recovery. Blood plasma and T cells were characterised for IL-7/sIL-7R and mIL-7R expression, respectively. IL-7-dependent T-cell functions were determined by analysing STAT5 phosphorylation, antigen-specific cytokine release and by analysing markers of T-cell exhaustion and inflammation. Tuberculosis patients had lower soluble IL-7R (p < 0.001 and higher IL-7 (p < 0.001 plasma concentrations as compared to healthy contacts. Both markers were largely independent and aberrant expression normalised during therapy and recovery. Furthermore, tuberculosis patients had lower levels of mIL-7R in T cells caused by post-transcriptional mechanisms. Functional in vitro tests indicated diminished IL-7-induced STAT5 phosphorylation and impaired IL-7-promoted cytokine release of Mycobacterium tuberculosis-specific CD4+ T cells from tuberculosis patients. Finally, we determined T-cell exhaustion markers PD-1 and SOCS3 and detected increased SOCS3 expression during therapy. Only moderate correlation of PD-1 and SOCS3 with IL-7 expression was observed. We conclude that diminished soluble IL-7R and increased IL-7 plasma concentrations, as well as decreased membrane-associated IL-7R expression in T cells, reflect impaired T

  1. Carbohydrate-deficient transferrin--a valid marker of alcoholism in population studies? Results from the Copenhagen City Heart Study

    DEFF Research Database (Denmark)

    Grønbaek, M; Becker, U; Henriksen, Jens Henrik Sahl

    1995-01-01

    Carbohydrate-deficient transferrin (CDT) was analyzed by a modified radioimmunoassay test in a random population sample of 400 individuals, and results were compared with reported alcohol intake derived from a structured questionnaire. Among the 180 men, the test was found to be acceptable...

  2. The induction of nitric oxide response of carp macrophages by transferrin is influenced by the allelic diversity of the molecule

    NARCIS (Netherlands)

    Jurecka, P.M.; Irnazarow, I.; Stafford, J.L.; Ruszczyk, A.; Taverne, N.; Belosevic, M.; Savelkoul, H.F.J.; Wiegertjes, G.F.

    2009-01-01

    The central role of transferrin (Tf) as an iron transporting protein has been extended by observations that modified versions of Tf also participate in the regulation of innate immunity. We report on the isolation of two carp Tf proteins (alleles D and G) to purity using rivanol precipitation and io

  3. Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells

    DEFF Research Database (Denmark)

    Reuter, Lauri J.; Shahbazi, Mohammad-Ali; Makila, Ermei M.

    2017-01-01

    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...

  4. [FECAL NONINVASIVE TESTS (CALPROTECTIN, TRANSFERRIN, HEMOGLOBIN) IN COMPLEX DIAGNOSIS OF DISEASES OF INTESTINES].

    Science.gov (United States)

    Livzan, M A; Lyalukova, E A; Nechaeva, G; Osipenko, M F; Dolgih, T I

    2015-01-01

    A research objective was the assessment of informational content of fecal noninvasive tests (calprotectin, transferrin, hemoglobin) in complex diagnosis of diseases of intestines. Open kogortny research by method of a cross cut included 52 patients (middle age - 38,6 years) with IBS-like symptoms (abdominal pain or discomfort, change of frequency and/or character of a chair). Sensitivity of dough on calprotectin for diagnosis of organic pathology of intestines made (89%), for dough on calprotectin and hemoglobin - also 89%. At patients at incomplete compliance of clinical signs to diagnostic criteria of IBS and lack of endoscopic signs of damage of a large intestine research on fecal biomarkers allows to increase efficiency of diagnostics.

  5. Transferrin Sialylation in Smoking and Non-Smoking Pregnant Women with Intrauterine Growth Restriction.

    Science.gov (United States)

    Wrześniak, Marta; Kepinska, Marta; Bizoń, Anna; Milnerowicz-Nabzdyk, Ewa; Milnerowicz, Halina

    2015-01-01

    Transferrin (Tf) is a glycosylated protein responsible for transporting iron. Various sialylation levels of Tf are observed during physiological and pathological processes. We studied if the changes in iron stores as well as tobacco smoke may have an impact on foetal development and in consequence lead to intrauterine growth restriction (IUGR). In the third trimester of pregnancy, lower levels of 4-sialoTf isoform and higher levels of 5-sialoTf were observed in the serum of non-smoking women with IUGR in comparison to the control group. On the day of labour, level of 2-sialoTf was significantly lower and level of 3-sialo was Tf higher in the serum of non-smoking women. Level of 4-sialo was found lower in the serum of smoking women with IUGR than in the control group. The observed changes may suggest a connection between iron stores, transport of iron to the foetus and foetal development.

  6. Independence of carbohydrate-deficient isoforms of transferrin and cyclic citrullinated peptides in rheumatoid arthritis.

    Science.gov (United States)

    Gudowska, Monika; Gindzienska-Sieskiewicz, Ewa; Gruszewska, Ewa; Cylwik, Bogdan; Sierakowski, Stanislaw; Szmitkowski, Maciej; Chrostek, Lech

    The aim of this study was to assess the relationship between the two types of posttranslational modifications of proteins in RA: glycosylation on the example of carbohydrate-deficient transferrin and citrullination by means of autoantibodies to cyclic citrullinated peptides. The study was carried out in 50 RA patients. CDT was measured using N Latex CDT immunonephelometric test, the results were presented in absolute and relative units. Anti-CCP were measured using the chemiluminescent method and rheumatoid factor by immunoturbidimetric method. 80% of RA patients were positive for anti-CCP, 70% for RF and 62% for both, anti-CCP and RF. The level of %CDT was significantly elevated, but absolute CDT level was not changed. The mean absolute CDT concentration was higher in anti-CCP positive patients than that in anti-CCP negative. CDT (absolute and relative concentration) did not correlate with anti-CCP and RF. However, serum RF significantly correlated with anti-CCP. %CDT did not correlate with anti-CCP, but absolute level correlated with anti-CCP only in anti-CCP negative and RF negative patients. CDT did not correlate with RF, but solely with anti-CCP in anti-CCP negative patients. Anti-CCP correlated with DAS 28 only in anti-CCP negative RA, but CDT (absolute and relative units) correlated with DAS 28 in all patients and in anti-CCP positive RA. These results suggest that the changes in CDT and anti-CCP concentrations are not associated with oneself and indicate on the independence of these posttranslational modifications in rheumatoid arthritis. Only the alterations in transferrin glycosylation reflected the activity of RA. Copyright © 2016 Elsevier Editora Ltda. All rights reserved.

  7. Immunocytochemical study with an anti-transferrin binding protein serum: a marker for avian oligodendrocytes.

    Science.gov (United States)

    Cho, S S; Lucas, J J

    1995-03-13

    We have investigated immunocytochemically the localization of a transferrin binding protein (TfBP) in adult CNS of avian and mammalian species using a polyclonal antibody raised against the protein purified from hen oviduct membranes (alpha OV-TfBP). TfBP has recently been shown to be HSP108. An overall strong immunoreactivity was revealed in most parts of the avian brains, especially in the white matter. The main immunoreactivity originated in small, intensely reacting cells interpreted as oligodendrocytes. The density of TfBP-labeled oligodendrocytes of the avian brains was generally proportional to the degree of myelination. There were no marked differences in TfBP-immunostaining pattern between avian species (chick, pigeon and lovebird). On the other hand, in rat, rabbit and cat brains we could not find any TfBP-immunoreactivity. Immunoelectron microscopy has further revealed that TfBP is present in the light and medium types of oligodendrocytes which are known to have high metabolic activities. TfBP reaction product was homogeneously dispersed throughout the perinuclear cytoplasm and fine processes of oligodendrocytes. The intracytoplasmic organelles such as mitochondria and Golgi apparatus were devoid of reaction product. The presence of TfBP in oligodendrocytes implies that this protein may play an important role in transferrin-mediated iron metabolism in the CNS. The complete lack of cross-reactivity between alpha OV-TfBP and mammalian tissues suggests that there is species variability in TfBP structure. We conclude that this chick TfBP antiserum will prove useful in studies of oligodendrocytes and myelination in the avian CNS.

  8. Interaction of VO2+ ion with human serum transferrin and albumin.

    Science.gov (United States)

    Sanna, Daniele; Garribba, Eugenio; Micera, Giovanni

    2009-04-01

    The complexation of VO(2+) ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO(2+) ion occupies three different binding sites, A, B(1) and B(2), distinguishable in the X-band anisotropic spectrum recorded in D(2)O. With albumin the results show that a dinuclear complex (VO)(2)(d)HSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO(2+), the multinuclear complex (VO)(x)(m)HSA is the prevalent species, where x=5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named "strong" site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the (51)V A(z) values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO(2+)/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO(2+) concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.

  9. Clinical utility of KRAS status in circulating plasma DNA compared to archival tumour tissue from patients with metastatic colorectal cancer treated with anti-epidermal growth factor receptor therapy

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Pallisgaard, Niels; Appelt, Ane Lindegaard

    2015-01-01

    by an in-house qPCR method. Results are presented according to REMARK. RESULTS: One-hundred-and-forty patients were included. Thirty-four percent had detectable KRAS mutations in the tumour, compared to 23% in plasma. KRAS detection in archival tumour tissue showed no correlation to survival, whereas...... in patients from metastatic colorectal cancer (mCRC) prior to anti-epidermal growth factor receptor (anti-EGFR) therapy. Secondly, we investigated the concentration of total cfDNA in relation to clinical outcome. PATIENTS AND METHODS: Patients were resistant to 5-FU, oxaliplatin and irinotecan and treated......BACKGROUND: Circulating cell-free DNA (cfDNA) in plasma is a mixture of DNA from malignant and normal cells, and can be used as a liquid biopsy to detect and quantify tumour specific mutations e.g. KRAS. We investigated the clinical value of KRAS mutations when detected in plasma compared to tumour...

  10. Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistanc