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Sample records for human transferrin receptor-1

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

    PubMed Central

    Zong, Min; Fofana, Isabel

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    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

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

    SciTech Connect

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

    2008-11-25

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

  5. Transferrin facilitates the formation of DNA double-strand breaks via transferrin receptor 1: the possible involvement of transferrin in carcinogenesis of high-grade serous ovarian cancer.

    PubMed

    Shigeta, S; Toyoshima, M; Kitatani, K; Ishibashi, M; Usui, T; Yaegashi, N

    2016-07-01

    Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (γH2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of γH2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivo study with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of

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

    PubMed

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

    2016-02-01

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

  7. Interaction of human diferric transferrin with reticulocytes.

    PubMed Central

    Huebers, H; Csiba, E; Josephson, B; Huebers, E; Finch, C

    1981-01-01

    Methods have been devised for preparing human transferrin with a different isotope of iron selectively labeling each of the two iron binding sites and for determining the distribution of radioiron among transferrin molecules. When diferric human transferrin was exposed to human or animal reticulocytes, there was an equal contribution of radioiron from the acid-stable and acid-labile sites. In this delivery, both atoms of iron were removed simultaneously from the diferric transferrin molecule, converting it to apotransferrin. At similar iron concentrations the amount of iron delivered by diferric transferrin was twice that delivered by monoferric transferrin. PMID:6264452

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

    PubMed

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

    2016-01-12

    Over-expression of transferrin receptor 1 (TFRC) is observed in hepatocellular carcinoma (HCC); however, there is a lack of conclusive information regarding the mechanisms of this dysregulation. In the present study, we demonstrated a significant increase in the levels of TFRC mRNA and protein in preneoplastic livers from relevant experimental models of human hepatocarcinogenesis and in human HCC cells. Additionally, using the TCGA database, we demonstrated an over-expression of TFRC in human HCC tissue samples and a markedly decreased level of microRNA-152 (miR-152) when compared to non-tumor liver tissue. The results indicated that the increase in levels of TFRC in human HCC cells and human HCC tissue samples may be attributed, in part, to a post-transcriptional mechanism mediated by a down-regulation of miR-152. This was evidenced by a strong inverse correlation between the level of TFRC and the expression of miR-152 in human HCC cells (r = -0.99, p = 4. 7 × 10-9), and was confirmed by in vitro experiments showing that transfection of human HCC cell lines with miR-152 effectively suppressed TFRC expression. This suggests that miR-152-specific targeting of TFRC may provide a selective anticancer therapeutic approach for the treatment of HCC. PMID:26657500

  9. Inhibition of transferrin receptor 1 transcription by a cell density response element

    PubMed Central

    2005-01-01

    TfR1 (transferrin receptor 1) mediates the uptake of transferrin-bound iron and thereby plays a critical role in cellular iron metabolism. Its expression is coupled to cell proliferation/differentiation and controlled in response to iron levels and other signals by transcriptional and post-transcriptional mechanisms. It is well established that TfR1 levels decline when cultured cells reach a high density and in the present study we have investigated the underlying mechanisms. Consistent with previous findings, we demonstrate that TfR1 expression is attenuated in a cell-density-dependent manner in human lung cancer H1299 cells and in murine B6 fibroblasts as the result of a marked decrease in mRNA content. This response is not associated with alterations in the RNA-binding activity of iron regulatory proteins that are indicative of a transcriptional mechanism. Reporter assays reveal that the human TfR1 promoters contains sequences mediating cell-density-dependent transcriptional inhibition. Mapping of the human and mouse TfR1 promoters identified a conserved hexa-nucleotide 5′-GAGGGC-3′ motif with notable sequence similarity to a previously described element within the IGF-2 (insulin-like growth factor-2) promoter. We show that this motif is necessary for the formation of specific complexes with nuclear extracts and for cell-density-dependent regulation in reporter gene assays. Thus the TfR1 promoter contains a functional ‘cell density response element’ (CDRE). PMID:16092918

  10. Noncanonical role of transferrin receptor 1 is essential for intestinal homeostasis

    PubMed Central

    Chen, Alan C.; Donovan, Adriana; Ned-Sykes, Renee; Andrews, Nancy C.

    2015-01-01

    Transferrin receptor 1 (Tfr1) facilitates cellular iron uptake through receptor-mediated endocytosis of iron-loaded transferrin. It is expressed in the intestinal epithelium but not involved in dietary iron absorption. To investigate its role, we inactivated the Tfr1 gene selectively in murine intestinal epithelial cells. The mutant mice had severe disruption of the epithelial barrier and early death. There was impaired proliferation of intestinal epithelial cell progenitors, aberrant lipid handling, increased mRNA expression of stem cell markers, and striking induction of many genes associated with epithelial-to-mesenchymal transition. Administration of parenteral iron did not improve the phenotype. Surprisingly, however, enforced expression of a mutant allele of Tfr1 that is unable to serve as a receptor for iron-loaded transferrin appeared to fully rescue most animals. Our results implicate Tfr1 in homeostatic maintenance of the intestinal epithelium, acting through a role that is independent of its iron-uptake function. PMID:26324903

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

    PubMed Central

    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.

    2015-01-01

    Patients with a combined immunodeficiency characterized by normal numbers, but impaired function, of T and B cells had a homozygous p.Tyr20His mutation in transferrin receptor 1 (TfR1), encoded by TFRC. The mutation disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 surface expression. Iron citrate rescued the lymphocyte defects and transduction of wild type, but not mutant, TfR1 rescued impaired transferrin uptake in patient fibroblasts. TfrcY20H/Y20H mice recapitulated the patients’ immunologic defects. Despite the critical role of TfR1 in erythrocyte development and function, the 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 fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares the patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity. PMID:26642240

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

    PubMed

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

    2016-01-01

    Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity. PMID:26642240

  13. Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

    PubMed Central

    Kim, Jonghan; Luo, Flora; Sanford, Jack; Chen, Juxing; Enns, Caroline; Wessling-Resnick, Marianne

    2013-01-01

    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. PMID:23894616

  14. A structural comparison of human serum transferrin and human lactoferrin.

    PubMed

    Wally, Jeremy; Buchanan, Susan K

    2007-06-01

    The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences. PMID:17216400

  15. Interlobe communication in 13C-methionine-labeled human transferrin.

    PubMed

    Beatty, E J; Cox, M C; Frenkiel, T A; Tam, B M; Mason, A B; MacGillivray, R T; Sadler, P J; Woodworth, R C

    1996-06-18

    [1H, 13C] NMR investigations of metal-induced conformational changes in the blood serum protein transferrin (80 kDa) are reported. These are thought to play an important role in the recognition of this protein by its cellular receptors. [1H, 13C] NMR resonance assignments are presented for all nine methionine 13CH3 groups of recombinant deglycosylated human transferrin on the basis of studies of recombinant N-lobe (40 kDa, five Met residues), NOESY-relayed [1H, 13C] HMQC spectra, and structural considerations. The first specific assignments for C-lobe resonances of transferrin are presented. Using methionine 13CH3 resonances as probes, it is shown that, with oxalate as the synergistic anion, Ga3+ binds preferentially to the C-lobe and subsequently to the N-lobe. The NMR shifts of Met464, which is in the Trp460-centered hydrophobic patch of helix 5 in the C-lobe in contact with the anion and metal binding site, show that Ga3+ binding causes movement of side chains within this helix, as is also the case in the N-lobe. The C-lobe residue Met382, which contacts the N-lobe hinge region, is perturbed when Ga3+ binds to the N-lobe, indicative of interlobe communication, a feature which may control the recognition of fully-metallated transferrin by its receptor. These results demonstrate that selective 13C labeling is a powerful method for probing the structure and dynamics of high-molecular-mass proteins. PMID:8672464

  16. Uptake and release of iron from human transferrin.

    PubMed Central

    Huebers, H; Josephson, B; Huebers, E; Csiba, E; Finch, C

    1981-01-01

    Purified fractions of human apotransferrin, monoferric transferrins with iron on the acid-labile binding site and on the acid-stable binding site, and diferric transferrin have been prepared. The iron loading and unloading behavior of these preparations has been examined by isoelectric focusing. Iron release from the two monoferric transferrin preparations to human reticulocytes was of similar magnitude. In a mixture containing equal amounts of diferic and monoferric iron, approximately 4 times the amount of iron delivered by the monoferric species was delivered by the diferric species. Iron loading of transferrin in vitro showed a random distribution between monoferric and diferric transferrin. Among the monoferric transferrins, loading of the acid-labile binding sites was greater than that of the acid-stable binding sites. In vivo iron distribution in normal subjects, as evaluated by in vitro-added 50Fe, gave similar results. Absorption of a large dose of orally administered iron in iron-deficient subjects resulted in a somewhat greater amount of diferric transferrin at low saturation and a somewhat smaller amount of diferric transferrin at higher saturations than would have been anticipated by random loading. These data would indicate that in the human, iron loading of transferrin may be considered essentially random. Unloading from the two monoferric transferrin species is of similar magnitude but far less than that delivered by diferric transferrin. PMID:6941310

  17. TDPAC studies of181Hf-labelled transferrin: Comparison between human and rat serum transferrin

    NASA Astrophysics Data System (ADS)

    Appel, H.; Duffield, J.; Taylor, D. M.; Then, G. M.; Thies, W.-G.

    1987-04-01

    A fast BaF2 TDPAC setup was used to study the binding of181Hf to serum transferrin. Two well-defined binding configurations were observed, which are characterized by high EFGs and large asymetry parameter values. The distribution between these configurations depends essentially on the pH of the serum. Small but significant differences between human and rat serum transferrin can be deduced from the electric quadrupole interaction (QI) parameters.

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

    PubMed

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

    2013-02-01

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

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

    PubMed Central

    Booth, A G; Wilson, M J

    1981-01-01

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

  20. Nonrandom distribution of iron in circulating human transferrin.

    PubMed

    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. PMID:3719094

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-11-01

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

  3. A variant of human transferrin with abnormal properties.

    PubMed Central

    Evans, R W; Williams, J; Moreton, K

    1982-01-01

    Screening of human serum samples by polyacrylamide-gel electrophoresis in the presence of 6 M-urea revealed an individual who is heterozygous for a variant transferrin. The variant transferrin is able to bind two atoms of iron, but the iron in the C-terminal binding site is bound abnormally, as judged by its spectral properties, and is dissociated from the protein on electrophoresis in the presence of 6 M-urea. The iron-free C-terminal domain of the variant protein is less stable than normal to thermal and urea denaturation. Structural changes in the variant protein have not yet been characterized. Images Fig. 1. Fig. 2. Fig. 6. Fig. 9. PMID:7082283

  4. Physical characteristics of human transferrin from small angle neutron scattering.

    PubMed Central

    Martel, P; Kim, S M; Powell, B M

    1980-01-01

    The technique of small angle neutron scattering has been used to determine the molecular shape, the volume, and the molecular weight of pooled human transferrin in an aqueous solution isotonic with blood. Analysis of the measurements assuming a spheroidal molecular shape indicates that an oblate spheroid with semi-axes of length 46.6 +/- 1.4, 46.6 +/- 1.4 and 15.8 +/- 3.8 A, and a molecular volume of (144 +/- 45) X 10(3) A3 is the best simple approximation to the shape of the transferrin molecule. The radius of gyration, Rg, determined from a Guinier plot is 30.25 +/- 0.49 A, in agreement with Rg calculated for the oblate spheroidal shape. The molecular weight is determined to be (75 +/- 5) X 10(3). The shape-independent molecular volume is found to be (98 +/- 10) X 10(3) A3. The difference in the two volumes suggests that transferrin is not a uniform spheroid but may have a more complex shape. PMID:7260293

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

    SciTech Connect

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

    2007-05-01

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

  6. Receptor-Mediated Recognition and Uptake of Iron from Human Transferrin by Staphylococcus aureus and Staphylococcus epidermidis

    PubMed Central

    Modun, Belinda; Evans, Robert W.; Joannou, Christopher L.; Williams, Paul

    1998-01-01

    Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea–polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but not Staphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of 125I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and

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

    PubMed

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

    2006-04-20

    Transferrin constitutes the major protein involved in the transport of iron from the sites of absorption to the sites of storage and utilization. Despite the high affinity of transferrin for iron, most bacterial pathogens, such as the human restricted Neisseria meningitidis, have developed iron acquisition mechanisms. Several animal models of bacterial infection that include the exogenous supply of human transferrin have been implemented, and tests using transgenic mouse models are underway. Here we describe an ELISA sandwich procedure based on two monoclonal antibodies with negligible cross-reactivity to murine transferrin, to estimate human transferrin concentrations in mouse sera. The assay can detect as little as 10 ng/ml of human transferrin with coefficients of variation ranging from 1.6% to 4.4% (intra-assay) and 3.8% to 5% (inter-assay). The recovery values range from 90% to 110% in the assay working range (25-400 ng/ml). Human transferrin concentrations estimated in sera from 41 human transferrin transgenic mice ranged from 2 to 14 microg/ml. Further estimations of human transferrin levels in mouse sera of a previously described mouse model of N. meningitidis were also carried out. The intraperitoneal injection of 8 mg of human transferrin achieved a sustained value of human transferrin in mouse sera in the range of 1-2mg/ml over the first 24h, indicating that bacteria reaching the blood stream during this time would be exposed to levels of hTf found in normal human serum. PMID:16529768

  8. Transferrin receptors of human fibroblasts. Analysis of receptor properties and regulation.

    PubMed Central

    Ward, J H; Kushner, J P; Kaplan, J

    1982-01-01

    Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03 X 10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9 X 10(-2) X min-1. The dissociation constant (KD) was 5.1 X 10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9 X 10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding. PMID:6297460

  9. Occupancy of the iron binding sites of human transferrin.

    PubMed Central

    Huebers, H A; Josephson, B; Huebers, E; Csiba, E; Finch, C A

    1984-01-01

    The in vivo distribution of iron between the binding sites of transferrin was examined. Plasma was obtained from normal subjects under basal conditions and after in vitro and in vivo iron loading. Independent methods, including measurement of the transferrin profile after isoelectric focusing and cross immunoelectrophoresis, and determination of the iron content in the separated fractions were in agreement that there was a random distribution of iron on binding sites. This held true with in vitro loading, when iron was increased by intestinal absorption and with loading from the reticuloendothelial system. The data indicate that the distribution of apo-, monoferric, and diferric transferrins is predictable on the basis of the plasma transferrin saturation and negate the concept that iron loading of transferrin in vitro is a selective process with possible functional consequences in tissue iron delivery. PMID:6589596

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

    PubMed Central

    Galbraith, G M; Galbraith, R M

    1980-01-01

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

  11. Cleavage of Human Transferrin by Porphyromonas gingivalis Gingipains Promotes Growth and Formation of Hydroxyl Radicals

    PubMed Central

    Goulet, Véronique; Britigan, Bradley; Nakayama, Koji; Grenier, Daniel

    2004-01-01

    Porphyromonas gingivalis, a gram-negative anaerobic bacterium associated with active lesions of chronic periodontitis, produces several proteinases which are presumably involved in host colonization, perturbation of the immune system, and tissue destruction. The aims of this study were to investigate the degradation of human transferrin by gingipain cysteine proteinases of P. gingivalis and to demonstrate the production of toxic hydroxyl radicals (HO·) catalyzed by the iron-containing transferrin fragments generated or by release of iron itself. Analysis by polyacrylamide gel electrophoresis and Western immunoblotting showed that preparations of Arg- and Lys-gingipains of P. gingivalis cleave transferrin (iron-free and iron-saturated forms) into fragments of various sizes. Interestingly, gingival crevicular fluid samples from diseased periodontal sites but not samples from healthy periodontal sites contained fragments of transferrin. By using 55Fe-transferrin, it was found that degradation by P. gingivalis gingipains resulted in the production of free iron, as well as iron bound to lower-molecular-mass fragments. Subsequent to the degradation of transferrin, bacterial cells assimilated intracellularly the radiolabeled iron. Growth of P. gingivalis ATCC 33277, but not growth of an Arg-gingipain- and Lys-gingipain-deficient mutant, was possible in a chemically defined medium containing 30% iron-saturated transferrin as the only source of iron and peptides, suggesting that gingipains play a critical role in the acquisition of essential growth nutrients. Finally, the transferrin degradation products generated by Arg-gingipains A and B were capable of catalyzing the formation of HO·, as determined by a hypoxanthine/xanthine oxidase system and spin trapping-electron paramagnetic resonance spectrometry. Our study indicates that P. gingivalis gingipains degrade human transferrin, providing sources of iron and peptides. The iron-containing transferrin fragments or the

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

    PubMed

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

    2010-05-01

    Human serum transferrin (hTf) is the major iron-binding protein in human plasma, having a vital role in iron transport. Additionally, hTf has many other uses including antimicrobial functions and growth factor effects on mammalian cell proliferation and differentiation. The multitask nature of hTf makes it highly valuable for different therapeutic and commercial applications. However, the success of hTf in these applications is critically dependent on the availability of high-quality hTf in large amounts. In this study, we have developed plants as a novel platform for the production of recombinant (r)hTf. We show here that transgenic plants are an efficient system for rhTf production, with a maximum accumulation of 0.25% total soluble protein (TSP) (or up to 33.5 microg/g fresh leaf weight). Furthermore, plant-derived rhTf retains many of the biological activities synonymous with native hTf. In particular, rhTf reversibly binds iron in vitro, exhibits bacteriostatic activity, supports cell proliferation in serum-free medium and can be internalized into mammalian cells in vitro. The success of this study validates the future application of plant rhTf in a variety of fields. Of particular interest is the use of plant rhTf as a novel carrier for cell-specific or oral delivery of protein/peptide drugs for the treatment of human diseases such as diabetes.To demonstrate this hypothesis, we have additionally expressed an hTf fusion protein containing glucagon-like peptide 1 (GLP-1) or its derivative in plants. Here, we show that plant-derived hTf-GLP-1 fusion proteins retain the ability to be internalized by mammalian cells when added to culture medium in vitro. PMID:20432512

  13. Sulfonylurea Receptor 1 Expression in Human Cerebral Infarcts

    PubMed Central

    Mehta, Rupal I.; Ivanova, Svetlana; Tosun, Cigdem; Castellani, Rudy J.; Gerzanich, Volodymyr

    2013-01-01

    Abstract In animal models of stroke, sulfonylurea receptor 1 (Sur1), a member of the adenosine triphosphate binding cassette transporter gene family, is transcriptionally upregulated in neural and vascular cells in which it plays a leading role in edema formation and necrotic cell death. To date, expression of Sur1 in the brains of humans with cerebral infarcts has not been systematically evaluated. We examined Sur1 expression in postmortem specimens obtained from 13 patients within the first 31 days after focal infarcts, 5 patients with lacunar infarcts, and 6 normal control brains using immunohistochemistry. Elevated immunoreactivity for Sur1 was detected in all cases of focal infarcts, with 3 distinct temporal patterns of expression: 1) neurons and endothelium showed the greatest elevation during the first week, after which levels declined; 2) astrocytes and microglia/macrophages showed progressive increases during the first 31 days; and 3) neutrophils near the infarct showed prominent immunoreactivity that did not change over time. Upregulation of Sur1 was corroborated using in situ hybridization for Abcc8 mRNA. Sulfonylurea receptor 1 immunoreactivity in lacunar infarcts was less prominent and more sporadic than in nonlacunar infarcts. In conjunction with previous studies, these data suggest that Sur1 may be a promising treatment target in patients with acute cerebral infarction. PMID:23965746

  14. Sulfonylurea receptor 1 expression in human cerebral infarcts.

    PubMed

    Mehta, Rupal I; Ivanova, Svetlana; Tosun, Cigdem; Castellani, Rudy J; Gerzanich, Volodymyr; Simard, J Marc

    2013-09-01

    In animal models of stroke, sulfonylurea receptor 1 (Sur1), a member of the adenosine triphosphate binding cassette transporter gene family, is transcriptionally upregulated in neural and vascular cells in which it plays a leading role in edema formation and necrotic cell death. To date, expression of Sur1 in the brains of humans with cerebral infarcts has not been systematically evaluated. We examined Sur1 expression in postmortem specimens obtained from 13 patients within the first 31 days after focal infarcts, 5 patients with lacunar infarcts, and 6 normal control brains using immunohistochemistry. Elevated immunoreactivity for Sur1 was detected in all cases of focal infarcts, with 3 distinct temporal patterns of expression: 1) neurons and endothelium showed the greatest elevation during the first week, after which levels declined; 2) astrocytes and microglia/macrophages showed progressive increases during the first 31 days; and 3) neutrophils near the infarct showed prominent immunoreactivity that did not change over time. Upregulation of Sur1 was corroborated using in situ hybridization for Abcc8 mRNA. Sulfonylurea receptor 1 immunoreactivity in lacunar infarcts was less prominent and more sporadic than in nonlacunar infarcts. In conjunction with previous studies, these data suggest that Sur1 may be a promising treatment target in patients with acute cerebral infarction. PMID:23965746

  15. An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells

    PubMed Central

    Wilner, Samantha E; Wengerter, Brian; Maier, Keith; de Lourdes Borba Magalhães, Maria; Del Amo, David Soriano; Pai, Supriya; Opazo, Felipe; Rizzoli, Silvio O; Yan, Amy; Levy, Matthew

    2012-01-01

    The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery. PMID:23344001

  16. TDPAC studies of the metal-binding sites in serum transferrin: comparison between 181Hf-labeled human- and rat-serum transferrin.

    PubMed

    Appel, H; Duffield, J; Taylor, D M; Then, G M; Thies, W G

    1987-12-01

    The binding of hafnium to human serum transferrin was studied using the time differential perturbed angular correlation (TDPAC-) technique. The samples were prepared in vitro by adding 181Hf-NTA solution to human serum. Two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. Their relative intensities depend on the pH, salt- and hafnium-concentrations, and on the incubation time. The present data may be compared with the results of a previous rat serum study, where the hafnium binding to transferrin behaved rather similarly. Small but significant differences, however, can be deduced from the TDPAC-parameters for these human and rat transferrin species. For either binding configuration, the electric field gradient (EFG) is slightly higher in the case of rat transferrin. The most characteristic difference, however, concerns the asymmetry parameter eta 2 of the second binding configuration, which is about 10% smaller for rat serum transferrin. The TDPAC-technique might be used as a sensitive and reliable analytical method to study the metal-binding sites of different transferrin species. PMID:3437277

  17. TDPAC studies of the metal-binding sites in serum transferrin: comparison between /sup 181/Hf-labeled human- and rat-serum transferrin

    SciTech Connect

    Appel, H.; Duffield, J.; Taylor, D.M.; Then, G.M.; Thies, W.G.

    1987-12-01

    The binding of hafnium to human serum transferrin was studied using the time differential perturbed angular correlation (TDPAC-) technique. The samples were prepared in vitro by adding /sup 181/Hf-NTA solution to human serum. Two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. Their relative intensities depend on the pH, salt- and hafnium-concentrations, and on the incubation time. The present data may be compared with the results of a previous rat serum study, where the hafnium binding to transferrin behaved rather similarly. Small but significant differences, however, can be deduced from the TDPAC-parameters for these human and rat transferrin species. For either binding configuration, the electric field gradient (EFG) is slightly higher in the case of rat transferrin. The most characteristic difference, however, concerns the asymmetry parameter eta 2 of the second binding configuration, which is about 10% smaller for rat serum transferrin. The TDPAC-technique might be used as a sensitive and reliable analytical method to study the metal-binding sites of different transferrin species.

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

    ERIC Educational Resources Information Center

    Barnes, Grady; Frieden, Earl

    1982-01-01

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

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

    PubMed Central

    Young, S P; Garner, C

    1990-01-01

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

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

    SciTech Connect

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

    2011-12-31

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

  1. Human granulocyte/pollen-binding protein. Recognition and identification as transferrin.

    PubMed Central

    Sass-Kuhn, S P; Moqbel, R; Mackay, J A; Cromwell, O; Kay, A B

    1984-01-01

    Normal human serum was found to contain a heat-stable protein which promoted the binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]). GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1. By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was identified as transferrin. Monospecific antisera raised against either GPBP or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin gave lines of identity with these two antisera. The apparent heterogeneity in the molecular size and charge of GPBP observed during progressive purification was minimal when GPBP was saturated with ferric ions before the separation procedures. These experiments indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin which appears to be unrelated to iron transport and raises the possibility that transferrin might have a physiological role in the removal of certain organic matter. Images PMID:6690479

  2. Human Serum Transferrin Fibrils: Nanomineralisation in Bacteria and Destruction of Red Blood Cells

    PubMed Central

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

    2015-01-01

    Fibrils formed by human serum transferrin [(1–3 μm) apo-Tf, partially iron-saturated (Fe0.6-Tf) and holo-Tf (Fe2-Tf) forms], from dilute bicarbonate solutions, were deposited on formvar surfaces and studied by electron microscopy. We observed that possible bacterial contamination appears to give rise to long, pea-pod-like (PPL) structures for Fe2-Tf, attributable to the formation of polyhydroxybutyrate (PHB) storage granules, under the nutrient-limiting conditions used. These PPL structures contained periodic nanomineralisation sites susceptible to uranyl stain. Extended incubation of transferrin solutions (about four days) gave rise to extensive transferrin fibril structures. Optical microscopy and AFM studies showed that red blood cells (RBCs) readily adhere to these fibrils. Moreover, the fibrils appear to penetrate RBC membranes and to induce rapid cell destruction (within about 5 h). It is speculated that in situations in vivo where transferrin fibrils can form, such interactions might have adverse physiological consequences, and further studies could aid the understanding of related pathological events. PMID:25476866

  3. X-ray small angle scattering of the human transferrin protein aggregates. A fractal study.

    PubMed Central

    Castellano, A C; Barteri, M; Bianconi, A; Borghi, E; Cassiano, L; Castagnola, M; Della Longa, S

    1993-01-01

    X-ray small angle scattering experiments, using a pin hole SAXS camera with Synchrotron radiation source, have been performed to study the conformational changes of lyophilized samples of Apo-, Mono-, and Diferric- human transferrin. We report the experimental evidence that the analysis of the scattered intensity through the fractal theory may give information on the particle size and its variation upon iron binding. PMID:8457675

  4. Effect of synthetic carrier ampholytes on saturation of human serum transferrin.

    PubMed Central

    Oratore, A; D'Alessandro, A M; D'Andrea, G

    1989-01-01

    We have investigated the effect in solution of synthetic carrier ampholytes on the saturation of human serum transferrin. By spectrophotometric titrations of human serum transferrin with various Fe3+-carrier ampholyte solutions, we demonstrated that under these conditions carrier ampholytes behave as typical chelators, their binding curves being very similar to that obtained with disodium nitrilotriacetate. On performing titration experiments at three different pH values, carrier ampholytes act like nitrilotriacetate at pH 7.5, but the former are more effective iron donors at pH 8.4 and worse iron donors at pH 5.2. Spectrophotometric titrations of isolated C-terminal and N-terminal fragments obtained from human serum transferrin by thermolysin cleavage show no differences between them, and no differences with respect to the whole protein except that they contain half the number of binding sites. In order to determine a site-specificity of iron in the presence of ampholytes, the classical urea/polyacrylamide-gel-electrophoresis technique was adopted. Under saturating conditions carrier ampholyte solutions act mostly on the C-terminal site, whereas desaturating agents remove iron preferentially from the N-terminal site. Our findings support the hypothesis that Ampholine may chelate Fe3+ as well as many other compounds. Images Fig. 3. PMID:2730592

  5. Killing of human tumor cells in culture with adriamycin conjugates of human transferrin

    SciTech Connect

    Yeh, C.J.; Faulk, W.P.

    1984-07-01

    Receptors for human transferrin (Trf) in high density are found on reticulocytes and syncytiotrophoblast, but most unstimulated, normal adult cells do not bind Trf. In contrast, leukemia and breast adenocarcinoma cells have been shown to manifest Trf receptors, raising the possibility that these receptors might be employed to bind cytotoxic Trf conjugates. Trf was conjugated with adriamycin (Adr) and it was shown that the conjugates are bound by Trf receptors on plasma membranes of Daudi and HL-60 cells, following which Adr is identified in the nuclei of these cells. The biological effect of Adr is quantitated by the inhibition of tritiated thymidine uptake, and subsequent cell death is measured by trypan blue exclusion. The killing correlates directly with both the time of exposure and the amount of conjugate employed. These results suggest that such cytotoxic Trf conjugates hold promise for selective in vivo killing of some malignant cells.

  6. Binding and release of iron by gel-encapsulated human transferrin: Evidence for a conformational search

    PubMed Central

    Navati, Mahantesh S.; Samuni, Uri; Aisen, Philip; Friedman, Joel M.

    2003-01-01

    Human transferrin is a single-chain bilobal protein with each of the two similar but not identical lobes in turn composed of two domains. Each lobe may assume one of two stable structural conformations, open or closed, determined by a rigid rotation of the domains with respect to each other. In solution, the transformation of a lobe between open and closed conformations is associated with the release or binding of an Fe(III) ion. The results of the present study indicate that encapsulation of transferrin within a porous sol-gel matrix allows for a dramatic expansion, to days or weeks, of this interconversion time period, thus providing an opportunity to probe heretofore inaccessible transient intermediates. Sol-gel-encapsulated iron-free transferrin samples are prepared by using two protocols. In the first protocol, the equilibrium form of apotransferrin is encapsulated in the sol-gel matrix, whereas in the second protocol holotransferrin is first encapsulated and then iron is removed from the protein. Results of kinetic and spectroscopic studies allow for distinguishing between two models for iron binding. In the first, iron is assumed to bind to amino acid ligands of one domain, inducing a rigid rotation of the second domain to effect closure of the interdomain cleft. In the second, iron undertakes a conformational search among the thermally accessible states of the lobe, “choosing” the state which most nearly approximates the stable closed state when iron is bound. Our experimental results support the second mechanism. PMID:12486226

  7. Equilibrium studies on the binding of cadmium(II) to human serum transferrin

    SciTech Connect

    Harris, W.R.; Madsen, L.J.

    1988-01-12

    The binding of cadmium(II) to human serum transferrin in 0.01 M N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid with 5 mM bicarbonate at 25/sup 0/C has been evaluated by difference ultraviolet spectroscopy. Equilibrium constants were determined by competition versus three different low molecular weight chelating agents: nitrilotriacetic acid, ethylenediamine-N,N'-diacetic acid, and triethylenetetramine. Conditional equilibrium constants for the sequential binding of two cadmium ions to transferrin under the stated experimental conditions are log K/sub 1/ = 5.95 +/- 0.10 and log K/sub 2/ = 4.86 +/- 0.13. A linear free energy relationship for the complexation of cadmium and zinc has been prepared by using equilibrium data on 243 complexes of these metal ions with low molecular weight ligands. The transferrin binding constants for cadmium and zinc are in good agreement with this linear free energy relationship. This indicates that the larger size of the cadmium(II) ion does not significantly hinder its binding to the protein.

  8. In vitro interaction between ceruloplasmin and human serum transferrin.

    PubMed

    Ha-Duong, Nguyêt-Thanh; Eid, Chantal; Hémadi, Miryana; El Hage Chahine, Jean-Michel

    2010-12-01

    The thermodynamics of the interactions of serum apotransferrin (T) and holotransferrin (TFe(2)) with ceruloplasmin (Cp), as well as those of human lactoferrin (Lf), were assessed by fluorescence emission spectroscopy. Cp interacts with two Lf molecules. The first interaction depends on pH and μ, whereas the second does not. Dissociation constants were as follows: K(11Lf) = 1.5 ± 0.2 μM, and K(12Lf) = 11 ± 2 μM. Two slightly different interactions of T or TFe(2) with Cp are detected for the first time. They are both independent of pH and μ and occur with 1:1 stoichiometry: K(1T) = 19 ± 7 μM, and K(1TFe2) = 12 ± 4 μM. These results can improve our understanding of the probable process of the transfer of iron from Cp to T in iron and copper transport and homeostasis. PMID:21049900

  9. Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes

    PubMed Central

    Kawakami, Yuki; Matsuo, Kosuke; Murata, Minako; Yudoh, Kazuo; Nakamura, Hiroshi; Shimizu, Hiroyuki; Beppu, Moroe; Inaba, Yutaka; Saito, Tomoyuki; Kato, Tomohiro; Masuko, Kayo

    2012-01-01

    Background. Besides its involvement in the cardiovascular system, the renin-angiotensin-aldosterone (RAS) system has also been suggested to play an important role in inflammation. To explore the role of this system in cartilage damage in arthritis, we investigated the expression of angiotensin II receptors in chondrocytes. Methods. Articular cartilage was obtained from patients with osteoarthritis, rheumatoid arthritis, and traumatic fractures who were undergoing arthroplasty. Chondrocytes were isolated and cultured in vitro with or without interleukin (IL-1). The expression of angiotensin II receptor types 1 (AT1R) and 2 (AT2R) mRNA by the chondrocytes was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). AT1R expression in cartilage tissue was analyzed using immunohistochemistry. The effect of IL-1 on AT1R/AT2R expression in the chondrocytes was analyzed by quantitative PCR and flow cytometry. Results. Chondrocytes from all patient types expressed AT1R/AT2R mRNA, though considerable variation was found between samples. Immunohistochemical analysis confirmed AT1R expression at the protein level. Stimulation with IL-1 enhanced the expression of AT1R/AT2R mRNA in OA and RA chondrocytes. Conclusions. Human articular chondrocytes, at least partially, express angiotensin II receptors, and IL-1 stimulation induced AT1R/AT2R mRNA expression significantly. PMID:23346400

  10. Kallikrein Promotes Inflammation in Human Dental Pulp Cells Via Protease-Activated Receptor-1.

    PubMed

    Hayama, Tomomi; Kamio, Naoto; Okabe, Tatsu; Muromachi, Koichiro; Matsushima, Kiyoshi

    2016-07-01

    Plasma kallikrein (KLKB1), a serine protease, cleaves high-molecular weight kininogen to produce bradykinin, a potent vasodilator and pro-inflammatory peptide. In addition, KLKB1 activates plasminogen and other leukocyte and blood coagulation factors and processes pro-enkephalin, prorenin, and C3. KLKB1 has also been shown to cleave protease-activated receptors in vascular smooth muscle cells to regulate the expression of epidermal growth factor receptor. In this study, we investigated KLKB1-dependent inflammation and activation of protease-activated receptor-1 in human dental pulp cells. These cells responded to KLKB1 stimulation by increasing intracellular Ca(2+) , upregulating cyclooxygenase-2, and secreting prostaglandin E2 . Remarkably, SCH79797, an antagonist of protease-activated receptor-1, blocked these effects. Thus, these data indicate that KLKB1 induces inflammatory reactions in human dental tissues via protease-activated receptor 1. J. Cell. Biochem. 117: 1522-1528, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566265

  11. In-111 chelate conjugates of human transferrin (HTr) and mouse monoclonal anti human transferrin receptor antibody (. cap alpha. HTrR MoAb) for tumor imaging

    SciTech Connect

    Goodwin, D.A.; Meares, C.F.; Diamanti, C.I.; McCall, M.; McTigue, M.; Torti, F.; Martin, B.

    1984-01-01

    At least one of the major pathways of uptake of the commonly used tumor scanning agent Ga-67 is via the transferrin receptor. This suggested the use of stably radio-labeled HTr, and ..cap alpha..HTrR MoAb for tumor imaging in humans. HTr and mouse ..cap alpha..HTrR MoAb were alkylated with 1-(parabromacetamidobenzyl)-EDTA. The mM Alkylproteins, approx. =1 chelate/molecule were labeled with 1-3 mCi In-111 citrate pH/sub 5/ (Sp Act approx. = 100-300 Ci/m mole). Images were made 24 hours after 1 mCi IV and in some patients blood levels, urine excretion and digitized whole body scans were obtained at 1, 24,48 and 96 hours post injection. Ten patients with biopsy proven prostate cancer were studied with In-111 HTr, and four with In-111 ..cap alpha.. HTrR MoAb; all had positive mets on bone scan. In-111 HTr persisted in the circulation with a T1/2 of approx. = four days, approx. = 5%/day being excreted in the urine, to a total of approx. = 60% in 21 days. Nine of ten scans were false negative due to the high blood background. In-111 ..cap alpha..HTrR disappeared rapidly from the blood; with most in the bone marrow at 24 hours. ROI analysis of three patients showed whole body 94% at 24 hours, 89% at 48 hours, and 82% at 96 hours (T1/2 = 10.7 days); liver 19% at 1 hour, 25% at 24 hours, and 21% at 96 hours; spleen 3% at 1 hour, 8% at 24 hours, 7.3% at 48 hours, and 3% at 96 hours. The high bone marrow background allowed only a few of the bone mets seen as bone scan to be visualized. Other tumor types not located in bone may be more easily seen.

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

    PubMed Central

    Galbraith, R M; Galbraith, G M

    1981-01-01

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

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

    PubMed

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

    2012-11-01

    The Fe(3+) binding protein human serum transferrin (hTF) is well known for its role in cellular iron delivery via the transferrin receptor (TFR). A new application is the use of hTF as a therapy and targeted drug delivery system for a number of diseases. Recently, production of hTF in plants has been reported; such systems provide a relatively inexpensive, animal-free (eliminating potential contamination by animal pathogens) method to produce large amounts of recombinant proteins for such biopharmaceutical applications. Specifically, the production of Optiferrin (hTF produced in rice, Oryza sativa, from InVitria) has been shown to yield large amounts of functional protein for use in culture medium for cellular iron delivery to promote growth. In the present work we describe further purification (by gel filtration) and characterization of hTF produced in rice (purified Optiferrin) to determine its suitability in biopharmaceutical applications. The spectral, mass spectrometric, urea gel and kinetic analysis shows that purified Optiferrin is similar to recombinant nonglycosylated N-His tagged hTF expressed by baby hamster kidney cells and/or serum derived glycosylated hTF. Additionally, in a competitive immunoassay, iron-loaded Optiferrin is equivalent to iron-loaded N-His hTF in its ability to bind to the soluble portion of the TFR immobilized in an assay plate. As an essential requirement for any functional hTF, both lobes of purified Optiferrin bind Fe(3+) tightly yet reversibly. Although previously shown to be capable of delivering Fe(3+) to cells, the kinetics of iron release from iron-loaded Optiferrin™/sTFR and iron-loaded N-His hTF/sTFR complexes differ somewhat. We conclude that the purified Optiferrin might be suitable for consideration in biopharmaceutical applications. PMID:23010327

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

    PubMed Central

    Byrne, Shaina L.

    2009-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  16. Inhibition of gallium-67 uptake in melanoma by an anti-human transferrin receptor monoclonal antibody

    SciTech Connect

    Chan, S.M.; Hoffer, P.B.; Maric, N.; Duray, P.

    1987-08-01

    The effect of an anti-human transferrin receptor (anti-TFR) monoclonal antibody (MoAb), designated B3/25, and an anti-melanoma antibody, designated 96.5, on the uptake of gallium-67 (/sup 67/Ga) by tumor was studied. Three groups of six athymic mice bearing a human melanoma were injected via tail vein with (a) 0.55 mg human serum albumin (HSA) (control group), (b) 0.5 mg MoAb B3/25 + 0.55 mg HSA, and (c) 0.5 mg MoAb 96.5 + 0.55 mg HSA, respectively. Twenty-four hours later, each mouse was given an intravenous dose of 5 microCi (/sup 67/Ga) citrate. Biodistribution of activity (percent injected dose per gram) determined 48 hr after injection of /sup 67/Ga showed a 75% decrease in tumor uptake in the group of mice that received B3/25 (anti-TFR MoAb) compared with the control group. In contrast, MoAb 96.5 did not show any effect on melanoma uptake of /sup 67/Ga. Histologic findings suggest that the decreased uptake was not due to cellular damage resulting from binding of B3/25 to TFR. The results of this study strongly suggest the involvement of TFR in the in vivo tumor uptake of /sup 67/Ga.

  17. Failure to extinguish fear and genetic variability in the human cannabinoid receptor 1.

    PubMed

    Heitland, I; Klumpers, F; Oosting, R S; Evers, D J J; Leon Kenemans, J; Baas, J M P

    2012-01-01

    Failure to extinguish fear can lead to persevering anxiety and has been postulated as an important mechanism in the pathogenesis of human anxiety disorders. In animals, it is well documented that the endogenous cannabinoid system has a pivotal role in the successful extinction of fear, most importantly through the cannabinoid receptor 1. However, no human studies have reported a translation of this preclinical evidence yet. Healthy medication-free human subjects (N=150) underwent a fear conditioning and extinction procedure in a virtual reality environment. Fear potentiation of the eyeblink startle reflex was measured to assess fear-conditioned responding, and subjective fear ratings were collected. Participants were genotyped for two polymorphisms located within the promoter region (rs2180619) and the coding region (rs1049353) of cannabinoid receptor 1. As predicted from the preclinical literature, acquisition and expression of conditioned fear did not differ between genotypes. Crucially, whereas both homozygote (G/G, N=23) and heterozygote (A/G, N=68) G-allele carriers of rs2180619 displayed robust extinction of fear, extinction of fear-potentiated startle was absent in A/A homozygotes (N=51). Additionally, this resistance to extinguish fear left A/A carriers of rs2180619 with significantly higher levels of fear-potentiated startle at the end of the extinction training. No effects of rs1049353 genotype were observed regarding fear acquisition and extinction. These results suggest for the first time involvement of the human endocannabinoid system in fear extinction. Implications are that genetic variability in this system may underlie individual differences in anxiety, rendering cannabinoid receptor 1 a potential target for novel pharmacological treatments of anxiety disorders. PMID:23010766

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

    PubMed

    Kanodia, J S; Gadkar, K; Bumbaca, D; Zhang, Y; Tong, R K; Luk, W; Hoyte, K; Lu, Y; Wildsmith, K R; Couch, J A; Watts, R J; Dennis, M S; Ernst, J A; Scearce-Levie, K; Atwal, J K; Ramanujan, S; Joseph, S

    2016-05-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. The distribution of iron between the metal-binding sites of transferrin human serum.

    PubMed

    Williams, J; Moreton, K

    1980-02-01

    The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction. PMID:7396826

  1. The distribution of iron between the metal-binding sites of transferrin human serum.

    PubMed Central

    Williams, J; Moreton, K

    1980-01-01

    The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction. Images Fig. 1. PMID:7396826

  2. The transmembrane segment of the human transferrin receptor functions as a signal peptide.

    PubMed Central

    Zerial, M; Melancon, P; Schneider, C; Garoff, H

    1986-01-01

    The human transferrin receptor (TR) is a protein comprising 760 amino acid residues that spans the membrane once with its N terminus towards the cytoplasm. It is synthesized without a cleavable signal peptide. We have tested whether the signal responsible for its membrane insertion is present within its transmembrane peptide using a combined recombinant DNA/in vitro translation approach. The complete TR coding region was first reconstructed from overlapping TR cDNA clones and then engineered into an SP6-based transcription vector. In vitro transcription and subsequent translation in the presence of rough microsomes yielded TR molecules that were glycosylated and correctly inserted into the membrane. Two kinds of experiments demonstrated that the spanning region of the TR polypeptide contained the signal for translocation across the membrane of the rough endoplasmic reticulum. First, we deleted the spanning region of TR and showed that this deletion mutant could not be inserted. Second, we showed that two cytoplasmic proteins (the mouse dihydrofolate reductase and the chimpanzee alpha-globin) could be inserted into the microsomal membrane in the expected orientation when the TR transmembrane segment was added to their N termini. Thus, the spanning peptide was shown to be both necessary and sufficient for chain translocation. Further analyses demonstrated that the translocation event was dependent on the signal recognition particle. Images Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3017701

  3. Fungistatic mechanism of human transferrin for Rhizopus oryzae and Trichophyton mentagrophytes: alternative to simple iron deprivation.

    PubMed Central

    Artis, W M; Patrusky, E; Rastinejad, F; Duncan, R L

    1983-01-01

    Human serum, human transferrin (TF), and the iron chelator 1,10-phenanthroline (OP) produce iron-reversible fungistatic activity which has been attributed to simple iron deprivation. In this study, the influence of the size of the inoculum on the inhibitory activity of serum, TF, and OP prepared with the same iron-binding capacity (2.5 micrograms/ml) for Rhizopus oryzae and Trichophyton mentagrophytes was examined. Inhibition was monitored in liquid microcultures maintained at 37 degrees C and pH 7.4 to 7.5 by measuring the change in absorbance density. Increasing the number of spores in the inoculum disrupted the fungistatic activity of serum and TF, but not that of OP. The dilution at which OP lost fungistatic activity was not affected by the number of spores in the inoculum and was the same for both fungi. The dilution at which TF and serum lost fungistatic activity was dependent upon both the quantity of the inoculum and the species of fungus. The number of viable spores, rather than the total number of spores in the inoculum, was determined to be important in overcoming the inhibition of fungal growth by serum and TF. The fungistatic activity of serum and TF could be diminished by the preexposure of the serum to viable but nongrowing spores. Direct and indirect fluorescence studies indicated that both T. mentagrophytes and R. oryzae absorbed TF. Glucose uptake by R. oryzae was inhibited by a 4-h exposure to 5.0 to 0.15 mg of apotransferrin per ml. These results suggest that the fungistatic activity of TF for R. oryzae and T. mentagrophytes may not be attributable to simple iron deprivation and raise the possibility of a requirement for a direct interaction. Images PMID:6885162

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

    PubMed Central

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

    2012-01-01

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

  5. Separation of tryptophan-derivative enantiomers with iron-free human serum transferrin by capillary zone electrophoresis.

    PubMed

    Kilár, F; Fanali, S

    1995-08-01

    Enantiomers can be separated by using human serum transferrin as a chiral phase. With the help of the native protein we were able to separate enantiomers with high efficiency, using a low ionic strength 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6, in capillary zone electrophoresis. Tryptophan methyl, ethyl and butyl ester enantiomers-moving towards the cathode at pH 6-were resolved by passing through an iron-free transferrin zone in coated capillaries. Since the isoelectric point of the iron-free transferrin is a little higher than 6, the protein zone is either not moving in the experiment or is slowly moving towards the anode. Under the simplest experimental conditions the highest resolution was obtained for the butyl ester enantiomers and the lowest for the methyl ester ones. By changing the experimental conditions, however, this order could be reversed. The results indicate that the lengths of the alkyl chains in the enantiomers have a significant effect on the resolution, i.e., on the interaction between the protein and the separands. PMID:8529623

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

    SciTech Connect

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

    2012-03-28

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  9. Antagonism of human formyl peptide receptor 1 with natural compounds and their synthetic derivatives.

    PubMed

    Schepetkin, Igor A; Khlebnikov, Andrei I; Kirpotina, Liliya N; Quinn, Mark T

    2016-08-01

    Formyl peptide receptor 1 (FPR1) regulates a wide variety of neutrophil functional responses and plays an important role in inflammation and the pathogenesis of various diseases. To date, a variety of natural and synthetic molecules have been identified as FPR1 ligands. Here, we review current knowledge on natural products and natural product-inspired small molecules reported to antagonize and/or inhibit the FPR1-mediated responses. Based on this literature, additional screening of selected commercially available natural compounds for their ability to inhibit fMLF-induced Ca(2+) mobilization in human neutrophils and FPR1 transfected HL-60 cells, and pharmacophore modeling, natural products with potential as FPR1 antagonists are considered and discussed in this review. The identification and characterization of natural products that antagonize FPR1 activity may have potential for the development of novel therapeutics to limit or alter the outcome of inflammatory processes. PMID:26382576

  10. Immune responses in humans and animals to meningococcal transferrin-binding proteins: implications for vaccine design.

    PubMed Central

    Ala'Aldeen, D A; Stevenson, P; Griffiths, E; Gorringe, A R; Irons, L I; Robinson, A; Hyde, S; Borriello, S P

    1994-01-01

    The results reported here show that the two meningococcal transferrin-binding proteins (TBP1 and TBP2) generate different immune responses in different host species and that there is variation in response dependent on the method of antigen preparation and possibly the route of administration. Mice immunized with either whole cells of Neisseria meningitidis SD (B:15:P1.16) or the isolated TBP1-TBP2 complex from the same strain produced antisera which, when tested against a representative panel of meningococcal isolates by Western blotting (immunoblotting), recognized some but not all heterologous TBP2 molecules. In contrast, rabbit antisera raised to the same preparations were cross-reactive with almost all the TBP2 molecules. The immune response to TBP1 was also host species dependent. Western blot analysis with denatured TBP1 failed to detect antibodies in antisera raised in mice to whole cells or in a rabbit to the TBP1-TBP2 complex but detected broadly cross-reactive antibodies in mouse anti-TBP1-TBP2 complex sera and strain-specific antibodies in rabbit anti-whole-cell serum. Human convalescent-phase sera obtained from five patients infected with meningococci of different serogroups and serotypes contained fully cross-reactive antibodies to TBP2 but no anti-TBP1 antibodies, when examined on Western blots. However, on dot immunoblots, the same patients' sera, as well as the mouse anti-whole cell and the rabbit anti-TBP1-TBP2 complex sera, reacted with purified biologically active TBP1 of strain SD. This indicates that native TBP1, a protein which loses its biological and some of its immunological activities when denatured, is immunogenic and that humans generate cross-reactive antibodies to native epitopes. These observations have important implications for assessing the vaccine potential of TBPs and other meningococcal antigens. Conclusions regarding the usefulness of TBPs as candidate components of meningococcal serogroup B vaccines based on results from

  11. Genetic variability in the human cannabinoid receptor 1 is associated with resting state EEG theta power in humans.

    PubMed

    Heitland, I; Kenemans, J L; Böcker, K B E; Baas, J M P

    2014-11-01

    It has long been postulated that exogenous cannabinoids have a profound effect on human cognitive functioning. These cannabinoid effects are thought to depend, at least in parts, on alterations of phase-locking of local field potential neuronal firing. The latter can be measured as activity in the theta frequency band (4-7Hz) by electroencephalogram. Theta oscillations are supposed to serve as a mechanism in neural representations of behaviorally relevant information. However, it remains unknown whether variability in endogenous cannabinoid activity is involved in theta rhythms and therefore, may serve as an individual differences index of human cognitive functioning. To clarify this issue, we recorded resting state EEG activity in 164 healthy human subjects and extracted EEG power across frequency bands (δ, θ, α, and β). To assess variability in the endocannabinoid system, two genetic polymorphisms (rs1049353, rs2180619) within the cannabinoid receptor 1 (CB1) were determined in all participants. As expected, we observed significant effects of rs1049353 on EEG power in the theta band at frontal, central and parietal electrode regions. Crucially, these effects were specific for the theta band, with no effects on activity in the other frequency bands. Rs2180619 showed no significant associations with theta power after Bonferroni correction. Taken together, we provide novel evidence in humans showing that genetic variability in the cannabinoid receptor 1 is associated with resting state EEG power in the theta frequency band. This extends prior findings of exogenous cannabinoid effects on theta power to the endogenous cannabinoid system. PMID:25116250

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

    PubMed

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

    2016-06-01

    Organophosphorus agents (OPs) like sarin, VX, or soman could inhibit acetylcholinesterase activity and cause poisoning. OPs could bind many proteins, such as butyrylcholinesterase and albumin, and the adducts formed could identify the exposure. In this paper, we studied human transferrin, which was one of the proteins that could be labeled by OPs. Pure human transferrin was incubated with an overdose of organophosphorus agents, including sarin, soman, VX, tabun, cyclosarin, ethyl tabun, and propyl tabun, and then additional OPs was removed through dialysis. Trypsin was used to cleave the OP-treated proteins and Q Exactive liquid chromatography tandem mass spectrometry (Q Exactive LC-MS/MS) was used to identify them. The present study set out to accomplish two goals. The first goal was to find a good method for identifying multiple binding sites on a given protein through Q Exactive LC-MS/MS. The second goal was to investigate the labeled peptides when transferrin was incubated with a numerous molar excess of OPs. Results showed that tyrosine, lysine, and serine formed covalent bonds with OPs. Twenty OP-labeled sites were found: ten tyrosine sites (including two reported sites), seven lysine sites, and three serine sites. Characteristic fragments for labeled-tyrosine and labeled-lysine adducts were summarized in detail. In conclusion, the method by Q Exactive LC-MS/MS using in this present work is a good way to diagnose exposure to OPs accurately when the binding sites of OPs are uncertain. Novel modified peptides and the characteristic ions found in this work could help investigators assess exposure to OPs. PMID:27128859

  13. Roles of transferrin receptors in erythropoiesis.

    PubMed

    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. PMID:27498743

  14. Genetic Polymorphisms Affect Mouse and Human Trace Amine-Associated Receptor 1 Function

    PubMed Central

    Shi, Xiao; Walter, Nicole A. R.; Harkness, John H.; Neve, Kim A.; Williams, Robert W.; Lu, Lu; Belknap, John K.; Eshleman, Amy J.; Phillips, Tamara J.; Janowsky, Aaron

    2016-01-01

    Methamphetamine (MA) and neurotransmitter precursors and metabolites such as tyramine, octopamine, and β-phenethylamine stimulate the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 has been implicated in human conditions including obesity, schizophrenia, depression, fibromyalgia, migraine, and addiction. Additionally TAAR1 is expressed on lymphocytes and astrocytes involved in inflammation and response to infection. In brain, TAAR1 stimulation reduces synaptic dopamine availability and alters glutamatergic function. TAAR1 is also expressed at low levels in heart, and may regulate cardiovascular tone. Taar1 knockout mice orally self-administer more MA than wild type and are insensitive to its aversive effects. DBA/2J (D2) mice express a non-synonymous single nucleotide polymorphism (SNP) in Taar1 that does not respond to MA, and D2 mice are predisposed to high MA intake, compared to C57BL/6 (B6) mice. Here we demonstrate that endogenous agonists stimulate the recombinant B6 mouse TAAR1, but do not activate the D2 mouse receptor. Progeny of the B6XD2 (BxD) family of recombinant inbred (RI) strains have been used to characterize the genetic etiology of diseases, but contrary to expectations, BXDs derived 30–40 years ago express only the functional B6 Taar1 allele whereas some more recently derived BXD RI strains express the D2 allele. Data indicate that the D2 mutation arose subsequent to derivation of the original RIs. Finally, we demonstrate that SNPs in human TAAR1 alter its function, resulting in expressed, but functional, sub-functional and non-functional receptors. Our findings are important for identifying a predisposition to human diseases, as well as for developing personalized treatment options. PMID:27031617

  15. Genetic Polymorphisms Affect Mouse and Human Trace Amine-Associated Receptor 1 Function.

    PubMed

    Shi, Xiao; Walter, Nicole A R; Harkness, John H; Neve, Kim A; Williams, Robert W; Lu, Lu; Belknap, John K; Eshleman, Amy J; Phillips, Tamara J; Janowsky, Aaron

    2016-01-01

    Methamphetamine (MA) and neurotransmitter precursors and metabolites such as tyramine, octopamine, and β-phenethylamine stimulate the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 has been implicated in human conditions including obesity, schizophrenia, depression, fibromyalgia, migraine, and addiction. Additionally TAAR1 is expressed on lymphocytes and astrocytes involved in inflammation and response to infection. In brain, TAAR1 stimulation reduces synaptic dopamine availability and alters glutamatergic function. TAAR1 is also expressed at low levels in heart, and may regulate cardiovascular tone. Taar1 knockout mice orally self-administer more MA than wild type and are insensitive to its aversive effects. DBA/2J (D2) mice express a non-synonymous single nucleotide polymorphism (SNP) in Taar1 that does not respond to MA, and D2 mice are predisposed to high MA intake, compared to C57BL/6 (B6) mice. Here we demonstrate that endogenous agonists stimulate the recombinant B6 mouse TAAR1, but do not activate the D2 mouse receptor. Progeny of the B6XD2 (BxD) family of recombinant inbred (RI) strains have been used to characterize the genetic etiology of diseases, but contrary to expectations, BXDs derived 30-40 years ago express only the functional B6 Taar1 allele whereas some more recently derived BXD RI strains express the D2 allele. Data indicate that the D2 mutation arose subsequent to derivation of the original RIs. Finally, we demonstrate that SNPs in human TAAR1 alter its function, resulting in expressed, but functional, sub-functional and non-functional receptors. Our findings are important for identifying a predisposition to human diseases, as well as for developing personalized treatment options. PMID:27031617

  16. Shear stress reduces protease activated receptor-1 expression in human endothelial cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

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

    SciTech Connect

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

    2009-05-27

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

  18. Structure of the membrane proximal oxidoreductase domain of human Steap3, the dominant ferrireductase of the erythroid transferrin cycle

    PubMed Central

    Sendamarai, Anoop K.; Ohgami, Robert S.; Fleming, Mark D.; Lawrence, C. Martin

    2008-01-01

    The daily production of 200 billion erythrocytes requires 20 mg of iron, accounting for nearly 80% of the iron demand in humans. Thus, erythroid precursor cells possess an efficient mechanism for iron uptake in which iron loaded transferrin (Tf) binds to the transferrin receptor (TfR) at the cell surface. The Tf:TfR complex then enters the endosome via receptor-mediated endocytosis. Upon endosomal acidification, iron is released from Tf, reduced to Fe2+ by Steap3, and transported across the endosomal membrane by divalent metal iron transporter 1. Steap3, the major ferrireductase in erythrocyte endosomes, is a member of a unique family of reductases. Steap3 is comprised of an N-terminal cytosolic oxidoreductase domain and a C-terminal heme-containing transmembrane domain. Cytosolic NADPH and a flavin are predicted cofactors, but the NADPH/flavin binding domain differs significantly from those in other eukaryotic reductases. Instead, Steap3 shows remarkable, although limited homology to FNO, an archaeal oxidoreductase. We have determined the crystal structure of the human Steap3 oxidoreductase domain in the absence and presence of NADPH. The structure reveals an FNO-like domain with an unexpected dimer interface and substrate binding sites that are well positioned to direct electron transfer from the cytosol to a heme moiety predicted to be fixed within the transmembrane domain. Here, we discuss possible gating mechanisms for electron transfer across the endosomal membrane. PMID:18495927

  19. Expression and characterization of recombinant human eosinophil-derived neurotoxin and eosinophil-derived neurotoxin-anti-transferrin receptor sFv.

    PubMed

    Newton, D L; Nicholls, P J; Rybak, S M; Youle, R J

    1994-10-28

    The gene for the human recombinant eosinophil-derived neurotoxin (rEDN) was synthesized and fused to the gene encoding a single chain antibody (sFv) to the human transferrin receptor (EDNsFv). Both rEDN and EDNsFv were expressed as insoluble proteins in inclusion bodies in Escherichia coli BL21(DE3). Following denaturation and renaturation, EDN and EDNsFv were partially purified by chromatography on heparin-Sepharose. Final purification of EDN was achieved by Sephadex G-100, whereas EDNsFv which contained a 6-histidyl residue carboxyl terminus was highly purified using the metal chelate resin, Ni(2+)-nitriloacetic acid. Whereas the recombinant EDN had ribonuclease activity that was similar to the native protein, the fusion protein had enzymatic activity that was 6-13% that of native EDN. The fusion protein was able to bind to the human transferrin receptor. In contrast to rEDN that had no inherent cytotoxicity to human tumor cells, the EDNsFv fusion protein was cytotoxic to human leukemia cells that express the human transferrin receptor with an IC50, 0.2-1 nM. At 1.3 nM EDNsFv, no cytotoxicity was observed on cells that lack the human transferrin receptor. Free antibody to the human transferrin receptor, E6, inhibited the cytotoxicity of the EDNsFv. Human enzymes may be engineered to acquire cytotoxic properties by fusing them to antibodies. Thus, they may be candidates for the construction of immunofusion proteins that may be less immunogenic than immunotoxins containing bacterial- or plant-derived toxin moieties. PMID:7929408

  20. New DNA polymorphisms define ethnically distinct haplotypes in the human transferrin receptor gene.

    PubMed

    Van Landeghem, G F; Beckman, L E; Sikström, C; Saha, N; Kucinskas, V; Beckman, L

    1998-01-01

    In a study of transferrin receptor (TFR) polymorphism in different ethnic groups using PCR and restriction cleavage we found a new Hin6I polymorphism in intron 7 and confirmed a tentative BanI polymorphism in exon 4 reported by Evans and Kemp [Gene 1997;199:123-131]. In all ethnic groups there was a complete and highly significant (p < 10(-10)) linkage disequilibrium where all BanI 1 alleles were linked to Hin6I 1 alleles. Furthermore in the European populations, but not in the Chinese, there was a close correlation between the three BanI-Hin6I haplotypes and the alleles of a previously described three-allelic RsaI polymorphism in the TFR gene studied by Southern blotting. There were distinct ethnic differences in TFR allele and haplotype frequencies. Thus the Saamis were significantly different from the other European ethnic groups, and the Lithuanians had a significantly increased frequency of the BanI 2-Hin6I 1 haplotype, suggesting that this marker may be informative in tracing prehistoric migrations and admixture by Baltic peoples. The new TFR polymorphisms and haplotypes may also be useful markers in studies of interactions with the transferrin and hemochromatosis genes, the genetic influence on body iron stores and disease associations. PMID:9748693

  1. 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

    PubMed Central

    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

  2. Silver vanadate nanoribbons: A label-free bioindicator in the conversion between human serum transferrin and apotransferrin via surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Zhou, Qing; Shao, Mingwang; Que, Ronghui; Cheng, Liang; Zhuo, Shujuan; Tong, Yanhua; Lee, Shuit-Tong

    2011-05-01

    Silver vanadate nanoribbons were synthesized via a hydrothermal process, which exhibited surface-enhanced Raman scattering effect. This surface-enhanced substrate was stable and reproducible for identifying human serum transferrin and human serum apotransferrin in the concentration of 1×10-5 M, which further exhibited significant sensitivity in monitoring the conversion of these two proteins in turn. This result showed that the silver vanadate nanoribbon might be employed as biomonitor in such systems.

  3. Production and characterization of chimeric transferrins for the determination of the binding domains for bacterial transferrin receptors.

    PubMed

    Retzer, M D; Kabani, A; Button, L L; Yu, R H; Schryvers, A B

    1996-01-12

    Pathogenic bacteria in the Neisseriaceae and Pasteurellaceae possess outer membrane proteins that specifically bind transferrin from the host as the first step in the iron acquisition process. As a logical progression from prior studies of the ligand-receptor interaction using biochemical approaches, we have initiated an approach involving the production of recombinant chimeric transferrins to further identify the regions of transferrin involved in receptor binding. In order to prepare bovine/human hybrids, the bovine transferrin gene was cloned, sequenced, and compared with the existing human transferrin gene sequence. After identification of potential splice sites, hybrid transferrin genes were constructed using the polymerase chain reaction-based approach of splicing by overlap extension. Five hybrid genes containing sequences from both bovine and human transferrin were constructed. Recombinant transferrins were produced in a baculovirus expression vector system and affinity-purified using concanavalin A-Sepharose. The recombinant proteins were analyzed for reactivity against polyclonal and monoclonal antibodies and assessed for binding to Neisseria meningitidis transferrin receptor proteins in solid-phase binding assays and affinity isolation experiments. These experiments enabled us to localize the regions of human transferrin predominantly involved in binding to the N. meningitidis receptor to amino acid residues 346-588. The construction of these chimeras provides unique tools for the investigation of transferrin binding to receptors from both human and bovine bacterial pathogens. PMID:8557646

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

    PubMed

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

    2011-06-01

    Staphylococcus aureus is a frequent cause of bloodstream, respiratory tract, and skin and soft tissue infections. In the bloodstream, the iron-binding glycoprotein transferrin circulates to provide iron to cells throughout the body, but its iron-binding properties make it an important component of innate immunity. It is well established that siderophores, with their high affinity for iron, in many instances can remove iron from transferrin as a means to promote proliferation of bacterial pathogens. It is also established that catecholamine hormones can interfere with the iron-binding properties of transferrin, thus allowing infectious bacteria access to this iron pool. The present study demonstrates that S. aureus can use either of two carboxylate-type siderophores, staphyloferrin A and staphyloferrin B, via the transporters Hts and Sir, respectively, to access the transferrin iron pool. Growth of staphyloferrin-producing S. aureus in serum or in the presence of holotransferrin was not enhanced in the presence of catecholamines. However, catecholamines significantly enhanced the growth of staphyloferrin-deficient S. aureus in human serum or in the presence of human holotransferrin. It was further demonstrated that the Sst transporter was essential for this activity as well as for the utilization of bacterial catechol siderophores. The substrate binding protein SstD was shown to interact with ferrated catecholamines and catechol siderophores, with low to submicromolar affinities. Experiments involving mice challenged intravenously with wild-type S. aureus and isogenic mutants demonstrated that the combination of Hts, Sir, and Sst transport systems was required for full virulence of S. aureus. PMID:21402762

  5. Lymphatic vessel endothelial hyaluronan receptor-1 is a novel prognostic indicator for human hepatocellular carcinoma.

    PubMed

    Kitagawa, Koichi; Nakajima, Go; Kuramochi, Hidekazu; Ariizumi, Shun-Ichi; Yamamoto, Masakazu

    2013-11-01

    Angiogenesis is an important mechanism of tumor development, growth and metastasis in hepatocellular carcinoma (HCC). The poor prognosis of HCC patients has been associated with a failure to detect recurrences following surgery. In the present study, we investigated the association between the patient characteristics and the expression of angiogenic genes to identify early biomarkers of HCC. A comprehensive angiogenic gene expression profile was obtained by paired TaqMan gene array analysis of primary HCC nodules and adjacent non-HCC liver tissue from 12 patients. A total of 14 genes were found to be differentially expressed in HCC liver nodules (>2-fold change); the genes encoding collagen type XVα1, IVα1 and IVα2 were upregulated and the genes associated with vessel growth, neuropilin 2 (NRP2) and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) were downregulated. The histopathological analysis revealed that the evolution of HCC nodules from well to poorly differentiated was associated with a 5-fold decrease in LYVE-1 expression, reaching its lowest level early during the transition. The significance of this gene as a biomarker of postoperative survival was demonstrated by a 2-fold decrease in overall survival (OS) rates in the low expression group compared to the high expression group. The multivariate and univariate Cox regression analyses identified LYVE-1 expression as a significant independent prognostic parameter of OS [hazard ratio (HR)=3.067; 95% confidence interval (CI): 1.507-6.273; P=0.0021]. Thus, the results of this study suggested that LYVE-1 expression may constitute a novel early biomarker of postoperative survival in HCC patients. PMID:24649290

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

    PubMed Central

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

    2001-01-01

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

  7. Structural and functional consequences of the substitution of glycine 65 by arginine in the N-lobe of human transferrin

    PubMed Central

    Mason, Anne B.; Halbrooks, Peter J.; James, Nicholas G.; Byrne, Shaina L.; Grady, John K.; Chasteen, N. Dennis; Bobst, Cedric E.; Kaltashov, Igor A.; Smith, Valerie C.; MacGillivray, Ross T. A.; Everse, Stephen J.

    2009-01-01

    The G65R mutation in the N-lobe of human transferrin was created to mimic a naturally occurring variant (G394R) found in the homologous C-lobe. Because Gly65 is hydrogen-bonded to the iron-binding ligand Asp63, it comprises part of the second shell hydrogen bond network surrounding the iron within the metal binding cleft of the protein. Substitution with an arginine residue at this position disrupts the network, resulting in much more facile removal of iron from the G65R mutant. As shown by UV-vis and EPR spectroscopy, and by kinetic assays measuring the release of iron, the G65R mutant can exist in three forms. Two of the forms (yellow and pink in color) are inter-convertible. The yellow form predominates in 1 M bicarbonate; the pink form is generated from the yellow form upon exchange into 1 M HEPES buffer, pH 7.4. The third form (also pink in color) is produced by the addition of Fe3+-(nitrilotriacetate)2 to apo-G65R. Hydrogen/deuterium exchange experiments are consistent with all forms of the G65R mutant assuming a more open conformation. Additionally, mass spectroscopic analysis reveals the presence of nitrilotriacetate in the third form. The inability to obtain crystals of the G65R mutant, led to development of a novel crystallization strategy in which the double mutation G65R/K206E stabilizes a single closed pink conformer and captures Arg65 in a single position. Collectively, these studies highlight the importance of the hydrogen bond network in the cleft, as well as the inherent flexibility of the N-lobe which although able to adapt to accommodate the large arginine substitution exists in multiple conformations. PMID:19219998

  8. Generation and Characterization of Small Single Domain Antibodies Inhibiting Human Tumor Necrosis Factor Receptor 1*

    PubMed Central

    Steeland, Sophie; Puimège, Leen; Vandenbroucke, Roosmarijn E.; Van Hauwermeiren, Filip; Haustraete, Jurgen; Devoogdt, Nick; Hulpiau, Paco; Leroux-Roels, Geert; Laukens, Debby; Meuleman, Philip; De Vos, Martine; Libert, Claude

    2015-01-01

    The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named “TNF Receptor-One Silencer” (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients. PMID:25538244

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

    PubMed Central

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

    1992-01-01

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

  10. Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells.

    PubMed

    Gelb, Tara; Pshenichkin, Sergey; Hathaway, Hannah A; Grajkowska, Ewa; Dalley, Carrie Bowman; Wolfe, Barry B; Wroblewski, Jarda T

    2015-11-01

    The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability. PMID:26291396

  11. Recombinant human vascular endothelial growth factor receptor 1 effectively inhibits angiogenesis in vivo.

    PubMed

    Wang, Jinliang; Shi, Minglei; Xi, Yongyi; Gao, Lihua; Zhang, Guanyi; Shao, Yong; Chen, Huipeng; Hu, Xianwen

    2015-05-01

    Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF receptor‑1 (VEGFR‑1) acts as a decoy VEGF receptor that enables the regulation of VEGF on the vascular endothelium. In the present study, the recombinant human VEGFR1D1‑3/Fc (rhVEGFR‑1), which contains key domains for VEGF binding, was cloned and expressed in Chinese hamster ovary (CHO) cells. The rhVEGFR‑1 protein was purified using protein‑A affinity chromatography. The molecular weight of rhVEGFR‑1 was found to be ~162 and 81 kD in non‑reducing and reducing SDS‑PAGE, respectively. The majority of the final protein products were in the dimeric conformation. Western blot analysis revealed that rhVEGFR‑1 was only capable of binding to the full glycan form of rhVEGF‑165 and rhVEGF‑121. The dissociation constant for the binding of rhVEGFR‑1 to VEGF‑165, detected using Biacore, was 285 pM. In addition, rhVEGFR‑1 inhibited the proliferation and migration of human microvascular endothelial cells. In vivo experiments also demonstrated that rhVEGFR‑1 inhibited chicken chorioallantoic membrane neovascularization and angiogenesis in nude mice. In conclusion, an anti‑angiogenic recombinant soluble VEGFR was expressed (up to 5 mg/l) in CHO cells and was shown to be capable of inhibiting neovascularization in vivo and in vitro. PMID:25607471

  12. Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis.

    PubMed

    Wang, Yue; Okazaki, Yasumasa; Shi, Lei; Kohda, Hiro; Tanaka, Minoru; Taki, Kentaro; Nishioka, Tomoki; Hirayama, Tasuku; Nagasawa, Hideko; Yamashita, Yoriko; Toyokuni, Shinya

    2016-03-01

    Multi-wall carbon nanotubes (MWCNT) are a form of flexible fibrous nanomaterial with high electrical and thermal conductivity. However, 50-nm MWCNT in diameter causes malignant mesothelioma (MM) in rodents and, thus, the International Agency of Research on Cancer has designated them as a possible human carcinogen. Little is known about the molecular mechanism through which MWCNT causes MM. To elucidate the carcinogenic mechanisms of MWCNT in mesothelial cells, we used a variety of lysates to comprehensively identify proteins specifically adsorbed on pristine MWCNT of different diameters (50 nm, NT50; 100 nm, NT100; 150 nm, NT150; and 15 nm/tangled, NTtngl) using mass spectrometry. We identified >400 proteins, which included hemoglobin, histone, transferrin and various proteins associated with oxidative stress, among which we selected hemoglobin and transferrin for coating MWCNT to further evaluate cytotoxicity, wound healing, intracellular catalytic ferrous iron and oxidative stress in rat peritoneal mesothelial cells (RPMC). Cytotoxicity to RPMC was observed with pristine NT50 but not with NTtngl. Coating NT50 with hemoglobin or transferrin significantly aggravated cytotoxicity to RPMC, with an increase in cellular catalytic ferrous iron and DNA damage also observed. Knockdown of transferrin receptor with ferristatin II decreased not only NT50 uptake but also cellular catalytic ferrous iron. Our results suggest that adsorption of hemoglobin and transferrin on the surface of NT50 play a role in causing mesothelial iron overload, contributing to oxidative damage and possibly subsequent carcinogenesis in mesothelial cells. Uptake of NT50 at least partially depends on transferrin receptor 1. Modifications of NT50 surface may decrease this human risk. PMID:26679080

  13. Lectin-Like Oxidized LDL Receptor-1 Is an Enhancer of Tumor Angiogenesis in Human Prostate Cancer Cells

    PubMed Central

    González-Chavarría, Iván; Cerro, Rita P.; Parra, Natalie P.; Sandoval, Felipe A.; Zuñiga, Felipe A.; Omazábal, Valeska A.; Lamperti, Liliana I.; Jiménez, Silvana P.; Fernandez, Edelmira A.; Gutiérrez, Nicolas A.; Rodriguez, Federico S.; Onate, Sergio A.; Sánchez, Oliberto; Vera, Juan C.; Toledo, Jorge R.

    2014-01-01

    Altered expression and function of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been associated with several diseases such as endothelial dysfunction, atherosclerosis and obesity. In these pathologies, oxLDL/LOX-1 activates signaling pathways that promote cell proliferation, cell motility and angiogenesis. Recent studies have indicated that olr1 mRNA is over-expressed in stage III and IV of human prostatic adenocarcinomas. However, the function of LOX-1 in prostate cancer angiogenesis remains to be determined. Our aim was to analyze the contribution of oxLDL and LOX-1 to tumor angiogenesis using C4-2 prostate cancer cells. We analyzed the expression of pro-angiogenic molecules and angiogenesis on prostate cancer tumor xenografts, using prostate cancer cell models with overexpression or knockdown of LOX-1 receptor. Our results demonstrate that the activation of LOX-1 using oxLDL increases cell proliferation, and the expression of the pro-angiogenic molecules VEGF, MMP-2, and MMP-9 in a dose-dependent manner. Noticeably, these effects were prevented in the C4-2 prostate cancer model when LOX-1 expression was knocked down. The angiogenic effect of LOX-1 activated with oxLDL was further demonstrated using the aortic ring assay and the xenograft model of tumor growth on chorioallantoic membrane of chicken embryos. Consequently, we propose that LOX-1 activation by oxLDL is an important event that enhances tumor angiogenesis in human prostate cancer cells. PMID:25170920

  14. Glutaminolysis and Transferrin Regulate Ferroptosis.

    PubMed

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

    2015-07-16

    Ferroptosis has emerged as a new form of regulated necrosis that is implicated in various human diseases. However, the mechanisms of ferroptosis are not well defined. This study reports the discovery of multiple molecular components of ferroptosis and its intimate interplay with cellular metabolism and redox machinery. Nutrient starvation often leads to sporadic apoptosis. Strikingly, we found that upon deprivation of amino acids, a more rapid and potent necrosis process can be induced in a serum-dependent manner, which was subsequently determined to be ferroptosis. Two serum factors, the iron-carrier protein transferrin and amino acid glutamine, were identified as the inducers of ferroptosis. We further found that the cell surface transferrin receptor and the glutamine-fueled intracellular metabolic pathway, glutaminolysis, played crucial roles in the death process. Inhibition of glutaminolysis, the essential component of ferroptosis, can reduce heart injury triggered by ischemia/reperfusion, suggesting a potential therapeutic approach for treating related diseases. PMID:26166707

  15. In Vitro Characterization of Psychoactive Substances at Rat, Mouse, and Human Trace Amine-Associated Receptor 1.

    PubMed

    Simmler, Linda D; Buchy, Danièle; Chaboz, Sylvie; Hoener, Marius C; Liechti, Matthias E

    2016-04-01

    Trace amine-associated receptor 1 (TAAR1) has been implicated in the behavioral effects of amphetamine-type stimulant drugs in rodents. TAAR1 has also been suggested as a target for novel medications to treat psychostimulant addiction. We previously reported that binding affinities at TAAR1 can differ between structural analogs of psychostimulants, and species differences have been observed. In this study, we complement our previous findings with additional substances and the determination of functional activation potencies. In summary, we present here pharmacological in vitro profiles of 101 psychoactive substances at human, rat, and mouse TAAR1. p-Tyramine, β-phenylethylamine, and tryptamine were included as endogenous comparator compounds. Functional cAMP measurements and radioligand displacement assays were conducted with human embryonic kidney 293 cells that expressed human, rat, or mouse TAAR1. Most amphetamines, phenethylamine, and aminoindanes exhibited potentially physiologically relevant rat and mouse TAAR1 activation (EC50 < 5 µM) and showed full or partial (Emax < 80%) agonist properties. Cathinone derivatives, including mephedrone and methylenedioxypyrovalerone, exhibited weak (EC50 = 5-10 µM) to negligible (EC50 > 10 µM) binding properties at TAAR1. Pipradrols, including methylphenidate, exhibited no affinity for TAAR1. We found considerable species differences in activity at TAAR1 among the highly active ligands, with a rank order of rat > mouse > human. This characterization provides information about the pharmacological profile of psychoactive substances. The species differences emphasize the relevance of clinical studies to translationally complement rodent studies on the role of TAAR1 activity for psychoactive substances. PMID:26791601

  16. Reevaluation of Fatty Acid Receptor 1 as a Drug Target for the Stimulation of Insulin Secretion in Humans

    PubMed Central

    Wagner, Robert; Kaiser, Gabriele; Gerst, Felicia; Christiansen, Elisabeth; Due-Hansen, Maria E.; Grundmann, Manuel; Machicao, Fausto; Peter, Andreas; Kostenis, Evi; Ulven, Trond; Fritsche, Andreas; Häring, Hans-Ulrich; Ullrich, Susanne

    2013-01-01

    The role of free fatty acid receptor 1 (FFAR1/GPR40) in glucose homeostasis is still incompletely understood. Small receptor agonists stimulating insulin secretion are undergoing investigation for the treatment of type 2 diabetes. Surprisingly, genome-wide association studies did not discover diabetes risk variants in FFAR1. We reevaluated the role of FFAR1 in insulin secretion using a specific agonist, FFAR1-knockout mice and human islets. Nondiabetic individuals were metabolically phenotyped and genotyped. In vitro experiments indicated that palmitate and a specific FFAR1 agonist, TUG-469, stimulate glucose-induced insulin secretion through FFAR1. The proapoptotic effect of chronic exposure of β-cells to palmitate was independent of FFAR1. TUG-469 was protective, whereas inhibition of FFAR1 promoted apoptosis. In accordance with the proapoptotic effect of palmitate, in vivo cross-sectional observations demonstrated a negative association between fasting free fatty acids (NEFAs) and insulin secretion. Because NEFAs stimulate secretion through FFAR1, we examined the interaction of genetic variation in FFAR1 with NEFA and insulin secretion. The inverse association of NEFA and secretion was modulated by rs1573611 and became steeper for carriers of the minor allele. In conclusion, FFAR1 agonists support β-cell function, but variation in FFAR1 influences NEFA effects on insulin secretion and therefore could affect therapeutic efficacy of FFAR1 agonists. PMID:23378609

  17. Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.

    PubMed

    Peters, Derek T; Henderson, Christopher A; Warren, Curtis R; Friesen, Max; Xia, Fang; Becker, Caroline E; Musunuru, Kiran; Cowan, Chad A

    2016-05-01

    Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. PMID:27143754

  18. Expression and Prognostic Significance of Human Epidermal Growth Factor Receptors 1 and 3 in Gastric and Esophageal Adenocarcinoma

    PubMed Central

    Hedner, Charlotta; Borg, David; Nodin, Björn; Karnevi, Emelie; Jirström, Karin; Eberhard, Jakob

    2016-01-01

    Background Gastric and esophageal adenocarcinomas are major global cancer burdens. These cancer forms are characterized by a poor prognosis and a modest response to chemo- radio- and targeted treatment. Hence there is an obvious need for further enhanced diagnostic and treatment strategies. The aim of this study was to examine the expression and prognostic impact of human epidermal growth factor receptor 1 (HER1/EGFR) and 3 (HER3), as well as the occurrence of EGFR and KRAS mutations in gastric and esophageal adenocarcinoma. Methods Immunohistochemical expression of EGFR and HER3 was analysed in all primary tumours and a subset of lymph node metastases in a consecutive cohort of 174 patients with adenocarcinoma of the stomach, cardia and esophagus. The anti-HER3 antibody used was validated by siRNA-mediated knockdown, immunohistochemistry and quantitative real-time PCR. EGFR and KRAS mutation status was analysed by pyrosequencing tecchnology. Results and Discussion High EGFR expression was an independent risk factor for shorter overall survival (OS), whereas high HER3 expression was associated with a borderline significant trend towards a longer OS. KRAS mutations were present in only 4% of the tumours and had no prognostic impact. All tumours were EGFR wild-type. These findings contribute to the ongoing efforts to decide on the potential clinical value of different HERs and druggable mutations in gastric and esophageal adenocarcinomas, and attention is drawn to the need for more standardised investigational methods. PMID:26844548

  19. Reevaluation of fatty acid receptor 1 as a drug target for the stimulation of insulin secretion in humans.

    PubMed

    Wagner, Robert; Kaiser, Gabriele; Gerst, Felicia; Christiansen, Elisabeth; Due-Hansen, Maria E; Grundmann, Manuel; Machicao, Fausto; Peter, Andreas; Kostenis, Evi; Ulven, Trond; Fritsche, Andreas; Häring, Hans-Ulrich; Ullrich, Susanne

    2013-06-01

    The role of free fatty acid receptor 1 (FFAR1/GPR40) in glucose homeostasis is still incompletely understood. Small receptor agonists stimulating insulin secretion are undergoing investigation for the treatment of type 2 diabetes. Surprisingly, genome-wide association studies did not discover diabetes risk variants in FFAR1. We reevaluated the role of FFAR1 in insulin secretion using a specific agonist, FFAR1-knockout mice and human islets. Nondiabetic individuals were metabolically phenotyped and genotyped. In vitro experiments indicated that palmitate and a specific FFAR1 agonist, TUG-469, stimulate glucose-induced insulin secretion through FFAR1. The proapoptotic effect of chronic exposure of β-cells to palmitate was independent of FFAR1. TUG-469 was protective, whereas inhibition of FFAR1 promoted apoptosis. In accordance with the proapoptotic effect of palmitate, in vivo cross-sectional observations demonstrated a negative association between fasting free fatty acids (NEFAs) and insulin secretion. Because NEFAs stimulate secretion through FFAR1, we examined the interaction of genetic variation in FFAR1 with NEFA and insulin secretion. The inverse association of NEFA and secretion was modulated by rs1573611 and became steeper for carriers of the minor allele. In conclusion, FFAR1 agonists support β-cell function, but variation in FFAR1 influences NEFA effects on insulin secretion and therefore could affect therapeutic efficacy of FFAR1 agonists. PMID:23378609

  20. Substance P (SP) induces expression of functional corticotropin-releasing hormone receptor-1 (CRHR-1) in human mast cells.

    PubMed

    Asadi, Shahrzad; Alysandratos, Konstantinos-Dionysios; Angelidou, Asimenia; Miniati, Alexandra; Sismanopoulos, Nikolaos; Vasiadi, Magdalini; Zhang, Bodi; Kalogeromitros, Dimitrios; Theoharides, Theoharis C

    2012-02-01

    Corticotropin-releasing hormone (CRH) is secreted under stress and regulates the hypothalamic-pituitary-adrenal axis. However, CRH is also secreted outside the brain where it exerts proinflammatory effects through activation of mast cells, which are increasingly implicated in immunity and inflammation. Substance P (SP) is also involved in inflammatory diseases. Human LAD2 leukemic mast cells express only CRHR-1 mRNA weakly. Treatment of LAD2 cells with SP (0.5-2 μM) for 6 hours significantly increases corticotropin-releasing hormone receptor-1 (CRHR-1) mRNA and protein expression. Addition of CRH (1 μM) to LAD2 cells, which are "primed" with SP for 48 hours and then washed, induces synthesis and release of IL-8, tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF) 24 hours later. These effects are blocked by pretreatment with an NK-1 receptor antagonist. Treatment of LAD2 cells with CRH (1 μM) for 6 hours induces gene expression of NK-1 as compared with controls. However, repeated stimulation of mast cells with CRH (1 μM) leads to downregulation of CRHR-1 and upregulation in NK-1 gene expression. These results indicate that SP can stimulate mast cells and also increase expression of functional CRHR-1, whereas CRH induces NK-1 gene expression. These results may explain CRHR-1 and NK-1 expression in lesional skin of psoriatic patients. PMID:22089831

  1. TIBC, UIBC and Transferrin

    MedlinePlus

    ... suspected of having either iron deficiency or iron overload. These two tests are used to calculate the ... thus transferrin saturation becomes very low. In iron overload states, such as hemochromatosis , the iron level will ...

  2. Iron, transferrin and myelinogenesis

    NASA Astrophysics Data System (ADS)

    Sergeant, C.; Vesvres, M. H.; Devès, G.; Baron, B.; Guillou, F.

    2003-09-01

    Transferrin (Tf), the iron binding protein of vertebrates serum, is known to be synthesized by oligodendrocytes (Ols) in the central nervous system. It has been postulated that Tf is involved in Ols maturation and myelinogenesis. This link is particularly important in the understanding of a severe human pathology: the multiple sclerosis, which remains without efficient treatment. We generated transgenic mice containing the complete human Tf gene and extensive regulatory sequences from the 5 ' and 3 ' untranslated regions that specifically overexpress Tf in Ols. Brain cytoarchitecture of the transgenic mice appears to be normal in all brain regions examined, total myelin content is increased by 30% and motor coordination is significantly improved when compared with non-transgenic littermates. Tf role in the central nervous system may be related to its affinity for metallic cations. Normal and transgenic mice were used for determination of trace metals (iron, copper and zinc) and minerals (potassium and calcium) concentration in cerebellum and corpus callosum. The freeze-dried samples were prepared to allow proton-induced X-ray emission and Rutherford backscattering spectrometry analyses with the nuclear microprobe in Bordeaux. Preliminary results were obtained and carbon distribution was revealed as a very good analysis to distinguish precisely the white matter region. A comparison of metallic and mineral elements contents in brain between normal and transgenic mice shows that iron, copper and zinc levels remained constant. This result provides evidence that effects of Tf overexpression in the brain do not solely relate to iron transport.

  3. Binding and surface exposure characteristics of the gonococcal transferrin receptor are dependent on both transferrin-binding proteins.

    PubMed Central

    Cornelissen, C N; Sparling, P F

    1996-01-01

    Neisseria gonorrhoeae is capable of iron utilization from human transferrin in a receptor-mediated event. Transferrin-binding protein 1 (Tbp1) and Tbp2 have been implicated in transferrin receptor function, but their specific roles in transferrin binding and transferrin iron utilization have not yet been defined. We utilized specific gonococcal mutants lacking Tbp1 or Tbp2 to assess the relative transferrin-binding properties of each protein independently of the other. The apparent affinities of the wild-type transferrin receptor and of Tbp1 and Tbp2 individually were much higher than previously estimated for the gonococcal receptor and similar to the estimates for the mammalian transferrin receptor. The binding parameters of both of the mutants were distinct from those of the parent, which expressed two transferrin-binding sites. Tbp2 discriminated between ferrated transferrin and apotransferrin, while Tbp1 did not. Results of transferrin-binding affinity purification, and protease accessibility experiments were consistent with the hypothesis that Tbp1 and Tbp2 interact in the wild-type strain, although both proteins were capable of binding to transferrin independently when separated in the mutants. The presence of Tbp1 partially protected Tbp2 from trypsin proteolysis, and Tbp2 also protected Tbp1 from trypsin exposure. Addition of transferrin to wild-type but not mutant cells protected Tbp1 from trypsin but increased the trypsin susceptibility of Tbp2. These observations indicate that Tbp1 and Tbp2 function together in the wild-type strain to evoke binding conformations that are distinct from those expressed by the mutants lacking either protein. PMID:8631722

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

    SciTech Connect

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

    1987-06-01

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

  5. Methamphetamine induces trace amine-associated receptor 1 (TAAR1) expression in human T lymphocytes: role in immunomodulation.

    PubMed

    Sriram, Uma; Cenna, Jonathan M; Haldar, Bijayesh; Fernandes, Nicole C; Razmpour, Roshanak; Fan, Shongshan; Ramirez, Servio H; Potula, Raghava

    2016-01-01

    The novel transmembrane G protein-coupled receptor, trace amine-associated receptor 1 (TAAR1), represents a potential, direct target for drugs of abuse and monoaminergic compounds, including amphetamines. For the first time, our studies have illustrated that there is an induction of TAAR1 mRNA expression in resting T lymphocytes in response to methamphetamine. Methamphetamine treatment for 6 h significantly increased TAAR1 mRNA expression (P < 0.001) and protein expression (P < 0.01) at 24 h. With the use of TAAR1 gene silencing, we demonstrate that methamphetamine-induced cAMP, a classic response to methamphetamine stimulation, is regulated via TAAR1. We also show by TAAR1 knockdown that the down-regulation of IL-2 in T cells by methamphetamine, which we reported earlier, is indeed regulated by TAAR1. Our results also show the presence of TAAR1 in human lymph nodes from HIV-1-infected patients, with or without a history of methamphetamine abuse. TAAR1 expression on lymphocytes was largely in the paracortical lymphoid area of the lymph nodes with enhanced expression in lymph nodes of HIV-1-infected methamphetamine abusers rather than infected-only subjects. In vitro analysis of HIV-1 infection of human PBMCs revealed increased TAAR1 expression in the presence of methamphetamine. In summary, the ability of methamphetamine to activate trace TAAR1 in vitro and to regulate important T cell functions, such as cAMP activation and IL-2 production; the expression of TAAR1 in T lymphocytes in peripheral lymphoid organs, such as lymph nodes; and our in vitro HIV-1 infection model in PBMCs suggests that TAAR1 may play an important role in methamphetamine -mediated immune-modulatory responses. PMID:26302754

  6. Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood.

    PubMed

    Brekke, Ole-Lars; Hellerud, Bernt Christian; Christiansen, Dorte; Fure, Hilde; Castellheim, Albert; Nielsen, Erik Waage; Pharo, Anne; Lindstad, Julie Katrine; Bergseth, Grethe; Leslie, Graham; Lambris, John D; Brandtzaeg, Petter; Mollnes, Tom Eirik

    2011-09-01

    The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood. PMID:21839519

  7. Calorimetric, spectroscopic, and model studies provide insight into the transport of Ti(IV) by human serum transferrin.

    PubMed

    Tinoco, Arthur D; Incarvito, Christopher D; Valentine, Ann M

    2007-03-21

    Evidence suggests that transferrin can bind Ti(IV) in an unhydrolyzed form (without bound hydroxide or oxide) or in a hydrolyzed form. Ti(IV) coordination by N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) at different pH values models the two forms of Ti(IV)-loaded transferrin spectrally and structurally. 13C NMR and stopped-flow kinetic experiments reveal that when the metal is delivered to the protein using an unhydrolyzed source, Ti(IV) can coordinate in the typical distorted octahedral environment with a bound synergistic anion. The crystal structure of TiHBED obtained at low pH models this type of coordination. The solution structure of the complex compares favorably with the solid state from pH 3.0 to 4.0, and the complex can be reduced with E1/2 = -641 mV vs NHE. Kinetic and thermodynamic competition studies at pH 3.0 reveal that Ti(citrate)3 reacts with HBED via a dissociative mechanism and that the stability of TiHBED (log beta = 34.024) is weaker than that of the Fe(III) complex. pH stability studies show that Ti(IV) hydrolyzes ligand waters at higher pH but still remains bound to HBED until pH 9.5. Similarly, at a pH greater than 8.0 the synergistic anion that binds Ti(IV) in transferrin is readily displaced by irreversible metal hydrolysis although the metal remains bound to the protein until pH 9.5. Thermal denaturation studies conducted optically and by differential scanning calorimetry reveal that Ti(IV)-bound transferrin experiences only minimal enhanced thermal stability unlike when Fe(III) is bound. The C- and N-lobe transition Tm values shift to a few degrees higher. The stability, competition, and redox studies performed provide insight into the possible mechanism of Ti2-Tf transport in cells. PMID:17315875

  8. Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists.

    PubMed

    Hu, Xin; Myhr, Courtney; Huang, Zaohua; Xiao, Jingbo; Barnaeva, Elena; Ho, Brian A; Agoulnik, Irina U; Ferrer, Marc; Marugan, Juan J; Southall, Noel; Agoulnik, Alexander I

    2016-03-29

    The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone, including its tissue remodeling and antifibrotic effects. The peptide has a short half-life in plasma, limiting its therapeutic utility. However, small-molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach, especially for chronic diseases such as heart failure and fibrosis. The first small-molecule agonists of RXFP1 were recently identified from a high-throughput screening, using a homogeneous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent in vitro ADME and pharmacokinetic properties. Here, we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small-molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seventh transmembrane domain (TM7) and the third extracellular loop (ECL3). Several residues were determined to play an important role in the agonist binding and receptor activation, including a hydrophobic region at TM7 consisting of W664, F668, and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule ML290 were compared with the cognate ligand, relaxin, providing valuable insights on the structural basis and molecular mechanism of receptor activation and selectivity for RXFP1. PMID:26866459

  9. Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells*

    PubMed Central

    D'Alessio, Alessio; Kluger, Martin S.; Li, Jie H.; Al-Lamki, Rafia; Bradley, John R.; Pober, Jordan S.

    2010-01-01

    TNFR1 (tumor necrosis factor receptor 1) localizes to caveolae of human endothelial-derived EA.hy926 cells. Transduced TNFR1 molecules lacking amino acid residues 229–244 (spanning the transmembrane/intercellular boundary) are expressed on the cell surface equivalently to full-length TNFR1 molecules but incompletely localize to caveolae. A peptide containing this sequence pulls down CAV-1 (caveolin-1) and TNFR1 from cell lysates but fails to do so following disruption of caveolae with methyl-β-cyclodextrin. We previously reported that methyl-β-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-κB in EA.hy926 cells. Both CAV-1 and FLOT-2 (flotillin-2), organizing proteins of caveolae and lipid rafts, respectively, associate with caveolae in EA.hy926 cells. Small interfering RNA-mediated knockdown of CAV-1 but not FLOT-2 strikingly reduces caveolae number. Both knockdowns reduce total TNFR1 protein expression, but neither prevents TNFR1 localization to low density membrane domains, TNF-induced internalization of TNFR1, or NF-κB activation by TNF. Both CAV-1 and FLOT-2 knockdowns reduce TNF-mediated activation of stress-activated protein kinase (SAPK). However, both knockdowns reduce expression of TRAF2 (TNF receptor-associated factor-2) protein, and small interfering RNA targeting of TRAF2 also selectively inhibits SAPK activation. We conclude that TNFR1 contains a membrane-proximal sequence that targets the receptor to caveolae/lipid rafts. Neither TNFR1 targeting to nor internalization from these low density membrane domains depends upon CAV-1 or FLOT-2. Furthermore, both NF-κB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization. PMID:20511226

  10. Distinct cellular responses induced by saporin and a transferrin-saporin conjugate in two different human glioblastoma cell lines.

    PubMed

    Cimini, A; Mei, S; Benedetti, E; Laurenti, G; Koutris, I; Cinque, B; Cifone, M G; Galzio, R; Pitari, G; Di Leandro, L; Giansanti, F; Lombardi, A; Fabbrini, M S; Ippoliti, R

    2012-03-01

    Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ~12-18 months post-diagnosis. GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4-Pseudomonas aeruginosa exotoxin A (IL4-PE, NBI-3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha-TP-38, IL13-PE38, and a transferrin-C diphtheriae toxin mutant (Tf-CRM107). In this work, we studied the effects of the plant ribosome-inactivating saporin and of its chimera transferrin-saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways. PMID:21503892

  11. Transferrin D1: identity in Australian aborigines and American Negroes.

    PubMed

    Wang, A C; Sutton, H E; Scott, I D

    1967-05-19

    Human transferrin D(1) obtained from an Australian aborigine was found to have the same substitution of glycine for aspartic acid in peptide 1C previously shown in transferrin D(1) from an American Negro. This finding is relevant to formation of distinct Australoid and African populations. PMID:6023254

  12. Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-terminal regions of the protein.

    PubMed Central

    Evans, R W; Williams, J

    1978-01-01

    1. Trypsin digestion of human serum transferrin partially saturated with iron(III)-nitrilotriacetate at pH 5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCl3, iron (III) ascorabate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560--564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined. Images Fig. 1. Fig. 3. Fig. 5. Fig. 6. PMID:100104

  13. Fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes

    PubMed Central

    2011-01-01

    Introduction Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA. Methods Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity. Results Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and

  14. Thermodynamic binding constants for gallium transferrin

    SciTech Connect

    Harris, W.R.; Pecoraro, V.L.

    1983-01-18

    Gallium-67 is widely used as an imaging agent for tumors and inflammatory abscesses. It is well stablished that Ga/sup 3 +/ travels through the circulatory system bound to the serum iron transport protein transferrin and that this protein binding is an essential step in tumor localization. However, there have been conflicting reports on the magnitude of the gallium-transferrin binding constants. Therefore, thermodynamic binding constants for gallium complexation at the two specific metal binding sites of human serum transferrin at pH 7.4 and 5 mM NaHCO/sub 3/ have been determined by UV difference spectroscopy. The conditional constants calculated for 27 mM NaHCO/sub 3/ are log K/sub 1/* = 20.3 and log K/sub 2/* = 19.3. These results are discussed in relation to the thermodynamics of transferrin binding of Fe/sup 3 +/ and to previous reports on gallium binding. The strength of transferrin complexation is also compared to that of a series of low molecular weight ligands by using calculated pM values (pM = -log (Ga(H/sub 2/O)/sub 6/)) to express the effective binding strength at pH 7.4.

  15. Insights into endosomal maturation of human holo-transferrin in the enteric parasite Entamoeba histolytica: essential roles of Rab7A and Rab5 in biogenesis of giant early endocytic vacuoles.

    PubMed

    Verma, Kuldeep; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2015-12-01

    The pathogenic amoeba Entamoeba histolytica is one of the causative agents of health hazards in tropical countries. It causes amoebic dysentery, colitis and liver abscesses in human. Iron is one of the essential nutritional resources for survival and chronic infection caused by the amoeba. The parasite has developed multiple ways to import, sequester and utilize iron from various iron-binding proteins from its host. In spite of its central role in pathogenesis, the mechanism of iron uptake by the parasite is largely unknown. Here, we carried out a systematic study to understand the role of some of the amoebic homologues of mammalian endocytic Rab GTPases (Rab5 and Rab21, Rab7A and Rab7B) in intracellular transport of human holo-transferrin by the parasite. Flow cytometry and quantitative microscopic image analysis revealed that Rab5 and Rab7A are required for the biogenesis of amoebic giant endocytic vacuoles (GEVs) and regulate the early phase of intracellular trafficking of transferrin. Rab7B is involved in the late phase, leading to the degradation of transferrin in the amoebic lysosome-like compartments. Using time-lapse fluorescence imaging in fixed trophozoites, we determined the kinetics of the vesicular transport of transferrin through Rab5-, Rab7A- and Rab7B-positive compartments. The involvement of Rab7A in the early phase of endocytosis by the parasite marks a significant divergence from its host in terms of spatiotemporal regulation by the Rab GTPases. PMID:26096601

  16. Transferrin synthesis by small cell lung cancer cells acts as an autocrine regulator of cellular proliferation.

    PubMed Central

    Vostrejs, M; Moran, P L; Seligman, P A

    1988-01-01

    Since transferrin is required for cellular proliferation, we investigated transferrin synthesis by a small cell lung cancer line (NCI-H510) that survives in serum-free media without added transferrin. Immunoassays for human transferrin demonstrated that these cells contained immunoreactive human transferrin. Immunofluorescence studies showed that the protein is expressed on the surface of cells, presumably bound to transferrin receptor. Media conditioned by NCI-H510 cells support proliferation of human leukemic cells that would not survive in media lacking transferrin. [35S]Methionine incorporation documented transferrin synthesis by NCI-H510 cells as well as three other small cell lines. Transferrin synthesis by NCI-H510 cells increased more than 10-fold when cells entered active phases of the cell cycle, and this increase was seen before large increases in transferrin-receptor expression. Further experiments examining the effects of agents that affect iron metabolism show that the addition of transferrin-iron or hemin to the media is associated with a more rapid initial rate of proliferation and lower rates of transferrin synthesis than control cells. Gallium salts, which inhibit iron uptake, inhibited proliferation of these cells. If the cells recovered from this effect, transferrin synthesis remained greatly increased compared to control. We conclude that transferrin synthesis by these malignant cells is ultimately related to an iron requirement for cellular proliferation. It appears that this synthesized transferrin acts as part of an important autocrine mechanism permitting proliferation of these cells, and perhaps permitting tumor cell growth in vivo in areas not well vascularized. Images PMID:2839550

  17. Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation

    SciTech Connect

    Hirata, T.; Bitterman, P.B.; Mornex, J.; Crystal, R.G.

    1986-02-15

    The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, (/sup 125/I)-labeled human transferrin and a (/sup 32/P)-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (K/sub a/) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 x 10/sup 8/ M/sup -1/, and the number of receptors per cell was 4.4 +/- 1.2 x 10/sup 4/. In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, > 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.

  18. Molecular modeling of human serum transferrin for rationalizing the changes in its physicochemical properties induced by iron binding. Implication of the mechanism of binding to its receptor.

    PubMed

    Yajima, H; Sakajiri, T; Kikuchi, T; Morita, M; Ishii, T

    2000-04-01

    In order to rationalize the physicochemical properties of human serum-transferrin (STf) and the STf-receptor (TfR) recognition process, we have tried to predict the 3D structures of apo- and iron-loaded STf using a homology modeling technique to study the changes in the structural characteristics that take place upon the uptake of iron by STf in solution. The crystal structures of both forms for ovotransferrin were used as templates for the STf modeling. The modeled structure of STf gave a satisfactory interpretation for the typical physicochemical properties such that (1) STf has a negative electrophoretic mobility and its value increases with iron uptake, and (2) the radius of gyration Rg of Tf decreases with iron uptake. It was found that upon iron binding, interdomain closures take place with large movements of the NII and CII subdomains comprising the N- and C-lobes in STf through a hinge-bending motion, accompanied by the opening of the bridge region with a displacement of more than 15 A. Moreover, in view of the findings from our capillary electrophoresis experiments that the electrostatic interactions significantly contribute to a specific binding of Fe2-STf with TfR, it is inferred that the connecting (bridge) and its neighboring region associated with a surface exposure of negative charge play an important role in the STf-receptor recognition process. PMID:10981814

  19. The electrophoresis of transferrins in urea/polyacrylamide gels.

    PubMed Central

    Evans, R W; Williams, J

    1980-01-01

    The denaturation of transferrin by urea has been studied by (a) electrophoresis in polyacrylamide gels incorporating a urea gradient, (b) measurements of the loss of iron-binding capacity and (c) u.v. difference spectrometry. In human serum transferrin and hen ovotransferrin the N-terminal and C-terminal domains of the iron-free protein were found to denature at different urea concentrations. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 7. PMID:7213345

  20. Nogo Receptor 1 (RTN4R) as a Candidate Gene for Schizophrenia: Analysis Using Human and Mouse Genetic Approaches

    PubMed Central

    Hsu, Ruby; Woodroffe, Abigail; Lai, Wen-Sung; Cook, Melloni N.; Mukai, Jun; Dunning, Jonathan P.; Swanson, Douglas J.; Roos, J. Louw; Abecasis, Gonçalo R.; Karayiorgou, Maria; Gogos, Joseph A.

    2007-01-01

    Background NOGO Receptor 1 (RTN4R) regulates axonal growth, as well as axon regeneration after injury. The gene maps to the 22q11.2 schizophrenia susceptibility locus and is thus a strong functional and positional candidate gene. Methodology/Principal Findings We evaluate evidence for genetic association between common RTN4R polymorphisms and schizophrenia in a large family sample of Afrikaner origin and screen the exonic sequence of RTN4R for rare variants in an independent sample from the U.S. We also employ animal model studies to assay a panel of schizophrenia-related behavioral tasks in an Rtn4r-deficient mouse model. We found weak sex-specific evidence for association between common RTN4R polymorphisms and schizophrenia in the Afrikaner patients. In the U.S. sample, we identified two novel non-conservative RTN4R coding variants in two patients with schizophrenia that were absent in 600 control chromosomes. In our complementary mouse model studies, we identified a haploinsufficient effect of Rtn4r on locomotor activity, but normal performance in schizophrenia-related behavioral tasks. We also provide evidence that Rtn4r deficiency can modulate the long-term behavioral effects of transient postnatal N-methyl-D-aspartate (NMDA) receptor hypofunction. Conclusions Our results do not support a major role of RTN4R in susceptibility to schizophrenia or the cognitive and behavioral deficits observed in individuals with 22q11 microdeletions. However, they suggest that RTN4R may modulate the genetic risk or clinical expression of schizophrenia in a subset of patients and identify additional studies that will be necessary to clarify the role of RTN4R in psychiatric phenotypes. In addition, our results raise interesting issues about evaluating the significance of rare genetic variants in disease and their role in causation. PMID:18043741

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

    PubMed Central

    Barber, Matthew F.; Elde, Nels C.

    2015-01-01

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

  2. Binding of trivalent chromium to serum transferrin is sufficiently rapid to be physiologically relevant.

    PubMed

    Deng, Ge; Wu, Kristi; Cruce, Alex A; Bowman, Michael K; Vincent, John B

    2015-02-01

    Transferrin, the major iron transport protein in the blood, also transports trivalent chromium in vivo. Recent in vitro studies have, however, suggested that the binding of chromic ions to apotransferrin is too slow to be biologically relevant. Nevertheless, the in vitro studies have generally failed to adequately take physiological bicarbonate concentrations into account. In aqueous buffer (with ambient (bi)carbonate concentrations), the binding of chromium to transferrin is too slow to be physiologically relevant, taking days to reach equilibrium with the protein's associated conformational changes. However, in the presence of 25mM (bi)carbonate, the concentration in human blood, chromic ions bind rapidly and tightly to transferrin. Details of the kinetics of chromium binding to human serum transferrin and conalbumin (egg white transferrin) in the presence of bicarbonate and other major potential chromium ligands are described and are consistent with transferrin being the major chromic ion transporter from the blood to tissues. PMID:25528477

  3. Hafnium binding to rat serum transferrin

    NASA Astrophysics Data System (ADS)

    Then, G.; Zell, I.; Appel, H.; Thies, W.-G.; Duffield, J.; Taylor, D. M.

    1983-12-01

    Using the TDPAC-technique binding parameters for Hf were determined after in vivo uptake of181Hf in rat plasma. As much as 98.5% of the metal ions proved to be bound to protein, essentially to transferrin. The main fraction of the181Hf ions experiences a well defined electric quadrupole perturbation frequency (vQ1=(1516 ± 15)MHz, δ1=(5.3 ± 0.8)%) connected with a marked relaxation damping (λ = (46 ± 8)MHz). The remaining Hf nuclei are subject to a fairly broad distribution of electric field gradients (vQ2=(1014 ± 37)MHz, δ2=(16±3)%). The results are compared with data obtained with in vitro 181Hf-labeled human transferrin.

  4. The association between fructosamine-3 kinase 900C/G polymorphism, transferrin polymorphism and human herpesvirus-8 infection in diabetics living in South Kivu.

    PubMed

    Cikomola, Justin C; Vandepoele, Karl; Katchunga, Philippe B; Kishabongo, Antoine S; Padalko, Elizaveta Y; Speeckaert, Marijn M; Delanghe, Joris R

    2016-11-01

    Prevalences of human herpesvirus-8 (HHV-8) infection and diabetes mellitus are very common in certain parts of Africa, containing iron-rich soils. We hypothesized that some genetic factors could have a link with susceptibility to HHV-8 infection. We focused on ferroportin Q248H mutation (rs11568350), transferrin (TF) polymorphism and fructosamine-3 kinase (FN3K) 900C/G polymorphism (rs1056534). The study population consisted of 210 type 2 diabetic adults and 125 healthy controls recruited in Bukavu (South Kivu). In the whole study population (diabetics+healthy controls), ferroportin Q248H mutation was detected in 47 subjects (14.0%) with 43 heterozygotes and 4 homozygotes. TF phenotype frequencies were 88.1% (CC), 10.4% (CD) and 1.5% (BC). Genotype frequencies of FN3K 900C/G polymorphism were respectively 9,3% (CC), 43.3% (GC) and 47.4% (GG). Prevalence of HHV8-infection in the study population was 77.3%. HHV-8 infection rate and HHV-8 IgG antibody titer were significantly higher in diabetics then in controls (p<0.0001). Significant differences were observed in HHV-8 infection rate and in HHV-8 IgG antibody titer according to FN3K rs1056534 (p<0.05 and p<0.05, respectively) and TF polymorphism (p<0.05 and p=0.005, respectively). No significant differences in HHV-8 infection rate and in HHV-8 IgG antibody titer were observed in the ferroportin Q248H mutation carriers (rs11568350) in comparison with ferroportin wild type. In a multiple regression analysis, FN3K rs1056534, TF polymorphism and presence of diabetes mellitus were predictors for HHV-8 infection. In contrast to these findings, ferroportin Q248H mutation (rs11568350) did not influence the susceptibility for an HHV-8 infection in sub-Saharan Africans. PMID:27461879

  5. A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS).

    PubMed

    Feng, Liuxing; Zhang, Dan; Wang, Jun; Shen, Dairui; Li, Hongmei

    2015-07-16

    Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with (34)S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and (34)S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (m(sp)/m(sam)) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5-3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations. PMID:26073803

  6. Genetic variation in the vasopressin receptor 1a gene (AVPR1A) associates with pair-bonding behavior in humans

    PubMed Central

    Walum, Hasse; Westberg, Lars; Henningsson, Susanne; Neiderhiser, Jenae M.; Reiss, David; Igl, Wilmar; Ganiban, Jody M.; Spotts, Erica L.; Pedersen, Nancy L.; Eriksson, Elias; Lichtenstein, Paul

    2008-01-01

    Pair-bonding has been suggested to be a critical factor in the evolutionary development of the social brain. The brain neuropeptide arginine vasopressin (AVP) exerts an important influence on pair-bonding behavior in voles. There is a strong association between a polymorphic repeat sequence in the 5′ flanking region of the gene (avpr1a) encoding one of the AVP receptor subtypes (V1aR), and proneness for monogamous behavior in males of this species. It is not yet known whether similar mechanisms are important also for human pair-bonding. Here, we report an association between one of the human AVPR1A repeat polymorphisms (RS3) and traits reflecting pair-bonding behavior in men, including partner bonding, perceived marital problems, and marital status, and show that the RS3 genotype of the males also affects marital quality as perceived by their spouses. These results suggest an association between a single gene and pair-bonding behavior in humans, and indicate that the well characterized influence of AVP on pair-bonding in voles may be of relevance also for humans. PMID:18765804

  7. Preclinical and first-in-human phase I studies of KW-2450, an oral tyrosine kinase inhibitor with insulin-like growth factor receptor-1/insulin receptor selectivity.

    PubMed

    Schwartz, Gary K; Dickson, Mark A; LoRusso, Patricia M; Sausville, Edward A; Maekawa, Yoshimi; Watanabe, Yasuo; Kashima, Naomi; Nakashima, Daisuke; Akinaga, Shiro

    2016-04-01

    Numerous solid tumors overexpress or have excessively activated insulin-like growth factor receptor-1 (IGF-1R). We summarize preclinical studies and the first-in-human study of KW-2450, an oral tyrosine kinase inhibitor with IGF-1R and insulin receptor (IR) inhibitory activity. Preclinical activity of KW-2450 was evaluated in various in vitro and in vivo models. It was then evaluated in a phase I clinical trial in 13 patients with advanced solid tumors (NCT00921336). In vitro, KW-2450 inhibited human IGF-1R and IR kinases (IC50 7.39 and 5.64 nmol/L, respectively) and the growth of various human malignant cell lines. KW-2450 40 mg/kg showed modest growth inhibitory activity and inhibited IGF-1-induced signal transduction in the murine HT-29/GFP colon carcinoma xenograft model. The maximum tolerated dose of KW-2450 was 37.5 mg once daily continuously; dose-limiting toxicity occurred in two of six patients at 50 mg/day (both grade 3 hyperglycemia) and in one of seven patients at 37.5 mg/day (grade 3 rash). Four of 10 evaluable patients showed stable disease. Single-agent KW-2450 was associated with modest antitumor activity in heavily pretreated patients with solid tumors and is being further investigated in combination therapy with lapatinib/letrozole in patients with human epidermal growth factor receptor 2-postive metastatic breast cancer. PMID:26850678

  8. Bioactive secondary metabolites of a marine Bacillus sp. inhibit superoxide generation and elastase release in human neutrophils by blocking formyl peptide receptor 1.

    PubMed

    Yang, Shun-Chin; Lin, Chwan-Fwu; Chang, Wen-Yi; Kuo, Jimmy; Huang, Yin-Ting; Chung, Pei-Jen; Hwang, Tsong-Long

    2013-01-01

    It is well known that overwhelming neutrophil activation is closely related to acute and chronic inflammatory injuries. Formyl peptide receptor 1 (FPR1) plays an important role in activation of neutrophils and may represent a potent therapeutic target in inflammatory diseases. In the present study, we demonstrated that IA-LBI07-1 (IA), an extract of bioactive secondary metabolites from a marine Bacillus sp., has anti-inflammatory effects in human neutrophils. IA significantly inhibited superoxide generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated neutrophils, but failed to suppress the cell responses activated by non-FPR1 agonists. IA did not alter superoxide production and elastase activity in cell-free systems. IA also attenuated the downstream signaling from FPR1, such as the Ca2+, MAP kinases and AKT pathways. In addition, IA inhibited the binding of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein, a fluorescent analogue of FMLP, to FPR1 in human neutrophils and FPR1-transfected HEK293 cells. Taken together, these results show that the anti-inflammatory effects of IA in human neutrophils are through the inhibition of FPR1. Also, our data suggest that IA may have therapeutic potential to decrease tissue damage induced by human neutrophils. PMID:23736784

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-01

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

  12. Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens.

    PubMed

    Smith, L Cody; Clark, Jessica C; Bisesi, Joseph H; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-09-01

    The diverse biological effects of xenoestrogens may be explained by their ability to differentially recruit co-regulatory proteins to the estrogen receptor (ER). We employed high-throughput receptor affinity binding and co-regulatory protein recruitment screening assays based on fluorescence polarization and time resolved florescence resonance energy transfer (TR-FRET), respectively, to assess xenoestrogen-specific binding and co-regulatory protein recruitment to the ER. Then we used a functional proteomic assay based on co-immunoprecipitation of ER-bound proteins to isolate and identify intact co-regulatory proteins recruited to a ligand-activated ER. Through these approaches, we revealed differential binding affinity of bisphenol-A (BPA) and genistein (GEN) to the human ERα (ESR1) and ligand-dependent recruitment of SRC-1 and SRC-3 peptides. Recruitment profiles were variable for each ligand and in some cases were distinct compared to 17β-estradiol (E2). For example, E2 and GEN recruited both SRC-1 and -3 peptides whereas BPA recruited only SRC-1 peptides. Results of the functional proteomic assay showed differential recruitment between ligands where E2 recruited the greatest number of proteins followed by BPA then GEN. A number of proteins share previously identified relationships with ESR1 as determined by STRING analysis. Although there was limited overlap in proteins identified between treatments, all ligands recruited proteins involved in cell growth as determined by subnetwork enrichment analysis (p<0.05). A comparative, in silico analysis revealed that fewer interactions exist between zebrafish (Danio rerio) esr1 and zebrafish orthologs of proteins identified in our functional proteomic analysis. Taken together these results identify recruitment of known and previously unknown co-regulatory proteins to ESR1 and highlight new methods to assay recruitment of low abundant and intact, endogenous co-regulatory proteins to ESR1 or other nuclear receptors, in

  13. Discovery of potent hexapeptide agonists to human neuromedin u receptor 1 and identification of their serum metabolites.

    PubMed

    Takayama, Kentaro; Mori, Kenji; Sohma, Yuko; Taketa, Koji; Taguchi, Akihiro; Yakushiji, Fumika; Minamino, Naoto; Miyazato, Mikiya; Kangawa, Kenji; Hayashi, Yoshio

    2015-03-12

    Neuromedin U (NMU) and S (NMS) display various physiological activities, including an anorexigenic effect, and share a common C-terminal heptapeptide-amide sequence that is necessary to activate two NMU receptors (NMUR1 and NMUR2). On the basis of this knowledge, we recently developed hexapeptide agonists 2 and 3, which are highly selective to human NMUR1 and NMUR2, respectively. However, the agonists are still less potent than the endogenous ligand, hNMU. Therefore, we performed an additional structure-activity relationship study, which led to the identification of the more potent hexapeptide 5d that exhibits similar NMUR1-agonistic activity as compared to hNMU. Additionally, we studied the stability of synthesized agonists, including 5d, in rat serum, and identified two major biodegradation sites: Phe(2)-Arg(3) and Arg(5)-Asn(6). The latter was more predominantly cleaved than the former. Moreover, substitution with 4-fluorophenylalanine, as in 5d, enhanced the metabolic stability at Phe(2)-Arg(3). These results provide important information to guide the development of practical hNMU agonists. PMID:25815150

  14. Interferon Regulatory Factor 6 (IRF6) and Fibroblast Growth Factor Receptor 1 (FGFR1) Contribute to Human Tooth Agenesis

    PubMed Central

    Vieira, Alexandre R.; Modesto, Adriana; Meira, Raquel; Barbosa, Anna Renata Schneider; Lidral, Andrew C.; Murray, Jeffrey C.

    2008-01-01

    Phenotypic characteristics expressed in syndromes give clues to the factors involved in the cause of isolated forms of the same defects. We investigated two genes responsible for craniofacial syndromes, FGFR1 and IRF6, in a collection of families with isolated tooth agenesis. Cheek swab samples were obtained for DNA analysis from 116 case/parent trios. Probands had at least one developmentally missing tooth, excluding third molars. In addition, we studied 89 cases and 50 controls from Ohio to replicate any positive findings. Genotyping was performed by kinetic polymerase chain-reaction or TaqMan assays. Linkage disequilibrium analysis and transmission distortion of the marker alleles were performed. The same variants in the IRF6 gene that are associated with isolated orofacial clefts are also associated with human tooth agenesis (rs861019, P = 0.058; rs17015215—V274I, P = 0.0006; rs7802, P = 0.004). Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. The craniofacial phenotypic characteristics of these syndromes include oral clefts and preferential tooth agenesis of incisors and premolars, besides pits on the lower lips. Also it appears that preferential premolar agenesis is associated with FGFR1 (P = 0.014) and IRF6 (P = 0.002) markers. There were statistically significant data suggesting that IRF6 interacts not only with MSX1 (P = 0.001), but also with TGFA (P = 0.03). PMID:17318851

  15. Substance P (SP) induces expression of functional corticotropin-releasing hormone receptor-1 (CRHR-1) in human mast cells

    PubMed Central

    Asadi, Shahrzad; Alysandratos, Konstantinos-Dionysios; Angelidou, Asimenia; Miniati, Alexandra; Sismanopoulos, Nikolaos; Vasiadi, Magdalini; Zhang, Bodi; Kalogeromitros, Dimitrios; Theoharides, Theoharis C.

    2012-01-01

    Corticotropin-releasing hormone (CRH) is secreted under stress and regulates the hypothalamic-pituitary-adrenal (HPA) axis. However, CRH is also secreted outside the brain where it exerts pro-inflammatory effects through activation of mast cells, which are increasingly implicated in immunity and inflammation. Substance P (SP) is also involved in inflammatory diseases. Human LAD2 leukemic mast cells express only CRHR-1 mRNA weakly. Treatment of LAD2 cells with SP (0.5–2 µM) for 6 hr significantly increases CRHR-1 mRNA and protein expression. Addition of CRH (1 µM) to LAD2 cells “primed” with SP for 48 hr and then washed, induces synthesis and release of IL-8, tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) 24 hr later. These effects are blocked by pretreatment with an NK-1 receptor antagonist. Treatment of LAD2 cells with CRH (1 µM) for 6 hr induces gene expression of NK-1 as compared to controls. However, repeated stimulation of mast cells with CRH (1 µM) leads to downregulation of CRHR-1 and upregulation in NK-1 gene expression. These results indicate that SP can stimulate mast cells and also increase expression of functional CRHR-1, while CRH induces NK-1 gene expression. These results may explain CRHR-1 and NK-1 expression in lesional skin of psoriatic patients. PMID:22089831

  16. Expression and Prognostic Significance of Human Epidermal Growth Factor Receptors 1, 2 and 3 in Periampullary Adenocarcinoma

    PubMed Central

    Heby, Margareta; Warfvinge, Carl Fredrik; Nodin, Björn; Eberhard, Jakob; Jirström, Karin

    2016-01-01

    Periampullary adenocarcinoma, including pancreatic cancer, is a heterogeneous group of tumours with dismal prognosis, for which there is an urgent need to identify novel treatment strategies. The human epithelial growth factor receptors EGFR, HER2 and HER3 have been studied in several tumour types, and HER-targeting drugs have a beneficial effect on survival in selected types of cancer. However, these effects have not been evident in pancreatic cancer, and remain unexplored in other types of periampullary cancer. The prognostic impact of HER-expression in these cancers also remains unclear. The aim of this study was therefore to examine the expression and prognostic value of EGFR, HER2 and HER3 in periampullary cancer, with particular reference to histological subtype. To this end, protein expression of EGFR, HER2 and HER3, and HER2 gene amplification was assessed by immunohistochemistry and silver in situ hybridization, respectively, on tissue microarrays with tumours from 175 periampullary adenocarcinomas, with follow-up data on recurrence-free survival (RFS) and overall survival (OS) for up to 5 years. EGFR expression was similar in pancreatobiliary (PB) and intestinal (I) type tumours, but high HER2 and HER3 expression was significantly more common in I-type tumours. In PB-type cases receiving adjuvant gemcitabine, but not in untreated cases, high EGFR expression was significantly associated with a shorter OS and RFS, with a significant treatment interaction in relation to OS (pinteraction = 0.042). In I-type cases, high EGFR expression was associated with a shorter OS and RFS in univariable, but not in multivariable, analysis. High HER3 expression was associated with a prolonged RFS in univariable, but not in multivariable, analysis. Neither HER2 protein expression nor gene amplification was prognostic. The finding of a potential interaction between the expression of EGFR and response to adjuvant chemotherapy in PB-type tumours needs validation, and merits

  17. Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa.

    PubMed

    Reyes-López, Magda; Piña-Vázquez, Carolina; Serrano-Luna, Jesús

    2015-01-01

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

  18. Transferrin: Endocytosis and Cell Signaling in Parasitic Protozoa

    PubMed Central

    Serrano-Luna, Jesús

    2015-01-01

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

  19. Leukocyte-associated immunoglobulin-like receptor-1 is expressed on human megakaryocytes and negatively regulates the maturation of primary megakaryocytic progenitors and cell line

    SciTech Connect

    Xue, Jiangnan; Zhang, Xiaoshu; Zhao, Haiya; Fu, Qiang; Cao, Yanning; Wang, Yuesi; Feng, Xiaoying; Fu, Aili

    2011-02-04

    Research highlights: {yields} LAIR-1 is expressed on human megakaryocytes from an early stage. {yields} Up-regulation of LAIR-1 negatively regulates megakaryocytic differentiation of cell line. {yields} LAIR-1 negatively regulates the differentiation of primary megakaryocytic progenitors. -- Abstract: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an inhibitory collagen receptor which belongs to the immunoglobulin (Ig) superfamily. Although the inhibitory function of LAIR-1 has been extensively described in multiple leukocytes, its role in megakaryocyte (MK) has not been explored so far. Here, we show that LAIR-1 is expressed on human bone marrow CD34{sup +}CD41a{sup +} and CD41a{sup +}CD42b{sup +} cells. LAIR-1 is also detectable in a fraction of human cord blood CD34{sup +} cell-derived MK that has morphological characteristics of immature MK. In megakaryoblastic cell line Dami, the membrane protein expression of LAIR-1 is up-regulated significantly when cells are treated with phorbol ester phorbol 12-myristate 13-acetate (PMA). Furthermore, cross-linking of LAIR-1 in Dami cells with its natural ligand or anti-LAIR-1 antibody leads to the inhibition of cell proliferation and PMA-promoted differentiation when examined by the MK lineage-specific markers (CD41a and CD42b) and polyploidization. In addition, we also observed that cross-linking of LAIR-1 results in decreased MK generation from primary human CD34{sup +} cells cultured in a cytokines cocktail that contains TPO. These results suggest that LAIR-1 is a likely candidate for an early marker of MK differentiation, and provide initial evidence indicating that LAIR-1 serves as a negative regulator of megakaryocytopoiesis.

  20. Iron release from transferrin by pyoverdin and elastase from Pseudomonas aeruginosa.

    PubMed Central

    Wolz, C; Hohloch, K; Ocaktan, A; Poole, K; Evans, R W; Rochel, N; Albrecht-Gary, A M; Abdallah, M A; Döring, G

    1994-01-01

    Pseudomonas aeruginosa produces the siderophores pyoverdin and pyochelin as well as receptors for siderophores in response to iron deprivation. Previously, it has been shown in vitro that at neutral pH purified pyoverdin acquires iron from transferrin only in the presence of P. aeruginosa elastase (LasB), which proteolytically degrades transferrin. We constructed a LasB-negative mutant, PAO1E, by insertional mutagenesis to investigate whether this mutant differs in growth from the parental strain PAO1 in an iron-depleted medium supplemented with transferrin or human serum. PAO1 and PAO1E did not differ in growth with 1.25 microM Fe2-transferrin as the only iron source. Urea gel electrophoresis indicated iron release from intact transferrin during the logarithmic growth phase of PAO1 and PAO1E. A total of 333 microM LasB was synthesized from PAO1 after onset of stationary-phase growth. Quantification of pyoverdin by spectroscopy revealed that up to 900 microM pyroverdin was produced during growth of the strains in medium supplemented with Fe2-transferrin or 10% human serum. Incubation of Fe2-transferrin and purified pyoverdin in concentrations similar to those found in the culture supernatant resulted in release iron from transferrin after 10 h at 37 degrees C. However, LasB significantly enhanced the rate constant for iron acquisition of pyoverdin from transferrin. We conclude that P. aeruginosa can use transferrin as an iron source without further need of LasB or pH changes. This is further supported by experiments with P. aeruginosa K437, which has a defective iron uptake system, and its LasB-negative mutant, K437E. Though K437 and K437E did not differ in growth with Fe2-transferrin as the only iron source, their growth was significantly reduced relative to that of PAO1 and PAO1E. Images PMID:8063422

  1. Transferrin treatment corrects aging-related immunologic and hormonal decay in old mice.

    PubMed

    Pierpaoli, W; Bulian, D; Arrighi, S

    2000-05-01

    Experiments were conducted to study the effect of heterologous plasma transferrins separated and purified from human plasma pools on endocrine and immune functions of old, aging mice. Two similar experiments have shown that parenteral treatment with iron and zinc-free human transferrins produces a significant improvement of immunological and endocrine functions in the aging mice toward more juvenile values. Those changes occur in the thymus and its cell subsets, in peripheral blood lymphocytes, in the restoration of juvenile levels of thyroxine, in the increase of testis weight, and in the normalization of plasma zinc levels. These totally unsuspected effects of transferrin in aging mice suggest a most important role of endogenous transferrins in the maintenance of neuroendocrine and immune functions. The mechanism remains unexplained although the basic immunoenhancing and anti-apoptotic effect of transferrin-vehiculated zinc may be relevant. PMID:10832059

  2. Complex of transferrin with ruthenium for medical applications

    DOEpatents

    Richards, P.; Srivastava, S.C.; Meinken, G.E.

    1984-05-15

    A novel ruthenium-transferrin complex is disclosed which is prepared by reacting iron-free human transferrin dissolved in a sodium acetate solution at pH 7 with ruthenium by heating at about 40 C for about 2 hours. The complex is purified by means of gel chromotography with pH 7 sodium acetate as eluent. The mono- or di-metal complex produced can be used in nuclear medicine in the diagnosis and/or treatment of tumors and abscesses. Comparative results with Ga-67-citrate, which is the most widely used tumor-localizing agent in nuclear medicine, indicate increased sensitivity of detection and greater tumor uptake with the Ru-transferrin complex. No Drawings

  3. Complex of transferrin with ruthenium for medical applications

    DOEpatents

    Richards, Powell; Srivastava, Suresh C.; Meinken, George E.

    1984-05-15

    A novel Ruthenium-transferrin complex, prepared by reacting iron-free human transferrin dissolved in a sodium acetate solution at pH 7 with ruthenium by heating at about 40.degree. C. for about 2 hours, and purifying said complex by means of gel chromotography with pH 7 sodium acetate as eluent. The mono- or di-metal complex produced can be used in nuclear medicine in the diagnosis and/or treatment of tumors and abscesses. Comparative results with Ga-67-citrate, which is the most widely used tumor-localizing agent in nuclear medicine, indicate increased sensitivity of detection and greater tumor uptake with the Ru-transferrin complex.

  4. The PET Radioligand 18F-FIMX Images and Quantifies Metabotropic Glutamate Receptor 1 in Proportion to the Regional Density of Its Gene Transcript in Human Brain

    PubMed Central

    Zanotti-Fregonara, Paolo; Xu, Rong; Zoghbi, Sami S.; Liow, Jeih-San; Fujita, Masahiro; Veronese, Mattia; Gladding, Robert L.; Rallis-Frutos, Denise; Hong, Jinsoo; Pike, Victor W.; Innis, Robert B.

    2016-01-01

    A recent study from our laboratory found that 18F-FIMX is an excellent PET radioligand for quantifying metabotropic glutamate receptor 1 (mGluR1) in monkey brain. This study evaluated the ability of 18F-FIMX to quantify mGluR1 in humans. A second goal was to use the relative density of mGluR1 gene transcripts in brain regions to estimate specific uptake and nondisplaceable uptake (VND) in each brain region. Methods After injection of 189 ± 3 MBq of 18F-FIMX, 12 healthy volunteers underwent a dynamic PET scan over 120 min. For 6 volunteers, images were acquired until 210 min. A metabolite-corrected arterial input function was measured from the radial artery. Four other subjects underwent whole-body scanning to estimate radiation exposure. Results 18F-FIMX uptake into the human brain was high (SUV = 4–6 in the cerebellum), peaked at about 10 min, and washed out rapidly. An unconstrained 2-tissue-compartment model fitted the data well, and distribution volume (VT) (mL·cm−3) values ranged from 1.5 in the caudate to 11 in the cerebellum. A 120-min scan provided stable VT values in all regions except the cerebellum, for which an acquisition time of at least 170 min was necessary. VT values in brain regions correlated well with mGluR1 transcript density, and the correlation suggested that VND of 18F-FIMX was quite low (0.5 mL·cm−3). This measure of VND in humans was similar to that from a receptor blocking study in monkeys, after correcting for differences in plasma protein binding. Similar to other 18F-labeled ligands, the effective dose was about 23 µSv/MBq. Conclusion 18F-FIMX can quantify mGluR1 in the human brain with a 120- to 170-min scan. Correlation of brain uptake with the relative density of mGluR1 transcript allows specific receptor binding of a radioligand to be quantified without injecting pharmacologic doses of a blocking agent. PMID:26514176

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

    PubMed

    Ghanbari, Z; Housaindokht, M R; Bozorgmehr, M R; Izadyar, M

    2016-09-01

    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. PMID:27235585

  6. 21 CFR 866.5880 - Transferrin immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Transferrin immunological test system. 866.5880 Section 866.5880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES...-binding and transporting serum protein) in serum, plasma, and other body fluids. Measurement...

  7. 21 CFR 866.5880 - Transferrin immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Transferrin immunological test system. 866.5880 Section 866.5880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES...-binding and transporting serum protein) in serum, plasma, and other body fluids. Measurement...

  8. 21 CFR 866.5880 - Transferrin immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Transferrin immunological test system. 866.5880 Section 866.5880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES...-binding and transporting serum protein) in serum, plasma, and other body fluids. Measurement...

  9. 21 CFR 866.5880 - Transferrin immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Transferrin immunological test system. 866.5880 Section 866.5880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES...-binding and transporting serum protein) in serum, plasma, and other body fluids. Measurement...

  10. 21 CFR 866.5880 - Transferrin immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Transferrin immunological test system. 866.5880 Section 866.5880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES...-binding and transporting serum protein) in serum, plasma, and other body fluids. Measurement...

  11. A Compartment Model of VEGF Distribution in Humans in the Presence of Soluble VEGF Receptor-1 Acting as a Ligand Trap

    PubMed Central

    Wu, Florence T. H.; Stefanini, Marianne O.; Mac Gabhann, Feilim; Popel, Aleksander S.

    2009-01-01

    Vascular endothelial growth factor (VEGF), through its activation of cell surface receptor tyrosine kinases including VEGFR1 and VEGFR2, is a vital regulator of stimulatory and inhibitory processes that keep angiogenesis – new capillary growth from existing microvasculature – at a dynamic balance in normal physiology. Soluble VEGF receptor-1 (sVEGFR1) – a naturally-occurring truncated version of VEGFR1 lacking the transmembrane and intracellular signaling domains – has been postulated to exert inhibitory effects on angiogenic signaling via two mechanisms: direct sequestration of angiogenic ligands such as VEGF; or dominant-negative heterodimerization with surface VEGFRs. In pre-clinical studies, sVEGFR1 gene and protein therapy have demonstrated efficacy in inhibiting tumor angiogenesis; while in clinical studies, sVEGFR1 has shown utility as a diagnostic or prognostic marker in a widening array of angiogenesis–dependent diseases. Here we developed a novel computational multi-tissue model for recapitulating the dynamic systemic distributions of VEGF and sVEGFR1. Model features included: physiologically-based multi-scale compartmentalization of the human body; inter-compartmental macromolecular biotransport processes (vascular permeability, lymphatic drainage); and molecularly-detailed binding interactions between the ligand isoforms VEGF121 and VEGF165, signaling receptors VEGFR1 and VEGFR2, non-signaling co-receptor neuropilin-1 (NRP1), as well as sVEGFR1. The model was parameterized to represent a healthy human subject, whereupon we investigated the effects of sVEGFR1 on the distribution and activation of VEGF ligands and receptors. We assessed the healthy baseline stability of circulating VEGF and sVEGFR1 levels in plasma, as well as their reliability in indicating tissue-level angiogenic signaling potential. Unexpectedly, simulated results showed that sVEGFR1 – acting as a diffusible VEGF sink alone, i.e., without sVEGFR1-VEGFR heterodimerization

  12. Expression of the Haemophilus influenzae transferrin receptor is repressible by hemin but not elemental iron alone.

    PubMed Central

    Morton, D J; Musser, J M; Stull, T L

    1993-01-01

    The absolute requirement for elemental iron and the porphyrin nucleus for growth of Haemophilus influenzae led us to investigate the role of iron and hemin in regulation of expression of the H. influenzae transferrin receptor. H. influenzae type b strain H1689 was grown in brain heart infusion broth supplemented with beta-NAD and either 10 or 0.1 microgram of hemin ml-1. Transferrin-binding ability was determined with a dot blot assay using human transferrin-horseradish peroxidase conjugate. Cells grown in media with 0.1 microgram of hemin ml-1 bound transferrin, but organisms grown in media with 10 micrograms ml-1 did not. In hemin-restricted media, transferrin binding occurred despite addition of up to 10 mM ferric nitrate, ferric citrate, or ferric PPi, whereas addition of 10 micrograms of hemoglobin ml-1 repressed expression. The breadth of species distribution of this mode of regulation was determined with strains previously characterized by multilocus enzyme electrophoresis. When grown in hemin-restricted media, 24 of 28 type b strains and 52 of 57 serologically nontypeable strains exhibited transferrin binding, although none did so in hemin- and iron-sufficient media. Strain H1689 and serologically nontypeable strain HI1423 grown in heat-inactivated pooled normal human serum, human cerebrospinal fluid, or human breast milk exhibited transferrin binding. Growth in these fluids with 10 micrograms of added hemin ml-1 abolished transferrin binding, whereas addition of 10 mM ferric nitrate did not. These data suggest that the transferrin receptor of H. influenzae is regulated by levels of hemin but not elemental iron alone and that this property is widely distributed among several major cloned families in the species. Images PMID:8406790

  13. Hafnium binding to comparison: comparison between lactoferrin and other transferrins

    NASA Astrophysics Data System (ADS)

    Becker, G.; Appel, H.; Neu, M.; Schwab, F. J.; Thies, W.-G.

    1993-03-01

    The TDPAC method was used to study the electric field gradients at the metal sites of human and bovine lactoferrin. Two specific binding configurations were observed. The distribution between these configurations depends on the phosphate content, the pH, and the temperature of the samples. The electric field gradients are compared with the results of previous studies for human and rat serum transferrin, and hen ovotransferrin.

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

    PubMed

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

    2014-04-01

    Growth differentiation factor 15 (GDF-15) is a bone marrow-derived cytokine whose ability to suppress iron regulator hepcidin in vitro and increased concentrations found in patients with ineffective erythropoiesis (IE)suggest that hepcidin deficiency mediated by GDF-15 may be the pathophysiological explanation for nontransfusional iron overload. We aimed to compare GDF-15 production in anemic states with different types of erythropoietic dysfunction. Complete blood counts, biochemical markers of iron status, plasma hepcidin, GDF-15, and known hepcidin regulators [interleukin-6 and erythropoietin (EPO)] were measured in 87 patients with red cell disorders comprising IE and hemolytic states: thalassemia, sickle cell anemia, and cobalamin deficiency. Healthy volunteers were also evaluated for comparison. Neither overall increased EPO,nor variable GDF-15 concentrations correlated with circulating hepcidin concentrations (P = 0.265 and P = 0.872). Relative hepcidin deficiency was found in disorders presenting with concurrent elevation of GDF-15 and soluble transferrin receptor (sTfR), a biomarker of erythropoiesis, and sTfR had the strongest correlation with hepcidin (r(s) = 0.584, P < 0.0001). Our data show that high concentrations of GDF-15 in vivo are not necessarily associated with pathological hepcidin reduction, and hepcidin deficiency was only found when associated with sTfR overproduction. sTfR elevation may be a necessary common denominator of erythropoiesis-driven mechanisms to favor iron absorption in anemic states and appears a suitable target for investigative approaches to iron disorders. PMID:24860871

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

    PubMed Central

    Cuthbert, J A; Lipsky, P E

    1984-01-01

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

  16. Transferrin: structure, function and potential therapeutic actions.

    PubMed

    Gomme, Peter T; McCann, Karl B; Bertolini, Joseph

    2005-02-15

    There are many proteins that can multi-task. Transferrin, widely known as an iron-binding protein, is one such example of a multi-tasking protein. In this review, the multiple biological actions of transferrin, including its growth and cytoprotective activities, are discussed with the view of highlighting the potential therapeutic applications of this protein. PMID:15708745

  17. Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.

    PubMed

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

    1999-07-27

    The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with

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

    PubMed

    Barber, Matthew F; Elde, Nels C

    2014-12-12

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

  19. Transferrin protein nanospheres: a nanoplatform for receptor-mediated cancer cell labeling and gene delivery

    NASA Astrophysics Data System (ADS)

    McDonald, Michael A.; Spurlin, Tighe A.; Tona, Alessandro; Elliott, John T.; Halter, Michael; Plant, Anne L.

    2010-02-01

    This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

  20. Identification of small-molecule agonists of human relaxin family receptor 1 (RXFP1) by using a homogenous cell-based cAMP assay.

    PubMed

    Chen, Catherine Z; Southall, Noel; Xiao, Jingbo; Marugan, Juan J; Ferrer, Marc; Hu, Xin; Jones, Raisa E; Feng, Shu; Agoulnik, Irina U; Zheng, Wei; Agoulnik, Alexander I

    2013-07-01

    The relaxin hormone is involved in a variety of biological functions, including female reproduction and parturition, as well as regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) relaxin family receptor 1 (RXFP1) is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. Although agonists of the receptor could have a wide range of pharmacologic utility, until now there have been no reported small-molecule agonists for relaxin receptors. Here, we report the development of a quantitative high-throughput platform for an RXFP1 agonist screen based on homogenous cell-based HTRF cyclic AMP (cAMP) assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365 677 compounds. Neither compound showed activity in a counterscreen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small-molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists. PMID:23212924

  1. Identification of small molecule agonists of human relaxin family receptor 1 (RXFP1) by utilizing a homogenous cell-based cAMP assay

    PubMed Central

    Chen, Catherine Z.; Southall, Noel; Xiao, Jingbo; Marugan, Juan J.; Ferrer, Marc; Hu, Xin; Jones, Raisa E.; Feng, Shu; Agoulnik, Irina U.

    2016-01-01

    The relaxin hormone is involved in a variety of biological functions including female reproduction and parturition, regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) RXFP1, relaxin family receptor 1, is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. While agonists of the receptor could have a wide range of pharmacological utility, up to date, there are no reported small molecule agonists for relaxin receptors. Here, we report the development of quantitative high-throughput platform for RXFP1 agonist screen based on homogenous cell-based HTRF cAMP assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365,677 compounds. Neither compound showed activity in a counter screen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists. PMID:23212924

  2. Transferrin-Polycation Conjugates as Carriers for DNA Uptake into Cells

    NASA Astrophysics Data System (ADS)

    Wagner, Ernst; Zenke, Martin; Cotten, Matt; Beug, Hartmut; Birnstiel, Max L.

    1990-05-01

    We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection."

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

    SciTech Connect

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

    2011-08-01

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

  4. Transferrin-polycation conjugates as carriers for DNA uptake into cells.

    PubMed Central

    Wagner, E; Zenke, M; Cotten, M; Beug, H; Birnstiel, M L

    1990-01-01

    We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection." Images PMID:2333290

  5. Leukocyte Immunoglobulin-Like Receptor 1-Expressing Human Natural Killer Cell Subsets Differentially Recognize Isolates of Human Cytomegalovirus through the Viral Major Histocompatibility Complex Class I Homolog UL18

    PubMed Central

    Chen, Kevin C.; Banat, Jareer J.

    2016-01-01

    ABSTRACT Immune responses of natural killer (NK) cell are controlled by the balance between activating and inhibitory receptors, but the expression of these receptors varies between cells within an individual. Although NK cells are a component of the innate immune system, particular NK cell subsets expressing Ly49H are positively selected and increase in frequency in response to cytomegalovirus infection in mice. Recent evidence suggests that in humans certain NK subsets also have an increased frequency in the blood of human cytomegalovirus (HCMV)-infected individuals. However, whether these subsets differ in their capacity of direct control of HCMV-infected cells remains unclear. In this study, we developed a novel in vitro assay to assess whether human NK cell subsets have differential abilities to inhibit HCMV growth and dissemination. NK cells expressing or lacking NKG2C did not display any differences in controlling viral dissemination. However, when in vitro-expanded NK cells were used, cells expressing or lacking the inhibitory receptor leukocyte immunoglobulin-like receptor 1 (LIR1) were differentially able to control dissemination. Surprisingly, the ability of LIR1+ NK cells to control virus spread differed between HCMV viral strains, and this phenomenon was dependent on amino acid sequences within the viral ligand UL18. Together, the results here outline an in vitro technique to compare the long-term immune responses of different human NK cell subsets and suggest, for the first time, that phenotypically defined human NK cell subsets may differentially recognize HCMV infections. IMPORTANCE HCMV infection is ubiquitous in most populations; it is not cleared by the host after primary infection but persists for life. The innate and adaptive immune systems control the spread of virus, for which natural killer (NK) cells play a pivotal role. NK cells can respond to HCMV infection by rapid, short-term, nonspecific innate responses, but evidence from murine

  6. Enhancement of p53 gene transfer efficiency in hepatic tumor mediated by transferrin receptor through trans-arterial delivery.

    PubMed

    Lu, Qin; Teng, Gao-Jun; Zhang, Yue; Niu, Huan-Zhang; Zhu, Guang-Yu; An, Yan-Li; Yu, Hui; Li, Guo-Zhao; Qiu, Ding-Hong; Wu, Chuan-Ging

    2008-02-01

    Transferrin-DNA complex mediated by transferrin receptor in combination with interventional trans-arterial injection into a target organ may be a duel-target-oriented delivery means to achieve an efficient gene therapy. In this study, transferrin receptor expression in normal human hepatocyte and two hepatocellular-carcinoma cells (Huh7/SK-Hep1) was determined. p53-LipofectAMINE with different amounts of transferrin was transfected into the cells and the gene transfection efficiency was evaluated. After VX2 rabbit hepatocarcinoma model was established, the transferrin-p53-LipofectAMINE complex was delivered into the hepatic artery via interventional techniques to analyze the therapeutic p53 gene transfer efficiency in vivo by Western blot, immunohistochemical/immunofluorescence staining analysis and survival time. The results were transferrin receptor expression in Huh7 and SK-Hep1 cells was higher than in normal hepatocyte. Transfection efficiency of p53 was increased in vitro in both Huh7 and SK-Hep1 cells with increasing transferrin in a dose-dependent manner. As compared to intravenous administration, interventional injection of p53-gene complex into hepatic tumor mediated by transferrin-receptor, could enhance the gene transfer efficiency in vivo as evaluated by Western blot, immunohistochemical/immunofluorenscence staining analyses and improved animal survival (H = 12.567, p = 0.0019). These findings show the transferrin-transferrin receptor system combined with interventional techniques enhanced p53-gene transfer to hepatic tumor and the duel-target-oriented gene delivery may be an effective approach for gene therapy. PMID:18347429

  7. Reptilian transferrins: evolution of disulphide bridges and conservation of iron-binding center.

    PubMed

    Ciuraszkiewicz, Justyna; Biczycki, Marian; Maluta, Aleksandra; Martin, Samuel; Watorek, Wiesław; Olczak, Mariusz

    2007-07-01

    Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to

  8. Developmental changes in the expression of somatostatin receptors (1-5) in the brain, hypothalamus, pituitary and spinal cord of the human fetus.

    PubMed

    Goodyer, C G; Grigorakis, S I; Patel, Y C; Kumar, U

    2004-01-01

    The actions of somatostatin (SST) in the nervous system are mediated by specific high affinity SST receptors (SSTR1-5). However, the role of this hormone and the distribution of its receptor subtypes have not yet been defined in neural structures of the human fetus. We have analyzed four neural tissues (CNS, hypothalamus, pituitary and spinal cord) from early to midgestation for the expression of five human SSTR mRNAs, using a reverse transcription-polymerase chain reaction and Southern blot approach. These fetal neural tissues all express mRNA for multiple SSTR subtypes from as early as 16 weeks of fetal life but the developmental patterns of expression vary considerably. Transcripts for SSTR1 and SSTR2A are the most widely distributed, being expressed in all four neural tissues. SSTR2A is often the earliest transcript to be detected (7.5 weeks in CNS). SSTR3 mRNA is confined to the pituitary, hypothalamus, and spinal cord. SSTR4 is expressed in fetal brain, hypothalamus and spinal cord but not pituitary. SSTR5 mRNA is detectable in the pituitary and spinal cord by 14-16 weeks of fetal life. This mapping of SSTR mRNA expression patterns in human fetal neural tissues is an important first step toward our goal of determining the role of SST in the nervous system during early stages in human development. PMID:15062986

  9. Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil.

    PubMed

    Beyer, U; Roth, T; Schumacher, P; Maier, G; Unold, A; Frahm, A W; Fiebig, H H; Unger, C; Kratz, F

    1998-07-16

    One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their p

  10. Investigation of transferrin polymorphism in Garole sheep.

    PubMed

    Yadav, Devesh K; Taraphder, Subhash; Sahoo, Ajit K; Dhara, K C

    2010-03-01

    The aim of this study was to determine the genetics of polymorph systems of Transferrin in Garole sheep breed. The present study was conducted on 95 adult Garole sheep comprising 52 ewes and 43 rams, maintained at Sheep and Goat Breeding Farm of West Bengal University of Animal and Fishery Sciences, West Bengal, during the period from April-September, 2009. The polymorphism of transferrin was determined through SDS-Polyacrylamide gel electrophoresis technique. It was found that the transferrin type was controlled by five codominant alleles (TfA, TfB, TfC TfD and TfE) in Garole sheep. These five alleles, because of co-dominant nature of inheritance, determined the occurrence of nine transferrin genotypes in the analyzed flock. Four (TfAA, TfBB, TfCC and TfDD) of these were homozygous and the remaining five (TfAD, TfBC, TfBD, TfCD and TfDE) heterozygous. It was found that the TfDD genotype (0.263) was predominant while TfDE genotype (0.042) was least common in the analyzed flock. Frequencies of other genotypes were as: TfCD(0.242), TfBD(0.126), TfCC(0.084), TfBB(0.074), TfAA(0.063), TfAD and TfBC (0.053 for each genotype ) in whole population. From the result it was found that in whole population combined, the heterozygotic genotypic frequency (0.516) was more than that of homozygotic genotypic frequency (0.484). Considerable variations were recognized in the frequencies of transferrin alleles. In the whole population frequencies of transferrin alleles were found to be TfA = 0.089, TfB = 0.163, TfC = 0.232, TfD = 0.495 and TfE = 0.021. Transferrin system has shown an absence of genetic equilibrium among the analyzed herd (chi2 value = 51.31). In conclusion, there were polymorphism in Transferrin types and the presence of differences among the frequencies of the five alleles by categories could be a source of genetic variation in Garole sheep. PMID:20349135

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Singh, Rahul Pratap; Sharma, Gunjan; Sonali; Singh, Sanjay; Patne, Shashikant C U; Pandey, Bajarangprasad L; Koch, Biplob; Muthu, Madaswamy S

    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 IC50 values demonstrated that the transferrin conjugated MWCNT could be 136-fold more efficient than Docel™ after 24h 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™. PMID:27287127

  13. Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex.

    PubMed

    Kolumam, Ganesh; Chen, Mark Z; Tong, Raymond; Zavala-Solorio, Jose; Kates, Lance; van Bruggen, Nicholas; Ross, Jed; Wyatt, Shelby K; Gandham, Vineela D; Carano, Richard A D; Dunshee, Diana Ronai; Wu, Ai-Luen; Haley, Benjamin; Anderson, Keith; Warming, Søren; Rairdan, Xin Y; Lewin-Koh, Nicholas; Zhang, Yingnan; Gutierrez, Johnny; Baruch, Amos; Gelzleichter, Thomas R; Stevens, Dale; Rajan, Sharmila; Bainbridge, Travis W; Vernes, Jean-Michel; Meng, Y Gloria; Ziai, James; Soriano, Robert H; Brauer, Matthew J; Chen, Yongmei; Stawicki, Scott; Kim, Hok Seon; Comps-Agrar, Laëtitia; Luis, Elizabeth; Spiess, Christoph; Wu, Yan; Ernst, James A; McGuinness, Owen P; Peterson, Andrew S; Sonoda, Junichiro

    2015-07-01

    Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin. PMID:26288846

  14. Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

    PubMed Central

    Kolumam, Ganesh; Chen, Mark Z.; Tong, Raymond; Zavala-Solorio, Jose; Kates, Lance; van Bruggen, Nicholas; Ross, Jed; Wyatt, Shelby K.; Gandham, Vineela D.; Carano, Richard A.D.; Dunshee, Diana Ronai; Wu, Ai-Luen; Haley, Benjamin; Anderson, Keith; Warming, Søren; Rairdan, Xin Y.; Lewin-Koh, Nicholas; Zhang, Yingnan; Gutierrez, Johnny; Baruch, Amos; Gelzleichter, Thomas R.; Stevens, Dale; Rajan, Sharmila; Bainbridge, Travis W.; Vernes, Jean-Michel; Meng, Y. Gloria; Ziai, James; Soriano, Robert H.; Brauer, Matthew J.; Chen, Yongmei; Stawicki, Scott; Kim, Hok Seon; Comps-Agrar, Laëtitia; Luis, Elizabeth; Spiess, Christoph; Wu, Yan; Ernst, James A.; McGuinness, Owen P.; Peterson, Andrew S.; Sonoda, Junichiro

    2015-01-01

    Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin. PMID:26288846

  15. Complex of transferrin with ruthenium for medical applications. [Ru 97, Ru 103

    DOEpatents

    Richards, P.; Srivastava, S.C.; Meinken, G.E.

    1980-11-03

    A novel Ruthenium-transferrin complex, prepared by reacting iron-free human transferrin dissolved in a sodium acetate solution at pH 7 with ruthenium by heating at about 40/sup 0/C for about 2 hours, and purifying said complex by means of gel chromatography with pH 7 sodium acetate as eluent. The mono- or di-metal complex produced can be used in nuclear medicine in the diagnosis and/or treatment of tumors and abscesses. Comparitive results with Ga-67-citrate, which is the most widely used tumor-localizing agent in nuclear medicine, indicate increased sensitivity of detection and greater tumor uptake with the Ru-transferrin complex.

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

    NASA Technical Reports Server (NTRS)

    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.

  17. A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand*

    PubMed Central

    Sun, Yi; Vandenbriele, Christophe; Kauskot, Alexandre; Verhamme, Peter; Hoylaerts, Marc F.; Wright, Gavin J.

    2015-01-01

    Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (KD ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation. PMID:25713122

  18. Conserved Regions of Gonococcal TbpB Are Critical for Surface Exposure and Transferrin Iron Utilization

    PubMed Central

    Ostberg, Karen L.; DeRocco, Amanda J.; Mistry, Shreni D.; Dickinson, Mary Kathryne

    2013-01-01

    The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system. PMID:23836816

  19. Identification of TbpA residues required for transferrin-iron utilization by Neisseria gonorrhoeae.

    PubMed

    Noto, Jennifer M; Cornelissen, Cynthia Nau

    2008-05-01

    Neisseria gonorrhoeae requires iron for survival in the human host and therefore expresses high-affinity receptors for iron acquisition from host iron-binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter critical for iron acquisition, while TbpB is a surface-exposed lipoprotein that increases the efficiency of iron uptake. The precise mechanism by which TbpA mediates iron acquisition has not been elucidated; however, the process is distinct from those of characterized siderophore transporters. Similar to these TonB-dependent transporters, TbpA is proposed to have two distinct domains, a beta-barrel and a plug domain. We hypothesize that the TbpA plug coordinates iron and therefore potentially functions in multiple steps of transferrin-mediated iron acquisition. To test this hypothesis, we targeted a conserved motif within the TbpA plug domain and generated single, double, and triple alanine substitution mutants. Mutagenized TbpAs were expressed on the gonococcal cell surface and maintained wild-type transferrin binding affinity. Single alanine substitution mutants internalized iron at wild-type levels, while the double and triple mutants showed a significant decrease in iron uptake. Moreover, the triple alanine substitution mutant was unable to grow on transferrin as a sole iron source; however, expression of TbpB compensated for this defect. These data indicate that the conserved motif between residues 120 and 122 of the TbpA plug domain is critical for transferrin-iron utilization, suggesting that this region plays a role in iron acquisition that is shared by both TbpA and TbpB. PMID:18347046

  20. Transferrin-a modulates hepcidin expression in zebrafish embryos

    PubMed Central

    Gibert, Yann; Holzheimer, Jason L.; Lattanzi, Victoria J.; Burnett, Sarah F.; Dooley, Kimberly A.; Wingert, Rebecca A.; Zon, Leonard I.

    2009-01-01

    The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a–dependent pathway in the zebrafish embryo. PMID:19047682

  1. Phylogenomic analysis of transferrin family from animals and plants.

    PubMed

    Bai, Lina; Qiao, Mu; Zheng, Rong; Deng, Changyan; Mei, Shuqi; Chen, Wanping

    2016-03-01

    Transferrins have been identified in animals and green algae, and they consist of a family of evolutionarily related proteins that play a central role in iron transport, immunity, growth and differentiation. This study assessed the transferrin genes among 100 genomes from a wide range of animal and plant kingdoms. The results showed that putative transferrins were widespread in animals, but their gene quantity and type differ greatly between animal groups. Generally, Mammalia possess abundant transferrin genes, whereas Trematoda contain few ones. Melanotransferrin and serotransferrin are widely distributed in vertebrates, while melanotransferrin-like and transferrin-like 1 are frequent in invertebrates. However, only a few plant species detected putative transferrins, and a novel transferrin member was first uncovered in Angiospermae and Pteridophyta. The structural comparison among transferrin family members revealed seven very well-repeated and conserved characteristic motifs, despite a considerable variation in the overall sequences. The phylogenetic analysis suggested that gene duplication, gene loss and horizontal transfer contributed to the diversification of transferrin family members, and their inferred evolutionary scenario was proposed. These findings help to the understanding of transferrin distribution, characteristic motifs and residues, and evolutionary process. PMID:26655280

  2. Antigenic and sequence diversity in gonococcal transferrin-binding protein A.

    PubMed

    Cornelissen, C N; Anderson, J E; Boulton, I C; Sparling, P F

    2000-08-01

    Neisseria gonorrhoeae is a gram-negative pathogen that is capable of satisfying its iron requirement with human iron-binding proteins such as transferrin and lactoferrin. Transferrin-iron utilization involves specific binding of human transferrin at the cell surface to what is believed to be a complex of two iron-regulated, transferrin-binding proteins, TbpA and TbpB. The genes encoding these proteins have been cloned and sequenced from a number of pathogenic, gram-negative bacteria. In the current study, we sequenced four additional tbpA genes from other N. gonorrhoeae strains to begin to assess the sequence diversity among gonococci. We compared these sequences to those from other pathogenic bacteria to identify conserved regions that might be important for the structure and function of these receptors. We generated polyclonal mouse sera against synthetic peptides deduced from the TbpA sequence from gonococcal strain FA19. Most of these synthetic peptides were predicted to correspond to surface-exposed regions of TbpA. We found that, while most reacted with denatured TbpA in Western blots, only one antipeptide serum reacted with native TbpA in the context of intact gonococci, consistent with surface exposure of the peptide to which this serum was raised. In addition, we evaluated a panel of gonococcal strains for antigenic diversity using these antipeptide sera. PMID:10899879

  3. High-level production of animal-free recombinant transferrin from saccharomyces cerevisiae

    PubMed Central

    2010-01-01

    Background Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin. Results The first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 μm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin. Conclusions A Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics. PMID:21083917

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

    PubMed Central

    Lesley, J F; Schulte, R J

    1984-01-01

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

  5. Construction and Characterization of Moraxella catarrhalis Mutants Defective in Expression of Transferrin Receptors

    PubMed Central

    Luke, Nicole R.; Campagnari, Anthony A.

    1999-01-01

    We have previously reported the construction of an isogenic mutant defective in expression of OmpB1, the TbpB homologue, in Moraxella catarrhalis 7169. In this report, we have extended these studies by constructing and characterizing two new isogenic mutants in this clinical isolate. One mutant is defective in expression of TbpA, and the other mutant is defective in expression of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sequencing. In vitro growth studies, comparing all three mutants, demonstrated that the tbpA mutant and the tbpAB mutant were severely limited in their ability to grow with human holotransferrin as the sole source of iron. In contrast, the ompB1 (tbpB) mutant was capable of utilizing iron from human transferrin, although not to the extent of the parental strain. While affinity chromatography with human holotransferrin showed that each Tbp was capable of binding independently to transferrin, solid-phase transferrin binding studies using whole cells demonstrated that the tbpA mutant exhibited binding characteristics similar to those seen with the wild-type bacteria. However, the ompB1 (tbpB) mutant exhibited a diminished capacity for binding transferrin, and no binding was detected with the double mutant. These data suggest that the M. catarrhalis TbpA is necessary for the acquisition of iron from transferrin. In contrast, TbpB is not essential but may serve as a facilitory protein that functions to optimize this process. Together these mutants are essential to provide a more thorough understanding of iron acquisition mechanisms utilized by M. catarrhalis. PMID:10531234

  6. The Latency-Associated UL138 Gene Product of Human Cytomegalovirus Sensitizes Cells to Tumor Necrosis Factor Alpha (TNF-α) Signaling by Upregulating TNF-α Receptor 1 Cell Surface Expression ▿

    PubMed Central

    Montag, Christina; Wagner, Jutta Annabella; Gruska, Iris; Vetter, Barbara; Wiebusch, Lüder; Hagemeier, Christian

    2011-01-01

    Many viruses antagonize tumor necrosis factor alpha (TNF-α) signaling in order to counteract its antiviral properties. One way viruses achieve this goal is to reduce TNF-α receptor 1 (TNFR1) on the surface of infected cells. Such a mechanism is also employed by human cytomegalovirus (HCMV), as recently reported by others and us. On the other hand, TNF-α has also been shown to foster reactivation of HCMV from latency. By characterizing a new variant of HCMV AD169, we show here that TNFR1 downregulation by HCMV only becomes apparent upon infection of cells with HCMV strains lacking the so-called ULb′ region. This region contains genes involved in regulating viral immune escape, cell tropism, or latency and is typically lost from laboratory strains but present in low-passage strains and clinical isolates. We further show that although ULb′-positive viruses also contain the TNFR1-antagonizing function, this activity is masked by a dominant TNFR1 upregulation mediated by the ULb′ gene product UL138. Isolated expression of UL138 in the absence of viral infection upregulates TNFR1 surface expression and can rescue both TNFR1 reexpression and TNF-α responsiveness of cells infected with an HCMV mutant lacking the UL138-containing transcription unit. Given that the UL138 gene product is one of the few genes recognized to be expressed during HCMV latency and the known positive effects of TNF-α on viral reactivation, we suggest that via upregulating TNFR1 surface expression UL138 may sensitize latently infected cells to TNF-α-mediated reactivation of HCMV. PMID:21880774

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

    PubMed

    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. PMID:27187189

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

    PubMed Central

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

    1980-01-01

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

  9. Transferrin conjugates of doxorubicin: synthesis, characterization, cellular uptake, and in vitro efficacy.

    PubMed

    Kratz, F; Beyer, U; Roth, T; Tarasova, N; Collery, P; Lechenault, F; Cazabat, A; Schumacher, P; Unger, C; Falken, U

    1998-03-01

    One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing suitable carrier proteins. Thus, thiolated human serum transferrin was conjugated with four maleimide derivatives of doxorubicin that differed in the stability of the chemical link between drug and spacer. Of the maleimide derivatives, 3-maleimidobenzoic or 4-maleimidophenylacetic acid was bound to the 3'-amino position of doxorubicin through a benzoyl or phenylacetyl amide bond, and 3-maleimidobenzoic acid hydrazide or 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto position through a benzoyl hydrazone or phenylacetyl hydrazone bond. The acid-sensitive transferrin conjugates prepared with the carboxylic hydrazone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line comparable to that of the free drug (employing the BrdU (5-bromo-2'-deoxyuridine) incorporation assay and tritiated thymidine incorporation assay, respectively, IC50 approximately 0.1-1 mM), whereas conjugates with the amide derivatives showed no activity. Furthermore, antiproliferative activity of the most active transferrin conjugate (i.e. the conjugate containing a benzoyl hydrazone link) was demonstrated in the LXFL 529 lung carcinoma cell line employing a sulforhodamine B assay. In contrast to in vitro studies in tumor cells, cell culture experiments performed with human endothelial cells (HUVEC) showed that the acid-sensitive transferrin conjugates of doxorubicin were significantly less active than free doxorubicin (IC50 values approximately 10-40 higher by the BrdU incorporation assay), indicating selectivity of the doxorubicin-transferrin conjugates for tumor cells. Fluorescence microscopy studies in the MDA-MB-468 breast cancer cell showed that free doxorubicin accumulates in the cell nucleus, whereas doxorubicin of the transferrin conjugates is found localized primarily

  10. GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

    PubMed

    Wei, M L; Bonzelius, F; Scully, R M; Kelly, R B; Herman, G A

    1998-02-01

    The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at

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

    PubMed

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

    2016-01-01

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

  12. Iterative endocytosis of transferrin by K562 cells.

    PubMed Central

    Young, S P; Bomford, A

    1994-01-01

    The effect of iron on the exocytosis of transferrin by K562 cells was studied by first allowing the cells to endocytose apotransferrin or diferric transferrin. Subsequent release of the apotransferrin was very rapid with a t 1/2 of 3.01 min, compared with 5.5 min for diferric transferrin. Release of apotransferrin was slowed by the weak base methylamine, t 1/2 8.0 min, but the effect of this agent was substantially greater when iron-transferrin was used, t 1/2 18.65 min, suggesting that methylamine affects both iron removal and receptor recycling. Release of iron-transferrin could be accelerated to a rate comparable with that of apotransferrin by addition of the permeant iron-chelator desferrioxamine. The difference in the rates of release of different forms of the protein could be explained by the re-endocytosis of the iron-rich protein, a process detected by the accelerated release of transferrin when the cells were washed in medium at pH 5.5 containing an iron-chelator or treated with a protease-containing medium to digest transferrin accessible at the cell surface. It appears that in cells incubated under control conditions, re-endocytosis of transferrin, which is incompletely depleted of iron, occurs and that a transferrin molecule may make two passes through the cell before all the iron is removed. This mechanism helps to explain why very little iron-transferrin is released from cells and why the efficiency of the iron uptake process is so high. PMID:8129715

  13. Monitoring pulmonary vascular permeability using radiolabeled transferrin

    SciTech Connect

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    PubMed Central

    Dixit, Suraj; Novak, Thomas; Miller, Kayla; Zhu, Yun; Kenney, Malcolm E.

    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 un-targeted 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. PMID:25519743

  16. The transferrin-iron import system from pathogenic Neisseria species.

    PubMed

    Noinaj, Nicholas; Buchanan, Susan K; Cornelissen, Cynthia Nau

    2012-10-01

    Two pathogenic species within the genus Neisseria cause the diseases gonorrhoea and meningitis. While vaccines are available to protect against four N. meningitidis serogroups, there is currently no commercial vaccine to protect against serogroup B or against N. gonorrhoeae. Moreover, the available vaccines have significant limitations and with antibiotic resistance becoming an alarming issue, the search for effective vaccine targets to elicit long-lasting protection against Neisseria species is becoming more urgent. One strategy for vaccine development has targeted the neisserial iron import systems. Without iron, the Neisseriae cannot survive and, therefore, these iron import systems tend to be relatively well conserved and are promising vaccine targets, having the potential to offer broad protection against both gonococcal and meningococcal infections. These efforts have been boosted by recent reports of the crystal structures of the neisserial receptor proteins TbpA and TbpB, each solved in complex with human transferrin, an iron binding protein normally responsible for delivering iron to human cells. Here, we review the recent structural reports and put them into perspective with available functional studies in order to derive the mechanism(s) for how the pathogenic Neisseriae are able to hijack human iron transport systems for their own survival and pathogenesis. PMID:22957710

  17. Evolutionary Diversification of the Vertebrate Transferrin Multi-gene Family

    PubMed Central

    Hughes, Austin L.; Friedman, Robert

    2014-01-01

    In a phylogenetic analysis of vertebrate transferrins (TFs), six major clades (subfamilies) were identified: (1) S, the mammalian serotransferrins; (2) ICA, the mammalian inhibitor of carbonic anhydrase (ICA) homologs; (3) L, the mammalian lactoferrins; (4) O, the ovotransferrins of birds and reptiles; (4) M, the melanotransferrins of bony fishes, amphibians, reptiles, birds, and mammals; and (5) 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: (1) in the common ancestor of the M subfamily; (2) in the common ancestor of the M-like subfamily; and (3) 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 a 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. PMID:25142446

  18. TRANSFERRIN: VARIATIONS IN BLOOD SERUM OF RED HOWLER MONKEYS.

    PubMed

    SCHOEN, M A; ARENDS, T

    1964-11-01

    Blood serum samples from 33 red howler monkeys (Alouatta seniculus) were examined. Three different phenotypes were found and denominated A, B, and C. Four serums could not be classified because their transferrin apparently did not bind iron-59, possibly owing to saturation. A difference was observed in the electrophoretic migration and pattern of the transferrins in these monkeys compared with those of other primates. PMID:14197564

  19. Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT).

    PubMed

    Helander, Anders; Wielders, Jos; Anton, Raymond; Arndt, Torsten; Bianchi, Vincenza; Deenmamode, Jean; Jeppsson, Jan-Olof; Whitfield, John B; Weykamp, Cas; Schellenberg, François

    2016-08-01

    Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures. PMID:27221205

  20. Pseudomonas and neutrophil products modify transferrin and lactoferrin to create conditions that favor hydroxyl radical formation.

    PubMed Central

    Britigan, B E; Edeker, B L

    1991-01-01

    In vivo most extracellular iron is bound to transferrin or lactoferrin in such a way as to be unable to catalyze the formation of hydroxyl radical from superoxide (.O2-) and hydrogen peroxide (H2O2). At sites of Pseudomonas aeruginosa infection bacterial and neutrophil products could possibly modify transferrin and/or lactoferrin forming catalytic iron complexes. To examine this possibility, diferrictransferrin and diferriclactoferrin which had been incubated with pseudomonas elastase, pseudomonas alkaline protease, human neutrophil elastase, trypsin, or the myeloperoxidase product HOCl were added to a hypoxanthine/xanthine oxidase .O2-/H2O2 generating system. Hydroxyl radical formation was only detected with pseudomonas elastase treated diferrictransferrin and, to a much lesser extent, diferriclactoferrin. This effect was enhanced by the combination of pseudomonas elastase with other proteases, most prominently neutrophil elastase. Addition of pseudomonas elastase-treated diferrictransferrin to stimulated neutrophils also resulted in hydroxyl radical generation. Incubation of pseudomonas elastase with transferrin which had been selectively iron loaded at either the NH2- or COOH-terminal binding site yielded iron chelates with similar efficacy for hydroxyl radical catalysis. Pseudomonas elastase and HOCl treatment also decreased the ability of apotransferrin to inhibit hydroxyl radical formation by a Fe-NTA supplemented hypoxanthine/xanthine oxidase system. However, apotransferrin could be protected from the effects of HOCl if bicarbonate anion was present during the incubation. Apolactoferrin inhibition of hydroxyl radical generation was unaffected by any of the four proteases or HOCl. Alteration of transferrin by enzymes and oxidants present at sites of pseudomonas and other bacterial infections may increase the potential for local hydroxyl radical generation thereby contributing to tissue injury. Images PMID:1655825

  1. Obtaining of pure transferrins D, M and R from equine serum and determination of transferrin level in relation to phenotype.

    PubMed

    Didkowski, S; Kaminski, M; Kerjan, P; Tomaszewska-Guszkiewicz, K; Zurkowski, M

    1984-01-01

    By the method of precipitation with Rivanol (2-ethoxy-6,9-diaminoacridine lactate) and ammonium sulphate followed by chromatography on DEAE cellulose three genetic variants of transferrin were purified from equine serum: D, M and R. Their molecular mass determined in this study was 80 000, and it was identical for all three variants, which differed slightly in their amino acid composition. The protein level was determined in the serum of 535 two-year-old thoroughbred English horses by the method of rocket immunoelectrophoresis using antibodies obtained against three transferrins. The individual variability of the protein level in horses of the same phenotype was fairly high (variability index 9-15%). No differences were observed in the transferrin level related to sex. It was found that the presence of D, F and H alleles was connected with a higher serum transferrin level, while O and R alleles were connected with a lower level. PMID:6545995

  2. Defective selection of thymic regulatory T cells accompanies autoimmunity and pulmonary infiltrates in Tcra-deficient mice double transgenic for human La/SS-B and human La-specific T cell receptor1

    PubMed Central

    Yaciuk, Jane C.; Pan, Yujun; Schwarz, Karen; Pan, Zi-jian; Maier-Moore, Jacen S.; Kosanke, Stanley D.; Lawrence, Christina; Farris, A. Darise

    2014-01-01

    A human La/SS-B (hLa)-specific TCR/hLa neo-self antigen double transgenic mouse model was developed and used to investigate cellular tolerance and autoimmunity to the ubiquitous RNA-binding La antigen often targeted in systemic lupus erythematosus and Sjögren's syndrome. Extensive thymic clonal deletion of CD4+ T cells occurred in H-2k/k double transgenic mice presenting high levels of the I-Ek-restricted hLa T cell epitope. In contrast, deletion was less extensive in H-2k/b double transgenic mice presenting lower levels of the epitope, and some surviving thymocytes were positively selected as thymic regulatory T cells (tTreg). These mice remained serologically tolerant to hLa and healthy. H-2k/b double transgenic mice deficient of all endogenous Tcra genes, a deficiency known to impair Treg development and function, produced IgG anti-hLa autoantibodies and displayed defective tTreg development. These autoimmune mice had interstitial lung disease characterized by lymphocytic aggregates containing transgenic T cells with an activated, effector memory phenotype. Salivary gland infiltrates were notably absent. Thus, expression of nuclear hLa antigen induces thymic clonal deletion and tTreg selection, and lymphocytic infiltration of the lung is a consequence of La-specific CD4+ T cell autoimmunity. PMID:25582858

  3. SIDEROPHILIN METAL COORDINATION. 1. COMPLEXATION OF THORIUM BY TRANSFERRIN: STRUCTURE-FUNCTION IMPLICATIONS

    SciTech Connect

    Harris, Wesley R.; Carrano, Carl J.; Pecoraro, Vincent L.; Raymond, Kenneth N.

    1980-08-01

    As part of a program to develop actinide-specific sequestering agents, the coordination of actinide ions by human transferrin is being investigated. Therapeutically useful synthetic ligands must be able to compete with this iron-transport protein for the bound actinide ion. As in the Fe(III) complex of the native protein, two Th(IV) ions bind at pH 7. This coordination has been monitored at several pH values by using difference ultraviolet spectroscopy. The corresponding coordination of a phenolic ligand, ethylene-bis-(o-hydroxyphenylglycine) [EHPG], has been used to determine {Delta}{epsilon} for a tyrosyl group coordinated to Th(IV), in contrast to the common practice of assuming the {Delta}{epsilon} for protons and all metal ions is the same. This in turn is used to determine, from the observed {Delta}{epsilon} upon protein coordination, the number of transferrin tyrosine residues that coordinate. Maxima in the Th(IV) + EHPG difference UV spectra occur at 292 and 238 nm, with corresponding {Delta}{epsilon} values per phenolic group of 2330 and 8680 cm{sup -1} M{sup -1}, respectively. At pH 7.2, the Th(IV) transferrin spectrum is closely similar to the TH(IV) EHPG spectrum, with maxima at 292 and 240 nm. The {Delta}{epsilon} at 240 nm reaches a maximum of 24700 cm{sup -1} M{sup -1}, which corresponds to coordination of three tyrosine residues in the dithorium-transferrin complex; the stronger binding site (“A” or C-terminal) coordinates via two tyrosines and the weaker (“B” or N-terminal) via one. There is evidence suggesting that the N-terminal site is slightly smaller than the C-terminal site; while Th(IV) easily fits into the C-terminal site, the large ionic radius of Th(IV) makes this ion of borderline size to fit into the N-terminal site. This may be an important biological difference between Th(IV) and the slightly smaller Pu(IV), which should easily fit into both sites. At pH values below 7, the complexation of Th(IV) by transferrin decreases

  4. The Intracellular Trafficking Pathway of Transferrin

    PubMed Central

    Mayle, Kristine M.; Le, Alexander M.; Kamei, Daniel T.

    2011-01-01

    Background Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. Scope of Review We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). Major Conclusions Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. General Significance Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs. PMID:21968002

  5. Galectin-4-mediated transcytosis of transferrin receptor

    PubMed Central

    Perez Bay, Andres E.; Schreiner, Ryan; Benedicto, Ignacio; Rodriguez-Boulan, Enrique J.

    2014-01-01

    ABSTRACT Some native epithelia, for example, retinal pigment epithelium (RPE) and kidney proximal tubule (KPT), constitutively lack the basolateral sorting adaptor AP-1B; this results in many basolateral plasma membrane proteins being repositioned to the apical domain, where they perform essential functions for their host organs. We recently reported the underlying apical polarity reversal mechanism: in the absence of AP-1B-mediated basolateral sorting, basolateral proteins are shuttled to the apical plasma membrane through a transcytotic pathway mediated by the plus-end kinesin KIF16B. Here, we demonstrate that this apical transcytotic pathway requires apical sorting of basolateral proteins, which is mediated by apical signals and galectin-4. Using RPE and KPT cell lines, and AP-1B-knockdown MDCK cells, we show that mutation of the N-glycan linked to N727 in the basolateral marker transferrin receptor (TfR) or knockdown of galectin-4 inhibits TfR transcytosis to apical recycling endosomes and the apical plasma membrane, and promotes TfR lysosomal targeting and subsequent degradation. Our results report a new role of galectins in basolateral to apical epithelial transcytosis. PMID:25179596

  6. The protective role of transferrin in Müller glial cells after iron-induced toxicity

    PubMed Central

    Fontaine, Isabelle; Jonet, Laurent; Guillou, Florian; Behar-Cohen, Francine; Courtois, Yves; Jeanny, Jean-Claude

    2008-01-01

    Purpose Transferrin (Tf) expression is enhanced by aging and inflammation in humans. We investigated the role of transferrin in glial protection. Methods 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 FeCl3-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. Results 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. Conclusions 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

  7. HFE and transferrin directly compete for transferrin receptor in solution and at the cell surface.

    PubMed

    Giannetti, Anthony M; Björkman, Pamela J

    2004-06-11

    Transferrin receptor (TfR) is a dimeric cell surface protein that binds both the serum iron transport protein transferrin (Fe-Tf) and HFE, the protein mutated in patients with the iron overload disorder hereditary hemochromatosis. HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, but HFE binding to TfR lowers the apparent affinity of the Fe-Tf/TfR interaction. This apparent affinity reduction could result from direct competition between HFE and Fe-Tf for their overlapping binding sites on each TfR polypeptide chain, from negative cooperativity, or from a combination of both. To explore the mechanism of the affinity reduction, we constructed a heterodimeric TfR that contains mutations such that one TfR chain binds only HFE and the other binds only Fe-Tf. Binding studies using a heterodimeric form of soluble TfR demonstrate that TfR does not exhibit cooperativity in heterotropic ligand binding, suggesting that some or all of the effects of HFE on iron homeostasis result from competition with Fe-Tf for TfR binding. Experiments using transfected cell lines demonstrate a physiological role for this competition in altering HFE trafficking patterns. PMID:15056661

  8. Transferrin Promotes Endothelial Cell Migration and Invasion: Implication in Cartilage Neovascularization

    PubMed Central

    Carlevaro, Mariella F.; Albini, Adriana; Ribatti, Domenico; Gentili, Chiara; Benelli, Roberto; Cermelli, Silvia; Cancedda, Ranieri; Cancedda, Fiorella Descalzi

    1997-01-01

    During endochondral bone formation, avascular cartilage differentiates to hypertrophic cartilage that then undergoes erosion and vascularization leading to bone deposition. Resting cartilage produces inhibitors of angiogenesis, shifting to production of angiogenic stimulators in hypertrophic cartilage. A major protein synthesized by hypertrophic cartilage both in vivo and in vitro is transferrin. Here we show that transferrin is a major angiogenic molecule released by hypertrophic cartilage. Endothelial cell migration and invasion is stimulated by transferrins from a number of different sources, including hypertrophic cartilage. Checkerboard analysis demonstrates that transferrin is a chemotactic and chemokinetic molecule. Chondrocyte-conditioned media show similar properties. Polyclonal anti-transferrin antibodies completely block endothelial cell migration and invasion induced by purified transferrin and inhibit the activity produced by hypertrophic chondrocytes by 50–70% as compared with controls. Function-blocking mAbs directed against the transferrin receptor similarly reduce the endothelial migratory response. Chondrocytes differentiating in the presence of serum produce transferrin, whereas those that differentiate in the absence of serum do not. Conditioned media from differentiated chondrocytes not producing transferrin have only 30% of the endothelial cell migratory activity of parallel cultures that synthesize transferrin. The angiogenic activity of transferrins was confirmed by in vivo assays on chicken egg chorioallantoic membrane, showing promotion of neovascularization by transferrins purified from different sources including conditioned culture medium. Based on the above results, we suggest that transferrin is a major angiogenic molecule produced by hypertrophic chondrocytes during endochondral bone formation. PMID:9087450

  9. Development and Evaluation of Transferrin-Stabilized Paclitaxel Nanocrystal Formulation

    PubMed Central

    Lu, Ying; Wang, Zhao-hui; Li, Tonglei; McNally, Helen; Park, Kinam; Sturek, Michael

    2014-01-01

    The aim of the present study was to prepare and evaluate a paclitaxel nanocrystal-based formulation stabilized by serum protein transferrin in a non-covalent manner. The pure paclitaxel nanocrystals were first prepared using an antisolvent precipitation method augmented by sonication. The serum protein transferrin was selected for use after evaluating the stabilizing effect of several serum proteins including albumin and immunoglobulin G. The formulation contained approximately 55~60% drug and was stable for at least 3 months at 4 °C. In vivo antitumor efficacy studies using mice inoculated with KB cells demonstrate significantly higher tumor inhibition rate of 45.1% for paclitaxel-transferrin formulation compared to 28.8% for paclitaxel nanosuspension treatment alone. Interestingly, the Taxol® formulation showed higher antitumor activity than the paclitaxel-transferrin formulation, achieving a 93.3% tumor inhibition rate 12 days post initial dosing. However, the paclitaxel-transferrin formulation showed a lower level of toxicity, which is indicated by steady increase in body weight of mice over the treatment period. In comparison, treatment with Taxol® resulted in toxicity issues as body weight decreased. These results suggest the potential benefit of using a serum protein in a non-covalent manner in conjunction with paclitaxel nanocrystals as a promising drug delivery model for anticancer therapy. PMID:24378441

  10. Manganese Transport via the Transferrin Mechanism

    PubMed Central

    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. Mn2+ is transported into cells via a number of mechanisms, while Mn3+ 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 Mn2+, Mn3+ and their complexes to which these cells are exposed; this complicates understanding the contributions of each transporter to Mn toxicity. While uptake of Fe3+ via the Tf mechanism is well understood, uptake of Mn3+ via this mechanism has not been systematically studied. The stability of the Mn3+Tf complex allowed us to form and purify this complex and label it with a fluorescent (Alexa green) tag. Using purified and labeled Mn3+Tf and biophysical tools, we have developed a novel approach to study Mn3+Tf transport independently of other Mn transport mechanisms. This approach was used to compare the uptake of Mn3+Tf into neuronal cell lines with published descriptions of Fe3+ 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 Mn3+ is transported by the Tf mechanism similarly to Fe3+Tf transport; although Mn3+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. PMID:23146871

  11. Common antigenic domains in transferrin-binding protein 2 of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae type b.

    PubMed

    Stevenson, P; Williams, P; Griffiths, E

    1992-06-01

    There is now considerable evidence to show that in the Neisseria and Haemophilus species, membrane receptors specific for either transferrin or lactoferrin are involved in the acquisition of iron from these glycoproteins. In Neisseria meningitidis, the transferrin receptor appears to consist of two proteins, one of which (TBP 1) has an M(r) of 95,000 and the other of which (TBP 2) has an M(r) ranging from 68,000 to 85,000, depending on the strain; TBP 2 binds transferrin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, but TBP 1 does not do so. The relative contributions of these two proteins to the binding reaction observed with intact cells and to iron uptake are presently unknown. However, they are being considered as potential components of a group B meningococcal vaccine. Analogous higher- and lower-molecular-weight proteins associated with transferrin binding have been found in N. gonorrhoeae and Haemophilus influenzae. Previous work with polyclonal antibodies raised in mice with whole cells of iron-restricted N. meningitidis showed that the meningococcal TBP 2 exhibits considerable antigenic heterogeneity. Here, we report that antiserum against purified TBP 2 from one strain of N. meningitidis cross-reacts on immunoblotting with the TBP 2 of all meningococcal isolates examined, as well as with the TBP 2 of N. gonorrhoeae. This antiserum also cross-reacted with the TBP 2 of several strains of H. influenzae type b, thus showing the presence of common antigenic domains among these functionally equivalent proteins in different pathogens; no cross-reaction was detected with a purified sample of the human transferrin receptor. PMID:1587606

  12. Serum Immunoglobulin and Transferrin Levels After Childhood Splenectomy

    PubMed Central

    Schumacher, M. J.

    1970-01-01

    IgG, IgA, IgM, and transferrin levels were measured in sera from a group of children who had been subjected to splenectomy in the previous 10 years. In those children splenectomized for hereditary spherocytosis, idiopathic thrombocytopenic purpura, or traumatic rupture, mean IgM levels were significantly lower and mean transferrin levels were significantly higher than in sera from control children. Children splenectomized for thalassaemia major had a significantly raised mean IgG level, while children splenectomized for portal hypertension had a raised mean IgA level. However, IgG and IgA levels in patients who had suffered splenic rupture did not differ from control values. The finding of high transferrin levels after splenectomy supports the concept of an immunological function for this protein in addition to its iron-binding capacity. PMID:4191614

  13. Biological variability of transferrin saturation and unsaturated iron binding capacity

    PubMed Central

    Adams, PC; Reboussin, DM; Press, RD; Barton, JC; Acton, RT; Moses, GC; Leiendecker-Foster, C; McLaren, GD; Dawkins, FW; Gordeuk, VR; Lovato, L; Eckfeldt, JH

    2007-01-01

    Background Transferrin saturation is widely considered the preferred screening test for hemochromatosis. Unsaturated iron binding capacity has similar performance at lower cost. However, the within-person biological variability of both these tests may limit their ability at commonly used cut points to detect HFE C282Y homozygous patients. Methods The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 101,168 primary care participants for iron overload using tansferrin saturation, unsaturated iron binding capacity, ferritin and HFE C282Y and H63D genotyping. Transferrin saturation and unsaturated iron binding capacity were performed at initial screening and again when selected participants and controls returned for a clinical examination several months later. A missed case was defined as a C282Y homozygote who had transferrin saturation below cut point (45 % women, 50 % men) or unsaturated iron binding capacity above cut point (150 μmol/L women, 125 μmol/L men) at either the initial screening or clinical examination, or both, regardless of serum ferritin. Results There were 209 C282Y previously undiagnosed homozygotes with transferrin saturation and unsaturated iron binding capacity testing done at initial screening and clinical examination. Sixty-eight C282Y homozygotes (33%) would have been missed at these transferrin saturation cut points (19 men, 49 women, median SF 170 μg/L, first and third quartiles 50 and 474 μg/L), and 58 homozygotes (28 %) would have been missed at the unsaturated iron binding capacity cut points (20 men, 38 women, median SF 168 μg/L, quartiles 38 and 454 μg/L). There was no advantage to using fasting samples. Conclusions The within-person biological variability of transferrin saturation and unsaturated iron binding capacity limit their usefulness as an initial screening test for expressing C282Y homozygotes. PMID:17976429

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

    SciTech Connect

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

    1986-12-01

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

  15. The measurement of serum transferrin by iron-binding capacity

    PubMed Central

    Ramsay, W. N. M.

    1973-01-01

    Two criteria which ought to be satisfied by an acceptable method for the estimation of serum transferrin by iron-binding capacity are enunciated. A screening procedure is described which involves the sequential quantitative use of ion exchange and gel filtration. Its use in testing the extent to which two published methods fulfil the recommended criteria is described. Both methods give results for total iron-binding capacity which are 1-10% high because of the inclusion of non-transferrin iron. PMID:4752411

  16. A natural kinase-deficient variant of fibroblast growth factor receptor 1.

    PubMed

    Wang, L Y; Edenson, S P; Yu, Y L; Senderowicz, L; Turck, C W

    1996-08-01

    A fibroblast growth factor receptor 1 variant missing 37 amino acids from the carboxy-terminal tyrosine kinase catalytic domain was discovered in human lung fibroblasts and several other human cell lines. The receptor variant binds specifically to acidic fibroblast growth factor but has no tyrosine kinase activity. It was found that cellular transfectants expressing the fibroblast growth factor receptor 1 variant are mitogenically inactive and ligand binding to the receptor causes neither receptor autophosphorylation nor phospholipase C-gamma transphosphorylation. The fibroblast growth factor receptor 1 variant therefore represents an inactive receptor for acidic fibroblast growth factor. Since both kinase and kinase-deficient receptor forms are expressed in cells, it is conceivable that the kinase-deficient receptor plays an important role in regulating cellular responses elicited by acidic fibroblast growth factor stimulation. PMID:8756477

  17. Conservation and antigenic cross-reactivity of the transferrin-binding proteins of Haemophilus influenzae, Actinobacillus pleuropneumoniae and Neisseria meningitidis.

    PubMed

    Holland, J; Parsons, T R; Hasan, A A; Cook, S M; Stevenson, P; Griffiths, E; Williams, P

    1996-12-01

    Haemophilus influenzae acquires iron from the iron-transporting glycoprotein transferrin via a receptor-mediated process. This involves two outer-membrane transferrin-binding proteins (Tbps) termed Tbp1 and Tbp2 which show considerable preference for the human form of transferrin. Since the Tbps are attracting considerable attention as potential vaccine components, we used transferrin affinity chromatography to examine their conservation amongst 28 H. influenzae type b strains belonging to different outer-membrane-protein subtypes as well as six non-typable strains. Whole cells of all type b and non-typable strains examined bound human transferrin; whilst most strains possessed a Tbp1 of approximately 105 kDa, the molecular mass of Tbp2 varied from 79 to 94 kDa. Antisera raised against affinity-purified native H. influenzae Tbp1/Tbp2 receptor complex cross-reacted on Western blots with the respective Tbps of all the Haemophilus strains examined. When used to probe Neisseria meningitidis Tbps, sera from each of four mice immunized with the Haemophilus Tbp1/2 complex recognized the 68 kDa Tbp2 of N. meningitidis strain B16B6 but not the 78 kDa Tbp2 of N. meningitidis strain 70942. Serum from one mouse also reacted weakly with Tbp1 of strain B16B6. Apart from a weak reaction with the Tbp2 of a serotype 5 strain, this mouse antiserum failed to recognize the Tbps of the porcine pathogen A. pleuropneumoniae. However, a monospecific polyclonal antiserum raised against the denatured Tbp2 of Neisseria meningitidis B16B6 recognized the Tbps of all Haemophilus and Actinobacillus strains examined. Since H. influenzae forms part of the natural flora of the upper respiratory tract, human sera were screened for the presence of antibodies to the Tbps. Sera from healthy adults contained antibodies which recognized both Tbp1 and Tbp2 from H. influenzae but not N. meningitidis. Convalescent sera from meningococcal meningitis patients contained antibodies which, on Western blots

  18. Chemotherapy-induced antitumor immunity requires formyl peptide receptor 1.

    PubMed

    Vacchelli, Erika; Ma, Yuting; Baracco, Elisa E; Sistigu, Antonella; Enot, David P; Pietrocola, Federico; Yang, Heng; Adjemian, Sandy; Chaba, Kariman; Semeraro, Michaela; Signore, Michele; De Ninno, Adele; Lucarini, Valeria; Peschiaroli, Francesca; Businaro, Luca; Gerardino, Annamaria; Manic, Gwenola; Ulas, Thomas; Günther, Patrick; Schultze, Joachim L; Kepp, Oliver; Stoll, Gautier; Lefebvre, Céline; Mulot, Claire; Castoldi, Francesca; Rusakiewicz, Sylvie; Ladoire, Sylvain; Apetoh, Lionel; Bravo-San Pedro, José Manuel; Lucattelli, Monica; Delarasse, Cécile; Boige, Valérie; Ducreux, Michel; Delaloge, Suzette; Borg, Christophe; André, Fabrice; Schiavoni, Giovanna; Vitale, Ilio; Laurent-Puig, Pierre; Mattei, Fabrizio; Zitvogel, Laurence; Kroemer, Guido

    2015-11-20

    Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer. We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1(-/-) mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity. Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes. Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses. PMID:26516201

  19. Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity

    PubMed Central

    Biville, Francis; Brézillon, Christophe; Giorgini, Dario; Taha, Muhamed-Kheir

    2014-01-01

    The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival. PMID:25290693

  20. Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors.

    PubMed

    Cornelissen, C N; Biswas, G D; Tsai, J; Paruchuri, D K; Thompson, S A; Sparling, P F

    1992-09-01

    The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor. PMID:1325963

  1. Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity.

    PubMed

    Zheng, Qing; Xiong, Yao-Ling; Su, Zhi-Jian; Zhang, Qi-Hao; Dai, Xiao-Yong; Li, Lin-Yan; Xiao, Xue; Huang, Ya-Dong

    2013-06-01

    Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells. PMID:23545225

  2. Nonbinding Site-Directed Mutants of Transferrin Binding Protein B Exhibit Enhanced Immunogenicity and Protective Capabilities

    PubMed Central

    Frandoloso, Rafael; Martínez-Martínez, Sonia; Calmettes, Charles; Fegan, Jamie; Costa, Estela; Curran, Dave; Yu, Rong-hua; Gutiérrez-Martín, César B.; Rodríguez-Ferri, Elías F.; Moraes, Trevor F.

    2014-01-01

    Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines. PMID:25547790

  3. A New Transferrin Receptor Aptamer Inhibits New World Hemorrhagic Fever Mammarenavirus Entry.

    PubMed

    Maier, Keith E; Jangra, Rohit K; Shieh, Kevin R; Cureton, David K; Xiao, Hui; Snapp, Erik L; Whelan, Sean P; Chandran, Kartik; Levy, Matthew

    2016-01-01

    Pathogenic New World hemorrhagic fever mammarenaviruses (NWM) utilize Glycoprotein 1 (GP1) to target the apical domain of the human transferrin receptor (hTfR) for facilitating cell entry. However, the conservation between their GP1s is low. Considering this and the slow evolutionary progression of mammals compared to viruses, therapeutic targeting of hTfR provides an attractive avenue for cross-strain inhibition and diminishing the likelihood of escape mutants. Aptamers present unique advantages for the development of inhibitors to vial entry, including ease of synthesis, lack of immunogenicity, and potentially cold-chain breaking solutions to diseases endemic to South America. Here, recognizing that in vivo competition with the natural ligand, transferrin (Tf), likely drove the evolution of GP1 to recognize the apical domain, we performed competitive in vitro selections against hTfR-expressing cells with supplemented Tf. The resultant minimized aptamer, Waz, binds the apical domain of the receptor and inhibits infection of human cells by recombinant NWM in culture (EC50 ~400 nmol/l). Aptamer multimerization further enhanced inhibition >10-fold (EC50 ~30 nmol/l). Together, our results highlight the ability to use a competitor to bias the outcome of a selection and demonstrate how avidity effects can be leveraged to enhance both aptamer binding and the potency of viral inhibition. PMID:27219515

  4. Targeted quantum dots fluorescence probes functionalized with aptamer and peptide for transferrin receptor on tumor cells

    NASA Astrophysics Data System (ADS)

    Zhang, Ming-Zhen; Yu, Rong-Na; Chen, Jun; Ma, Zhi-Ya; Zhao, Yuan-Di

    2012-12-01

    Quantum dots (QDs) fluorescent probes based on oligonucleotide aptamers and peptides with specific molecular recognition have attracted much attention. In this paper, CdSe/ZnS QDs probes for targeted delivery to mouse and human cells using aptamer GS24 and peptide T7 specific to mouse/human transferrin receptors were developed. Capillary electrophoresis analyses indicated that the optimal molar ratios of QDs to aptamer or peptide were 1:5. Fluorescence and confocal microscope imaging revealed QD-GS24 and QD-T7 probes were able to specifically recognize B16 cells and HeLa cells respectively. Quantitative flow cytometry analysis indicated the transportation of QD-GS24 or QD-T7 into cells could be promoted by corresponding free transferrin. Transmission electron microscopy confirmed the uptake of probes in cells and the effective intracellular delivery. MTT assay suggested the cytotoxicity of probes was related to the surface ligand, and aptamer GS24 (or peptide T7) could reduce the cytotoxicity of probes to a certain degree. The study has great significance for preparing QDs fluorescent probes using non-antibody target molecules.

  5. Molecular mechanism investigation of the neutralization of cadmium toxicity by transferrin.

    PubMed

    Wang, Jing; Wang, Jinhu; Song, Wei; Yang, Xinping; Zong, Wansong; Liu, Rutao

    2016-02-01

    Cadmium adversely affects the biological function of the liver. Transferrin might be involved in the detoxification system of cadmium. However, owing to the lack of investigation of the molecular mechanism of cadmium conjugating to transferrin, the role of transferrin in cadmium detoxification in the liver and how transferrin undergoes conformational and functional changes upon cadmium binding are not clear. In this article, we demonstrated the potential role of transferrin in the protection of the mouse primary hepatocytes against cadmium toxicity. After the incubation of hepatocytes with 10 and 100 μM CdCl2, pretreatment with transferrin significantly attenuated the reduction of cell viability in comparison with the samples treated with CdCl2 alone. Furthermore, a detailed molecular mechanism investigation of the interaction of CdCl2 with transferrin was reported using biophysical methods. Multi-spectroscopic measurements showed that CdCl2 formed complexes with transferrin and caused structural and conformational changes of transferrin. Isothermal titration calorimetry measurements revealed that transferrin has two classes of binding sites with different binding constants for CdCl2 binding. Hydrophobic forces and electrostatic forces are the major driving forces of the interaction. Preferred specific binding sites on transferrin were identified by dialysis experiments, molecular docking studies and molecular dynamics simulations. Upon low CdCl2 concentration exposure, no content of iron was released from transferrin because CdCl2 preferentially binds to the surface of transferrin molecules. Upon higher CdCl2 concentration exposure, the release of iron content from transferrin was observed due to the interaction of CdCl2 with the key residues around iron binding sites. PMID:26750974

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

    PubMed Central

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

    2015-01-01

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

  7. Transferrin In Korean Children With Attention Deficit Hyperactivity Disorder

    PubMed Central

    Kwon, Ho Jang; Ha, Mina; Kim, Eun Jung; Yoo, Seung Jin; Kim, Jong Wan; Paik, Ki Chung

    2011-01-01

    Objective The aim of the present study was to investigate the relationship between iron, ferritin, transferrin, total iron binding capacity (TIBC), hemoglobin, mean corpuscular volume (MCV) mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in children with ADHD. Methods MethodsaaThe sample consisted of 48 ADHD children and sex and age matched control children (a couple of 28 boys, 22 girls; age 6-8 years; mean±SD, 6.98±0.39). We diagnosed ADHD according to DSM-IV. ADHD symptoms were evaluated subjectively with Conners' Parent Rating Scales, Dupaul Parent ADHD Rating Scales. Subjects with ADHD and control were evaluated the hematology test including the iron, transferrin, MCV etc. Paired t test were used to evaluate the relation of a lot of hematology findings between ADHD and control group. Results The serum iron, ferritin, transferrin, TIBC, hemoglobin, MCV, MCH, and MCHC of ADHD group were respectively 80.92±33.33 ug/dL, 35.81±16.59 ng/mL, 248.42±44.15 mg/dL, 351.69±102.13 ug/dL, 12.78±0.71 g/dL, 82.94±2.58 fL, 27.18±1.12 uug, 32.79±1.12%. Otherwise the serum iron, ferritin, transferrin, TIBC, hemoglobin, MCV, MCH, and MCHC of control group were respectively 82.04±28.14 ug/dL, 37.05±18.28 ng/mL, 266.27±25.40 mg/dL, 352.77±89.54 ug/dL, 12.77±0.70 g/dL, 81.81±2.96 fL, 26.69±0.99 uug, 32.66±0.96%. A significant difference were found in the transferrin(t=2.63, p=0.011), MCV (t=2.19, p=0.034), and MCH (t=2.18, p=0.034). Conclusion These results suggested that lower transferrin levels might be related with ADHD symptoms. PMID:22216048

  8. Receptor-mediated uptake of labeled transferrin by embryonic chicken dorsal root ganglion neurons in culture.

    PubMed

    Markelonis, G J; Oh, T H; Park, L P; Azari, P; Max, S R

    1985-01-01

    Transferrin is a growth-promoting plasma protein which is known to occur within developing neurons. Since little information exists on the process by which transferrin is internalized by neurons, we studied this process using dissociated embryonic chicken dorsal root ganglion neurons in culture. Cultured dorsal root ganglion neurons were incubated in the presence of 3.75 nM (125)I-transferrin at 37°C, the cultures were extensively washed, the neurons were solubilized in a Triton-containing buffer and internalized (125)I-transferrin was quantified with a gamma counter. (125)I-transferrin was internalized in a linear fashion for at least 60 min, and this uptake was abolished by the presence of 1.25 μM unlabeled transferrin. No competition for the uptake of (125)I-transferrin was observed in the presence of 1.25 μM ovalbumin, cytochrome c, hemoglobin, insulin, horseradish peroxidase, aldolase or the carboxyl-terminal fragment ('half-site') of transferrin. By contrast, uptake was inhibited by approximately 50% in the presence of the ammo-terminal fragment ('half-site') of transferrin (1.25 μM) or in the presence of concanavalin A (1.25 μM). The binding of transferrin conjugated to fluorescein isothiocyanate to neurons at 4°C and its subsequent internalization at 37°C was demonstrated by fluorescence microscopy of unfixed cells following incubation of the neurons in the presence of the fluorescently labeled protein. Furthermore, the transferrin receptors were visualized immunocytochemically on the surface membranes of dorsal root ganglion neurons using rabbit antibodies directed against transferrin receptors from chicken reticulocytes. From these data, we conclude that transferrin is internalized by neurons via receptor-mediated endocytosis, and suggest that this protein may serve an important role in the development and survival of dorsal root ganglion neurons. PMID:24874753

  9. Bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant.

    PubMed

    Bergström, Moa Andresen; Isin, Emre M; Castagnoli, Neal; Milne, Claire E

    2011-10-01

    In the present work, the characterization of the biotransformation and bioactivation pathways of the cannabinoid receptor 1 antagonist rimonabant (Acomplia) is described. Rimonabant was approved in Europe in 2006 for the treatment of obesity but was withdrawn in 2008 because of a significant drug-related risk of serious psychiatric disorders. The aim of the present work is to characterize the biotransformation and potential bioactivation pathways of rimonabant in vitro in human and rat liver microsomes. The observation of a major iminium ion metabolite led us to perform reactive metabolite trapping, covalent binding to proteins, and time-dependent inhibition of cytochrome P450 3A4 studies. The major biotransformation pathways were oxidative dehydrogenation of the piperidinyl ring to an iminium ion, hydroxylation of the 3 position of the piperidinyl ring, and cleavage of the amide linkage. In coincubations with potassium cyanide, three cyanide adducts were detected. A high level of covalent binding of rimonabant in human liver microsomes was observed (920 pmol equivalents/mg protein). In coincubations with potassium cyanide and methoxylamine, the covalent binding was reduced by approximately 40 and 30%, respectively, whereas GSH had no significant effect on covalent binding levels. Rimonabant was also found to inhibit cytochrome P450 3A4 irreversibly in a time-dependent manner. In view of these findings, it is noteworthy that, to date, no toxicity findings related to the formation of reactive metabolites from rimonabant have been reported. PMID:21733882

  10. Quantitative synchrotron X-ray fluorescence study of the penetration of transferrin-conjugated gold nanoparticles inside model tumour tissues

    NASA Astrophysics Data System (ADS)

    Liu, Tianqing; Kempson, Ivan; de Jonge, Martin; Howard, Daryl L.; Thierry, Benjamin

    2014-07-01

    The next generation of therapeutic nanoparticles in the treatment of cancer incorporate specific targeting. There is implicit importance in understanding penetration of targeted nanomedicines within tumour tissues via accurate and quantitative temporospatial measurements. In this study we demonstrate the potential of state-of-the-art synchrotron X-ray fluorescence microscopy (XFM) to provide such insights. To this end, quantitative mapping of the distribution of transferrin-conjugated gold nanoparticles inside multicellular tumour spheroids was achieved using XFM and compared with qualitative data obtained using reflectance confocal microscopy. Gold nanoparticles conjugated with human transferrin with a narrow size-distribution and high binding affinity to tumour cells were prepared as confirmed by cellular uptake studies performed on 2D monolayers. Although the prepared 100 nm transferrin-conjugated gold nanoparticles had high targeting capability to cancer cells, penetration inside multicellular spheroids was limited even after 48 hours as shown by the quantitative XFM measurements. The rapid, quantitative and label-free nature of state-of-the-art synchrotron XFM make it an ideal technology to provide the structure-activity relationship understanding urgently required for developing the next generation of immuno-targeted nanomedicines.

  11. Applying the Fe(III) binding property of a chemical transferrin mimetic to Ti(IV) anticancer drug design.

    PubMed

    Parks, Timothy B; Cruz, Yahaira M; Tinoco, Arthur D

    2014-02-01

    As an endogenous serum protein binder of Ti(IV), transferrin (Tf) serves as an excellent vehicle to stabilize the hydrolysis prone metal ion and successfully transport it into cells. This transporting role is thought to be central to Ti(IV)'s anticancer function, but efforts to synthesize Ti(IV) compounds targeting transferrin have not produced a drug. Nonetheless, the Ti(IV) transferrin complex (Ti2Tf) greatly informs on a new Ti(IV)-based anticancer drug design strategy. Ti2Tf interferes with cellular uptake of Fe(III), which is particularly detrimental to cancer cells because of their higher requirement for iron. Ti(IV) compounds of chemical transferrin mimetic (cTfm) ligands were designed to facilitate Ti(IV) activity by attenuating Fe(III) intracellular levels. In having a higher affinity for Fe(III) than Ti(IV), these ligands feature the appropriate balance between stability and lability to effectively transport Ti(IV) into cancer cells, release Ti(IV) via displacement by Fe(III), and deplete the intracellular Fe(III) levels. The cTfm ligand N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) was selected to explore the feasibility of the design strategy. Kinetic studies on the Fe(III) displacement process revealed that Ti(IV) can be transported and released into cells by HBED on a physiologically relevant time scale. Cell viability studies using A549 cancerous and MRC5 normal human lung cells and testing the cytotoxicity of HBED and its Ti(IV), Fe(III), and Ga(III) compounds demonstrate the importance of Fe(III) depletion in the proposed drug design strategy and the specificity of the strategy for Ti(IV) activity. The readily derivatized cTfm ligands demonstrate great promise for improved Ti(IV) anticancer drugs. PMID:24422475

  12. Stress reticulocytes lose transferrin receptors by an extrinsic process involving spleen and macrophages.

    PubMed

    Rhodes, Melissa M; Koury, Stephen T; Kopsombut, Prapaporn; Alford, Catherine E; Price, James O; Koury, Mark J

    2016-09-01

    As they mature into erythrocytes during normal erythropoiesis, reticulocytes lose surface transferrin receptors before or concurrently with reticulin. Exosome release accounts for most of the loss of transferrin receptors from reticulocytes. During erythropoietic stress, reticulocytes are released early from hematopoietic tissues and have increased reticulin staining and transferrin receptors. Flow cytometry of dually stained erythrocytes of mice recovering from phlebotomy demonstrated delayed loss of reticulin and transferrin receptors during in vitro maturation compared to in vivo maturation, indicating that an in vivo process extrinsic to the reticulocytes facilitates their maturation. Splenectomy or macrophage depletion by liposomal clodronate inhibited in vivo maturation of reticulocytes and increased the numbers of reticulin-negative, transferrin receptor-positive cells during and after recovery from phlebotomy. This reticulin-negative, transferrin receptor-positive population was rarely found in normal mice. Transmission electron microscopy demonstrated that the reticulin-negative, transferrin receptor-positive cells were elongated and discoid erythrocytes, but they had intracellular and surface structures that appeared to be partially degraded organelles. The results indicate that maturation of circulating stress reticulocytes is enhanced by an extrinsic process that occurs in the spleen and involves macrophage activity. Complete loss of reticulin with incomplete loss of surface transferrin receptors in this process produces a reticulin-negative, transferrin receptor-positive erythrocyte population that has potential utility for detecting prior erythropoietic stresses including bleeding, hemolysis and erythropoietin administration, even after recovery has been completed. Am. J. Hematol. 91:875-882, 2016. © 2016 Wiley Periodicals, Inc. PMID:27194638

  13. Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1

    PubMed Central

    Chrencik, Jill E.; Roth, Christopher B.; Terakado, Masahiko; Kurata, Haruto; Omi, Rie; Kihara, Yasuyuki; Warshaviak, Dora; Nakade, Shinji; Asmar-Rovira, Guillermo; Mileni, Mauro; Mizuno, Hirotaka; Griffith, Mark T.; Rodgers, Caroline; Han, Gye Won; Velasquez, Jeffrey; Chun, Jerold; Stevens, Raymond C.

    2015-01-01

    Summary Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with six cognate G protein-coupled receptors. Herein we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analysis. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease. PMID:26091040

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

    PubMed

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

    2013-12-01

    Iron homeostasis in the mammalian host limits the availability of iron to invading pathogens and is thought to restrict iron availability for microbes inhabiting mucosal surfaces. The presence of surface receptors for the host iron-binding glycoproteins transferrin (Tf) and lactoferrin (Lf) in globally important Gram-negative bacterial pathogens of humans and food production animals suggests that Tf and Lf are important sources of iron in the upper respiratory or genitourinary tracts, where they exclusively reside. Lf receptors have the additional function of protecting against host cationic antimicrobial peptides, suggesting that the bacteria expressing these receptors reside in a niche where exposure is likely. In this review we compare Tf and Lf receptors with respect to their structural and functional features, their role in colonization and infection, and their distribution among pathogenic and commensal bacteria. PMID:24266357

  15. The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

    PubMed Central

    Young, S P; Bomford, A; Williams, R

    1984-01-01

    Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin. PMID:6743230

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

    NASA Astrophysics Data System (ADS)

    Ding, Hong; Sagar, Vidya; Agudelo, Marisela; Pilakka-Kanthikeel, Sudheesh; Subba Rao Atluri, Venkata; Raymond, Andrea; Samikkannu, Thangavel; Nair, Madhavan P.

    2014-02-01

    The blood-brain barrier (BBB) is considered as the primary impediment barrier for most drugs. Delivering therapeutic agents to the brain is still a big challenge to date. In our study, a dual mechanism, receptor mediation combined with external non-invasive magnetic force, was incorporated into ferrous magnet-based liposomes for BBB transmigration enhancement. The homogenous magnetic nanoparticles (MNPs), with a size of ˜10 nm, were synthesized and confirmed by TEM and XRD respectively. The classical magnetism assay showed the presence of the characteristic superparamagnetic property. These MNPs encapsulated in PEGylated fluorescent liposomes as magneto-liposomes (MLs) showed mono-dispersion, ˜130 ± 10 nm diameter, by dynamic laser scattering (DLS) using the lipid-extrusion technique. Remarkably, a magnetite encapsulation efficiency of nearly 60% was achieved. Moreover, the luminescence and hydrodynamic size of the MLs was stable for over two months at 4 ° C. Additionally, the integrity of the ML structure remained unaffected through 120 rounds of circulation mimicking human blood fluid. After biocompatibility confirmation by cytotoxicity evaluation, these fluorescent MLs were further embedded with transferrin and applied to an in vitro BBB transmigration study in the presence or absence of external magnetic force. Comparing with magnetic force- or transferrin receptor-mediated transportation alone, their synergy resulted in 50-100% increased transmigration without affecting the BBB integrity. Consequently, confocal microscopy and iron concentration in BBB-composed cells further confirmed the higher cellular uptake of ML particles due to the synergic effect. Thus, our multifunctional liposomal magnetic nanocarriers possess great potential in particle transmigration across the BBB and may have a bright future in drug delivery to the brain.

  17. Endocytosis of a Functionally Enhanced GFP-Tagged Transferrin Receptor in CHO Cells

    PubMed Central

    Chu, Chong; Jiang, Qing; Zhu, Huifen; He, Yong; Yue, Tingting; Wang, Ruibo; Lei, Ping; Shen, Guanxin

    2015-01-01

    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. PMID:25803700

  18. The mammalian transferrin-independent iron transport system may involve a surface ferrireductase activity.

    PubMed Central

    Jordan, I; Kaplan, J

    1994-01-01

    Mammalian cells accumulate iron from ferric citrate or ferric nitrilotriacetate through the activity of a transferrin-independent iron transport system [Sturrock, Alexander, Lamb, Craven and Kaplan (1990) J. Biol. Chem. 265, 3139-3145]. The uptake system might recognize and transport ferric-anion complexes, or cells may reduce ferric iron at the surface and then transport ferrous iron. To distinguish between these possibilities we exposed cells to either [59Fe]ferric citrate or ferric [14C]citrate and determined whether accumulation of iron was accompanied by the obligatory accumulation of citrate. In HeLa cells and human skin fibroblasts the rate of accumulation of iron was three to five times greater than that of citrate. Incubation of fibroblasts with ferric citrate or ferric ammonium citrate resulted in an enhanced accumulation of iron and citrate; the molar ratio of accumulation approaching unity. A similar rate of citrate accumulation, however, was observed when ferric citrate-incubated cells were exposed to [14C]citrate alone. Further studies demonstrated the independence of iron and citrate accumulation: addition of unlabelled citrate to cells decreased the uptake of labelled citrate without affecting the accumulation of 59Fe; iron uptake was decreased by the addition of ferrous chelators whereas the uptake of citrate was unaffected; reduction of ferric iron by ascorbate increased the uptake of iron but had no effect on the uptake of citrate. When HeLa cells were depleted of calcium, iron uptake decreased, but there was little effect on citrate uptake. These results indicate that transport of iron does not require the obligatory transport of citrate and vice versa. The mammalian transferrin-independent iron transport system appears functionally similar to iron transport systems in both the bacterial and plant kingdoms which require the activities of both a surface reductase and a ferrous metal transporter. PMID:7945215

  19. Therapeutic effects of dihydroartemisinin and transferrin against glioblastoma

    PubMed Central

    Kim, Suk Hee; Kang, Seong Hee

    2016-01-01

    BACKGROUND/OBJECTIVES Artemisinin, a natural product isolated from Gaeddongssuk (artemisia annua L.) and its main active derivative, dihydroartemisinin (DHA), have long been used as antimalarial drugs. Recent studies reported that artemisinin is efficacious for curing diseases, including cancers, and for improving the immune system. Many researchers have shown the therapeutic effects of artemisinin on tumors such as breast cancer, liver cancer and kidney cancer, but there is still insufficient data regarding glioblastoma (GBM). Glioblastoma accounts for 12-15% of brain cancer, and the median survival is less than a year, despite medical treatments such as surgery, radiation therapy, and chemotherapy. In this study, we investigated the anti-cancer effects of DHA and transferrin against glioblastoma (glioblastoma multiforme, GBM). MATERIALS/METHODS This study was performed through in vitro experiments using C6 cells. The toxicity dependence of DHA and transferrin (TF) on time and concentration was analyzed by MTT assay and cell cycle assay. Observations of cellular morphology were recorded with an optical microscope and color digital camera. The anti-cancer mechanism of DHA and TF against GBM were studied by flow cytometry with Annexin V and caspase 3/7. RESULTS MTT assay revealed that TF enhanced the cytotoxicity of DHA against C6 cells. An Annexin V immune-precipitation assay showed that the percentages of apoptosis of cells treated with TF, DHA alone, DHA in combination with TF, and the control group were 7.15 ± 4.15%, 34.3 ± 5.15%, 66.42 ± 5.98%, and 1.2 ± 0.15%, respectively. The results of the Annexin V assay were consistent with those of the MTT assay. DHA induced apoptosis in C6 cells through DNA damage, and TF enhanced the effects of DHA. CONCLUSION The results of this study demonstrated that DHA, the derivative of the active ingredient in Gaeddongssuk, is effective against GBM, apparently via inhibition of cancer cell proliferation by a pharmacological

  20. Unusual Synergism of Transferrin and Citrate in the Regulation of Ti(IV) Speciation, Transport, and Toxicity.

    PubMed

    Tinoco, Arthur D; Saxena, Manoj; Sharma, Shweta; Noinaj, Nicholas; Delgado, Yamixa; Quiñones González, Ernesto P; Conklin, Steven E; Zambrana, Nicole; Loza-Rosas, Sergio A; Parks, Timothy B

    2016-05-01

    Human serum transferrin (sTf) is a protein that mediates the transport of iron from blood to cells. Assisted by the synergistic anion carbonate, sTf transports Fe(III) by binding the metal ion in a closed conformation. Previous studies suggest sTf's role as a potential transporter of other metals such as titanium. Ti is a widely used metal in colorants, foods, and implants. A substantial amount of Ti is leached into blood from these implants. However, the fate of the leached Ti and its transport into the cells is not known. Understanding Ti interaction with sTf assumes a greater significance with our ever increasing exposure to Ti in the form of implants. On the basis of in vitro studies, it was speculated that transferrin can bind Ti(IV) assisted by a synergistic anion. However, the role and identity of the synergistic anion(s) and the conformational state in which sTf binds Ti(IV) are not known. Here we have solved the first X-ray crystal structure of a Ti(IV)-bound sTf. We find that sTf binds Ti(IV) in an open conformation with both carbonate and citrate as synergistic anions at the metal binding sites, an unprecedented role for citrate. Studies with cell lines suggest that Ti(IV)-sTf is transported into cells and that sTf and citrate regulate the metal's blood speciation and attenuate its cytotoxic property. Our results provide the first glimpse into the citrate-transferrin synergism in the regulation of Ti(IV) bioactivity and offers insight into the future design of Ti(IV)-based anticancer drugs. PMID:27070073

  1. Phylogenetic Analyses Uncover a Novel Clade of Transferrin in Nonmammalian Vertebrates

    PubMed Central

    Mohd-Adnan, Adura; Gabaldón, Toni

    2013-01-01

    Transferrin is a protein super-family involved in iron transport, a central process in cellular homeostasis. Throughout the evolution of vertebrates, transferrin members have diversified into distinct subfamilies including serotransferrin, ovotransferrin, lactoferrin, melanotransferrin, the inhibitor of carbonic anhydrase, pacifastin, and the major yolk protein in sea urchin. Previous phylogenetic analyses have established the branching order of the diverse transferrin subfamilies but were mostly focused on the transferrin repertoire present in mammals. Here, we conduct a comprehensive phylogenetic analysis of transferrin protein sequences in sequenced vertebrates, placing a special focus on the less-studied nonmammalian vertebrates. Our analyses uncover a novel transferrin clade present across fish, sauropsid, and amphibian genomes but strikingly absent from mammals. Our reconstructed scenario implies that this novel class emerged through a duplication event at the vertebrate ancestor, and that it was subsequently lost in the lineage leading to mammals. We detect footprints of accelerated evolution following the duplication event, which suggest positive selection and early functional divergence of this novel clade. Interestingly, the loss of this novel class of transferrin in mammals coincided with the divergence by duplication of lactoferrin and serotransferrin in this lineage. Altogether, our results provide novel insights on the evolution of iron-binding proteins in the various vertebrate groups. PMID:23258311

  2. Transferrin-immune complex disease: a potentially overlooked gammopathy mediated by IgM and IgG.

    PubMed

    Forni, Gian Luca; Pinto, Valeria; Musso, Marco; Mori, Marco; Girelli, Domenico; Caldarelli, Ilaria; Borriello, Adriana; Ragione, Fulvio Della

    2013-12-01

    The combination of marked hypersideremia, hypertransferrinemia, and monoclonal gammopathy of underdetermined significance (MGUS) should alert clinicians to the possible presence of an anti-transferrin immunoglobulin, an uncommon acquired disorder also defined as transferrin-immune complex disease (TICD). The authors have previously described a case of TICD with 100% transferrin saturation and liver iron overload. However, the findings in the few cases so far reported are heterogeneous, and the presence of high transferrin saturation and liver iron overload is not universal. In this article, the authors have described the identification of two additional patients with anti-transferrin monoclonal gammopathy, hypersideremia, and hypertransferrinemia, but with incomplete transferrin saturation and no hepatic iron overload. The autoantibodies were purified by using transferrin as affinity bait and characterized. One subject showed a high-titer monoclonal anti-transferrin IgM with a κ-type light chain. This finding is the first observation of IgM autoantibodies against transferrin. The other patient developed the disease after pregnancy. In this study, monoclonal antibody was an IgG mounting a κ-type light chain with altered molecular weight. These results highlight that transferrin might induce the development of a monoclonal immune response of different classes and specificity. The identification, in a single hematologic center, of three different subjects with anti-transferrin monoclonal gammopathy suggests that the disease probably represents a still underdiagnosed condition. From a clinical standpoint, these patients must be followed up both as MGUS and as hemochromatosis. PMID:23913829

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

    PubMed Central

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

    1985-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Metabolic Catastrophe in Mice Lacking Transferrin Receptor in Muscle

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-20

    Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure. PMID:26456827

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-06-01

    Following an internal contamination event, the transport of actinide (An) and lanthanide (Ln) metal ions through the body is facilitated by endogenous ligands such as the human iron-transport protein transferrin (Tf). The recognition of resulting metallo-transferrin complexes (M2Tf) by the cognate transferrin receptor (TfR) is therefore a critical step for cellular uptake of these metal ions. A high performance liquid chromatography-based method has been used to probe the binding of M2Tf with TfR, yielding a direct measurement of the successive thermodynamic constants that correspond to the dissociation of TfR(M2Tf)2 and TfR(M2Tf) complexes for Fe(3+), Ga(3+), La(3+), Nd(3+), Gd(3+), Yb(3+), Lu(3+), (232)Th(4+), (238)UO2(2+), and (242)Pu(4+). Important features of this method are (i) its ability to distinguish both 1 : 1 and 1 : 2 complexes formed between the receptor and the metal-bound transferrin, and (ii) the requirement for very small amounts of each binding partner (<1 nmol of protein per assay). Consistent with previous reports, the strongest receptor affinity is found for Fe2Tf (Kd1 = 5 nM and Kd2 = 20 nM), while the lowest affinity was measured for Pu2Tf (Kd1 = 0.28 μM and Kd2 = 1.8 μM) binding to the TfR. Other toxic metal ions such as Th(IV) and U(VI), when bound to Tf, are well recognized by the TfR. Under the described experimental conditions, the relative stabilities of TfR:(MxTf)y adducts follow the order Fe(3+) > Th(4+) ~ UO2(2+) ~ Cm(3+) > Ln(3+) ~ Ga(3+) > Yb(3+) ~ Pu(4+). This study substantiates a role for Tf in binding lanthanide fission products and actinides, and transporting them into cells by receptor-mediated endocytosis. PMID:23446908

  9. Transferrin receptor and ferritin-H are developmentally regulated in oligodendrocyte lineage cells.

    PubMed

    Li, Yunxia; Guan, Qiang; Chen, Yuhui; Han, Hongjie; Liu, Wuchao; Nie, Zhiyu

    2013-01-01

    Iron is an essential trophic element that is required for cell viability and differentiation, especially in oligodendrocytes, which consume relatively high rates of energy to produce myelin. Multiple iron metabolism proteins are expressed in the brain including transferrin receptor and ferritin-H. However, it is still unknown whether they are developmentally regulated in oligodendrocyte lineage cells for myelination. Here, using an in vitro cultured differentiation model of oligodendrocytes, we found that both transferrin receptor and ferritin-H are significantly upregulated during oligodendrocyte maturation, implying the essential role of iron in the development of oligodendrocytes. Additional different doses of Fe(3+) in the cultured medium did not affect oligodendrocyte precursor cell maturation or ferritin-H expression but decreased the expression of the transferrin receptor. These results indicate that upregulation of both transferrin receptor and ferritin-H contributes to maturation and myelination of oligodendrocyte precursor cells. PMID:25206366

  10. Acquisition of heme iron by Neisseria meningitidis does not involve meningococcal transferrin-binding proteins.

    PubMed

    Martel, N; Lee, B C

    1994-02-01

    Similarities in size between hemin-binding protein 1 (HmBP1) and transferrin-binding protein 1 (TBP1) of Neisseria meningitidis suggest that these proteins are functionally homologous. However, a meningococcal mutant lacking the transferrin-binding proteins retained the capacity to acquire iron from heme and hemoglobin. In immunoblots, hyperimmune polyclonal antiserum against TBP1 did not react with HmBP1. PMID:8300227

  11. Immunohistochemical distribution of ferritin, lactoferrin, and transferrin in granulomas of bovine paratuberculosis.

    PubMed Central

    Momotani, E; Furugouri, K; Obara, Y; Miyata, Y; Ishikawa, Y; Yoshino, T

    1986-01-01

    Granulomatous lesions of bovine paratuberculosis contained ferritin, lactoferrin, and a small amount of transferrin, as demonstrated by the immunohistochemical method. Macrophages in the normal bovine ileum did not contain lactoferrin and transferrin; however, ferritin was found in individual macrophages of Peyer's patches. These results may help elucidate the relationship between intracellular growth of Mycobacterium paratuberculosis and the presence of iron-binding proteins in the granulomas. Images PMID:3699898

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

    PubMed Central

    2016-01-01

    Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that 89Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with 89Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors. PMID:26725682

  13. Molecular cloning of bullfrog saxiphilin: a unique relative of the transferrin family that binds saxitoxin.

    PubMed Central

    Morabito, M A; Moczydlowski, E

    1994-01-01

    Plasma and tissue of certain vertebrates contain a protein called saxiphilin that specifically binds the neurotoxin saxitoxin with nanomolar affinity. We describe the isolation of a cDNA clone of saxiphilin from liver of the North American bullfrog (Rana catesbeiana). The cDNA sequence encodes a protein that is evolutionarily related to members of the transferrin family of Fe(3+)-binding proteins. Pairwise sequence alignment of saxiphilin with various transferrins reveals amino acid identity as high as 51% and predicts 14 disulfide bonds that are highly conserved. The larger size of saxiphilin (91 kDa) versus serum transferrin (approximately 78 kDa) is primarily due to a unique insertion of 144 residues. This insertion contains a 49-residue domain classified as a type 1 repetitive element of thyroglobulin, which is shared by a variety of membrane, secreted, and extracellular matrix proteins. Saxiphilin also differs from transferrins in 9 of 10 highly conserved amino acids in the two homologous Fe3+/HCO3-binding sites of transferrin. Identification of saxiphilin implies that transferrin-like proteins comprise a diverse superfamily with functions other than iron binding. Images PMID:8146142

  14. [Preparation of epitope imprinted particles for transferrin recognition by reversible addition fragmentation chain transfer strategy].

    PubMed

    Li, Qinran; Yang, Kaiguang; Li, Senwu; Liu, Jianxi; Zhang, Lihua; Liang, Zhen; Zhang, Yukui

    2014-10-01

    A kind of novel epitope surface imprinted particles was prepared by the reversible addition fragmentation chain transfer (RAFT) strategy. The epitope of transferrin, N-terminal peptide of the protein with nine amino acid residues, was chosen as the template and immobi- lized with covalent interaction on the surface of silica particles through the truss arm glutaraldehyde. The living/controlled polymerization was initialed by 2,2'-azobisisobutyronitrile (AIBN) at 70 °C in the solution of N,N-dimethylformamide, with the regulation by triothioester agent 2-(dodecylthiocarbonothioylthio)-2-methylpropanoic acid. Methacrylic acid and 2-hydroxyethyl methacrylate were chosen as the functional monomers and N, N-methylenebisacrylamide was chosen as the cross-linker in this polymerization. For this material, the binding capacity of the nine residue peptide could reach 2.36 mg/g with the imprinting factor (IF) of 1.89, while that for transferrin could reach 4.98 mg/g with IF of 1.61. The equilibrium could be achieved in 120 min for the transferrin recognition. In multi-protein competitive recognition, the imprinted factor of transferrin was the highest in the mixture of transferrin and other competitive proteins, such as cytochrome C and β-lactoglobulin. The results indicated that these epitope surface imprinted particles with RAFT strategy could recognize not only the nine residue peptide but also the transferrin with good selectivity, high binding capacity and fast mass transfer. PMID:25739262

  15. The mRNA-binding protein which controls ferritin and transferrin receptor expression is conserved during evolution.

    PubMed Central

    Rothenberger, S; Müllner, E W; Kühn, L C

    1990-01-01

    A post-transcriptional regulatory protein, termed iron regulatory factor (IRF), that binds specifically to the iron-responsive elements of ferritin and transferrin receptor mRNA, has recently been identified in the cytoplasm of human and mouse cells. Activation of this factor by low intracellular iron levels leads to inhibition of ferritin translation and an increase of TR mRNA stability. To investigate whether these feedback regulatory mechanisms are conserved during evolution, we analysed cytoplasmic extracts from 12 different species for a specific IRE-binding activity. We found mRNA-binding proteins in chicken, frog, fish and fly, which are equivalent to human and mouse IRF in gel-retardation assays with radiolabeled RNA transcripts. Competition experiments, molecular weight determinations, and modulation of the mRNA-binding activity in response to intracellular iron levels or reduction by beta-mercaptoethanol indicate that IRF has similar structural and functional properties in these different species. Images PMID:2157191

  16. Neoplastic transformation of rat liver epithelial cells is enhanced by non-transferrin-bound iron

    PubMed Central

    Messner, Donald J; Kowdley, Kris V

    2008-01-01

    Background Iron overload is associated with liver toxicity, cirrhosis, and hepatocellular carcinoma in humans. While most iron circulates in blood as transferrin-bound iron, non-transferrin-bound iron (NTBI) also becomes elevated and contributes to toxicity in the setting of iron overload. The mechanism for iron-related carcinogenesis is not well understood, in part due to a shortage of suitable experimental models. The primary aim of this study was to investigate NTBI-related hepatic carcinogenesis using T51B rat liver epithelial cells, a non-neoplastic cell line previously developed for carcinogenicity and tumor promotion studies. Methods T51B cells were loaded with iron by repeated addition of ferric ammonium citrate (FAC) to the culture medium. Iron internalization was documented by chemical assay, ferritin induction, and loss of calcein fluorescence. Proliferative effects were determined by cell count, toxicity was determined by MTT assay, and neoplastic transformation was assessed by measuring colony formation in soft agar. Cyclin levels were measured by western blot. Results T51B cells readily internalized NTBI given as FAC. Within 1 week of treatment at 200 μM, there were significant but well-tolerated toxic effects including a decrease in cell proliferation (30% decrease, p < 0.01). FAC alone induced little or no colony formation in soft agar. In contrast, FAC addition to cells previously initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in a concentration dependent increase in colony formation. This was first detected at 12 weeks of FAC treatment and increased at longer times. At 16 weeks, colony formation increased more than 10 fold in cells treated with 200 μM FAC (p < 0.001). The iron chelator desferoxamine reduced both iron uptake and colony formation. Cells cultured with 200 μM FAC showed decreased cyclin D1, decreased cyclin A, and increased cyclin B1. Conclusion These results establish NTBI as a tumor promoter in T51B rat liver

  17. Downregulation of transferrin receptor surface expression by intracellular antibody

    SciTech Connect

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

    2007-03-23

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

  18. New state markers for alcoholism. Comparison of carbohydrate deficient transferrin (CDT) and alcohol mediated (triantennary) transferrin (AMT).

    PubMed

    Kanitz, R D; Wood, W G; Wetterling, T; Forster, J; Oehler, G

    1994-05-01

    Carbohydrate deficient transferrin (CDTect-RIA, Pharmacia) was compared with an Immunoluminometric assay for isotransferrin separated by a short column Con-A sepharose which we have called alcohol mediated triantennary transferrin (AMT). 101 in-patients with alcohol dependency syndrome (alcohol consumption of more than 60 g/day) were grouped according to the time of abstinence A1 (0-7 days), A2 (8-14 days), A3 (> or = 15 days). Serum samples were obtained at admission (U0) and under abstinent conditions after 10-20 days (U1). All groups were controlled for AMT, CDT, GGT, MCV. Control groups were 30 in-patients with non alcoholic liver disease (NALD) and 31 healthy volunteers (alcohol consumption of less than 20g/day). Results showed for CDT and AMT highly significant differences between short abstinence period (group A1) and more than two weeks abstinence (group A3) alcoholics and between group A1 and healthy controls. In group A1 CDT was significantly elevated (P < or = .001) compared to NALD group whereas AMT showed no differences. CDT (cut off 22 mg/l) showed a high diagnostic specificity (A1/controls 97%, A1/NALD 83%, A1/A3 78%) but only a diagnostic sensitivity of 61%. AMT (cut off 260 mg/l) revealed a diagnostic test sensitivity of 74%. The diagnostic test specificity of AMT was inferior to CDT (A1/controls 74%, A1/NALD 50%, A1/A3 70%). Initial CDT and AMT values in alcoholics were highly correlated (P < or = .001) with time of abstinence. CDT and AMT decline was correlated with time of abstinence. CDT was proved for high significant (P < or = .001) decline over a longer period of abstinence (11-30 days) while AMT decline was significant (P = .008) only in early abstinence (0-10 days). Presence of a withdrawal syndrome was highly correlated (P < or = .01) with CDT values above 22 mg/l and AMT values above 260 mg/l. Furthermore in selected follow up cases it was shown that AMT seemed to be a more sensitive indicator for short alcoholic relapses than CDT. PMID

  19. Amorphous carbon nanoparticle used as novel resonance energy transfer acceptor for chemiluminescent immunoassay of transferrin.

    PubMed

    Gao, Hongfei; Wang, Wenwen; Wang, Zhenxing; Han, Jing; Fu, Zhifeng

    2014-03-28

    Amorphous carbon nanoparticles (ACNPs) showing highly efficient quenching of chemiluminescence (CL) were prepared from candle soot with a very simple protocol. The prepared ACNP was employed as the novel energy acceptor for a chemiluminescence resonance energy transfer (CRET)-based immunoassay. In this work, ACNP was linked with transferrin (TRF), and horseradish peroxidase (HRP) was conjugated to TRF antibody (HRP-anti-TRF). The immunoreaction rendered the distance between the ACNP acceptor and the HRP-catalyzed CL emitter to be short enough for CRET occurring. In the presence of TRF, this antigen competed with ACNP-TRF for HRP-anti-TRF, thus led to the decreased occurrence of CRET. A linear range of 20-400 ng mL(-1) and a limit of detection of 20 ng mL(-1) were obtained in this immunoassay. The proposed method was successfully applied for detection of TRF levels in human sera, and the results were in good agreement with ELISA method. Moreover, the ACNPs show higher energy transfer efficiency than other conventional nano-scaled energy acceptors such as graphene oxide in CRET assay. It is anticipated that this approach can be developed for determination of other analytes with low cost, simple manipulation and high specificity. PMID:24636417

  20. Synthesis and anti-cancer activity of covalent conjugates of artemisinin and a transferrin-receptor targeting peptide.

    PubMed

    Oh, Steve; Kim, Byung Ju; Singh, Narendra P; Lai, Henry; Sasaki, Tomikazu

    2009-02-01

    Artemisinin, a natural product isolated from Artemisia annua L., shows a unique anti-cancer activity by an iron dependent mechanism. Artemisinin was covalently conjugated to a transferrin-receptor targeting peptide, HAIYPRH that binds to a cavity on the surface of transferrin receptor. This enables artemisinin to be co-internalized with receptor-bound transferrin. The iron released from transferrin can activate artemisinin to generate toxic radical species to kill cells. The artemisinin-peptide conjugates showed potent anti-cancer activity against Molt-4 leukemia cells with a significantly improved cancer/normal cells selectivity. PMID:18838215

  1. Preparation, characterization and in vitro efficacy of magnetic nanoliposomes containing the artemisinin and transferrin

    PubMed Central

    2014-01-01

    Background Artemisinin is the major sesquiterpene lactones in sweet wormwood (Artemisia annua L.), and its combination with transferrin exhibits versatile anti-cancer activities. Their non-selective targeting for cancer cells, however, limits their application. The aim of this study was to prepare the artemisinin and transferrin-loaded magnetic nanoliposomes in thermosensitive and non-thermosensitive forms and evaluate their antiproliferative activity against MCF-7 and MDA-MB-231 cells for better tumor-targeted therapy. Methods Artemisinin and transferrin-loaded magnetic nanoliposomes was prepared by extrusion method using various concentrations of lipids. These formulations were characterized for particle size, zeta potential, polydispersity index and shape morphology. The artemisinin and transferrin-loading efficiencies were determined using HPLC. The content of magnetic iron oxide in the nanoliposomes was analysed by spectrophotometry. The in vitro release of artemisinin, transferrin and magnetic iron oxide from vesicles was assessed by keeping of the nanoliposomes at 37°C for 12 h. The in vitro cytotoxicity of prepared nanoliposomes was investigated against MCF-7 and MDA-MB-231 cells using MTT assay. Results The entrapment efficiencies of artemisinin, transferrin and magnetic iron oxide in the non-thermosensitive nanoliposomes were 89.11% ± 0.23, 85.09% ± 0.31 and 78.10% ± 0.24, respectively. Moreover, the thermosensitive formulation showed a suitable condition for thermal drug release at 42°C and exhibited high antiproliferative activity against MCF-7 and MDA-MB-231 cells in the presence of a magnetic field. Conclusions Our results showed that the thermosensitive artemisinin and transferrin-loaded magnetic nanoliposomes would be an effective choice for tumor-targeted therapy, due to its suitable stability and high effectiveness. PMID:24887240

  2. Lectin-dependent inhibition of antigen-antibody reaction: application for measuring α2,6-sialylated glycoforms of transferrin.

    PubMed

    Hoshi, Kyoka; Kariya, Yoshinobu; Nara, Kiyomitsu; Ito, Hiromi; Matsumoto, Kana; Nagae, Masamichi; Yamaguchi, Yoshiki; Nakajima, Madoka; Miyajima, Masakazu; Arai, Hajime; Kuno, Atsushi; Narimatsu, Hisashi; Shirotani, Keiro; Hashimoto, Yasuhiro

    2013-09-01

    We developed a high-throughput Enzyme-linked immunosorbent assay (ELISA) for measuring α2,6-sialylated transferrin (Tf), based on inhibition of anti-Tf antibody binding to α2,6-sialylated Tf by a lectin, Sambucus sieboldiana Agglutinin (SSA). The inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform specific. This finding was applied to an automated latex-agglutination immunoassay, using SSA lectin as an inhibitor (SSA-ALI). The concentration of α2,6-sialylated Tf measured by SSA-ALI in human cerebrospinal fluid was correlated with that of ELISA (r2 = 0.8554), previously developed for measuring α2,6-sialylated Tf. PMID:23921500

  3. Adenocarcinoma cells isolated from patients in the presence of cerium and transferrin in vitro

    PubMed Central

    Zende-Del, A; Gholami, MR; Abdollahpour, F; Ahmadvand, H

    2015-01-01

    Aim: Cerium as a trace element in the periodic table is a member of the lanthanide group. Cerium ionic radius and its binding properties are similar to ferric ions, which may be bound to transferrin. So it can be considered as a competitive element to iron and can interfere with iron absorption. The aim of this study was to investigate the inhibitory effect of Cerium in presence of transferrin on gastric adenocarcinoma cells in vitro. Methods: The adenocarcinoma cells were obtained from patients after a pathological confirmation, then they were cultured in DMEM environment and cytotoxic effect of different concentrations of cerium were measured (0.1, 1, 10 and 100 µM) in the presence and absence of transferrin, on periods 24 and 48 hours by MTT and LDH cytotoxic assay. Results: The results of MTT and LDH measurements showed that Cerium itself has a cytotoxic effect on cancer cells isolated from the patient as well as it increases significantly in the presence of transferrin carrying a mortality rate of cancer cells (P <.05). Conclusion: Cerium is competitive element in the mechanism of iron absorption and can interfere and inhibit the growth of adenocarcinoma cancer cells; also, the use of Cerium and transferrin simultaneously may cause a greater inhibitory effect. PMID:26664465

  4. Pseudechis australis venomics: adaptation for a defense against microbial pathogens and recruitment of body transferrin.

    PubMed

    Georgieva, Dessislava; Seifert, Jana; Öhler, Michaela; von Bergen, Martin; Spencer, Patrick; Arni, Raghuvir K; Genov, Nicolay; Betzel, Christian

    2011-05-01

    The venom composition of Pseudechis australis, a widely distributed in Australia reptile, was analyzed by 2-DE and mass spectrometric analysis. In total, 102 protein spots were identified as venom toxins. The gel is dominated by horizontal trains of spots with identical or very similar molecular masses but differing in the pI values. This suggests possible post-translational modifications of toxins, changing their electrostatic charge. The results demonstrate a highly specialized biosynthesis of toxins destroying the hemostasis (P-III metalloproteases, SVMPs), antimicrobial proteins (L-amino acid oxidases, LAAOs, and transferrin-like proteins, TFLPs), and myotoxins (phospholipase A(2)s, PLA(2)s). The three transferrin isoforms of the Australian P. australis (Elapidae snake) venom are highly homologous to the body transferrin of the African Lamprophis fuliginosus (Colubridae), an indication for the recruitment of body transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey. Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-5'-nucleotidases (5'-NTDs), nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymatic activities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, and biotechnological purposes. PMID:21417486

  5. Ractopamine, a Livestock Feed Additive, Is a Full Agonist at Trace Amine–Associated Receptor 1

    PubMed Central

    Grandy, David K.; Janowsky, Aaron

    2014-01-01

    Ractopamine (RAC) is fed to an estimated 80% of all beef, swine, and turkey raised in the United States. It promotes muscle mass development, limits fat deposition, and reduces feed consumption. However, it has several undesirable behavioral side effects in livestock, especially pigs, including restlessness, agitation, excessive oral-facial movements, and aggressive behavior. Numerous in vitro and in vivo studies suggest RAC’s physiological actions begin with its stimulation of β1- and β2-adrenergic receptor–mediated signaling in skeletal muscle and adipose tissue; however, the molecular pharmacology of RAC’s psychoactive effects is poorly understood. Using human cystic fibrosis transmembrane conductance regulator (hCFTR) chloride channels as a sensor for intracellular cAMP, we found that RAC and p-tyramine (TYR) produced concentration-dependent increases in chloride conductance in oocytes coexpressing hCFTR and mouse trace amine–associated receptor 1 (mTAAR1), which was completely reversed by the trace amine–associated receptor 1 (TAAR1)–selective antagonist EPPTB [N-(3-ethoxyphenyl)-4-pyrrolidin-1-yl-3-trifluoromethylbenzamide]. Oocytes coexpressing hCFTR and the human β2-adrenergic receptor showed no response to RAC or TYR. These studies demonstrate that, contrary to expectations, RAC is not an agonist of the human β2-adrenergic receptor but rather a full agonist for mTAAR1. Since TAAR1-mediated signaling can influence cardiovascular tone and behavior in several animal models, our finding that RAC is a full mTAAR1 agonist supports the idea that this novel mechanism of action influences the physiology and behavior of pigs and other species. These findings should stimulate future studies to characterize the pharmacological, physiological, and behavioral actions of RAC in humans and other species exposed to this drug. PMID:24799633

  6. Ractopamine, a livestock feed additive, is a full agonist at trace amine-associated receptor 1.

    PubMed

    Liu, Xuehong; Grandy, David K; Janowsky, Aaron

    2014-07-01

    Ractopamine (RAC) is fed to an estimated 80% of all beef, swine, and turkey raised in the United States. It promotes muscle mass development, limits fat deposition, and reduces feed consumption. However, it has several undesirable behavioral side effects in livestock, especially pigs, including restlessness, agitation, excessive oral-facial movements, and aggressive behavior. Numerous in vitro and in vivo studies suggest RAC's physiological actions begin with its stimulation of β1- and β2-adrenergic receptor-mediated signaling in skeletal muscle and adipose tissue; however, the molecular pharmacology of RAC's psychoactive effects is poorly understood. Using human cystic fibrosis transmembrane conductance regulator (hCFTR) chloride channels as a sensor for intracellular cAMP, we found that RAC and p-tyramine (TYR) produced concentration-dependent increases in chloride conductance in oocytes coexpressing hCFTR and mouse trace amine-associated receptor 1 (mTAAR1), which was completely reversed by the trace amine-associated receptor 1 (TAAR1)-selective antagonist EPPTB [N-(3-ethoxyphenyl)-4-pyrrolidin-1-yl-3-trifluoromethylbenzamide]. Oocytes coexpressing hCFTR and the human β2-adrenergic receptor showed no response to RAC or TYR. These studies demonstrate that, contrary to expectations, RAC is not an agonist of the human β2-adrenergic receptor but rather a full agonist for mTAAR1. Since TAAR1-mediated signaling can influence cardiovascular tone and behavior in several animal models, our finding that RAC is a full mTAAR1 agonist supports the idea that this novel mechanism of action influences the physiology and behavior of pigs and other species. These findings should stimulate future studies to characterize the pharmacological, physiological, and behavioral actions of RAC in humans and other species exposed to this drug. PMID:24799633

  7. Iron release is reduced by mutations of lysines 206 and 296 in recombinant N-terminal half-transferrin.

    PubMed

    Steinlein, L M; Ligman, C M; Kessler, S; Ikeda, R A

    1998-09-29

    Human serum transferrin consists of two iron-binding lobes connected by a short peptide linker. While the high homology and structural similarity between the two halves of the molecule would suggest similar characteristics, it has been shown that the pH-dependent rate of release of iron from the N-terminal lobe is quite different from that of its C-terminal counterpart. This suggests that the N-lobe of human serum transferrin has a specific, pH-dependent, molecular mechanism for releasing iron. Sacchettini and co-workers using structural information have hypothesized that two lysines in the N-terminal lobe of ovotransferrin create a dilysine interaction and suggest that this is the trigger for pH-dependent iron release. To investigate this hypothesis, we used a Pichia pastoris expression system to produce large amounts of wild-type nTf, the single point mutants, nTfK206A (Lys 206 to alanine) and nTfK296A (Lys 296 to alanine), and the double mutant, nTfK206/296A. The purified recombinant proteins were then used to measure rates of iron release to pyrophosphate. It was found that the rate of iron release from all three mutant proteins at pH 5.7 (the pH at which nTf would normally release iron) was too slow to measure. Only when the pH was reduced to 5.0 could the rates of iron release from the mutant proteins be reliably determined. Although this precludes a direct comparison to wild-type nTf (the rate of iron release from nTf at pH 5.0 is too fast to measure), it implicates lysines 206 and 296 in the pH-dependent release of iron from nTf. PMID:9753457

  8. Effect of gestational age and intrauterine nutrition on plasma transferrin and iron in the newborn.

    PubMed Central

    Scott, P H; Berger, H M; Kenward, C; Scott, P; Wharton, B A

    1975-01-01

    Plasma concentrations of transferrin and iron were measured in the cord blood of babies of varying gestational age and birthweight. Tranferrin and iron concentrations rose with gestational age; values in light-for-dates babies did not differ from those in babies of appropriate weight. In the last trimester of pregnancy plasma transferrin and iron concentrations in the fetus are affected by the maturity of the pregnancy but are independent of the nutritional status of the fetus. The low transferrin levels, particularly in preterm babies, may caution the use of iron especially by the parenteral route in the neonatal period, but we are wary of abandoning on this evidence alone the well tried clinical custom of giving oral iron to preterm babies who are not breast fed. PMID:1236569

  9. Transferrin binding and iron uptake by mouse lymph node cells during transformation in response to concanavalin A.

    PubMed Central

    Brock, J H; Rankin, M C

    1981-01-01

    Mouse lymph node cells cultured with Concanavalin A (Con A) in serum-free medium containing 59Fe-transferrin took up 59Fe more rapidly than cells cultured without Con-A. Uptake of iron commenced rapidly and preceded the onset of DNA synthesis in stimulated cells. Total uptake of transferrin during culture was much lower than that of iron, indicating that cells could remove iron from transferrin. The released transferrin appeared to be undergraded. Lymphoblasts bound six times more transferrin per cell than small lymphocytes. Lymphocytes also took up iron from citrate and nitrilotriacetate complexes, and iron so acquired was not readily removed by desferrioxamine, indicating that it was not bound extracellularly. PMID:7251060

  10. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB. PMID:10865941

  11. Rapid Lymphatic Dissemination of Encapsulated Group A Streptococci via Lymphatic Vessel Endothelial Receptor-1 Interaction

    PubMed Central

    Johnson, Louise A.; Holder, Kayla A.; Reglinski, Mark; Wing, Peter A. C.; Rigby, David; Jackson, David G.; Sriskandan, Shiranee

    2015-01-01

    The host lymphatic network represents an important conduit for pathogen dissemination. Indeed, the lethal human pathogen group A streptococcus has a predilection to induce pathology in the lymphatic system and draining lymph nodes, however the underlying basis and subsequent consequences for disease outcome are currently unknown. Here we report that the hyaluronan capsule of group A streptococci is a crucial virulence determinant for lymphatic tropism in vivo, and further, we identify the lymphatic vessel endothelial receptor-1 as the critical host receptor for capsular hyaluronan in the lymphatic system. Interference with this interaction in vivo impeded bacterial dissemination to local draining lymph nodes and, in the case of a hyper-encapsulated M18 strain, redirected streptococcal entry into the blood circulation, suggesting a pivotal role in the manifestation of streptococcal infections. Our results reveal a novel function for bacterial capsular polysaccharide in directing lymphatic tropism, with potential implications for disease pathology. PMID:26352587

  12. Detection and measurement of carbohydrate deficient transferrin in serum using immuno-capture mass spectrometry: diagnostic applications for annual ryegrass toxicity and corynetoxin exposure.

    PubMed

    Penno, M A S; Colegate, S M; Michalski, W P; Hoffmann, P

    2012-10-01

    The neurological livestock disease annual ryegrass toxicity (ARGT) is caused by the ingestion of the naturally occurring glycolipid toxins - the corynetoxins. Corynetoxins also threaten human health as potential contaminants of the food supply. Presently, there are no routine diagnostic tests for corynetoxins-exposure in humans or livestock. Chronic ingestion of corynetoxins has been modeled in rats exposed to dietary tunicamycins for 12 months and carbohydrate deficient transferrin (CDT) has been previously identified as a candidate disease biomarker. Here, the technique of immuno-capture mass spectrometry (icMS) was used to evaluate serum levels of CDT, discriminating between control and tunicamycins-exposed rats with 85% accuracy. The icMS approach is based on the combination of specific transferrin enrichment with functionalized magnetic beads and automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). With no other clinically-relevant diagnostic tests available icMS could be readily adapted for high-throughput clinical assessment of corynetoxins-exposure in humans or livestock. PMID:21963292

  13. Ehrlichia chaffeensis inclusions are early endosomes which selectively accumulate transferrin receptor.

    PubMed Central

    Barnewall, R E; Rikihisa, Y; Lee, E H

    1997-01-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium which infects macrophages and monocytes. Double immunofluorescence labeling was used to characterize the nature of E. chaffeensis inclusion in the human promyelocytic leukemia cell line THP-1. E. chaffeensis was labeled with dog anti-E. chaffeensis serum and fluorescein isothiocyanate-conjugated anti-dog immunoglobulin G (IgG). Lissamine rhodamine-conjugated anti-mouse IgG was used to label various mouse monoclonal antibodies. Ehrlichial inclusions did not fuse with lysosomes, since they were not labeled with anti-CD63 or anti-LAMP-1. The ehrlichial inclusions were slightly acidic, since they weakly accumulated 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and stained weakly positive for vacuolar type H+ ATPase. Some ehrlichial inclusions were labeled positive with antibodies against HLA-DR, HLA-ABC, and beta2 microglobulin, while other inclusions in the same cell were labeled negative. The inclusions were labeled strongly positive for transferrin receptors (TfRs) and negative for the clathrin heavy chain. Time course labeling for TfRs showed that up to 3 h postinfection, most of the ehrlichial inclusions were negative for TfRs. After 6 h postinfection, 100% of the ehrlichial inclusions became TfR positive and the intensity of labeling was increased during the subsequent 3 days. Reverse transcription-PCR showed a gradual increase in the level of TfR mRNA postinfection, which reached a peak at 24 h postinfection. These results suggest that ehrlichial inclusions are early endosomes which selectively accumulate TfRs and that the ehrlichiae up-regulate TfR mRNA expression. PMID:9119487

  14. Cannabinoid Receptor 1 Gene Association With Nicotine Dependence

    PubMed Central

    Chen, Xiangning; Williamson, Vernell S.; An, Seon-Sook; Hettema, John M.; Aggen, Steven H.; Neale, Michael C.; Kendler, Kenneth S.

    2009-01-01

    Context The endogenous cannabinoid system has been implicated in drug addiction in animal models. The cannabinoid receptor 1 (CNR1) gene is 1 of the 2 receptors expressed in the brain. It has been reported to be associated with alcoholism and multiple drug abuse and dependence. Objective To test the hypothesis that the CNR1 gene is associated with nicotine dependence. Design Genotype-phenotype association study. Ten single-nucleotide polymorphisms were genotyped in the CNR1 gene in 2 independent samples. For the first sample (n=688), a 3-group case-control design was used to test allele association with smoking initiation and nicotine dependence. For the second sample (n = 961), association was assessed with scores from the Fagerström Test for Nicotine Dependence (FTND). Settings Population samples selected from the Mid-Atlantic Twin Registry. Participants White patients aged 18 to 65 years who met the criteria of inclusion. Main Outcome Measures Fagerström Tolerance Questionnaire and FTND scores. Results Significant single-marker and haplotype associations were found in both samples, and the associations were female specific. Haplotype 1-1-2 of markers rs2023239-rs12720071-rs806368 was associated with nicotine dependence and FTND score in the 2 samples (P<.001 and P = .009, respectively). Conclusion Variants and haplotypes in the CNR1 gene may alter the risk for nicotine dependence, and the associations are likely sex specific. PMID:18606954

  15. Conformational thermostabilisation of corticotropin releasing factor receptor 1

    PubMed Central

    Kean, James; Bortolato, Andrea; Hollenstein, Kaspar; Marshall, Fiona H.; Jazayeri, Ali

    2015-01-01

    Recent technical advances have greatly facilitated G-protein coupled receptors crystallography as evidenced by the number of successful x-ray structures that have been reported recently. These technical advances include novel detergents, specialised crystallography techniques as well as protein engineering solutions such as fusions and conformational thermostabilisation. Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation. In this paper we describe for the first time the application of this technique to a member of a class B GPCR, the corticotropin releasing factor receptor 1 (CRF1R). Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents. We further describe the subsequent construct engineering steps that eventually yielded a crystallisation-ready construct which recently led to the solution of the first x-ray structure of a class B receptor. Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation. PMID:26159865

  16. Conformational thermostabilisation of corticotropin releasing factor receptor 1.

    PubMed

    Kean, James; Bortolato, Andrea; Hollenstein, Kaspar; Marshall, Fiona H; Jazayeri, Ali

    2015-01-01

    Recent technical advances have greatly facilitated G-protein coupled receptors crystallography as evidenced by the number of successful x-ray structures that have been reported recently. These technical advances include novel detergents, specialised crystallography techniques as well as protein engineering solutions such as fusions and conformational thermostabilisation. Using conformational thermostabilisation, it is possible to generate variants of GPCRs that exhibit significantly increased stability in detergent micelles whilst preferentially occupying a single conformation. In this paper we describe for the first time the application of this technique to a member of a class B GPCR, the corticotropin releasing factor receptor 1 (CRF1R). Mutational screening in the presence of the inverse agonist, CP-376395, resulted in the identification of a construct with twelve point mutations that exhibited significantly increased thermal stability in a range of detergents. We further describe the subsequent construct engineering steps that eventually yielded a crystallisation-ready construct which recently led to the solution of the first x-ray structure of a class B receptor. Finally, we have used molecular dynamic simulation to provide structural insight into CRF1R instability as well as the stabilising effects of the mutants, which may be extended to other class B receptors considering the high degree of structural conservation. PMID:26159865

  17. Delivery of Transferrin-Conjugated Polysaccharide Nanoparticles in 9L Gliosacoma Cells.

    PubMed

    Jeong, Young-Il; Kim, Young-Wook; Jung, Shin; Pei, Jian; Wen, Min; Li, Song-Yuan; Ryu, Hyang-Hwa; Lim, Jung Cheol; Jang, Woo-Youl; Kim, In-Young; Moon, Kyung-Sub; Jung, Tae-Young

    2015-01-01

    To investigate the possibility of drug targeting via the transferrin receptor-mediated pathway, iron-saturated transferrin was conjugated with chitosan (Tr-chitosan) and complexed with doxorubicin-conjugated methoxy poly(ethylene glycol)-b-dextran succinate (DEX-DOX). DEX-DOX nanoparticles have spherical morphologies with less than 150 nm particle sizes. When Tr-chitosan was complexed with DEX-DOX nanoparticles (TR nanoparticle), particle sizes were increased to higher than 200 nm. Viability of 9L cells with treatment of doxorubicin (DOX) or DEX-DOX nanoparticle was dose-dependently decreased regardless of transferrin receptor blocking. However, cytotoxicity of TR nanoparticles was reduced by blocking of transferrin receptor. Flow cytometric analysis and confocal microscopic observation showed that fluorescence intensity of tumor cells with treatment of TR nanoparticles was significantly decreased by blocking of transferring receptor while DEX-DOX nanoparticles were not affected by blocking of transferring receptor. These results indicated that TR nanoparticles are promising candidates for brain tumor drug delivery. PMID:26328315

  18. Damage of the outer membrane of enteric gram-negative bacteria by lactoferrin and transferrin.

    PubMed Central

    Ellison, R T; Giehl, T J; LaForce, F M

    1988-01-01

    Many studies have shown that lactoferrin and transferrin have antimicrobial activity against gram-negative bacteria, but a mechanism of action has not been defined. We hypothesized that the iron-binding proteins could affect the gram-negative outer membrane in a manner similar to that of the chelator EDTA. The ability of lactoferrin and transferrin to release radiolabeled lipopolysaccharide (LPS) from a UDP-galactose epimerase-deficient Escherichia coli mutant and from wild-type Salmonella typhimurium strains was tested. Initial studies in barbital-acetate buffer showed that EDTA and lactoferrin cause significant release of LPS from all three strains. Further studies found that LPS release was blocked by iron saturation of lactoferrin, occurred between pH 6 and 7.5, was comparable for bacterial concentrations from 10(4) to 10(7) CFU/ml, and increased with increasing lactoferrin concentrations. Studies using Hanks balanced salt solution lacking calcium and magnesium showed that transferrin also could cause LPS release. Additionally, both lactoferrin and transferrin increased the antibacterial effect of a subinhibitory concentration of rifampin, a drug excluded by the bacterial outer membrane. This work demonstrates that these iron-binding proteins damage the gram-negative outer membrane and alter bacterial outer membrane permeability. Images PMID:3169987

  19. 181Hf-Labelled rat serum transferrin: Influence of temperature on the metal-binding configurations

    NASA Astrophysics Data System (ADS)

    Appel, H.; Duffield, J.; Taylor, D. M.; Then, G. M.; Thies, W.-G.

    1987-04-01

    A181Hf-labelled rat serum transferrin sample was studied at different temperatures between 100 K and 346 K using a fast BaF2 TDPAC setup. Relaxation effects were observed in the liquid serum as well as in the frozen serum sample. The temperature dependence of the correlation time has been determined. Different relaxation modes are discussed.

  20. Cannabinoid receptor 1 ligands revisited: Pharmacological assessment in the ACTOne system.

    PubMed

    Presley, Chaela S; Abidi, Ammaar H; Moore, Bob M

    2016-04-01

    In vitro cannabinoid pharmacology has evolved over time from simple receptor binding to include [(35)S]GTPγ, β-arrestin, and cAMP assays. Each assay has benefits and drawbacks; however, no single functional system has been used for high-throughput evaluation of compounds from binding to pharmacological functionality and antagonist assessment in a well-characterized human cell line. In this study, we evaluated and validated one system-ACTOne human embryonic kidney cells transfected with a cyclic nucleotide gated channel and cannabinoid receptor 1 (CB1)-and compared human CB1 affinity, functional, and antagonistic effects on cAMP with previously published results. The study was conducted on a diverse group of CB1 ligands, including endocannabinoids and related compounds, 2-AG, AEA, MAEA, and ACEA, the phytocannabinoid Δ(9) THC, and synthetic cannabinoids CP 55,940, WIN 55,212-2, SR 141716A, CP 945,598, and WIN 55,212-3. Our results were compared with literature values where human CB1 was used for affinity determination and cAMP was used as a functional readout. Here we report the first detailed evaluation of the ACTOne assay for the pharmacological evaluation of CB1 ligands. The results from the study reveal some interesting deviations from previously reported functional activities of the aforementioned ligands. PMID:26772161

  1. Humanization of immunotoxins.

    PubMed

    Rybak, S M; Hoogenboom, H R; Meade, H M; Raus, J C; Schwartz, D; Youle, R J

    1992-04-15

    The construction and expression of a chimeric gene encoding a mouse/human antibody to the human transferrin receptor fused to the gene for angiogenin, a human homolog of pancreatic RNase, are described. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human angiogenin to a chimeric anti-transferrin receptor heavy chain gene. The antibody-enzyme fusion gene was introduced into a transfectoma that secretes the chimeric light chain of the same antibody, and cell lines were cloned that synthesize and secrete the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/ml. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis of K562 cells that express the human transferrin receptor but not toward a non-human-derived cell line that lacks this receptor. Whereas excess antibody to the same receptor did not itself inhibit protein synthesis, it was able to completely prevent the protein synthesis inhibition caused by the fusion protein. These results indicate that the cytotoxicity is due to a transferrin receptor-mediated mechanism involving the angiogenin portion of the fusion protein and demonstrate the feasibility of constructing recombinant antibody-RNase molecules capable of killing tumor cells bearing the transferrin receptor. The significance of the acquired cytotoxicity of a mouse/human chimeric antibody linked to a human protein may bear importantly in human therapeutic strategies that use mouse antibodies linked to toxins from plants or bacteria to target tumor cells. It is expected that the humanization of immunotoxins will lead to less toxicity and immunogenicity than currently available reagents. PMID:1565609

  2. Evolution of the transferrin family: conservation of residues associated with iron and anion binding.

    PubMed

    Lambert, Lisa A; Perri, Holly; Halbrooks, Peter J; Mason, Anne B

    2005-10-01

    The transferrin family spans both vertebrates and invertebrates. It includes serum transferrin, ovotransferrin, lactoferrin, melanotransferrin, inhibitor of carbonic anhydrase, saxiphilin, the major yolk protein in sea urchins, the crayfish protein, pacifastin, and a protein from green algae. Most (but not all) contain two domains of around 340 residues, thought to have evolved from an ancient duplication event. For serum transferrin, ovotransferrin and lactoferrin each of the duplicated lobes binds one atom of Fe (III) and one carbonate anion. With a few notable exceptions each iron atom is coordinated to four conserved amino acid residues: an aspartic acid, two tyrosines, and a histidine, while anion binding is associated with an arginine and a threonine in close proximity. These six residues in each lobe were examined for their evolutionary conservation in the homologous N- and C-lobes of 82 complete transferrin sequences from 61 different species. Of the ligands in the N-lobe, the histidine ligand shows the most variability in sequence. Also, of note, four of the twelve insect transferrins have glutamic acid substituted for aspartic acid in the N-lobe (as seen in the bacterial ferric binding proteins). In addition, there is a wide spread substitution of lysine for the anion binding arginine in the N-lobe in many organisms including all of the fish, the sea squirt and many of the unusual family members i.e., saxiphilin and the green alga protein. It is hoped that this short analysis will provide the impetus to establish the true function of some of the TF family members that clearly lack the ability to bind iron in one or both lobes and additionally clarify the evolutionary history of this important family of proteins. PMID:16111909

  3. Binding of Chromium(III) to Transferrin Could Be Involved in Detoxification of Dietary Chromium(III) Rather than Transport of an Essential Trace Element.

    PubMed

    Levina, Aviva; Pham, T H Nguyen; Lay, Peter A

    2016-07-01

    Cr(III) binding to transferrin (Tf; the main Fe(III) transport protein) has been postulated to mediate cellular uptake of Cr(III) to facilitate a purported essential role for this element. Experiments using HepG2 (human hepatoma) cells, which were chosen because of high levels of the transferrin receptor, showed that Cr(III) binding to vacant Fe(III) -binding sites of human Tf effectively blocks cellular Cr(III) uptake. Through bio-layer interferometry studies of the Tf cycle, it was found that both exclusion and efflux of Cr2 (III) Tf from cells was caused by 1) relatively low Cr2 Tf affinity to cell-surface Tf receptors compared to Fe2 Tf, and 2) disruption of metal release under endosomal conditions and post-endosomal Tf dissociation from the receptor. These data support mounting evidence that Cr(III) is not essential and that Tf binding is likely to be a natural protective mechanism against the toxicity and potential genotoxicity of dietary Cr through blocking Cr(III) cellular accumulation. PMID:27197571

  4. Corneal avascularity is due to soluble VEGF receptor-1.

    PubMed

    Ambati, Balamurali K; Nozaki, Miho; Singh, Nirbhai; Takeda, Atsunobu; Jani, Pooja D; Suthar, Tushar; Albuquerque, Romulo J C; Richter, Elizabeth; Sakurai, Eiji; Newcomb, Michael T; Kleinman, Mark E; Caldwell, Ruth B; Lin, Qing; Ogura, Yuichiro; Orecchia, Angela; Samuelson, Don A; Agnew, Dalen W; St Leger, Judy; Green, W Richard; Mahasreshti, Parameshwar J; Curiel, David T; Kwan, Donna; Marsh, Helene; Ikeda, Sakae; Leiper, Lucy J; Collinson, J Martin; Bogdanovich, Sasha; Khurana, Tejvir S; Shibuya, Masabumi; Baldwin, Megan E; Ferrara, Napoleone; Gerber, Hans-Peter; De Falco, Sandro; Witta, Jassir; Baffi, Judit Z; Raisler, Brian J; Ambati, Jayakrishna

    2006-10-26

    Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea. PMID:17051153

  5. Interaction of lipids with the neurotensin receptor 1.

    PubMed

    Bolivar, Juan H; Muñoz-García, Juan C; Castro-Dopico, Tomas; Dijkman, Patricia M; Stansfeld, Phillip J; Watts, Anthony

    2016-06-01

    Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs. PMID:26926422

  6. Cannabinoid receptor 1-expressing neurons in the nucleus accumbens.

    PubMed

    Winters, Bradley D; Krüger, Juliane M; Huang, Xiaojie; Gallaher, Zachary R; Ishikawa, Masago; Czaja, Krzysztof; Krueger, James M; Huang, Yanhua H; Schlüter, Oliver M; Dong, Yan

    2012-10-01

    Endocannabinoid signaling critically regulates emotional and motivational states via activation of cannabinoid receptor 1 (CB1) in the brain. The nucleus accumbens (NAc) functions to gate emotional and motivational responses. Although expression of CB1 in the NAc is low, manipulation of CB1 signaling within the NAc triggers robust emotional/motivational alterations related to drug addiction and other psychiatric disorders, and these effects cannot be exclusively attributed to CB1 located at afferents to the NAc. Rather, CB1-expressing neurons in the NAc, although sparse, appear to be critical for emotional and motivational responses. However, the cellular properties of these neurons remain largely unknown. Here, we generated a knock-in mouse line in which CB1-expressing neurons expressed the fluorescent protein td-Tomato (tdT). Using these mice, we demonstrated that tdT-positive neurons within the NAc were exclusively fast-spiking interneurons (FSIs). These FSIs were electrically coupled with each other, and thus may help synchronize populations/ensembles of NAc neurons. CB1-expressing FSIs also form GABAergic synapses on adjacent medium spiny neurons (MSNs), providing feed-forward inhibition of NAc output. Furthermore, the membrane excitability of tdT-positive FSIs in the NAc was up-regulated after withdrawal from cocaine exposure, an effect that might increase FSI-to-MSN inhibition. Taken together with our previous findings that the membrane excitability of NAc MSNs is decreased during cocaine withdrawal, the present findings suggest that the basal functional output of the NAc is inhibited during cocaine withdrawal by multiple mechanisms. As such, CB1-expressing FSIs are targeted by cocaine exposure to influence the overall functional output of the NAc. PMID:23012412

  7. Effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study

    SciTech Connect

    Kilar, F.; Simon, I.

    1985-11-01

    Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion.

  8. The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study.

    PubMed Central

    Kilár, F; Simon, I

    1985-01-01

    Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion. PMID:4074838

  9. METAL-TYROSYL COORDINATION IN TRANSFERRIN. 2. DIFFERENCE ULTRAVIOLET SPECTROSCOPY OF DI-, TRI-, AND TETRAVALENT METAL IONS WITH ETHYLENE-BIS(0-DYDROXYPHENYLGLY-CINE).

    SciTech Connect

    Pecoraro, Vincent L.; Harris, Wesley R.; Carrano, Carl J.; Raymond, Kenneth N.

    1980-08-01

    In order to probe the metal ion coordination site in the human iron transport protein, transferrin, the complexation of a series of metal ions by the chelate analogue ethylene-bis(o-hydroxyphenylglycine) (EHPG) has been studied by difference uv spectroscopy, in which {Delta}{epsilon} values per coordinated phenol have been determined for the metal complex versus the protonated form of the ligand. With the exception of the.Cu {sup 2+} complex, maxima are observed at 242 nm and 290 nm with a minimum at 269 nm. The {Delta}{epsilon} values at 242 fall into two groups. Complexes of divalent metal ions (Zn{sup 2+}, Cu{sup 2+}, Cd{sup 2+}) have 6£ values ranging from 5000 to 6600 M{sup -1} cm{sup -1} whereas larger {Delta}{epsilon} values are observed for complexes of tri- and tetra- valent metal ions (Th{sup 4+}, Ga{sup 3+}, Fe{sup 3+}, • Ho{sup 3+}, Eu{sup 3+}, Er{sup 3+}, Tb{sup 2+}, VO{sup 2+}), 7400 - 8700 M{sup -1} cm{sup -1}. It is known that the transferrin binding sites contain tyrosyl residues, but there has been considerable debate concerning the precise number of tyrosine groups which bind to specific metal ions. Since it has been the common practice to assume the {Delta}{epsilon} values for coordination by all metal ions are identical, the larger range of {Delta}{epsilon} values actually observed here shows that such an assumption can actually lead to an erroneous tyrosine/metal site ratio. The difference spectra of transferrin and EHPG complexes are very similar, and we have taken the {Delta}{epsilon} values of the EHPG complexes as estimates for the intrinsic {Delta}{epsilon} for coordination of a single tyrosine ligand. The number of tyrosines bound per metal ion is then calculated based on previously reported total {Delta}{epsilon} values of several di(metallo)transferrin complexes. The results show that two tyrosines are coordinated per metal ion for all the transition metals and the smaller lanthanides. Very large metal ions have difficulty fitting

  10. The effect of AZT and chloroquine on the activities of ricin and a saporin-transferrin chimeric toxin.

    PubMed

    Lizzi, A R; D'Alessandro, A M; Zeolla, N; Brisdelli, F; D'Andrea, G; Pitari, G; Oratore, A; Bozzi, A; Ippoliti, R

    2005-08-15

    This study deals with the combination of chloroquine (CQ, an anti-malaric drug) and 3'-azido-3'-deoxythymidine (AZT, anti-human immuno-deficiency virus (HIV) drug) with a chimeric toxin (TS) obtained by chemical linking of saporin (a ribosome inactivating protein from the plant Saponaria officinalis) and human transferrin, in the intoxication of the human chronic myeloid leukaemia cells (K562). Our data demonstrate that AZT, at concentrations comparable to those reached in the blood of HIV-infected patients under pharmacological treatment with this drug, can increase the toxicity of TS in cooperation with CQ inducing an increased effect on protein synthesis in K562 cells ( approximately 50% inhibition of protein synthesis for TS alone, and TS with AZT and approximately 70% with both AZT and CQ). Furthermore, pre-treatment of cells with AZT alone can induce an increase of apoptosis in K562 cells intoxicated with TS. By comparing data obtained with the model toxin ricin, we get indications that the two toxins partially differ in their intracellular routes, also suggesting that chimeric constructs containing ricin-like toxins (i.e. immunotoxins) could be coupled with the use of common and cheap drugs for the treatment of cancer in HIV-infected patients. PMID:15982641

  11. Incretin-like effects of small molecule trace amine-associated receptor 1 agonists

    PubMed Central

    Raab, Susanne; Wang, Haiyan; Uhles, Sabine; Cole, Nadine; Alvarez-Sanchez, Ruben; Künnecke, Basil; Ullmer, Christoph; Matile, Hugues; Bedoucha, Marc; Norcross, Roger D.; Ottaway-Parker, Nickki; Perez-Tilve, Diego; Conde Knape, Karin; Tschöp, Matthias H.; Hoener, Marius C.; Sewing, Sabine

    2015-01-01

    Objective Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight. Methods We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice. Results TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls. Conclusions We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity. PMID:26844206

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

    PubMed

    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. PMID:25454663

  13. Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation

    PubMed Central

    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.

    2016-01-01

    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 Hbbth1/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. PMID:26635037

  14. The Mammalian Neuroendocrine Hormone Norepinephrine Supplies Iron for Bacterial Growth in the Presence of Transferrin or Lactoferrin

    PubMed Central

    Freestone, Primrose P. E.; Lyte, Mark; Neal, Christopher P.; Maggs, Anthony F.; Haigh, Richard D.; Williams, Peter H.

    2000-01-01

    Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum. Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of Escherichia coli. Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth. Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels. Norepinephrine also caused the loss of iron from lactoferrin. The pharmacologically inactive metabolite norepinephrine 3-O-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium. Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin. Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine. Norepinephrine-stimulated growth in medium containing 55Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells. Moreover, norepinephrine-stimulated growth in medium containing [3H]norepinephrine indicated concomitant uptake of norepinephrine. In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity (55Fe or 3H) associated with bacterial cells. A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed. PMID:11029429

  15. Experimental treatment of breast cancer-bearing BALB/c mice by artemisinin and transferrin-loaded magnetic nanoliposomes

    PubMed Central

    Gharib, Amir; Faezizadeh, Zohreh; Mesbah-Namin, Seyed Ali Reza; Saravani, Ramin

    2015-01-01

    Background: 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. Materials and Methods: 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. Results: 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. Conclusion: 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. PMID:26109756

  16. Anthrax Toxin Receptor 1 Is Essential for Arteriogenesis in a Mouse Model of Hindlimb Ischemia

    PubMed Central

    Andersen, N. J.; Boguslawski, E. A.; Naidu, A. S.; Szot, C.; Bromberg-White, J. L.; Kits, K.; Kuk, C. Y.; Holton, L. E.; St. Croix, B.; Chambers, C. M.; Duesbery, N. S.

    2016-01-01

    Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1-/- mice. Additional tests were performed by measuring gene expression in in vitro models of fluid shear stress and hypoxia, as well as in human muscle tissues obtained from patients having peripheral artery disease. We observed that Antxr1 expression transiently increased in ischemic tissues following femoral artery ligation and that its expression was necessary for arteriogenesis. In the absence of Antxr1, the mean arterial lumen area in ischemic tissues decreased. Antxr1 mRNA and protein expression was positively regulated by fluid shear stress, but not by hypoxia. Furthermore, Antxr1 expression was elevated in human peripheral artery disease requiring lower extremity bypass surgery. These findings demonstrate an essential physiologic role for Antxr1 in arteriogenesis and peripheral artery disease, with important implications for managing ischemia and other arteriogenesis-dependent vascular diseases. PMID:26785120

  17. Differential expression and tumorigenic function of neurotensin receptor 1 in neuroendocrine tumor cells

    PubMed Central

    Kim, Ji Tae; Li, Jing; Song, Jun; Lee, Eun Y.; Weiss, Heidi L.; Townsend, Courtney M.; Evers, B. Mark

    2015-01-01

    Neurotensin (NTS), localized predominantly to the small bowel, stimulates the growth of a variety of cancers, including neuroendocrine tumors (NETs), mainly through its interaction with the high-affinity NTS receptor 1 (NTSR1). Here, we observed increased expression of NTSR1 in almost all tested clinical NET samples, but not in normal tissues. Through RT-PCR analysis, we found that the expression of NTSR1 and NTSR2 was either variable (NTSR1) or absent (NTSR2) in human NET cell lines. In contrast, NTSR3 and NTS were expressed in all NET cells. Treatment with 5-aza-2′-deoxycytidine, a demethylating agent, increased levels of NTSR1 and NTSR2 suggesting that DNA methylation contributes to NTSR1/2 expression patterns, which was confirmed by methylation analyses. In addition, we found that knockdown of NTSR1 decreased proliferation, expression levels of growth-related proteins, and anchorage-independent growth of BON human carcinoid cells. Moreover, stable silencing of NTSR1 suppressed BON cell growth, adhesion, migration and invasion. Our results show that high expression of NTSR1 is found in clinical NETs and that promoter methylation is an important mechanism controlling the differential expression of NTSR1 and silencing of NTSR2 in NET cells. Furthermore, knockdown of NTSR1 in BON cells suppressed oncogenic functions suggesting that NTSR1 contributes to NET tumorigenesis. PMID:26298774

  18. Oxytocin receptor and vasopressin receptor 1a genes are respectively associated with emotional and cognitive empathy.

    PubMed

    Uzefovsky, F; Shalev, I; Israel, S; Edelman, S; Raz, Y; Mankuta, D; Knafo-Noam, A; Ebstein, R P

    2015-01-01

    Empathy is the ability to recognize and share in the emotions of others. It can be considered a multifaceted concept with cognitive and emotional aspects. Little is known regarding the underlying neurochemistry of empathy and in the current study we used a neurogenetic approach to explore possible brain neurotransmitter pathways contributing to cognitive and emotional empathy. Both the oxytocin receptor (OXTR) and the arginine vasopressin receptor 1a (AVPR1a) genes contribute to social cognition in both animals and humans and hence are prominent candidates for contributing to empathy. The following research examined the associations between polymorphisms in these two genes and individual differences in emotional and cognitive empathy in a sample of 367 young adults. Intriguingly, we found that emotional empathy was associated solely with OXTR, whereas cognitive empathy was associated solely with AVPR1a. Moreover, no interaction was observed between the two genes and measures of empathy. The current findings contribute to our understanding of the distinct neurogenetic pathways involved in cognitive and emotional empathy and underscore the pervasive role of both oxytocin and vasopressin in modulating human emotions. PMID:25476609

  19. The Discovery of Indole Full Agonists of the Neurotensin Receptor 1 (NTSR1)

    PubMed Central

    Di Fruscia, Paolo; He, Yuanjun; Koenig, Marcel; Tabrizifard, Sahba; Nieto, Ainhoa; McDonald, Patricia H.; Kamenecka, Theodore M.

    2014-01-01

    Neurotensin (NT) is an endogenous tridecapeptide found in the central nervous system (CNS) and in peripheral tissues. Neurotensin exerts a wide range of physiological effects and it has been found to play a critical role in a number of human diseases, such as schizophrenia, Parkinson’s disease and drug addiction. The discovery of small-molecule non-peptide neurotensin receptor (NTSR) modulators would represent an important breakthrough as such compounds could be used as pharmacological tools, to further decipher the cellular functions of neurotensin, and potentially as therapeutic agents to treat human disease. Herein, we report the identification of non-peptide low-micromolar neurotensin receptor 1 (NTSR1) full agonists, discovered through structural optimization of the known NTSR1 partial agonist 1. In vitro cellular screenings, based on an intracellular Ca2+ mobilization assay, revealed our best hit molecule 8 (SR-12062) to have an EC50 of 2 μM at NTSR1 with full agonist behaviour (Emax = 100%), showing a higher efficacy and ~ 90-fold potency improvement compared to parent compound 1 (EC50 = 178 μM; Emax = 17%). PMID:24997685

  20. Discoidin Domain Receptor 1 Protein Is a Novel Modulator of Megakaryocyte-Collagen Interactions*

    PubMed Central

    Abbonante, Vittorio; Gruppi, Cristian; Rubel, Diana; Gross, Oliver; Moratti, Remigio; Balduini, Alessandra

    2013-01-01

    Growing evidence demonstrates that extracellular matrices regulate many aspects of megakaryocyte (MK) development; however, among the different extracellular matrix receptors, integrin α2β1 and glycoprotein VI are the only collagen receptors studied in platelets and MKs. In this study, we demonstrate the expression of the novel collagen receptor discoidin domain receptor 1 (DDR1) by human MKs at both mRNA and protein levels and provide evidence of DDR1 involvement in the regulation of MK motility on type I collagen through a mechanism based on the activity of SHP1 phosphatase and spleen tyrosine kinase (Syk). Specifically, we demonstrated that inhibition of DDR1 binding to type I collagen, preserving the engagement of the other collagen receptors, glycoprotein VI, α2β1, and LAIR-1, determines a decrease in MK migration due to the reduction in SHP1 phosphatase activity and consequent increase in the phosphorylation level of its main substrate Syk. Consistently, inhibition of Syk activity restored MK migration on type I collagen. In conclusion, we report the expression and function of a novel collagen receptor on human MKs, and we point out that an increasing level of complexity is necessary to better understand MK-collagen interactions in the bone marrow environment. PMID:23530036

  1. Anthrax Toxin Receptor 1 Is Essential for Arteriogenesis in a Mouse Model of Hindlimb Ischemia.

    PubMed

    Andersen, N J; Boguslawski, E A; Naidu, A S; Szot, C; Bromberg-White, J L; Kits, K; Kuk, C Y; Holton, L E; St Croix, B; Chambers, C M; Duesbery, N S

    2016-01-01

    Anthrax toxin receptor 1/tumor endothelial marker 8 (Antxr1 or TEM8) is up-regulated in tumor vasculature and serves as a receptor for anthrax toxin, but its physiologic function is unclear. The objective of this study was to evaluate the role of Antxr1 in arteriogenesis. The role of Antxr1 in arteriogenesis was tested by measuring gene expression and immunohistochemistry in a mouse model of hindlimb ischemia using wild-type and ANTXR1(-/-) mice. Additional tests were performed by measuring gene expression in in vitro models of fluid shear stress and hypoxia, as well as in human muscle tissues obtained from patients having peripheral artery disease. We observed that Antxr1 expression transiently increased in ischemic tissues following femoral artery ligation and that its expression was necessary for arteriogenesis. In the absence of Antxr1, the mean arterial lumen area in ischemic tissues decreased. Antxr1 mRNA and protein expression was positively regulated by fluid shear stress, but not by hypoxia. Furthermore, Antxr1 expression was elevated in human peripheral artery disease requiring lower extremity bypass surgery. These findings demonstrate an essential physiologic role for Antxr1 in arteriogenesis and peripheral artery disease, with important implications for managing ischemia and other arteriogenesis-dependent vascular diseases. PMID:26785120

  2. NOVEL CHARACTERIZATION OF bEnd.3 CELLS THAT EXPRESS LYMPHATIC VESSEL ENDOTHELIAL HYALURONAN RECEPTOR-1

    PubMed Central

    Yuen, D.; Leu, R.; Tse, J.; Wang, S.; Chen, L.L.; Chen, L.

    2015-01-01

    Murine bEnd.3 endothelioma cell line has been widely used in vascular research and here we report the novel finding that bEnd.3 cells express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR-3). Moreover, these cells express progenitor cell markers of Sca-1 and CD133. Upon stimulation with tumor necrosis factor-alpha (TNF-α), the bEnd.3 cells demonstrate enhanced formation of capillary-type tubes, which express LYVE-1. As the bEnd.3 cell line is derived from murine endothelioma, we further examined human tissues of endothelioma and identified lymphatic vessels in the tumor samples which express both LYVE-1 and podoplanin. Moreover, a significantly higher number of lymphatic vessels were detected in the endothelioma samples compared with normal control. Taken together, this study not only redefines bEnd.3 cells for vascular research, but also indicates a broader category of human diseases that are associated with lymphatics, such as endothelioma. PMID:25282873

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

    PubMed

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

    2016-05-01

    DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface. PMID:27032044

  4. Enhancing surface interactions with colon cancer cells on a transferrin-conjugated 3D nanostructured substrate.

    PubMed

    Banerjee, Shashwat S; Paul, Debjani; Bhansali, Sujit G; Aher, Naval D; Jalota-Badhwar, Archana; Khandare, Jayant

    2012-06-11

    A transferrin-conjugated PEG-Fe(3) O(4) nanostructured matrix is developed to explore cellular responses in terms of enhanced cell adhesion, specific interactions between ligands in the matrix and molecular receptors on the cell membrane, comparison of cell shapes on 2D and 3D surfaces, and effect of polymer architecture on cell adhesion. Integration of such advanced synthetic nanomaterials into a functionalized 3D matrix to control cell behavior on surfaces will have implications in nanomedicine. PMID:22434693

  5. Specific ligand binding attributable to individual epitopes of gonococcal transferrin binding protein A.

    PubMed

    Masri, Heather P; Cornelissen, Cynthia Nau

    2002-02-01

    The gonococcal transferrin receptor complex comprises two iron-regulated proteins, TbpA and TbpB. TbpA is essential for transferrin-iron uptake and is a TonB-dependent integral outer membrane protein. TbpB is thought to increase the efficiency of iron uptake from transferrin and is lipid modified and surface exposed. To evaluate the structure-function relationships in one of the components of the receptor, TbpA, we created constructs that fused individual putative loops of TbpA with amino-terminal affinity tags. The recombinant proteins were then overexpressed in Escherichia coli, and the fusions were recovered predominately from inclusion bodies. Inclusion body proteins were solubilized, and the epitope fusions were renatured by slow dialysis. To assess transferrin binding capabilities, the constructs were tested in a solid-phase dot blot assay followed by confirmatory quantitative chemiluminescent enzyme-linked immunosorbent assays. The constructs with only loop 5 and with loops 4 and 5 demonstrated dose-dependent specific ligand binding in spite of being out of the context of the intact receptor. The immunogenicities of individual TbpA-specific epitopes were investigated by generating rabbit polyclonal antisera against the fusion proteins. Most of the fusion proteins were immunogenic under these conditions, and the resulting sera recognized full-length TbpA in immunoblots. These results suggest that individual epitopes of TbpA are both immunogenic and functional with respect to ligand binding capabilities, and the vaccine implications of these findings are discussed. PMID:11796606

  6. The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains.

    PubMed

    Ala'Aldeen, D A; Borriello, S P

    1996-01-01

    When grown in vivo, or under iron-restriction in vitro, Neisseria meningitidis expresses a number of iron-regulated outer membrane proteins, including two transferrin-binding proteins (Tbp1 and Tbp2). The Tbps are highly specific receptors for human transferrin and we have previously demonstrated their immunogenicity in humans and animals and their exposure on the surface of the organism. There is a growing interest in incorporating these Tbps in future outer membrane-based meningococcal vaccines. Protection against meningococcal infection has been correlated with serum bactericidal antibodies, therefore, it is important for these vaccine candidates to generate such antibodies. We have previously raised rabbit and murine polyclonal monospecific antisera against the Tbps of strain SD (B:15:P1.16) which showed varying degrees of cross-reactivity on immunoblots between the Tbp1 and/or Tbp2 molecules of different heterologous strains from various serogroups, types and subtypes. The ability of these antisera to kill meningococci were tested by incubating live organisms (grown to log phase under iron-restriction) with the antisera in the presence of a human complement source (serum from an agammaglobulinaemic patient). The antisera killed the homologous and the majority of the examined heterologous strains with varying efficiency, with no obvious correlation with the identity of the strains or the Tbp isotypes which vary between strains. Although the animal anti-Tbp antibodies failed to kill some meningococcal strains, it is not clear how human anti-Tbp antibodies would behave. The mouse antiserum was able to kill some heterologous stains against which it only had detectable anti-Tbp1 and not anti-Tbp2 antibodies, as seen on Western blots. Furthermore, the rabbit antiserum was able to kill both Tbp1 and Tbp2 mutants of strain B16B6 (B2a:P1.2) to almost the same level as the wild type strain, indicating that both components of the transferrin receptor (Tbp1 and Tbp2) are

  7. In vivo and in vitro studies of hafnium-binding to rat serum transferrin.

    PubMed

    Then, G M; Appel, H; Duffield, J; Taylor, D M; Thies, W G

    1986-08-01

    The binding of hafnium to rat serum transferrin was studied using the time differential perturbed angular correlation (TDPAC) technique. Hafnium is interesting as a toxic metal binding to transferrin because it behaves metabolically similarly to plutonium. The isotope 181Hf offers favorable access to the TDPAC-method. Samples were prepared in vivo by intravenous injection of Hf-NTA, Hf-citrate, and Hf-oxalate solutions, respectively, into Sprague-Dawley rats and in vitro by adding Hf-NTA solution to fresh rat serum. In both cases two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. They may be attributed to the N-terminal and the C-terminal binding site in the transferrin molecule. The 181Hf-distribution between these two binding states depends on pH, salt and hafnium concentrations, temperature, and incubation time. With a fast TDPAC-setup of four BaF2-detectors a time resolution of about 600 ps could be achieved. The specific binding configurations of 181Hf and the comparatively slow relaxation times lead to spectra of considerable accuracy. PMID:3746294

  8. In vivo and in vitro studies of hafnium-binding to rat serum transferrin

    SciTech Connect

    Then, G.M.; Appel, H.; Duffield, J.; Taylor, D.M.; Thies, W.G.

    1986-08-01

    The binding of hafnium to rat serum transferrin was studied using the time differential perturbed angular correlation (TDPAC) technique. Hafnium is interesting as a toxic metal binding to transferrin because it behaves metabolically similarly to plutonium. The isotope 181Hf offers favorable access to the TDPAC-method. Samples were prepared in vivo by intravenous injection of Hf-NTA, Hf-citrate, and Hf-oxalate solutions, respectively, into Sprague-Dawley rats and in vitro by adding Hf-NTA solution to fresh rat serum. In both cases two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. They may be attributed to the N-terminal and the C-terminal binding site in the transferrin molecule. The 181Hf-distribution between these two binding states depends on pH, salt and hafnium concentrations, temperature, and incubation time. With a fast TDPAC-setup of four BaF2-detectors a time resolution of about 600 ps could be achieved. The specific binding configurations of 181Hf and the comparatively slow relaxation times lead to spectra of considerable accuracy.

  9. Partial characterization of horse transferrin heterogeneity with respect to the atypical type, Tf C.

    PubMed

    Stratil, A; Glasnák, V

    1981-01-01

    In starch gel electrophoresis of horse sera each transferrin variant is formed by a strong anodal band and a weaker cathodal band. An 'atypical' Tf C, has two zones of about equal intensity. Family data show that Tf C is genetically controlled by an allele Tf C at the Tf locus. Frequencies of transferrin alleles in various horse breeds are also presented. After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephadex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of hands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid. PMID:7283207

  10. Juvenile hormone binding protein and transferrin from Galleria mellonella share a similar structural motif.

    PubMed

    Krzyzanowska, D; Ozyhar, A; Lalik, A; Parkitna, J M; Szkudlarek, J; Waśniowska, K; Lisowska, E; Kochman, M

    2001-07-01

    It has been previously suggested that juvenile hormone binding protein(s) (JHBP) belongs to a new class of proteins. In the search for other protein(s) that may contain structural motifs similar to those found in JHBP, hemolymph from Galleria mellonella (Lepidoptera) was chromatographed over a Sephadex G-200 column and resulting fractions were subjected to SDS-PAGE, transferred onto nitrocellulose membrane and scanned with a monoclonal antibody, mAb 104, against hemolymph JHBP. Two proteins yielded a positive reaction with mAb 104, one corresponding to JHBP and the second corresponding to a transferrin, as judged from N-terminal amino acid sequencing staining. Transferrin was purified to about 80% homogeneity using a two-step procedure including Sephadex G-200 gel filtration and HPLC MonoQ column chromatography. Panning of a random peptide display library and analysis with immobilized synthetic peptides were applied for finding a common epitope present in JHBP and the transferrin molecule. The postulated epitope motif recognized by mAb 104 in the JHBP sequence is RDTKAVN, and is localized at position 82-88. PMID:11530933

  11. Isolation of mouse transferrin using salting-out chromatography on Sepharose CL-6B.

    PubMed

    Sawatzki, G; Anselstetter, V; Kubanek, B

    1981-01-30

    A new method for the isolation of considerable quantities of mouse transferrin is described. This technique employs salting-out chromatography on Sepharose CL-6B, a new step in the preparation of plasma proteins. This step is followed by ion-exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on Sephacryl S-200. The isolated mouse transferrin was shown to be pure by immunoelectrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by the 465 nm/410 nm ratio of absorbances being 1.41. The molecular weight was determined to be about 77 500. The advantages of this procedure are that it is reproducible, gives a high recovery, and can be extended to a larger scale. The advantage over other protein purification techniques is its general utility, due to the fact that there is no need for species-specific antibodies. The application of this method offers a rapid purification of sufficient quantities of mouse transferrin essential for the elucidation of biological functions of this protein and investigations of its molecular structure. PMID:7213791

  12. Cloning and expression of a transferrin-binding protein from Actinobacillus pleuropneumoniae.

    PubMed Central

    Gerlach, G F; Anderson, C; Potter, A A; Klashinsky, S; Willson, P J

    1992-01-01

    An expression library was constructed from Actinobacillus pleuropneumoniae serotype 7. Escherichia coli transformants expressing recombinant proteins were identified by immunoscreening with porcine convalescent serum. One transformant expressing a 60-kDa protein (60K protein) in aggregated form was identified. Serum raised against the recombinant protein recognized a polypeptide with an indistinguishable electrophoretic mobility in the A. pleuropneumoniae wild type after iron-restricted growth only. The recombinant protein bound transferrin after blotting onto nitrocellulose. Using a competitive enzyme-linked immunosorbent assay (ELISA), the specificity of this binding for the amino-terminal half of iron-saturated porcine transferrin was established. Also, the 60K wild-type protein bound hemin as assessed by hemin-agarose chromatography. Hemin could inhibit transferrin binding of the recombinant protein in the competitive ELISA, whereas hemoglobin and synthetic iron chelators failed to do so. Southern blot analysis of several other A. pleuropneumoniae strains indicated that highly homologous sequence is present in eight of eight isolates of serotype 7 and in some isolates of serotypes 2, 3, and 4. Images PMID:1541562

  13. Changes in transferrin saturation after treatment with the oral iron chelator deferiprone in patients with iron overload.

    PubMed Central

    al-Refaie, F N; De Silva, C E; Wonke, B; Hoffbrand, A V

    1995-01-01

    AIMS--To evaluate the changes in transferrin saturation in patients with iron overload following the oral administration of the iron chelator deferiprone; to assess the correlation between the degree of transferrin desaturation, the deferiprone dose, and urinary iron excretion. METHODS--Serum samples were obtained from 16 patients with iron overload at different time intervals following the oral administration of deferiprone (50 mg/kg). These samples were analysed using 6M urea/polyacrylamide gel electrophoresis (UPAGE). This method is able to resolve serum transferrin into four different forms (free iron, two forms of monoferric, and diferric). The deferiprone concentration in these samples was estimated using high pressure liquid chromatography (HPLC). Zero time samples (t0) from 10 patients were incubated with 150 microM deferiprone or normal saline either at room temperature or at 37 degrees C for 30 minutes and 24 hours, and also at -20 degrees C for six weeks. Samples were then analysed using UPAGE. RESULTS--A maximum decrease in transferrin saturation from (mean (SD)) 93.0 (10.6)% to 54.5 (17.2)% was observed 72.5 (50.0) minutes after deferiprone administration and in most of the patients coincided with peak deferiprone concentration. This was associated with a maximum rise in the percentage of iron free transferrin (apotransferrin) from 2.9 (7.0)% to 27.3 (17.8)%. The total amount of iron estimated to be removed from transferrin constituted 21.3 (20.2)% of the 24 hour urinary iron excretion measured during the study. When deferiprone (150 mumol/l) was incubated in vitro with t0 samples from 10 patients for 30 minutes and 24 hours at room temperature, 37 degrees C, and at -20 degrees C for six weeks, deferiprone was more efficient at removing iron from transferrin at 37 degrees C, with maximum transferrin desaturation accomplished within 30 minutes compared with 24 hours at room temperature. CONCLUSIONS--The results confirm that deferiprone can remove iron

  14. Classification of congenital disorders of glycosylation based on analysis of transferrin glycopeptides by capillary liquid chromatography-mass spectrometry.

    PubMed

    Barroso, Albert; Giménez, Estela; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

    2016-11-01

    In this work, we describe a multivariate data analysis approach for data exploration and classification of the complex and large data sets generated to study the alteration of human transferrin (Tf) N-glycopeptides in patients with congenital disorders of glycosylation (CDG). Tf from healthy individuals and two types of CDG patients (CDG-I and CDG-II) is purified by immunoextraction from serum samples before trypsin digestion and separation by capillary liquid chromatography mass spectrometry (CapLC-MS). Following a targeted data analysis approach, partial least squares discriminant analysis (PLS-DA) is applied to the relative abundance of Tf glycopeptide glycoforms obtained after integration of the extracted ion chromatograms of the different samples. The performance of PLS-DA for classification of the different samples and for providing a novel insight into Tf glycopeptide glycoforms alteration in CDGs is demonstrated. Only six out of fourteen of the detected glycoforms are enough for an accurate classification. This small glycoform set may be considered a sensitive and specific novel biomarker panel for CDGs. PMID:27591658

  15. Role of phosphate-containing compounds in the transfer of indium-111 and gallium-67 from transferrin to ferritin.

    PubMed

    Weiner, R E

    1989-01-01

    Physiologic concentrations of ATP stimulate the translocation of gallium-67 (67Ga) from human transferrin (TF) to horse ferritin (HoFE). The mechanism of this translocation was examined. One millimolar ATP did not speed the binding of 67Ga or indium-111 (111In) to HoFE. ATP and pyrophosphate (PPi) at 1 mM, did not form high affinity complexes with 67Ga or 111In. ATP and PPi interacted directly with the [67Ga]TF complex and could within minutes increase the amount of nonprotein-bound 67Ga. Serum HCO3- concentration, 30 mM, prevented the ATP-induced dissociation of 67Ga from TF, whereas intracellular concentrations (0.4 and 5 mM) did not. Using a dialysis technique, ATP also stimulated the translocation of 111In from TF to HoFE; however, this process was much slower than with 67Ga. ATP caused an increase in the nonprotein-bound 111In compared to the control. These results suggest the formation of nonprotein-bound nuclide by these phosphate-containing compounds in a kinetically labile form is important to the translocation mechanism. PMID:2536083

  16. A Polyethylenimine-Containing and Transferrin-Conjugated Lipid Nanoparticle System for Antisense Oligonucleotide Delivery to AML

    PubMed Central

    Yuan, Yiming; Zhang, Lijing; Cao, Hua; Yang, Yi; Zheng, Yu; Yang, Xiao-juan

    2016-01-01

    Limited success of antisense oligonucleotides (ASO) in clinical anticancer therapy calls for more effective delivery carriers. The goal of this study was to develop a nanoparticle system for delivery of ASO G3139, which targets mRNA of antiapoptotic protein Bcl-2, to acute myeloid leukemia (AML) cells. The synthesized nanoparticle Tf-LPN-G3139 contained a small molecular weight polyethylenimine and two cationic lipids as condensing agents, with transferrin on its surface for selective binding and enhanced cellular uptake. The optimized nitrogen to phosphate (N/P) ratio was 4 to achieve small particle size and high G3139 entrapment efficiency. The Tf-LPN-G3139 exhibited excellent colloidal stability during storage for at least 12 weeks and remained intact for 4 hours in nuclease-containing serum. The cellular uptake results showed extensive internalization of fluorescence-labelled G3139 in MV4-11 cells through Tf-LPN. Following transfection, Tf-LPN-G3139 at 1 µM ASO level induced 54% Bcl-2 downregulation and >20-fold apoptosis compared to no treatment. When evaluated in mice bearing human xenograft AML tumors, Tf-LPN-G3139 suppressed tumor growth by ~60% at the end of treatment period, accompanied by remarkable pharmacological effect of Bcl-2 inhibition in tumor. In conclusion, Tf-LPN-G3139 is a promising nanoparticle system for ASO G3139 delivery to AML and warrants further investigations. PMID:27034925

  17. Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

    PubMed

    Chu, L-X; Zhou, S-X; Yang, F; Qin, Y-Q; Liang, Z-S; Mo, C-G; Wang, X-D; Xie, J; He, L-P

    2016-01-01

    Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway. PMID:26950455

  18. Cysteinyl Leukotriene Receptor-1 Antagonists as Modulators of Innate Immune Cell Function

    PubMed Central

    Theron, A. J.; Steel, H. C.; Tintinger, G. R.; Gravett, C. M.; Anderson, R.; Feldman, C.

    2014-01-01

    Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8+ cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5′-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies. PMID:24971371

  19. Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury

    SciTech Connect

    Eto, Hideyuki; Miyata, Masaaki . E-mail: miyatam@m3.kufm.kagoshima-u.ac.jp; Kume, Noriaki; Minami, Manabu; Itabe, Hiroyuki; Orihara, Koji; Hamasaki, Shuichi; Biro, Sadatoshi; Otsuji, Yutaka; Kita, Toru; Tei, Chuwa

    2006-03-10

    Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 {mu}g/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescense staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury.

  20. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

    PubMed Central

    Chambers, R C; Dabbagh, K; McAnulty, R J; Gray, A J; Blanc-Brude, O P; Laurent, G J

    1998-01-01

    Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production. PMID:9639571

  1. Molecular basis for antagonistic activity of anifrolumab, an anti-interferon-α receptor 1 antibody.

    PubMed

    Peng, Li; Oganesyan, Vaheh; Wu, Herren; Dall'Acqua, William F; Damschroder, Melissa M

    2015-01-01

    Anifrolumab (anifrolumab) is an antagonist human monoclonal antibody that targets interferon α receptor 1 (IFNAR1). Anifrolumab has been developed to treat autoimmune diseases and is currently in clinical trials. To decipher the molecular basis of its mechanism of action, we engaged in multiple epitope mapping approaches to determine how it interacts with IFNAR1 and antagonizes the receptor. We identified the epitope of anifrolumab using enzymatic fragmentation, phage-peptide library panning and mutagenesis approaches. Our studies revealed that anifrolumab recognizes the SD3 subdomain of IFNAR1 with the critical residue R(279). Further, we solved the crystal structure of anifrolumab Fab to a resolution of 2.3 Å. Guided by our epitope mapping studies, we then used in silico protein docking of the anifrolumab Fab crystal structure to IFNAR1 and characterized the corresponding mode of binding. We find that anifrolumab sterically inhibits the binding of IFN ligands to IFNAR1, thus blocking the formation of the ternary IFN/IFNAR1/IFNAR2 signaling complex. This report provides the molecular basis for the mechanism of action of anifrolumab and may provide insights toward designing antibody therapies against IFNAR1. PMID:25606664

  2. Optimization of cAMP fluorescence dataset from ACTOne cannabinoid receptor 1 cell line.

    PubMed

    Presley, Chaela S; Abidi, Ammaar H; Moore, Bob M

    2016-06-01

    The ACTOne cannabinoid receptor 1 functional system is comprised of transfected HEK cells with the parental cyclic nucleotide gated channel (CNG) co-transfected with cannabinoid receptor 1 (CB1). The ACTOne CB1 cell line was evaluated for cAMP driven fluorescence by optimizing experimental conditions for sensitivity to forskolin and CP 55,940, reading time point, reliability of cell passage number, and pertussis inactivation of Gi/o. PMID:27115025

  3. Optimization of cAMP fluorescence dataset from ACTOne cannabinoid receptor 1 cell line

    PubMed Central

    Presley, Chaela S.; Abidi, Ammaar H.; Moore, Bob M.

    2016-01-01

    The ACTOne cannabinoid receptor 1 functional system is comprised of transfected HEK cells with the parental cyclic nucleotide gated channel (CNG) co-transfected with cannabinoid receptor 1 (CB1). The ACTOne CB1 cell line was evaluated for cAMP driven fluorescence by optimizing experimental conditions for sensitivity to forskolin and CP 55,940, reading time point, reliability of cell passage number, and pertussis inactivation of Gi/o. PMID:27115025

  4. The Cytomegaloviral Protein pUL138 Acts as Potentiator of Tumor Necrosis Factor (TNF) Receptor 1 Surface Density To Enhance ULb′-Encoded Modulation of TNF-α Signaling ▿ †

    PubMed Central

    Le, Vu Thuy Khanh; Trilling, Mirko; Hengel, Hartmut

    2011-01-01

    Human cytomegalovirus is a ubiquitous herpesvirus that establishes lifelong latent infection. Changes in immune homeostasis induce the reactivation of lytic infection, which is mostly inapparent in healthy individuals but often causes overt disease in immunocompromised hosts. Based on discrepant tumor necrosis factor receptor 1 surface disposition between human cytomegalovirus AD169 variants differing in the ULb′ region, we identified the latency-associated gene product pUL138, which also is expressed during productive infection, as a selective potentiator of tumor necrosis factor receptor 1, one of the key receptors of innate immunity. Ectopically expressed pUL138 coprecipitated with tumor necrosis factor receptor 1, extended the protein half-life, and enhanced its signaling responses, thus leading to tumor necrosis factor receptor 1 hyperresponsiveness. Conversely, the targeted deletion of UL138 from the human cytomegaloviral genome strongly reduced tumor necrosis factor receptor 1 surface densities of infected cells. Remarkably, the comparison of UL138 deficiency to ULb′ deficiency revealed the presence of further positive modulators of tumor necrosis factor alpha signal transduction encoded within the human cytomegalovirus ULb′ region, identifying this region as a hub for multilayered tumor necrosis factor alpha signaling regulation. PMID:21976655

  5. Metabotropic Glutamate Receptor-1 Contributes to Progression in Triple Negative Breast Cancer

    PubMed Central

    Banda, Malathi; Speyer, Cecilia L.; Semma, Sara N.; Osuala, Kingsley O.; Kounalakis, Nicole; Torres Torres, Keila E.; Barnard, Nicola J.; Kim, Hyunjin J.; Sloane, Bonnie F.; Miller, Fred R.; Goydos, James S.; Gorski, David H.

    2014-01-01

    TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy. PMID:24404125

  6. The lectin-like oxidized LDL receptor-1: a new potential molecular target in colorectal cancer.

    PubMed

    Murdocca, Michela; Mango, Ruggiero; Pucci, Sabina; Biocca, Silvia; Testa, Barbara; Capuano, Rosamaria; Paolesse, Roberto; Sanchez, Massimo; Orlandi, Augusto; di Natale, Corrado; Novelli, Giuseppe; Sangiuolo, Federica

    2016-03-22

    The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P<0.001), and strongly overexpressed in 90% of highly aggressive and metastatic tumours (P<0.001), as compared to normal mucosa. Moreover LOX-1 results modulated since the early stage of the disease (adenomas vs normal mucosa; P<0.001) suggesting an involvement in tumor insurgence and progression. The in vitro knockdown of LOX-1 in DLD-1 and HCT-8 colon cancer cells by siRNA and anti-LOX-1 antibody triggers to an impaired proliferation rate and affects the maintenance of cell growth and tumorigenicity. The wound-healing assay reveals an evident impairment in closing the scratch. Lastly knockdown of LOX-1 delineates a specific pattern of volatile compounds characterized by the presence of a butyrate derivative, suggesting a potential role of LOX-1 in tumor-specific epigenetic regulation in neoplastic cells. The role of LOX-1 as a novel biomarker and molecular target represents a concrete opportunity to improve current therapeutic strategies for CRC. In addition, the innovative application of a technology focused to the identification of LOX-1 driven volatiles specific to colorectal cancer provides a promising diagnostic tool for CRC screening and for monitoring the response to therapy. PMID:26895376

  7. The lectin-like oxidized LDL receptor-1: a new potential molecular target in colorectal cancer

    PubMed Central

    Murdocca, Michela; Mango, Ruggiero; Pucci, Sabina; Biocca, Silvia; Testa, Barbara; Capuano, Rosamaria; Paolesse, Roberto; Sanchez, Massimo; Orlandi, Augusto; di Natale, Corrado; Novelli, Giuseppe; Sangiuolo, Federica

    2016-01-01

    The identification of new biomarkers and targets for tailored therapy in human colorectal cancer (CRC) onset and progression is an interesting challenge. CRC tissue produces an excess of ox-LDL, suggesting a close correlation between lipid dysfunction and malignant transformation. Lectin-like oxidized LDL receptor-1 (LOX-1) is involved in several mechanisms closely linked to tumorigenesis. Here we report a tumor specific LOX-1 overexpression in human colon cancers: LOX-1 results strongly increased in the 72% of carcinomas (P<0.001), and strongly overexpressed in 90% of highly aggressive and metastatic tumours (P<0.001), as compared to normal mucosa. Moreover LOX-1 results modulated since the early stage of the disease (adenomas vs normal mucosa; P<0.001) suggesting an involvement in tumor insurgence and progression. The in vitro knockdown of LOX-1 in DLD-1 and HCT-8 colon cancer cells by siRNA and anti-LOX-1 antibody triggers to an impaired proliferation rate and affects the maintenance of cell growth and tumorigenicity. The wound-healing assay reveals an evident impairment in closing the scratch. Lastly knockdown of LOX-1 delineates a specific pattern of volatile compounds characterized by the presence of a butyrate derivative, suggesting a potential role of LOX-1 in tumor-specific epigenetic regulation in neoplastic cells. The role of LOX-1 as a novel biomarker and molecular target represents a concrete opportunity to improve current therapeutic strategies for CRC. In addition, the innovative application of a technology focused to the identification of LOX-1 driven volatiles specific to colorectal cancer provides a promising diagnostic tool for CRC screening and for monitoring the response to therapy. PMID:26895376

  8. Enhancing Glioblastoma-Specific Penetration by Functionalization of Nanoparticles with an Iron-Mimic Peptide Targeting Transferrin/Transferrin Receptor Complex.

    PubMed

    Kang, Ting; Jiang, Mengyin; Jiang, Di; Feng, Xingye; Yao, Jianhui; Song, Qingxiang; Chen, Hongzhuan; Gao, Xiaoling; Chen, Jun

    2015-08-01

    Treatment of glioblastoma (GBM) remains to be the most formidable challenge because of the hindrance of the blood-brain barrier (BBB) along with the poor drug penetration into the glioma parenchyma. Nanoparticulate drug delivery systems (DDS) utilizing transferrin (Tf) as the targeting ligand to target the glioma-associated transferrin receptor (TfR) had met the problem of loss of specificity in biological environment due to the high level of endogenous Tf. Here we conjugated CRT peptide, an iron-mimicry moiety targeting the whole complex of Tf/TfR, to poly(ethylene glycol)-poly(l-lactic-co-glycolic acid) nanoparticles (CRT-NP), to open a new route to overcome such obstacle. High cellular associations, advanced transport ability through the BBB model, and penetration in 3-dimensional C6 glioma spheroids in vitro had preliminarily proved the advantages of CRT-NP over Tf-nanoparticle conjugates (Tf-NP). Compared with Tf-NP, NP, and Taxol, paclitaxel-loaded CRT-NP (CRT-NP-PTX) displayed a superior antiproliferation effect on C6 glioma cells and stronger inhibitory effect on glioma spheroids. Favored pharmacokinetics behavior and enhanced accumulation in glioma foci was observed, together with a much deeper distribution pattern in glioma parenchyma compared with unmodified nanoparticles and Tf-NP. Eventually, mice treated with CRT-NP-PTX showed a remarkably prolonged median survival compared to those treated with Taxol, NP, or Tf-NP. In conclusion, the modification of CRT to nanoparticles holds great promise for enhancement of antiglioma therapy. PMID:26149889

  9. Real-time observation of the effect of iron on receptor-mediated endocytosis of transferrin conjugated with quantum dots

    NASA Astrophysics Data System (ADS)

    Zhang, Hai-Li; Li, Yong-Qiang; Zhang, Ming-Zhen; Zhao, Yuan-Di

    2010-07-01

    The optical properties of antiphotobleaching and the advantage of long-term fluorescence observation of quantum dots are fully adopted to study the effects of iron on the endocytosis of transferrin. Quantum dots are labeled for transferrin and endocytosis of transferrin in HeLa cells is observed under the normal state, iron overloading, and an iron-deficient state. In these three states, the fluorescence undergoes a gradual process of first dark, then light, and finally dark, indicating the endocytosis of transferrin. The fluorescence intensity analysis shows that a platform emerges when fluorescence changes to a certain degree in the three states. Experienced a same period of time after platform, the fluorescence strength of cells in the normal state is 1.2 times the first value, and the iron-deficiency state is 1.4 times, but the iron overloading state was 0.85 times. We also find that the average fluorescence intensity in cells detected by the spectrophotometer in the iron-deficiency state is almost 7 times than that in a high iron state. All this proves that iron overloading would slow the process, but iron deficiency would accelerate endocytosis. We advance a direct observational method that may contribute to further study of the relationship of iron and transferrin.

  10. Molecular characterization of Ephestia kuehniella (Lepidoptera: Pyralidae) transferrin and its response to parasitoid Venturia canescens (Hymenoptera: Ichneumonidae Gravenhorst).

    PubMed

    Guz, Nurper; Kilincer, N; Aksoy, S

    2012-04-01

    In the present study, we characterized a full-length cDNA encoding a putative iron-binding protein transferrin from the lepidopteran Mediterranean flour moth (EkTrf, 2397 bp). The putative EkTrf is 683 amino acids with a molecular mass of approximately 76 kDa. The deduced amino acid sequence showed significant homology with other insect transferrins from Chilo suppressalis (76%), Galleria mellonella (75%), Plutella xylostella (72%), Manduca sexta (74%), Bombyx mori (73%), Spodoptera litura and (72%), Choristoneura fumiferana (71%). Northern blot analysis indicated that Ephestia transferrin mRNA was expressed in the last larval instars of both males and females and in the pupal developmental stages. EkTrf is expressed predominantly in the fat body and ovary tissues. Analysis of parasitized larva by the endoparasitoid Venturia canescens suggests that transferrin expression is induced following parasitoid challenge. Expression of EkTrf levels also increased upon bacterial infection at 6 h post treatment and remained high until 24 h. Similarly to other insect transferrins, EkTrf may play a role in immunity through its iron-binding capacity. PMID:22229520

  11. Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression.

    PubMed

    do Nascimento, Adriana Mendes; Cuvillier-Hot, Virginie; Barchuk, Angel Roberto; Simões, Zilá Luz Paulino; Hartfelder, Klaus

    2004-05-01

    Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species. PMID:15110862

  12. FCRL6 is an MHC class II receptor1

    PubMed Central

    Schreeder, Daniel M.; Cannon, John P.; Wu, Jiongru; Li, Ran; Shakhmatov, Mikhail A.; Davis, Randall S.

    2016-01-01

    Receptors for the Fc portion (FCR) of Ig have been extensively characterized and are known to regulate humoral responses, but members of the closely related FCR-like (FCRL) family have not been found to bind Ig and to date no ligand has been identified for any FCRL. Using a cell-based GFP reporter system and a recombinant Fc chimeric protein, we show that human FCRL6, a receptor selectively expressed by cytotoxic T and NK cells, directly binds HLA-DR, a major histocompatibility complex (MHC) class II molecule. Given the similarity among constant regions of Ig and MHC molecules, these findings suggest that representatives of the FCR and FCRL multigene families may have independently evolved to engage two ancestral elements fundamental to adaptive immunity. This discovery may offer new insight into the interaction between cytotoxic lymphocytes and antigen presenting cells and may have important implications for better understanding HLA disease susceptibility and pathogenesis. PMID:20519654

  13. Anaemia in Systemic Lupus Erythematosus Based on Iron Studies and Soluble Transferrin Receptor Levels

    PubMed Central

    Agarwal, Preeti; Wakhlu, Anupam; Kumar, Ashutosh; Mehrotra, Raj; Mittal, Saumya

    2016-01-01

    Introduction Haematological alterations such as anaemia, neutropenia and thrombocytopenia are frequent in Systemic Lupus Erythematosus (SLE). Ferritin being an acute phase reactant can be falsely elevated in lupus cases. Aim To evaluate the haematological alterations and to re-categorise the types of anemia by soluble transferrin receptor levels in diagnosed cases of SLE. Materials and Methods A sample of 30 newly diagnosed ANA positive SLE patients was taken. Complete blood counts, ESR, reticulocyte count, coagulation studies, diluted Russel Viper Venom Test (dRVVT), mixing studies, serological tests, high sensitivity CRP along with iron profile, transferrin saturation, soluble transferrin receptor (sol TFR) levels, anti-beta2 glycoprotein1, direct and indirect Coomb’s test were estimated in cases diagnosed as SLE. Clinical symptoms were co-related with and Systemic Lupus Erythaematosus Disease Activity Index (SLEDAI) was estimated. Results Anaemia was the most prevalent haematological alteration followed by thrombocytopenia. Further sub typing of anaemia was done by serum ferritin levels and using sol TFR assays. Ferritin is an acute phase reactant; it underestimated iron deficiency in patients of SLE. When sol TFR was used; patients with pure Anaemia of Chronic Disease (ACD) reduced from 68% to 26%, those with pure IDA reduced from 32% to 16% and a group with co-existing IDA & ACD (58%) was defined {Agreement=53%, p=0.09} by sol TFR which co-related with clinical response to Iron therapy in these patients. CRP was significantly raised in association with disease activity. Fever (p<0.0001), arthritis (p<0.03) were significantly related and CRP was elevated (p<0.04) in cases with high SLEDAI (severe flare). Conclusion Thus, in SLE, anaemia is the most frequent hematological alteration; iron deficiencies supercede in contrast to ACD and further autoimmune haemolytic anaemia. Sol TFR emerged as a better parameter to detect iron deficiency in patients of non

  14. Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1

    PubMed Central

    Luo, Ling; Uehara, Hironori; Zhang, Xiaohui; Das, Subrata K; Olsen, Thomas; Holt, Derick; Simonis, Jacquelyn M; Jackman, Kyle; Singh, Nirbhai; Miya, Tadashi R; Huang, Wei; Ahmed, Faisal; Bastos-Carvalho, Ana; Le, Yun Zheng; Mamalis, Christina; Chiodo, Vince A; Hauswirth, William W; Baffi, Judit; Lacal, Pedro M; Orecchia, Angela; Ferrara, Napoleone; Gao, Guangping; Young-hee, Kim; Fu, Yingbin; Owen, Leah; Albuquerque, Romulo; Baehr, Wolfgang; Thomas, Kirk; Li, Dean Y; Chalam, Kakarla V; Shibuya, Masabumi; Grisanti, Salvatore; Wilson, David J; Ambati, Jayakrishna; Ambati, Balamurali K

    2013-01-01

    Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI: http://dx.doi.org/10.7554/eLife.00324.001 PMID:23795287

  15. Identification of transferrin as the main binding site for protactinium in rat blood serum.

    PubMed

    Taylor, D M; Farrow, L C

    1987-01-01

    The distribution of 233Pa in rat serum at periods between 5 and 50 min after i.v. injection of a solution of protactinium chloride was studied by gel chromatography. Sequential analysis of sera on Sephacryl S-300 and DEAE-Sephadex showed that 233Pa was associated only with the transferrin fraction of the serum proteins. This finding was confirmed by iso-electric focusing electrophoresis. In the cytosol fractions prepared from the liver and kidneys of the 233Pa injected rats the nuclide was also shown to be protein bound. PMID:3583752

  16. Purification of transferrin from Cohn supernatant I using ion-exchange chromatography.

    PubMed

    McCann, Karl B; Hughes, Ben; Wu, John; Bertolini, Joseph; Gomme, Peter T

    2005-12-01

    The present paper describes an anion-exchange chromatography method to separate iron-free apo-Tf (apo-transferrin) from albumin and IgG in Cohn supernatant I. The method uses DEAE-fast flow Sepharose chromatography along with optimized protein concentration (5%, w/v) and column operation conditions (40 g/l, conductivity <1.0 mS/cm) to resolve apo-Tf from IgG and albumin. The single step purifies apo-Tf to >90% and provides an efficient means to obtain commercial quantities of the protein. PMID:15943579

  17. Enhanced adiponectin actions by overexpression of adiponectin receptor 1 in macrophages

    PubMed Central

    Luo, Nanlan; Chung, B Hong; Wang, Xiangdong; Klein, Richard L.; Tang, Chao-Ke; Garvey, W. Timothy; Fu, Yuchang

    2013-01-01

    Objective Adiponectin is one of several important, metabolically active cytokines secreted from adipose tissue. Epidemiologic studies have associated low circulating levels of this adipokine with multiple metabolic disorders including obesity, insulin resistance, type II diabetes, and cardiovascular disease. To investigate how enhanced adiponectin-mediated changes in metabolism in vivo, we generated transgenic mice which specifically overexpress the gene coding for adiponectin receptor 1 (AdipoR1) in mouse macrophages using the human scavenger receptor A-I gene (SR-AI) enhancer/promoter. We found that macrophage-specific AdipoR1 transgenic mice (AdR1-TG) presented reduced whole body weight, fat accumulation and liver steatosis when these transgenic mice were fed with a high fat diet. Moreover, these macrophage AdR1-TG mice exhibited enhanced whole-body glucose tolerance and insulin sensitivity with reduced proinflammatory cytokines, MCP-1 and TNF-α, both in the serum and in the insulin target metabolic tissues. Additional studies demonstrated that these macrophage AdR1-TG animals exhibited reduced macrophage foam cell formation in the arterial wall when these transgenic mice were crossed with a low-density lipoprotein receptor (Ldlr) deficient mouse model. Conclusions These results suggest that AdipoR1 overexpressed in macrophages can physiologically modulate metabolic activities in vivo by enhancing adiponectin actions in distal metabolically active tissues. The AdipoR1 modified macrophages provide unique interactions with the residented tissues/cells, suggesting a novel role of macrophage adiponectin receptor in improving metabolic disorders in vivo. PMID:23510830

  18. Prokineticin receptor 1 is required for mesenchymal-epithelial transition in kidney development.

    PubMed

    Arora, Himanshu; Boulberdaa, Mounia; Qureshi, Rehana; Bitirim, Verda; Messadeq, Nadia; Dolle, Pascal; Nebigil, Canan G

    2016-08-01

    Identification of factors regulating renal development is important to understand the pathogenesis of congenital kidney diseases. Little is known about the molecular mechanism of renal development and functions triggered by the angiogenic hormone prokineticin-2 and its receptor, PKR1. Utilizing the Gata5 (G5)-Cre and Wilms tumor 1 (Wt1)(GFP)cre transgenic lines, we generated mutant mice with targeted PKR1 gene disruptions in nephron progenitors. These mutant mice exhibited partial embryonic and postnatal lethality. Kidney developmental defects in PKR(G5-/-) mice are manifested in the adult stage as renal atrophy with glomerular defects, nephropathy, and uremia. PKR1(Wt1-/-) embryos exhibit hypoplastic kidneys with premature glomeruli and necrotic nephrons as a result of impaired proliferation and increased apoptosis in Wt1(+) renal mesenchymal cells. PKR1 regulates renal mesenchymal-epithelial transition (MET) that is involved in formation of renal progenitors, regulating glomerulogenesis toward forming nephrons during kidney development. In the isolated embryonic Wt1(+) renal cells, overexpression or activation of PKR1 promotes MET defined by the transition from elongated cell to octagonal cell morphology, and alteration of the expression of MET markers via activating NFATc3 signaling. Together, these results establish PKR1 via NFATc3 as a crucial modifier of MET processing to the development of nephron. Our study should facilitate new therapeutic opportunities in human renal disorders.-Arora, H., Boulberdaa, M., Qureshi, R., Bitirim, V., Messadeq, N., Dolle, P., Nebigil, C. G. Prokineticin receptor 1 is required for mesenchymal-epithelial transition in kidney development. PMID:27084889

  19. TNF receptor 1 signaling is critically involved in mediating angiotensin-II-induced cardiac fibrosis.

    PubMed

    Duerrschmid, Clemens; Crawford, Jeffrey R; Reineke, Erin; Taffet, George E; Trial, Joann; Entman, Mark L; Haudek, Sandra B

    2013-04-01

    Angiotensin-II (Ang-II) is associated with many conditions involving heart failure and pathologic hypertrophy. Ang-II induces the synthesis of monocyte chemoattractant protein-1 that mediates the uptake of CD34(+)CD45(+) monocytic cells into the heart. These precursor cells differentiate into collagen-producing fibroblasts and are responsible for the Ang-II-induced development of non-adaptive cardiac fibrosis. In this study, we demonstrate that in vitro, using a human monocyte-to-fibroblast differentiation model, Ang-II required the presence of tumor necrosis factor-alpha (TNF) to induce fibroblast maturation from monocytes. In vivo, mice deficient in both TNF receptors did not develop cardiac fibrosis in response to 1week Ang-II infusion. We then subjected mice deficient in either TNF receptor 1 (TNFR1-KO) or TNF receptor 2 (TNFR2-KO) to continuous Ang-II infusion. Compared to wild-type, in TNFR1-KO, but not in TNFR2-KO hearts, collagen deposition was greatly attenuated, and markedly fewer CD34(+)CD45(+) cells were present. Quantitative RT-PCR demonstrated a striking reduction of key fibrosis-related, as well as inflammation-related mRNA expression in Ang-II-treated TNFR1-KO hearts. TNFR1-KO animals also developed less cardiac remodeling, cardiac hypertrophy, and hypertension compared to wild-type and TNFR2-KO in response to Ang-II. Our data suggest that TNF induced Ang-II-dependent cardiac fibrosis by signaling through TNFR1, which enhances the generation of monocytic fibroblast precursors in the heart. PMID:23337087

  20. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1.

    PubMed

    Finetti, Federica; Solito, Raffaella; Morbidelli, Lucia; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2008-01-25

    Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth. PMID:18042549

  1. Evaluation of serum transferrin receptor assay in a centralized iron screening service.

    PubMed

    O'Broin, S; Kelleher, B; Balfe, A; Mc Mahon, C

    2005-06-01

    This study assesses the impact of permitting unrestricted access to requests for soluble transferrin receptor (sTfR) analysis in screening for iron deficiency (ID). Biochemical data including sTfR, serum ferritin (sFn), transferrin saturation, zinc protoporphyrins (ZPP) and also erythrocyte indices are used to highlight the differences between hospital (H) and general practitioner (GP) patient groups. A significantly higher number of abnormal sFn values (40%) over abnormal sTfR values (25%) occurred in GP patients. This trend was reversed in the H patient group where high sTfR values predominated. Consequently, screening with sFn, exclusively, missed ID (sTfR > 28.1 nmol/l) in 5% of GP patients and in 20% of H patients. Some 40% of H patients had elevated CRP values (CRP > 10 mg/l) indicating inflammatory disease, however, ZPP was more efficient than CRP at screening the validity of normal sFn values in the group. Unrestricted access to sTfR, sFn and ZPP analyses should expedite diagnosis in all patients, particularly H patients, but may be costly. The high specificity (>90%) of the mean cell haemoglobin for ID may be under-utilized diagnostically. PMID:15938725

  2. Polymorphism of the Transferrin Gene in Eye Diseases: Keratoconus and Fuchs Endothelial Corneal Dystrophy

    PubMed Central

    Wójcik, Katarzyna A.; Jiménez-García, Manuel P.; Kaminska, Anna; Polakowski, Piotr; Szaflik, Jerzy; Szaflik, Jacek P.

    2013-01-01

    Oxidative stress may play a role in the pathogenesis of keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Iron may promote the stress by the Fenton reaction, so its homeostasis should be strictly controlled. Transferrin is essential for iron homeostasis because it transports iron from plasma into cells. The malfunction of transferrin, which may be caused by variation in its gene (TF) variation, may contribute to oxidative stress and change KC and FECD risk. To verify this hypothesis we investigated the association between three polymorphisms of the TF gene, g.3296G>A (rs8177178), g.3481A>G (rs8177179), and c.–2G>A (rs1130459), and KC and FECD occurrence. Genotyping was performed in blood lymphocytes in 216 patients with KC, 130 patients with FECD and 228 controls by PCR-RFLP. We studied also the influence of other risk factors. The A/A genotype and the A allele of the g.3296G>A polymorphism were associated with KC occurrence, while the G allele was negatively correlated with it. We observed a decrease in KC occurrence associated with the A/G genotype of the g.3481A>G polymorphism. We did not find any association between the c.–2G>A polymorphism and KC. No association was found between all three polymorphisms and FECD occurrence. PMID:24350254

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

    NASA Astrophysics Data System (ADS)

    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.

    2011-09-01

    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.

  4. Transferrin and cell-penetrating peptide dual-functioned liposome for targeted drug delivery to glioma

    PubMed Central

    Zheng, Chuanyi; Ma, Chunyang; Bai, Enqi; Yang, Kun; Xu, Ruxiang

    2015-01-01

    A brain drug delivery system for glioma chemotherapy based on transferrin and cell-penetrating peptide dual-functioned liposome, Tf/TAT-lip, was made and evaluated with doxorubicin (DOX) as a model drug. TAT conjugated liposome (TAT-lip) loaded with doxorubicin (DOX) were prepared by the thin film hydration methods (lip-DOX) and then conjugated with transferrin (Tf) to yield Tf/TAT-lip-DOX which was characterized for their various physicochemical and pharmaceutical properties. Cellular uptakes were explored in both brain capillary endothelial cells (BCECs) of rats and U87 cells. The blood brain barrier model in vitro was established to evaluate the trans-endothelial ability crossing the BBB. The biodistribution of each formulation was further identified. The Tf/TAT-lip-DOX presents the best anti-proliferative activity against U87 cells. The orthotropic glioma model was established for the evaluation of anti-glioma effect. In conclusion, the experimental data in vitro and in vivo indicated that the Tf/TAT-lip was a promising brain drug delivery system due to its high delivery efficiency across the BBB. PMID:25932094

  5. Effect of cadmium on Fe/sup +3/-transferrin formation in the rat intestinal mucosa

    SciTech Connect

    Sugawara, N.; Chen, B.Q.; Sugawara, C.; Miyake, H.

    1988-07-01

    The effect of cadmium (Cd) on iron (Fe) metabolism has been an important subject for Cd toxicity, since anemia is usually observed in Itai-Itai patients who are exposed for long periods to Cd from the surrounding environment. It was previously accepted that Cd-induced anemia was not dependent on the route of administration. Thereafter, however, it was shown that oral Cd administration was essential for the development of anemia. Studies suggest that one of the possible sensitive sites of competition between Cd and Fe is in the gastrointestinal tract. Cd competes with Fe at one or more steps in the transport system and these metals undergo the same step(s) during their absorption. An hypothesis implies that these two metals possess a common carrier. Two Fe-binding proteins, ferritin and transferrin are well documented in the case of Fe deficiency but not in Cd exposed animals. Recently, the authors reported that the status of mucosal Fe-binding proteins in rats fed with Cd was similar to that in the Fe deficient rats. The present work was performed in an attempt to clarify the effect of in vivo and in vitro Cd on mucosal transferrin formation, which is one of two main Fe-binding proteins.

  6. Interaction of transferrin and its iron-binding fragments with heparin.

    PubMed Central

    Regoeczi, E; Chindemi, P A; Hu, W L

    1994-01-01

    The interaction of heparin with transferrin (Tf; bovine and rat) and the isolated iron-binding lobes of bovine Tf were investigated. Affinity chromatography of rat Tf on heparin-agarose showed that interaction depended on both the iron content of Tf and the pH of the medium. Both the iron-free and iron-saturated forms of Tf were strongly bound by the column at pH 5.6, but only the iron-free form revealed significant affinity at pH 7.4. Desialylation of Tf moderately promoted interaction, treatment with cyclohexanedione moderately reduced interaction, and succinylation abolished it altogether. In the presence of heparin, iron release from the N-terminal lobe of native bovine Tf was accelerated and from the C-terminal lobe it was slightly reduced. The heparin effect remained qualitatively the same on each lobe after their separation by tryptic digestion and DEAE-cellulose chromatography. The affinity of native bovine Tf for heparin was very close to that of its isolated N-terminal lobe, thus suggesting that it is this portion of the molecule that binds to the glycosaminoglycan. It is concluded that the consequences for iron-binding strength of the two transferrin lobes are diagonally opposite when Tf is bound to heparin as opposed to its natural cell-surface receptor. PMID:8192672

  7. Genetic changes at the transferrin locus in the red-backed vole (Clethrionomys gapperi)

    SciTech Connect

    Mihok, S.; Fuller, W.A.; Canham, R.P.; McPhee, E.C.

    1983-01-01

    Genetic changes at the transferrin locus in Clethrionomys gapperi were intermittently monitored in a subarctic population from 1966 to 1978. Over this 13-year period, only minor fluctuations in gene frequency were observed. Gene frequency of Tf/sup J/ increased over winter during declines from high nonbreeding density in autumn. This phenomenon may have been responsible for a general negative correlation between the frequency of Tf/sup J/ and population density. Outside of winter, no frequency changes were detected within trappable age-classes of voles from relatively discrete seasonal generations. Excess of Tf/sup M/J/ heterozygotes occurred in three of four samples of young voles that matured in the year of their birth. A similar heterozygote excess occurred in one of six samples of overwintered voles taken in a year characterized by a high rate of population growth. These results suggest that selection may occur during ecologically different conditions of high density or population growth. A heterozygote advantage in early-season cohorts may account for the maintenance of transferrin polymorphism. This hypothesis requires further data on the breeding structure and early life history of voles.

  8. Anti-cancer activity of doxorubicin-loaded liposomes co-modified with transferrin and folic acid.

    PubMed

    Sriraman, Shravan Kumar; Salzano, Giusseppina; Sarisozen, Can; Torchilin, Vladimir

    2016-08-01

    Cancer-specific drug delivery represents an attractive approach to prevent undesirable side-effects and increase the accumulation of the drug in the tumor. Surface modification of nanoparticles such as liposomes with targeting moieties specific to the up-regulated receptors on the surface of tumor cells thus represents an effective strategy. Furthermore, since this receptor expression can be heterogeneous, using a dual-combination of targeting moieties may prove advantageous. With this in mind, the anti-cancer activity of PEGylated doxorubicin-loaded liposomes targeted with folic acid (F), transferrin (Tf) or both (F+Tf) was evaluated. The dual-targeted liposomes showed a 7-fold increase in cell association compared to either of the single-ligand targeted ones in human cervical carcinoma (HeLa) cell monolayers. The increased penetration and cell association of the dual-targeted liposomes were also demonstrated using HeLa cell spheroids. The in vitro cytotoxicity of the doxorubicin liposomes (LD) was then evaluated using HeLa and A2780-ADR ovarian carcinoma cell monolayers. In both these cell lines, the (F+Tf) LD showed significantly higher cytotoxic effects than the untargeted, or single-ligand targeted liposomes. In a HeLa xenograft model in nude mice, compared to the untreated group, though the untargeted LD showed 42% tumor growth inhibition, both the (F) LD and (F+Tf) LD showed 75% and 79% tumor growth inhibition respectively. These results thus highlight that though the dual-targeted liposomes represent an effective cytotoxic formulation in the in vitro setting, they were equally effective as the folic acid-targeted liposomes in reducing tumor burden in the more complex in vivo setting in this particular model. PMID:27264717

  9. Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons

    PubMed Central

    Ji, Changyi; Kosman, Daniel J.

    2015-01-01

    The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and DMT1, and ferrireductases Steap2 and SDR2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co-localize, indicating these two proteins may function in Fe3+ reduction prior to Fe2+ permeation. Zip8, DMT1 and Steap2 co-localize with the transferrin receptor (TfR)/transferrin (Tf) complex, suggesting they may be involved in TfR/Tf-mediated iron assimilation. In brain interstitial fluid, transferring-bound iron (TBI) and non-transferrin-bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin-59Fe3+) and NTBI, whether presented as 59Fe2+-citrate or 59Fe3+-citrate; reductase-independent 59Fe2+ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn2+ inhibition of Fe2+ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of 59Fe from TBI relies at least in part on an endocytosis-independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons. PMID:25649872

  10. Mass spectrometry of transferrin and apolipoprotein C-III for diagnosis and screening of congenital disorder of glycosylation.

    PubMed

    Wada, Yoshinao

    2016-06-01

    Congenital disorder of glycosylation (CDG), formerly representing a group of diseases due to defects in the biosynthetic pathway of protein N-glycosylation, currently covers a wide range of disorders affecting glycoconjugates. Since its first application to serum transferrin from a CDG patient with phosphomannomutase-2 deficiency in 1992, mass spectrometry (MS) has been playing a key role in identification and characterization of glycosylation defects affecting glycoproteins. MS of native transferrin detects a lack of glycans characteristic to the classical CDG-I type of molecular abnormality. Electrospray ionization MS of native transferrin, especially, allows glycoforms to be analyzed precisely but requires basic knowledge regarding deconvolution of multiply-charged ions which may generate ghost signals upon transformation into a singly-charged form. MS of glycopeptides from tryptic digestion of transferrin delineates site-specific glycoforms and reveals a delicate balance of donor/acceptor substrates or the conformational effect of nascent proteins in cells. Matrix-assisted laser desorption ionization MS of apolipoprotein C-III is a simple method of elucidating the profiles of mucin-type core 1 O-glycans including site occupancy and glycoforms. In this technological review, the principle and pitfalls of MS for CDG are discussed and mass spectra of various types of CDG are presented. PMID:26873821

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

    PubMed

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

    2016-06-01

    The effective treatment of brain cancer is hindered by the poor transport across the blood-brain barrier (BBB) and the low penetration across the blood-tumor barrier (BTB). The objective of this work was to formulate transferrin-conjugated docetaxel (DTX)-loaded d-alpha-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS) micelles for targeted brain cancer therapy. The micelles with and without transferrin conjugation were prepared by the solvent casting method and characterized for their particle size, polydispersity, drug encapsulation efficiency, drug loading, in vitro release study and brain distribution study. Particle sizes of prepared micelles were determined at 25 °C by dynamic light scattering technique. The external surface morphology was determined by transmission electron microscopy analysis and atomic force microscopy. The encapsulation efficiency was determined by spectrophotometery. In vitro release studies of micelles and control formulations were carried out by dialysis bag diffusion method. The particle sizes of the non-targeted and targeted micelles were <20 nm. About 85% of drug encapsulation efficiency was achieved with micelles. The drug release from transferrin-conjugated micelles was sustained for >24 h with 50% of drug release. The in vivo results indicated that transferrin-targeted TPGS micelles could be a promising carrier for brain targeting due to nano-sized drug delivery, solubility enhancement and permeability which provided an improved and prolonged brain targeting of DTX in comparison to the non-targeted micelles and marketed formulation. PMID:26431064

  12. [Disseminated BCG disease revealing a partial deficiency in receptor 1 interferon gamma].

    PubMed

    Antonietti, J; Retornaz, K; Bernasconi, A; Laporte, R-J; Minodier, P; Bustamante, J-C; Dubus, J-C

    2015-09-01

    We report on a case of disseminated BCGitis with an unusual presentation in a 4-month-old infant revealing a syndrome of Mendelian susceptibility to mycobacteria due to a partial dominant mutation of the interferon gamma receptor 1 gene. PMID:26251056

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

    PubMed

    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. PMID:26385212

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

    PubMed Central

    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. PMID:26385212

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

    PubMed Central

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

    1985-01-01

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

  16. Maxi-circles, glycosomes, gene transposition, expression sites, transsplicing, transferrin receptors and base J.

    PubMed

    Borst, Piet

    2016-01-01

    This is a personal story of the author of his research on trypanosomatids, covering a period of 1970-2015. Some of the highlights include the discovery of new aspects of kDNA, the mini-circle heterogeneity and the maxi-circle; the glycosome; the discovery of gene transposition as a major mechanism for antigenic variation; trans-splicing as an essential step in the synthesis of all trypanosome mRNAs; Pulsed Field Gradient gels to size-fractionate chromosome-sized DNA molecules of protozoa; the sequence of trypanosome telomeres and their growth and contraction; the first ABC-transporter of trypanosomatids, LtpgpA; the variable transferrin receptor of T. brucei and its role in Fe uptake; and base J, its structure, biosynthesis and function. PMID:27021571

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

    PubMed Central

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

    2016-01-01

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

  18. Paving the way for adequate myelination: The contribution of galectin-3, transferrin and iron.

    PubMed

    Franco, Paula G; Pasquini, Laura A; Pérez, María J; Rosato-Siri, María V; Silvestroff, Lucas; Pasquini, Juana M

    2015-11-14

    Considering the worldwide incidence of well characterized demyelinating disorders such as Multiple Sclerosis (MS) and the increasing number of pathologies recently found to involve hypomyelinating factors such as micronutrient deficits, elucidating the molecular basis of central nervous system (CNS) demyelination, remyelination and hypomyelination becomes essential to the development of future neuroregenerative therapies. In this context, this review discusses novel findings on the contribution of galectin-3 (Gal-3), transferrin (Tf) and iron to the processes of myelination and remyelination and their potentially positive regulation of oligodendroglial precursor cell (OPC) differentiation. Studies were conducted in cuprizone (CPZ)-induced demyelination and iron deficiency (ID)-induced hypomyelination, and the participation of glial and neural stem cells (NSC) in the remyelination process was evaluated by means of both in vivo and in vitro assays on primary cell cultures. PMID:26296311

  19. Transferrin variants as markers of migrations and admixture between populations in the Baltic Sea region.

    PubMed

    Beckman, L; Sikström, C; Mikelsaar, A V; Krumina, A; Ambrasiene, D; Kucinskas, V; Beckman, G

    1998-01-01

    Transferrin (TF) types were examined by isoelectric focusing in an attempt to elucidate migrations and admixture between populations in the Baltic Sea region. A highly significant heterogeneity between populations was found with respect to TF*C subtypes as well as the rare TF variants B2, B0-1 and DCHI. With the exception for Estonia, increased frequencies of the TF*C3 allele were observed east of the Baltic Sea. The island of Gotland in the middle of the Baltic Sea also showed a high TF*C3 frequency indicating an eastern influence. The TF*DCHI allele, a marker of eastern (Finno-Ugric) influence, was found in Finland and Estonia and on the island of Gotland, but not in mainland Sweden and in the Baltic peoples (Latvians and Lithuanians). These results indicate the presence of a Finno-Ugric, most likely Estonian or Livonian, genetic influence in the Gotland population. PMID:9694249

  20. [FECAL NONINVASIVE TESTS (CALPROTECTIN, TRANSFERRIN, HEMOGLOBIN) IN COMPLEX DIAGNOSIS OF DISEASES OF INTESTINES].

    PubMed

    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. PMID:26281175

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

    PubMed

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

    2016-01-01

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

  2. Transferrin Decorated Thermoresponsive Nanogels as Magnetic Trap Devices for Circulating Tumor Cells.

    PubMed

    Asadian-Birjand, Mazdak; Biglione, Catalina; Bergueiro, Julian; Cappelletti, Ariel; Rahane, Chinmay; Chate, Govind; Khandare, Jayant; Klemke, Bastian; Strumia, Miriam C; Calderón, Marcelo

    2016-03-01

    A rational design of magnetic capturing nanodevices, based on a specific interaction with circulating tumor cells (CTCs), can advance the capturing efficiency and initiate the development of modern smart nanoformulations for rapid isolation and detection of these CTCs from the bloodstream. Therefore, the development and evaluation of magnetic nanogels (MNGs) based on magnetic nanoparticles and linear thermoresponsive polyglycerol for the capturing of CTCs with overexpressed transferrin (Tf(+) ) receptors has been presented in this study. The MNGs are synthesized using a strain-promoted "click" approach which has allowed the in situ surface decoration with Tf-polyethylene glycol (PEG) ligands of three different PEG chain lengths as targeting ligands. An optimal value of around 30% of cells captures is achieved with a linker of eight ethylene glycol units. This study shows the potential of MNGs for the capture of CTCs and the necessity of precise control over the linkage of the targeting moiety to the capturing device. PMID:26691543

  3. The preparation and partial characterization of N-terminal and C-terminal iron-binding fragments from rabbit serum transferrin.

    PubMed Central

    Heaphy, S; Williams, J

    1982-01-01

    Two iron-binding fragments of Mr 36 000 and 33 000 corresponding to the N-terminal domain of rabbit serum transferrin were prepared. One iron-binding fragment of Mr 39 000 corresponding to the C-terminal domain was prepared. The N-terminal amino acid sequence of rabbit serum transferrin is: Val-Thr-Glu-Lys-Thr-Val-Asn-Trp-?-Ala-Val-Ser. One glycan unit is presented in rabbit serum transferrin and it is located in the C-terminal domain. Images Fig. 2. Fig. 3. Fig. 4. PMID:6816218

  4. Metabotropic Glutamate Receptor 1 (Grm1) Is An Oncogene In Epithelial Cells

    PubMed Central

    Martino, Jeffrey J; Wall, Brian A; Mastrantoni, Elisa; Wilimczyk, Barbara J; La Cava, Stephanie N; Degenhardt, Kurt; White, E; Chen, Suzie

    2014-01-01

    Non-neuronal expression of components of the glutamatergic system has been increasingly observed, and our laboratory previously had demonstrated the etiological role of ectopically expressed metabotropic glutamate receptor 1 (Grm1/mGluR1) in mouse models of melanoma. We hypothesize that inappropriate glutamatergic signaling in other cell types can dysregulate growth leading to transformation and tumorigenesis. As most cancers are carcinomas, we selected an immortalized primary baby mouse kidney (iBMK) cell model to assess whether Grm1 can transform epithelial cells. These iBMK cells, engineered to be immortal yet non-tumorigenic and retaining normal epithelial characteristics, were used as recipients for exogenous Grm1 cDNA. Several stable Grm1 expressing clones were isolated and the Grm1-receptors were shown to be functional, as evidenced by the accumulation of second messengers in response to Grm1 agonist. Additionally activated by agonist were MAPK and AKT signaling cascades, major intracellular pathways shown by many investigators to be critical in melanomagenesis and other neoplasms. These Grm1-iBMK cells exhibited enhanced cell proliferation in in vitro MTT assays and significant tumorigenicity in in vivo allografts. Persistent Grm1 expression was required for the maintenance of the in vivo tumorigenic phenotype as demonstrated by an inducible Grm1-silencing RNA. These are the first results that indicate Grm1 can be an oncogene in epithelial cells. Additionally, relevance to human disease in the corresponding tumor type of renal cell carcinoma (RCC) may be suggested by observed expression of GRM1/mGluR1 in a number of RCC tumor biopsy samples and cell lines, and the effects of GRM1 modulation on tumorigenicity therein. Moreover RCC cell lines exhibited elevated levels of extracellular glutamate, and some lines responded to drugs which modulate the glutamatergic system. These findings imply a possible role for glutamate signaling apparatus in RCC cell growth

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

    PubMed Central

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

    2012-01-01

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

  6. Advanced Gestational Age Increases Serum Carbohydrate-Deficient Transferrin Levels in Abstinent Pregnant Women

    PubMed Central

    Bakhireva, Ludmila N.; Cano, Sandra; Rayburn, William F.; Savich, Renate D.; Leeman, Lawrence; Anton, Raymond F.; Savage, Daniel D.

    2012-01-01

    Aims: Carbohydrate-deficient transferrin (%CDT) is a well-established and highly specific biomarker for sustained heavy consumption of alcohol. However, in pregnant women, the specificity of this biomarker might be affected by advanced gestational age, even after accounting for increased transferrin concentrations in pregnancy. The goal of this prospective study was to assess the variability in %CDT during pregnancy among alcohol-abstaining patients. Methods: Patients were recruited during one of the first prenatal care visits and followed-up to term. Abstinence was confirmed by maternal self-report and by alcohol biomarkers. Biomarkers assessed in the mother included serum gamma-glutamyltranspeptidase, urine ethyl glucuronide and ethyl sulfate, and whole blood phosphatidylethanol (PEth). In addition, PEth was measured in a dry blood spot card obtained from a newborn. For %CDT analysis, serum samples were collected at baseline and at term and analyzed by an internationally validated high-performance liquid chromatography and spectrophotometric detection method. Results: At recruitment (mean gestational age 22.6 ± 7.3 weeks), the mean %CDT concentration was 1.49 ± 0.30%, while at term, it increased to 1.67 ± 0.28% (P = 0.001). Using a conventional cutoff concentration %CDT >1.7%, 22.9 and 45.7% of the sample would be classified as ‘positive’ for this biomarker at recruitment and at term, respectively (P = 0.011 ). Conclusion: These results suggest that a conventional cutoff of 1.7% might be too low for pregnant women and would generate false-positive results. We propose that %CDT >2.0% be used as a cutoff concentration indicative of alcohol exposure in pregnant women. The sensitivity of %CDT at this cutoff for heavy drinking during pregnancy needs to be assessed further. PMID:22878591

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

    PubMed

    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. Graphical Abstract ᅟ. PMID:26392277

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

    NASA Astrophysics Data System (ADS)

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

    PubMed

    Broadbent, Suzanne

    2011-01-01

    We investigated whether 12 months of chronic endurance training would affect haematology, CD4(+) lymphocyte transferrin receptor (CD71) expression, CD4(+) intracellular iron and the incidence of upper respiratory tract illnesses (URTI) in Ironman triathletes compared with untrained men. Resting venous blood samples were taken from 15 Ironman triathletes (TR 30 ± 5 year) and 12 untrained men (UT 30 ± 6 year) every 4 weeks for 12 months. Erythrocyte, leukocyte and platelet concentration, haematocrit, haemoglobin (Hb) and mean corpuscular haemoglobin (MCHC) were measured with a full blood count. CD4(+) lymphocytes were analysed for changes in transferrin receptor (CD71) expression (CD4(+)CD71(+)), and intracellular iron (Fe(3+)), by flow cytometry. The TR group had significantly lower Hb, MCHC, and platelets for 10, 9 and 11 months, respectively; lower CD4(+)CD71(+) (3 months) and Fe(3+) (1 month), respectively; higher CD4(+)CD71(+) (1 month); a higher lymphocyte count for 4 months. There were no between-group differences in other variables. In both groups haematology and lymphocytes increased during spring, early summer and winter and decreased during late summer/late winter, with an inverse relationship between CD4(+)CD71(+) and Fe(3+). The TR group reported significantly fewer URTI than the UT. Low Hb and MCHC suggest an iron deficiency which may affect triathlete performance. Monthly changes in lymphocytes, CD4(+)CD71(+) and Fe(3+) suggested that spring, summer and late autumn are associated with CD4(+) proliferation. There may be seasonal relationships between haematology and lymphocyte function, independent of endurance training, possibly affecting performance but not the incidence of URTI. PMID:20821024

  10. A novel fibroblast growth factor receptor 1 inhibitor protects against cartilage degradation in a murine model of osteoarthritis

    PubMed Central

    Xu, Wei; Xie, Yangli; Wang, Quan; Wang, Xiaofeng; Luo, Fengtao; Zhou, Siru; Wang, Zuqiang; Huang, Junlan; Tan, Qiaoyan; Jin, Min; Qi, Huabing; Tang, Junzhou; Chen, Liang; Du, Xiaolan; Zhao, Chengguang; Liang, Guang; Chen, Lin

    2016-01-01

    The attenuated degradation of articular cartilage by cartilage-specific deletion of fibroblast growth factor receptor 1 (FGFR1) in adult mice suggests that FGFR1 is a potential target for treating osteoarthritis (OA). The goal of the current study was to investigate the effect of a novel non-ATP-competitive FGFR1 inhibitor, G141, on the catabolic events in human articular chondrocytes and cartilage explants and on the progression of cartilage degradation in a murine model of OA. G141 was screened and identified via cell-free kinase-inhibition assay. In the in vitro study, G141 decreased the mRNA levels of catabolic markers ADAMTS-5 and MMP-13, the phosphorylation of Erk1/2, JNK and p38 MAPK, and the protein level of MMP-13 in human articular chondrocytes. In the ex vivo study, proteoglycan loss was markedly reduced in G141 treated human cartilage explants. For the in vivo study, intra-articular injection of G141 attenuated the surgical destabilization of the medial meniscus (DMM) induced cartilage destruction and chondrocyte hypertrophy and apoptosis in mice. Our data suggest that pharmacologically antagonize FGFR1 using G141 protects articular cartilage from osteoarthritic changes, and intra-articular injection of G141 is potentially an effective therapy to alleviate OA progression. PMID:27041213

  11. Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients.

    PubMed

    Porter, John B; Walter, Patrick B; Neumayr, Lynne D; Evans, Patricia; Bansal, Sukhvinder; Garbowski, Maciej; Weyhmiller, Marcela G; Harmatz, Paul R; Wood, John C; Miller, Jeffery L; Byrnes, Colleen; Weiss, Guenter; Seifert, Markus; Grosse, Regine; Grabowski, Dagmar; Schmidt, Angelica; Fischer, Roland; Nielsen, Peter; Niemeyer, Charlotte; Vichinsky, Elliott

    2014-12-01

    In transfusional iron overload, extra-hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non-transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond-Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin-iron utilization (DBA). PMID:25209728

  12. Neurotensin-induced miR-133α expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin

    PubMed Central

    Law, Ivy Ka Man; Jensen, Dane; Bunnett, Nigel W.; Pothoulakis, Charalabos

    2016-01-01

    Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH. PMID:26902265

  13. Neurotensin-induced miR-133α expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin.

    PubMed

    Law, Ivy Ka Man; Jensen, Dane; Bunnett, Nigel W; Pothoulakis, Charalabos

    2016-01-01

    Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH. PMID:26902265

  14. Facile preparation of hyaluronic acid and transferrin co-modified Fe3O4 nanoparticles with inherent biocompatibility for dual-targeting magnetic resonance imaging of tumors in vivo.

    PubMed

    Pan, Jinbin; Sun, Shao-Kai; Wang, Yaqiong; Fu, Yan-Yan; Zhang, Xuejun; Zhang, Yi; Yu, Chunshui

    2015-12-14

    Clinical diagnosis of malignant tumors using nanoprobes needs severe improvements in the aspects of sensitivity and biocompatibility. Integrating a dual-targeting strategy with the selection of human-inherent elements and molecules as raw materials shows great potential in the development of a biosafe and sensitive nanoplatform. To carry out the proposed design, we constructed a biocompatible, dual-targeting MR imaging nanoprobe, based on Fe3O4 nanoparticles (NPs) co-modified with inherently innoxious hyaluronic acid (HA) and transferrin (Tf). HA was used as both a template and a targeting molecule to form Fe3O4@HA NPs through a one-step co-precipitation method, which were then further modified with Tf to obtain the dual-targeting Fe3O4@HA@Tf NPs at room temperature. The excellent biocompatibility of the nanoprobe was demonstrated via toxicity assays in vitro and in vivo. The desirable dual-targeting ability towards tumor cells was confirmed by a cellular uptake test (Hela cells, overexpressing both CD44 and transferrin receptors), and the developed nanoprobe was successfully applied in tumor-targeted MR imaging in vivo. In summation, we developed a dual-targeting Fe3O4 nanoprobe, following a facile procedure at room temperature. The nanoprobe showed a high targeting ability towards tumor cells and excellent biocompatibility, which showed its great potential to be applied in the clinical diagnosis of tumors. PMID:26507890

  15. Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

    1998-01-01

    Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

  16. Luminescent quantum clusters of gold in transferrin family protein, lactoferrin exhibiting FRET

    NASA Astrophysics Data System (ADS)

    Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; Verma, Pramod Kumar; Pal, Samir Kumar; Pradeep, Thalappil

    2010-12-01

    We report the synthesis of highly luminescent, water soluble quantum clusters (QCs) of gold, which are stabilized by an iron binding transferrin family protein, lactoferrin (Lf). The synthesized AuQC@Lfclusters were characterized using UV-Visiblespectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), photoluminescence (PL), matrix assisted laser desorption ionizationmass spectrometry (MALDI-MS), FTIR spectroscopy and circular dichroism (CD) spectroscopy along with picosecond-resolved lifetime measurements. Detailed investigations with FTIR and CD spectroscopy have revealed changes in the secondary structure of the protein in the cluster. We have also studied Förster resonance energy transfer (FRET) occurring between the protein and the cluster. The ability of the clusters to sense cupric ions selectively at ppm concentrations was tested. The stability of clusters in widely varying pH conditions and their continued luminescence make it feasible for them to be used for intracellular imaging and molecular delivery, particularly in view of Lf protection.We report the synthesis of highly luminescent, water soluble quantum clusters (QCs) of gold, which are stabilized by an iron binding transferrin family protein, lactoferrin (Lf). The synthesized AuQC@Lfclusters were characterized using UV-Visiblespectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), photoluminescence (PL), matrix assisted laser desorption ionizationmass spectrometry (MALDI-MS), FTIR spectroscopy and circular dichroism (CD) spectroscopy along with picosecond-resolved lifetime measurements. Detailed investigations with FTIR and CD spectroscopy have revealed changes in the secondary structure of the protein in the cluster. We have also studied Förster resonance energy transfer (FRET) occurring between the protein and the cluster. The ability of the clusters to sense cupric ions selectively at ppm concentrations was tested. The

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

    PubMed Central

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

    2015-01-01

    Abstract Iron deficiency is common in patients with inflammatory bowel disease (IBD), but can be difficult to diagnose in the presence of inflammation because ferritin is an acute phase reactant. The transferrin receptor-ferritin index (TfR-F) has a high sensitivity and specificity for iron deficiency diagnosis in chronic diseases. The diagnostic efficacy of TfR-F is little known in patients with IBD. The aim of the study was to assess the added value of TfR-F to iron deficiency diagnosis in a prospective cohort of patients with IBD. Consecutive IBD patients were prospectively enrolled. Patients were excluded in case of blood transfusion, iron supplementation, or lack of consent. IBD activity was assessed on markers of inflammation (C-reactive protein, endoscopy, fecal calprotectin). Hemoglobin, ferritin, vitamin B9 and B12, Lactate dehydrogenase, haptoglobin, and soluble transferrin receptor (sTfR) were assayed. TfR-F was calculated as the ratio sTfR/log ferritin. Iron deficiency was defined by ferritin <30 ng/mL or TfR-F >2 in the presence of inflammation. One-hundred fifty patients with median age 38 years (16–78) and Crohn disease (n = 105), ulcerative colitis (n = 43), or unclassified colitis (n = 2) were included. Active disease was identified in 45.3%. Anemia was diagnosed in 28%. Thirty-six patients (24%) had ferritin <30 ng/mL. Thirty-two patients (21.3%) had ferritin levels from 30 to 100 ng/ml and inflammation: 2 had vitamin B12 deficiency excluding TfR-F analysis, 13 of 30 (43.3%) had TfR-F >2. Overall, iron deficiency was diagnosed in 32.7% of the patients. TfR-F in addition to ferritin <30 ng/mL criterion increased by 36% diagnosis rates of iron deficiency. TfR-F appeared as a useful biomarker that could help physicians to diagnose true iron deficiency in patients with active IBD. PMID:26131803

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

    PubMed

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

    2015-07-01

    Iron deficiency is common in patients with inflammatory bowel disease (IBD), but can be difficult to diagnose in the presence of inflammation because ferritin is an acute phase reactant. The transferrin receptor-ferritin index (TfR-F) has a high sensitivity and specificity for iron deficiency diagnosis in chronic diseases. The diagnostic efficacy of TfR-F is little known in patients with IBD. The aim of the study was to assess the added value of TfR-F to iron deficiency diagnosis in a prospective cohort of patients with IBD.Consecutive IBD patients were prospectively enrolled. Patients were excluded in case of blood transfusion, iron supplementation, or lack of consent. IBD activity was assessed on markers of inflammation (C-reactive protein, endoscopy, fecal calprotectin). Hemoglobin, ferritin, vitamin B9 and B12, Lactate dehydrogenase, haptoglobin, and soluble transferrin receptor (sTfR) were assayed. TfR-F was calculated as the ratio sTfR/log ferritin. Iron deficiency was defined by ferritin <30 ng/mL or TfR-F >2 in the presence of inflammation.One-hundred fifty patients with median age 38 years (16-78) and Crohn disease (n = 105), ulcerative colitis (n = 43), or unclassified colitis (n = 2) were included. Active disease was identified in 45.3%. Anemia was diagnosed in 28%. Thirty-six patients (24%) had ferritin <30 ng/mL. Thirty-two patients (21.3%) had ferritin levels from 30 to 100 ng/ml and inflammation: 2 had vitamin B12 deficiency excluding TfR-F analysis, 13 of 30 (43.3%) had TfR-F >2. Overall, iron deficiency was diagnosed in 32.7% of the patients.TfR-F in addition to ferritin <30 ng/mL criterion increased by 36% diagnosis rates of iron deficiency. TfR-F appeared as a useful biomarker that could help physicians to diagnose true iron deficiency in patients with active IBD. PMID:26131803

  19. Biodistribution of Ru-97-labeled DTPA, DMSA and transferrin. [Diagnostic potential

    SciTech Connect

    Som, P; Oster, Z H; Fairchild, R G; Atkins, H L; Brill, A B; Gil, M C; Srivastava, S C; Meinken, G E; Goldman, A G; Richards, P

    1980-01-01

    Ruthenium-97 is being produced at the Brookhaven Linac Isotope Producer (BLIP). The favorable physical properties of Ru-97 and chemical reactivity of ruthenium offer a potential for using this isotope to label compounds useful for delayed scanning. Diethylenetriamine pentaacetic acid (DTPA), 2,3-Dimercaptosuccinic acid (DMSA), and Transferrin (TF) were labeled with Ru-97-chloride. Ru-97-DTPA and In-111-DTPA, injected intravenously, showed similar organ distribution, kinetics, and more than 80% excretion by 0.5 h. Ru-97-DTPA and In-111-DTPA injected into the cisterna magna of dogs showed similar kinetics in brain, blood, and urinary bladder. The energy deposited by 1 mCi In-111-DTPA is twice that from 1 mCi Ru-97-DTPA. High quality camera images of the CSF space in the dog were obtained with both isotopes. Ru-97-DMSA was prepared with and without the addition of SnCl/sub 2/.2H/sub 2/O. Tin-free DMSA was rapidly excreted via the kidneys, whereas for maximum cortical deposition, the tin-containing preparation was superior. This compound is suitable for delayed imaging of both normal and impaired kidneys. Tissue distribution studies were performed in abscess-bearing rats with Ru-97-transferrin. Although blood levels were higher than with Ga-67-citrate, the abscess had twice as much Ru-97-TF as Ga-67-citrate and the Ru-97 muscle activity was one-third that of Ga-67. Imaging of abscess-bearing rabbits with Ru-97-TF visualized the abscesses as early as 1/2 hr after injection. Since the initial images visualize the abscess so clearly and since the TF portion of the compound binds to the abscess, Tc-99m-TF is being studied for the same purpose. Ru-97-labeled compounds are a promising replacement for In-111 and possibly also for Ga-67 compounds with the advantages of lower radiation dose and high quality image. (ERB)

  20. Effects of Trace Amine-associated Receptor 1 Agonists on the Expression, Reconsolidation, and Extinction of Cocaine Reward Memory

    PubMed Central

    Liu, Jian-Feng; Thorn, David A; Zhang, Yanan

    2016-01-01

    Background: As a modulator of dopaminergic system, trace amine-associated receptor 1 has been shown to play a critical role in regulating the rewarding properties of additive drugs. It has been demonstrated that activation of trace amine-associated receptor 1 decreased the abuse-related behaviors of cocaine in rats. However, the role of trace amine-associated receptor 1 in specific stages of cocaine reward memory is still unclear. Methods: Here, using a cocaine-induced conditioned place preference model, we tested the effects of a selective trace amine-associated receptor 1 agonist RO5166017 on the expression, reconsolidation, and extinction of cocaine reward memory. Results: We found that RO5166017 inhibited the expression but not retention of cocaine-induced conditioned place preference. RO5166017 had no effect on the reconsolidation of cocaine reward memory. Pretreatment with RO5166017 before extinction hindered the formation of extinction long-term memory. RO5166017 did not affect the movement during the conditioned place preference test, indicating the inhibitory effect of RO5166017 on the expression of cocaine-induced conditioned place preference was not caused by locomotion inhibition. Using a cocaine i.v. self-administration model, we found that the combined trace amine-associated receptor 1 partial agonist RO5263397 with extinction had no effect on the following cue- and drug-induced reinstatement of cocaine-seeking behavior. Repeated administration of the trace amine-associated receptor 1 agonist during extinction showed a continually inhibitory effect on the expression of cocaine reward memory both in cocaine-induced conditioned place preference and cocaine self-administration models. Conclusions: Taken together, these results indicate that activation of trace amine-associated receptor 1 specifically inhibited the expression of cocaine reward memory. The inhibitory effect of trace amine-associated receptor 1 agonists on cocaine reward memory suggests

  1. Supporting data for the MS identification of distinct transferrin glycopeptide glycoforms and citrullinated peptides associated with inflammation or autoimmunity.

    PubMed

    Rosal-Vela, A; Barroso, A; Giménez, E; García-Rodríguez, S; Longobardo, V; Postigo, J; Iglesias, M; Lario, A; Merino, J; Merino, R; Zubiaur, M; Sanz-Nebot, V; Sancho, J

    2016-03-01

    This data article presents the results of all the statistical analyses applied to the relative intensities of the detected 2D-DiGE protein spots for each of the 3 performed DiGE experiments. The data reveals specific subsets of protein spots with significant differences between WT and CD38-deficient mice with either Collagen-induced arthritis (CIA), or with chronic inflammation induced by CFA, or under steady-state conditions. This article also shows the MS data analyses that allowed the identification of the protein species which serve to discriminate the different experimental groups used in this study. Moreover, the article presents MS data on the citrullinated peptides linked to specific protein species that were generated in CIA(+) or CFA-treated mice. Lastly, this data article provides MS data on the efficiency of the analyses of the transferrin (Tf) glycopeptide glycosylation pattern in spleen and serum from CIA(+) mice and normal controls. The data supplied in this work is related to the research article entitled "identification of multiple transferrin species in spleen and serum from mice with collagen-induced arthritis which may reflect changes in transferrin glycosylation associated with disease activity: the role of CD38" [1]. All mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifiers PRIDE: PXD002644, PRIDE: PXD002643, PRIDE: PXD003183 and PRIDE: PXD003163. PMID:26909372

  2. Gene diversity for haptoglobin and transferrin classical markers among Hindu and Muslim populations of Aligarh City, India.

    PubMed

    Ara, G; Siddique, Y H; Afzal, M

    2011-06-01

    The present paper reports the distribution of serum protein markers viz. haptoglobin and transferrin in two major groups of Aligarh city of North India. In present study we have undertaken a survey of 538 individuals belonging to eight different populations, four from the Hindu community i.e. Brahmin, Bania, Rajput and Jatav, and the rest four among the Muslim community i.e. Syed, Sheikh, Pathan and Ansari. The heterozygosity ranged from 0.2939 (Ansari) to 0.4873 (Brahmin) for haptoglobin and from 0.000 (Rajput) to 0.1498 (Pathan) for transferrin. The values of D(ST) are 0.4122 and 0.4406, and that of G(ST) are 0.5059 and 0.9726 for haptoglobin and transferrin markers respectively. Through F(ST) test, it has been concluded that there is a high genetic differentiation of populations within Hindu and Muslim groups, though there is absence of any significant differences between these groups. PMID:21866866

  3. Supporting data for the MS identification of distinct transferrin glycopeptide glycoforms and citrullinated peptides associated with inflammation or autoimmunity

    PubMed Central

    Rosal-Vela, A.; Barroso, A.; Giménez, E.; García-Rodríguez, S.; Longobardo, V.; Postigo, J.; Iglesias, M.; Lario, A.; Merino, J.; Merino, R.; Zubiaur, M.; Sanz-Nebot, V.; Sancho, J.

    2016-01-01

    This data article presents the results of all the statistical analyses applied to the relative intensities of the detected 2D-DiGE protein spots for each of the 3 performed DiGE experiments. The data reveals specific subsets of protein spots with significant differences between WT and CD38-deficient mice with either Collagen-induced arthritis (CIA), or with chronic inflammation induced by CFA, or under steady-state conditions. This article also shows the MS data analyses that allowed the identification of the protein species which serve to discriminate the different experimental groups used in this study. Moreover, the article presents MS data on the citrullinated peptides linked to specific protein species that were generated in CIA+ or CFA-treated mice. Lastly, this data article provides MS data on the efficiency of the analyses of the transferrin (Tf) glycopeptide glycosylation pattern in spleen and serum from CIA+ mice and normal controls. The data supplied in this work is related to the research article entitled “identification of multiple transferrin species in spleen and serum from mice with collagen-induced arthritis which may reflect changes in transferrin glycosylation associated with disease activity: the role of CD38” [1]. All mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifiers PRIDE: PXD002644, PRIDE: PXD002643, PRIDE: PXD003183 and PRIDE: PXD003163. PMID:26909372

  4. Effects of Hypoxia and Transferrin on Toxicity and DNA Binding of Ruthenium Antitumor Agents in Hela Cells

    PubMed Central

    Frasca, D.; Ciampa, J.; Emerson, J.; Umans, R. S.

    1996-01-01

    Nuclear DNA binding and inhibition of growth of HeLa cells in culture were determined after 24 h incubation with the ruthenium anticancer agents cis-[Cl2(NH3)4Ru]Cl (CCR) and (ImH)trans-[(Im)2Cl4Ru] (ICR) as a function of [Ru], Po2, and added transferrin. Consistent with the “activation-by-reduction” hypothesis, cytotoxicity and DNA binding for both complexes increased under reduced oxygen conditions. Consistent with the “transferrin- transport” hypothesis, inhibition of cell growth also increased with added transferrin for both complexes. Despite their differences in charge, reduction potentials and substitution rates, both complexes behaved remarkably similarly indicating a common mechanism of action for both. Under atmospheric Conditions (Po2 = 159 torr), CCR inhibited HeLa cell growth with IC50 = 3.5 μM, while that for ICR was 2.0 μM. The binding of both complexes to DNA (RuDNA/PDNA) correlated with toxicity and was approximately linear in the concentration of the ruthenium complex in the culture medium, [Ru]. For both complexes, IC50 values decrease and DNA binding increases with decreasing log(Po2). In general, DNA binding at all oxygen pressures for both complexes is in the range of one Ru per 1000-2000 DNA base pairs at [Ru] = IC50. PMID:18475755

  5. Hepatitis E virus ORF1 encoded macro domain protein interacts with light chain subunit of human ferritin and inhibits its secretion.

    PubMed

    Ojha, Nishant Kumar; Lole, Kavita S

    2016-06-01

    Hepatitis E Virus (HEV) is the major causative agent of acute hepatitis in developing countries. Its genome has three open reading frames (ORFs)-called as ORF1, ORF2, and ORF3. ORF1 encodes nonstructural polyprotein having multiple domains, namely: Methyltransferase, Y domain, Protease, Macro domain, Helicase, and RNA-dependent RNA polymerase. In the present study, we show that HEV-macro domain specifically interacts with light chain subunit of human ferritin (FTL). In cultured hepatoma cells, HEV-macro domain reduces secretion of ferritin without causing any change in the expression levels of FTL. This inhibitory effect was further enhanced upon Brefeldin-A treatment. The levels of transferrin Receptor 1 or ferroportin, two important proteins in iron metabolism, remained unchanged in HEV-macro domain expressing cells. Similarly, there were no alterations in the levels of cellular labile iron pool and reactive oxygen species, indicating that HEV-macro domain does not influence cellular iron homeostasis/metabolism. As ferritin is an acute-phase protein, secreted in higher level in infected persons and HEV-macro domain has the property of reducing synthesis of inflammatory cytokines, we propose that by directly binding to FTL, macro domain prevents ferritin from entering into circulation and helps in further attenuation of the host immune response. PMID:27170377

  6. DNA Methylation at the Neonatal State and at the Time of Diagnosis: Preliminary Support for an Association with the Estrogen Receptor 1, Gamma-Aminobutyric Acid B Receptor 1, and Myelin Oligodendrocyte Glycoprotein in Female Adolescent Patients with OCD

    PubMed Central

    Nissen, Judith Becker; Hansen, Christine Søholm; Starnawska, Anna; Mattheisen, Manuel; Børglum, Anders Dupont; Buttenschøn, Henriette Nørmølle; Hollegaard, Mads

    2016-01-01

    Obsessive–compulsive disorder (OCD) is a neuropsychiatric disorder. Non-genetic factors and their interaction with genes have attracted increasing attention. Epigenetics is regarded an important interface between environmental signals and activation/repression of genomic responses. Epigenetic mechanisms have not previously been examined in OCD in children and adolescents. The aim of the present study was to examine the DNA methylation profile of selected genes in blood spots from neonates later diagnosed with OCD and in the same children/adolescents at the time of diagnosis compared with age- and sex-matched controls. Furthermore, we wanted to characterize the association of the differential methylation profiles with the severity of OCD and treatment outcome. Dried and new blood spot samples were obtained from 21 female children/adolescents with verified OCD and 12 female controls. The differential methylation was analyzed using a linear model and the correlation with the severity of OCD and treatment outcome was analyzed using the Pearson correlation. We evaluated selected Illumina Infinium HumanMethylation450 BeadChip probes within and up to 100,000 bp up- and downstream of 14 genes previously associated with OCD (SLC1A1, SLC25A12, GABBR1, GAD1, DLGAP1, MOG, BDNF, OLIG2, NTRK2 and 3, ESR1, SL6A4, TPH2, and COMT). The study found no significantly differential methylation. However, preliminary support for a difference was found for the gamma-aminobutyric acid (GABA) B receptor 1 (cg10234998, cg17099072) in blood samples at birth and for the estrogen receptor 1 (ESR1) (cg10939667), the myelin oligodendrocyte glycoprotein (MOG) (cg16650906), and the brain-derived neurotrophic factor (BDNF) (cg14080521) in blood samples at the time of diagnosis. Preliminary support for an association was observed between the methylation profiles of GABBR1 and MOG and baseline severity, treatment effect, and responder status; and between the methylation profile of ESR1 and baseline

  7. DNA Methylation at the Neonatal State and at the Time of Diagnosis: Preliminary Support for an Association with the Estrogen Receptor 1, Gamma-Aminobutyric Acid B Receptor 1, and Myelin Oligodendrocyte Glycoprotein in Female Adolescent Patients with OCD.

    PubMed

    Nissen, Judith Becker; Hansen, Christine Søholm; Starnawska, Anna; Mattheisen, Manuel; Børglum, Anders Dupont; Buttenschøn, Henriette Nørmølle; Hollegaard, Mads

    2016-01-01

    Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder. Non-genetic factors and their interaction with genes have attracted increasing attention. Epigenetics is regarded an important interface between environmental signals and activation/repression of genomic responses. Epigenetic mechanisms have not previously been examined in OCD in children and adolescents. The aim of the present study was to examine the DNA methylation profile of selected genes in blood spots from neonates later diagnosed with OCD and in the same children/adolescents at the time of diagnosis compared with age- and sex-matched controls. Furthermore, we wanted to characterize the association of the differential methylation profiles with the severity of OCD and treatment outcome. Dried and new blood spot samples were obtained from 21 female children/adolescents with verified OCD and 12 female controls. The differential methylation was analyzed using a linear model and the correlation with the severity of OCD and treatment outcome was analyzed using the Pearson correlation. We evaluated selected Illumina Infinium HumanMethylation450 BeadChip probes within and up to 100,000 bp up- and downstream of 14 genes previously associated with OCD (SLC1A1, SLC25A12, GABBR1, GAD1, DLGAP1, MOG, BDNF, OLIG2, NTRK2 and 3, ESR1, SL6A4, TPH2, and COMT). The study found no significantly differential methylation. However, preliminary support for a difference was found for the gamma-aminobutyric acid (GABA) B receptor 1 (cg10234998, cg17099072) in blood samples at birth and for the estrogen receptor 1 (ESR1) (cg10939667), the myelin oligodendrocyte glycoprotein (MOG) (cg16650906), and the brain-derived neurotrophic factor (BDNF) (cg14080521) in blood samples at the time of diagnosis. Preliminary support for an association was observed between the methylation profiles of GABBR1 and MOG and baseline severity, treatment effect, and responder status; and between the methylation profile of ESR1 and baseline

  8. Assignment of the lactotransferrin gene to human chromosome 3 and to mouse chromosome 9.

    PubMed

    Teng, C T; Pentecost, B T; Marshall, A; Solomon, A; Bowman, B H; Lalley, P A; Naylor, S L

    1987-11-01

    Lactotransferrin (LTF), a member of the transferrin family of genes, is the major iron-binding protein in milk and body secretions. The amino acid sequence of LTF consists of two homologous domains homologous to proteins in the transferrin family. Recent isolation of cDNA encoding mouse LTF has expedited the mapping of both mouse and human LTF genes. Southern blot analysis of DNA from mouse-Chinese hamster and human-mouse somatic cell hybrids maps the LTF gene to mouse chromosome 9 and to human chromosome 3, respectively. Furthermore, analysis of cell hybrids containing defined segments of human chromosome 3 demonstrates that the gene is located in the 3q21-qter region. These results suggest that LTF and associated genes of the transferrin family have existed together on the same chromosomal region for 300-500 million years. PMID:3478818

  9. Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells.

    PubMed Central

    Mwanjewe, James; Grover, Ashok K

    2004-01-01

    Cells take up transferrin-bound iron or NTBI (non-transferrin-bound iron). After treatment with NGF (nerve growth factor), PC12 cells exhibited a neuronal phenotype and an increase in the NTBI uptake (55Fe2+ or 55Fe3+). We loaded the cells with the dye calcein, whose fluorescence increases in the presence of Ca2+ but is quenched with Fe2+ or Fe3+. When examined using calcein fluorescence or radioactive iron, DAG (diacylglycerol)-stimulated NTBI entry was more in NGF-treated PC12 cells compared with untreated cells. All experiments were performed at 1.5 mM extracellular Ca2+. Nramp2 (natural-resistance-associated macrophage protein 2) mRNA expression did not change after the NGF treatment. Expression of the bivalent cation entry protein TRPC6 (transient receptor potential canonical 6) was detected only in the NGF-treated cells. To verify that increased NTBI uptake depended on TRPC6, we examined whether transfecting HEK-293 (human embryonic kidney 293) cells with TRPC6 also increased the NTBI (55Fe) uptake. We also cotransfected HEK-293 cells with two plasmids, one expressing TRPC6 and the other expressing the fluorescent protein DsRED2 to identify the transfected cells. Challenging the calcein-loaded HEK-293 cells (which intrinsically express the a1-adrenergic receptors) with phenylephrine or a cell-permeant DAG increased the fluorescence signal more rapidly in transfected cells compared with untransfected cells. However, when iron (Fe2+ and Fe3+) was added before adding phenylephrine or DAG, the fluorescence intensity decreased more rapidly in transfected cells compared with untransfected cells, thereby indicating a greater stimulation of the NTBI uptake in cells expressing TRPC6. We postulate that the increase in the NTBI entry into neuronal PC12 cells is through TRPC6, a pathway that is unique since it is receptor-stimulated. Since neuronal cells express TRPC6, this pathway may have a role in neurotoxicity. PMID:14640978

  10. Transgenic HFE-dependent induction of hepcidin in mice does not require transferrin receptor-2

    PubMed Central

    Schmidt, Paul J.; Fleming, Mark D.

    2012-01-01

    Hereditary hemochomatosis (HH) is caused by mutations in several genes, including HFE and transferrin receptor-2 (TFR2). Loss of either protein decreases expression of the iron regulatory hormone hepcidin by the liver, leading to inappropriately high iron uptake from the diet, and resulting in systemic iron overload. In tissue culture, overexpressed HFE and TFR2 physically interact. Hepatocellular overexpression of Hfe in vivo increases hepcidin expression, despite an associated decrease in Tfr2. On this basis, we hypothesized that Tfr2 would not be required for Hfe-dependent up-regulation of hepcidin. We show that hepatocellular overexpression of Hfe in Tfr2Y245X/Y245X mice leads to hepcidin induction eventuating in iron deficiency and a hypochromic, microcytic anemia. Furthermore, co-immunoprecipitation studies using liver lysates did not provide evidence for physical interaction between Hfe and Tfr2 in vivo. In conclusion, we demonstrate that Tfr2 is not essential for Hfe-mediated induction of hepcidin expression, supporting the possibility that TFR2 may regulate iron metabolism in an HFE-independent manner. PMID:22460705

  11. N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS.

    PubMed

    Quaranta, Alessandro; Sroka-Bartnicka, Anna; Tengstrand, Erik; Thorsén, Gunnar

    2016-07-01

    It has been known for a long time that diseases can be associated with changes to the glycosylation of specific proteins. This has been shown for cancer, immunological disorders, and neurodegenerative diseases. The possibility of using the glycosylation patterns of proteins as biomarkers for disease would be a great asset for clinical research or diagnosis. There is at present a lack of rapid, automated, and cost-efficient analytical techniques for the determination of the glycosylation of specific serum proteins. We have developed a method for determining the glycosylation pattern of proteins based on the affinity capture of a specific serum protein, the enzymatic release of the N-linked glycans, and the analysis of the glycan pattern using MALDI-MS. All sample preparation is performed in a disposable centrifugal microfluidic disc. The sample preparation is miniaturized, requiring only 1 μL of sample per determination, and automated with the possibility of processing 54 samples in parallel in 3.5 h. We have developed a method for the glycosylation pattern analysis of transferrin. The method has been tested on serum samples from chronic alcohol abusers and a control group. Also, a SIMCA model was created and evaluated to discriminate between the two groups. PMID:27137515

  12. The Role of Cell-Penetrating Peptide and Transferrin on Enhanced Delivery of Drug to Brain

    PubMed Central

    Sharma, Gitanjali; Lakkadwala, Sushant; Modgil, Amit; Singh, Jagdish

    2016-01-01

    The challenge of effectively delivering therapeutic agents to brain has led to an entire field of active research devoted to overcome the blood brain barrier (BBB) and efficiently deliver drugs to brain. This review focusses on exploring the facets of a novel platform designed for the delivery of drugs to brain. The platform was constructed based on the hypothesis that a combination of receptor-targeting agent, like transferrin protein, and a cell-penetrating peptide (CPP) will enhance the delivery of associated therapeutic cargo across the BBB. The combination of these two agents in a delivery vehicle has shown significantly improved (p < 0.05) translocation of small molecules and genes into brain as compared to the vehicle with only receptor-targeting agents. The comprehensive details of the uptake mechanisms and properties of various CPPs are illustrated here. The application of this technology, in conjunction with nanotechnology, can potentially open new horizons for the treatment of central nervous system disorders. PMID:27231900

  13. The Role of Cell-Penetrating Peptide and Transferrin on Enhanced Delivery of Drug to Brain.

    PubMed

    Sharma, Gitanjali; Lakkadwala, Sushant; Modgil, Amit; Singh, Jagdish

    2016-01-01

    The challenge of effectively delivering therapeutic agents to brain has led to an entire field of active research devoted to overcome the blood brain barrier (BBB) and efficiently deliver drugs to brain. This review focusses on exploring the facets of a novel platform designed for the delivery of drugs to brain. The platform was constructed based on the hypothesis that a combination of receptor-targeting agent, like transferrin protein, and a cell-penetrating peptide (CPP) will enhance the delivery of associated therapeutic cargo across the BBB. The combination of these two agents in a delivery vehicle has shown significantly improved (p < 0.05) translocation of small molecules and genes into brain as compared to the vehicle with only receptor-targeting agents. The comprehensive details of the uptake mechanisms and properties of various CPPs are illustrated here. The application of this technology, in conjunction with nanotechnology, can potentially open new horizons for the treatment of central nervous system disorders. PMID:27231900

  14. Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier

    SciTech Connect

    Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M. )

    1991-06-01

    Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate {approximately} 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.

  15. Plasmid pORF-hTRAIL targeting to glioma using transferrin-modified polyamidoamine dendrimer

    PubMed Central

    Gao, Song; Li, Jianfeng; Jiang, Chen; Hong, Bo; Hao, Bing

    2016-01-01

    A gene drug delivery system for glioma therapy based on transferrin (Tf)-modified polyamidoamine dendrimer (PAMAM) was prepared. Gene drug, tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL)-encoding plasmid open reading frame (pORF-hTRAIL, Trail), was condensed by Tf-modified PAMAM to form nanoparticles (NPs). PAMAM-PEG-Tf/DNA NPs showed higher cellular uptake, in vitro gene expression, and cytotoxicity than PAMAM-PEG/DNA NPs in C6 cells. The in vivo targeting efficacy of NPs was visualized by ex vivo fluorescence imaging. Tf-modified NPs showed obvious glioma-targeting trend. Plasmid encoding green fluorescence protein (GFP) was also condensed by modified or unmodified PAMAM to evaluate the in vivo gene expression level. The PAMAM-PEG-Tf/plasmid encoding enhanced green fluorescence protein (pEGFP) NPs exhibited higher GFP expression level than PAMAM-PEG/pEGFP NPs. TUNEL assay revealed that Tf-modified NPs could induce much more tumor apoptosis. The median survival time of PAMAM-PEG-Tf/Trail-treated rats (28.5 days) was longer than that of rats treated with PAMAM-PEG/Trail (25.5 days), temozolomide (24.5 days), PAMAM-PEG-Tf/pEGFP (19 days), or saline (17 days). The therapeutic effect was further confirmed by magnetic resonance imaging. This study demonstrated that targeting gene delivery system had potential application for the treatment of glioma. PMID:26719669

  16. Characteristics of sequential targeting of brain glioma for transferrin-modified cisplatin liposome.

    PubMed

    Lv, Qing; Li, Li-Min; Han, Min; Tang, Xin-Jiang; Yao, Jin-Na; Ying, Xiao-Ying; Li, Fan-Zhu; Gao, Jian-Qing

    2013-02-28

    Methods on how to improve the sequential targeting of glioma subsequent to passing of drug through the blood-brain barrier (BBB) have been occasionally reported. However, the characteristics involved are poorly understood. In the present study, cisplatin (Cis) liposome (lipo) was modified with transferrin (Tf) to investigate the characteristics of potential sequential targeting to glioma. In bEnd3/C6 co-culture BBB models, higher transport efficiency across the BBB and cytotoxicity in basal C6 cells induced by Cis-lipo(Tf) than Cis-lipo and Cis-solution, suggest its sequential targeting effect. Interestingly, similar liposomal morphology as that of donor compartment was first demonstrated in the receptor solution of BBB models. Meanwhile, a greater acquisition in the lysosome of bEnd3, distributed sequentially into the nucleus of C6 cells were found for the Cis-lipo(Tf). Pre-incubation of chlorpromazine and Tf inhibited this process, indicating that a clathrin-dependent endocytosis is involved in the transport of Cis-lipo(Tf) across the BBB. PMID:23347891

  17. Comparison of NIR FRET pairs for quantitative transferrin-based assay

    NASA Astrophysics Data System (ADS)

    Sinsuebphon, Nattawut; Bevington, Travis; Zhao, Lingling; Ken, Abe; Barroso, Margarida; Intes, Xavier

    2014-02-01

    Transferrin (Tfn) is commonly used as a drug delivery carrier for cancer treatment. Tfn cellular internalization can be observed by Förster resonance energy transfer (FRET), which occurs when two fluorophores - donor and acceptor - are a few nanometers apart. Donor fluorescence lifetime can be used to sense and quantify FRET occurrence. In FRET state, the donor is quenched leading to a significant reduction in its lifetime. In this study, donor and acceptor near-infrared (NIR) fluorophore-labeled Tfn were used to quantify cellular internalization in breast cancer cell line (T47D). Based on donor lifetime, quantum yield and spectral data, seven NIR FRET pairs were chosen for this comparison. Performance of the different NIR FRET pairs was evaluated in vitro in multiwell plate settings and by analyzing the relationship between quenched donor fraction and acceptor:donor ratio. Additionally, we performed brightness comparison between each pairs. Several parameters, such as brightness, lifetime, R0 and FRET donor population values are used to identify the most suitable NIR FRET pair for in vivo studies in preclinical settings.

  18. The expression of transferrin binding protein in the turtle nervous system.

    PubMed

    Park, Sang Wook; Lee, Ha Na; Jeon, Gye Sun; Sim, Ki-Bum; Cho, Ik-Hyun; Cho, Sa Sun

    2009-03-01

    Transferrin binding protein (TfBP) is a cytoplasmic glycoprotein that was originally isolated from the chick oviduct. As we previously demonstrated the constitutive expression of TfBP in the avian nervous system, in this study we examined whether TfBP is expressed in the reptilian nervous system. In accordance with previous findings in the chicken, oligodendrocytes were most prominently labeled by antiserum to TfBP. Great variability was observed between different regions of the central nervous system (CNS) in terms of TfBP-labeled oligodendrocyte numbers. In the retina, TfBP was localized specifically in the cells that are morphologically oligodendrocytes and present in the optic nerve and the ganglion cell layer. TfBP staining was also seen in the Schwann cells of peripheral nerves. Furthermore, choroid plexus cells and capillary endothelial cells similarly exhibited strong reactions. These results may reflect the fact that the homology of nervous system genes is conserved between close phylogenetic lines, and proove the potential of TfBP as a marker for oligodendrocytes in avian as well as reptile. PMID:19789413

  19. A transferrin gene associated with development and 2-tridecanone tolerance in Helicoverpa armigera

    PubMed Central

    Zhang, L; Shang, Q; Lu, Y; Zhao, Q; Gao, X

    2015-01-01

    The full-length cDNA (2320 bp) encoding a putative iron-binding transferrin protein from Helicoverpa armigera was cloned and named HaTrf. The putative HaTrf sequence included 670 amino acids with a molecular mass of approximately 76 kDa. Quantitative PCR results demonstrated that the transcriptional level of HaTrf was significantly higher in the sixth instar and pupa stages as compared with other developmental stages. HaTrf transcripts were more abundant in fat bodies and in the epidermis than in malpighian tubules. Compared with the control, the expression of HaTrf increased dramatically 24 h after treatment with 2-tridecanone. Apparent growth inhibition with a dramatic body weight decrease was observed in larvae fed with HaTrf double-stranded RNA (dsRNA), as compared with those fed with green fluorescent protein dsRNA. RNA interference of HaTrf also significantly increased the susceptibility of larvae to 2-tridecanone. These results indicate the possible involvement of HaTrf in tolerance to plant secondary chemicals. PMID:25430818

  20. Medical treatment of orthotopic glioblastoma with transferrin-conjugated nanoparticles encapsulating zoledronic acid

    PubMed Central

    Porru, Manuela; Zappavigna, Silvia; Salzano, Giuseppina; Luce, Amalia; Stoppacciaro, Antonella; Balestrieri, Maria Luisa; Artuso, Simona; Lusa, Sara; De Rosa, Giuseppe; Leonetti, Carlo; Caraglia, Michele

    2014-01-01

    Glioblastomas are highly aggressive adult brain tumors with poor clinical outcome. In the central nervous system (CNS) the blood-brain barrier (BBB) is the most important limiting factor for both development of new drugs and drug delivery. Here, we propose a new strategy to treat glioblastoma based on transferrin (Tf)-targeted self-assembled nanoparticles (NPs) incorporating zoledronic acid (ZOL) (NPs-ZOL-Tf). NPs-ZOL-Tf have been assessed on the glioblastoma cell line U373MG-LUC that showed a refractoriness in vitro to temozolomide (TMZ) and fotemustine (FTM). NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was superimposable to that one induced by NPs-ZOL (not armed with Tf). On the other hand, NPs-ZOL-Tf showed a higher antitumor efficacy if compared with that one caused by NPs-ZOL in immunosuppressed mice intramuscularly bearing U373MG-LUC xenografts, inducing a significant tumor weight inhibition (TWI). The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the efficacy of NPs-ZOL-Tf. These effects were paralleled by a higher intratumour localization of fluorescently-labeled-NPs-Tf both in intramuscular and intracranial xenografts. In conclusion, our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB. PMID:25431953

  1. Bionano interactions of mcf-7 breast tumor cells with a transferrin receptor targeted nanoparticle.

    PubMed

    Du, Wenwen; Fan, Yuchen; He, Bing; Zheng, Nan; Yuan, Lan; Dai, Wenbing; Zhang, Hua; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Zhang, Qiang

    2015-05-01

    Although transferrin receptor (TfR) is widely accepted as a target for cancer therapy, few studies have elaborated on delivery efficiency of TfR upon interactions with TfR-targeted nanomedicine. Here, a micellar system employing TfR-specific 7peptide (histidine-alanine-isoleucine-tyrosine- proline-arginine-histidine, HAIYPRH, 7pep) as the targeting moiety was constructed; and its endocytosis, intracellular trafficking as well as influence on TfR expression and in vivo tumor targeting were explored in the MCF-7 tumor model. In contrast to unmodified micelles, 7pep modification enhanced the cellular uptake of micelles without altering endocytic pathways, and slowed down the trafficking of micelles to lysosomes without changing the final intracellular colocalization. Interestingly, cellular TfR level was increased by 7pep-modified micelles. Furthermore, receptor saturation and recovery was observed in vivo. In conclusion, this study comprehensively investigated the bionano interaction between TfR positive tumors and 7pep-modified micelles, and provided scientific information for cancer therapy with receptor-mediated nanomedicines. PMID:25811613

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    1990-11-01

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

  4. Immune response to dna vaccine expressing transferrin binding protein a gene of Pasteurella multocida.

    PubMed

    Singh, Satparkash; Singh, Vijendra Pal; Cheema, Pawanjit Singh; Sandey, Maninder; Ranjan, Rajeev; Gupta, Santosh Kumar; Sharma, Bhaskar

    2011-04-01

    Haemorrhagic Septicaemia (HS), an acute and fatal disease of cattle and buffalo is primarily caused by serotype B:2 or E:2 of Pasteurella multocida. The transferrin binding protein A (TbpA) has been found to act as immunogen and potent vaccine candidate in various Gram negative bacteria including P. multocida. The present study was carried out to evaluate the potential of this antigen as a DNA vaccine against HS in mice model. The tbpA gene of P. multocida serotype B:2 was cloned in a mammalian expression vector alone and along with murine IL2 gene as immunological adjuvant to produce monocistronic and bicistronic DNA vaccine constructs, respectively. The immune response to DNA vaccines was evaluated based on serum antibody titres and lymphocyte proliferation assay. A significant increase in humoral and cell mediated immune responses was observed in mice vaccinated with DNA vaccines as compared to non immunized group. Additionally, the bicistronic DNA vaccine provided superior immune response and protection level following challenge as compared to monocistronic construct. The study revealed that DNA vaccine presents a promising approach for the prevention of HS. PMID:24031690

  5. Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

    PubMed Central

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Kim, Hyo Jeong; Ko, Myoung Seok; Seol, Wongi; Joe, Yeonsoo; Chung, Hun Taeg; Lee, Byung Ju; Moon, Chang Hoon; Cho, Wha Ja; Park, Jeong Woo

    2016-01-01

    The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling. PMID:26582392

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

    PubMed

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

    2015-02-12

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

  7. A transferrin gene associated with development and 2-tridecanone tolerance in Helicoverpa armigera.

    PubMed

    Zhang, L; Shang, Q; Lu, Y; Zhao, Q; Gao, X

    2015-04-01

    The full-length cDNA (2320 bp) encoding a putative iron-binding transferrin protein from Helicoverpa armigera was cloned and named HaTrf. The putative HaTrf sequence included 670 amino acids with a molecular mass of approximately 76 kDa. Quantitative PCR results demonstrated that the transcriptional level of HaTrf was significantly higher in the sixth instar and pupa stages as compared with other developmental stages. HaTrf transcripts were more abundant in fat bodies and in the epidermis than in malpighian tubules. Compared with the control, the expression of HaTrf increased dramatically 24 h after treatment with 2-tridecanone. Apparent growth inhibition with a dramatic body weight decrease was observed in larvae fed with HaTrf double-stranded RNA (dsRNA), as compared with those fed with green fluorescent protein dsRNA. RNA interference of HaTrf also significantly increased the susceptibility of larvae to 2-tridecanone. These results indicate the possible involvement of HaTrf in tolerance to plant secondary chemicals. PMID:25430818

  8. Plasmid pORF-hTRAIL targeting to glioma using transferrin-modified polyamidoamine dendrimer.

    PubMed

    Gao, Song; Li, Jianfeng; Jiang, Chen; Hong, Bo; Hao, Bing

    2016-01-01

    A gene drug delivery system for glioma therapy based on transferrin (Tf)-modified polyamidoamine dendrimer (PAMAM) was prepared. Gene drug, tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL)-encoding plasmid open reading frame (pORF-hTRAIL, Trail), was condensed by Tf-modified PAMAM to form nanoparticles (NPs). PAMAM-PEG-Tf/DNA NPs showed higher cellular uptake, in vitro gene expression, and cytotoxicity than PAMAM-PEG/DNA NPs in C6 cells. The in vivo targeting efficacy of NPs was visualized by ex vivo fluorescence imaging. Tf-modified NPs showed obvious glioma-targeting trend. Plasmid encoding green fluorescence protein (GFP) was also condensed by modified or unmodified PAMAM to evaluate the in vivo gene expression level. The PAMAM-PEG-Tf/plasmid encoding enhanced green fluorescence protein (pEGFP) NPs exhibited higher GFP expression level than PAMAM-PEG/pEGFP NPs. TUNEL assay revealed that Tf-modified NPs could induce much more tumor apoptosis. The median survival time of PAMAM-PEG-Tf/Trail-treated rats (28.5 days) was longer than that of rats treated with PAMAM-PEG/Trail (25.5 days), temozolomide (24.5 days), PAMAM-PEG-Tf/pEGFP (19 days), or saline (17 days). The therapeutic effect was further confirmed by magnetic resonance imaging. This study demonstrated that targeting gene delivery system had potential application for the treatment of glioma. PMID:26719669

  9. Targeting iron-mediated retinal degeneration by local delivery of transferrin.

    PubMed

    Picard, Emilie; Le Rouzic, Quentin; Oudar, Antonin; Berdugo, Marianne; El Sanharawi, Mohamed; Andrieu-Soler, Charlotte; Naud, Marie-Christine; Jonet, Laurent; Latour, Chloé; Klein, Christophe; Galiacy, Stéphane; Malecaze, François; Coppin, Hélène; Roth, Marie-Paule; Jeanny, Jean-Claude; Courtois, Yves; Behar-Cohen, Francine

    2015-12-01

    Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress. PMID:26454080

  10. Polymorphisms of Transferrin gene are associated with schizophrenia in Chinese Han population.

    PubMed

    Qu, Mei; Yue, Weihua; Tang, Fulei; Wang, Lifang; Han, Yonghua; Zhang, Dai

    2008-09-01

    Several recent studies have provided evidence that abnormalities in oligodendrocyte and myelin function may contribute to the etiopathology of schizophrenia. Transferrin (TF), an iron transport glycoprotein playing an important role in synthesis of myelin and the development of oligodendrocytes, has been identified as down-regulated expression in schizophrenia brain by microarray, quantitative PCR and in situ hybridization method. In order to further assess the role of TF in schizophrenia, we examined seven polymorphisms in TF region using a set sample of Chinese Han subjects consisting of 326 schizophrenia patients and 344 healthy controls. Four single nucleotide polymorphisms (SNPs) namely, rs4481157, rs3811655, rs6762415 and rs1405022 were analyzed in this study. Our results showed that one intronic SNP had strong association with schizophrenia (rs3811655: allele C>G, P=1.34E-6, OR=1.89, 95% CI=1.46-2.46; genotype P=3.72E-6). Two haplotypes A-C and G-G constructed of rs4481157-rs3811655 also revealed significant associations with schizophrenia (global P=0.0001). Our findings support that TF gene may be involved in susceptibility to schizophrenia in the Chinese Han population. However, further studies are needed to confirm these findings in other populations and to identify functional variants in TF that may be implicated in pathogenesis. PMID:18045615

  11. A combination of serum iron, ferritin and transferrin predicts outcome in patients with intracerebral hemorrhage

    PubMed Central

    Yang, Guang; Hu, Rong; Zhang, Chao; Qian, Christopher; Luo, Qian-Qian; Yung, Wing-Ho; Ke, Ya; Feng, Hua; Qian, Zhong-Ming

    2016-01-01

    Association of a high-serum ferritin with poor outcome showed that iron might play a detrimental role in the brain after intracerebral hemorrhage (ICH). Here, we investigated changes in serum iron, ferritin, transferrin (Tf) and ceruloplasmin (CP) in patients with ICH (n = 100) at day 1 (admission), 3, 7, 14 and 21 and those in control subjects (n = 75). The hematoma and edema volumes were also determined in ICH-patients on admission and at day 3. The Modified Rankin Scale (mRS) of 59 patients was ≥3 (poor outcome) and 41 < 3 (good outcome) at day 90. Serum ferritin was significantly higher and serum iron and Tf markedly lower in patients with poor-outcome than the corresponding values in patients with good-outcome at day 1 to 7 and those in the controls. There was a significant positive correlation between serum ferritin and relative edema volume or ratio at day 1 and 3 and hematoma volume at day 1 (n = 28), and a negative correlation between serum iron or Tf and hematoma volume at day 1 (n = 100). We concluded that not only increased serum ferritin but also reduced serum iron and Tf are associated with outcome as well as hematoma volume. PMID:26898550

  12. Binding studies of hydroxylated Multi-Walled Carbon Nanotubes to hemoglobin, gamma globulin and transferrin.

    PubMed

    Sekar, Gajalakshmi; Kandiyil, Shirona Thazae; Sivakumar, Amaravathy; Mukherjee, Amitava; Chandrasekaran, Natarajan

    2015-12-01

    Biocompatibility of nanoparticles depends on their binding behavior with biomolecules. Herein, we have reported the interaction of three different biological macromolecules such as hemoglobin, gamma globulin and transferrin with hydroxyl group functionalized Multi-Walled Carbon Nanotubes (OH-MWCNTs). Multiple spectroscopic methods were utilized to identify the binding cum structural changes in biomolecules upon their interaction. Hyperchromic effect observed in the UV-visible spectra, and the quenching behavior from fluorescence emission evidences the existence of bio-nanotube complex formation. Synchronous and three-dimensional fluorescence spectra of biomolecules, in correspondence with Trp and Tyr residues showed the possible disturbance towards their aromatic micro-environment. Changes observed in the FTIR and FT-Raman amide bands, and amino acid residue position of biomolecules upon interaction with CNTs showed the possible effect towards their secondary structure. Further studies with CD spectroscopy indicated the loss of alpha-helical structures quantitatively. The study remains significant in evaluating the biosafety profile of functionalized MWCNTs for their in vivo biomedical applications. PMID:26432959

  13. Methamphetamine and HIV-1-induced neurotoxicity: Role of trace amine associated receptor 1 cAMP signaling in astrocytes

    PubMed Central

    Cisneros, Irma E.

    2014-01-01

    Methamphetamine (METH) is abused by about 5% of the United States population with approximately 10–15% of human immunodeficiency virus-1 (HIV-1) patients reporting its use. METH abuse accelerates the onset and severity of HIV-associated neurocognitive disorders (HAND) and astrocyte-induced neurotoxicity. METH activates G-protein coupled receptors such as trace amine associated receptor 1 (TAAR1) increasing intracellular cyclic adenosine monophosphate (cAMP) levels in presynaptic cells of monoaminergic systems. In the present study, we investigated the effects of METH and HIV-1 on primary human astrocyte TAAR1 expression, function and glutamate clearance. Our results demonstrate combined conditions increased TAAR1 mRNA levels 7-fold and increased intracellular cAMP levels. METH and beta-phenylethylamine (β-PEA), known TAAR1 agonists, increased intracellular cAMP levels in astrocytes. Further, TAAR1 knockdown significantly reduced intracellular cAMP levels in response to METH/β-PEA, indicating signaling through astrocyte TAAR1. METH +/− HIV-1 decreased excitatory amino acid transporter-2 (EAAT-2) mRNA and significantly decreased glutamate clearance. RNA interference for TAAR1 prevented METH-mediated decreases in EAAT-2. TAAR1 knockdown significantly increased glutamate clearance, which was further heightened significantly by METH. Moreover, TAAR1 overexpression significantly decreased EAAT-2 levels and glutamate clearance that were further reduced by METH. Taken together, our data show that METH treatment activated TAAR1 leading to intracellular cAMP in human astrocytes and modulated glutamate clearance abilities. Furthermore, molecular alterations in astrocyte TAAR1 levels correspond to changes in astrocyte EAAT-2 levels and function. To our knowledge this is the first report implicating astrocyte TAAR1 as a novel receptor for METH during combined injury in the context of HAND. PMID:24950453

  14. New targets for renal interstitial fibrosis: relaxin family peptide receptor 1-angiotensin type 2 receptor heterodimers.

    PubMed

    Sasser, Jennifer M

    2014-07-01

    The signal transduction mechanisms involved in the renoprotective effects of relaxin are not well understood. Chow et al. demonstrate that relaxin family peptide receptor 1 (RXFP1) forms heterodimer complexes with the angiotensin type 2 receptor (AT2), even in the absence of ligand, and that these heterodimers are required for relaxin's antifibrotic effects. These findings identify a previously unknown link between relaxin and angiotensin II signaling that could be a potential new target for slowing the progression of fibrotic renal diseases. PMID:24978374

  15. Cannabinoid receptor 1 signaling in cardiovascular regulating nuclei in the brainstem: A review

    PubMed Central

    Ibrahim, Badr M.; Abdel-Rahman, Abdel A.

    2013-01-01

    Cannabinoids elicit complex hemodynamic responses in experimental animals that involve both peripheral and central sites. Centrally administered cannabinoids have been shown to predominantly cause pressor response. However, very little is known about the mechanism of the cannabinoid receptor 1 (CB1R)-centrally evoked pressor response. In this review, we provided an overview of the contemporary knowledge regarding the cannabinoids centrally elicited cardiovascular responses and the possible underlying signaling mechanisms. The current review focuses on the rostral ventrolateral medulla (RVLM) as the primary brainstem nucleus implicated in CB1R-evoked pressor response. PMID:25685481

  16. Trace Amine-Associated Receptor 1 (TAAR1) is Activated by Amiodarone Metabolites

    PubMed Central

    Snead, Aaron N.; Miyakawa, Motonori; Tan, Edwin S.; Scanlan, Thomas S.

    2012-01-01

    Amiodarone (Cordarone, Wyeth-Ayerst Pharmaceuticals) is a clinically available drug used to treat a wide variety of cardiac arrhythmias. We report here the synthesis and characterization of a panel of potential amiodarone metabolites that have significant structural similarity to thyroid hormone and its metabolites the iodothyronamines. Several of these amiodarone derivatives act as specific agonists of the G protein-coupled receptor (GPCR) trace amine-associated receptor 1 (TAAR1). This result demonstrates a novel molecular target for amiodarone derivatives with potential clinical significance. PMID:18752950

  17. The potential impacts of formyl peptide receptor 1 in inflammatory diseases.

    PubMed

    Yang, Shun-Chin; Hwang, Tsong-Long

    2016-01-01

    Neutrophils play a critical role in acute and chronic inflammatory diseases. N-formyl peptides, which originate from bacterial peptides or mitochondrial proteins bind with a high binding affinity to formyl peptide receptor 1 (FPR1). N-formyl peptide-FPR1 is involved in the pathogenesis of sterile and infectious inflammatory processes and causes phagocytosis of pathogens or injured cells by neutrophils. Excessive activation of neutrophils by binding of N-formyl peptides is associated with tissue injury requiring drugs that block FPR1-dependent signaling. Here, we review the roles of FPR1 as a critical regulator of inflammatory processes and its involvement in pathological conditions. PMID:27100350

  18. Targeted delivery of siRNA to activated T cells via transferrin-polyethylenimine (Tf-PEI) as a potential therapy of asthma.

    PubMed

    Xie, Yuran; Kim, Na Hyung; Nadithe, Venkatareddy; Schalk, Dana; Thakur, Archana; Kılıç, Ayşe; Lum, Lawrence G; Bassett, David J P; Merkel, Olivia M

    2016-05-10

    Asthma is a worldwide health problem. Activated T cells (ATCs) in the lung, particularly T helper 2 cells (Th2), are strongly associated with inducing airway inflammatory responses and chemoattraction of inflammatory cells in asthma. Small interfering RNA (siRNA) as a promising anti-sense molecule can specifically silence inflammation related genes in ATCs, however, lack of safe and efficient siRNA delivery systems limits the application of siRNA as a therapeutic molecule in asthma. Here, we designed a novel pulmonary delivery system of siRNA, transferrin-polyethylenimine (Tf-PEI), to selectively deliver siRNA to ATCs in the lung. Tf-PEI polyplexes demonstrated optimal physicochemical properties such as size, distribution, zeta-potential, and siRNA condensation efficiency. Moreover, in vitro studies showed significantly enhanced cellular uptake and gene knockdown mediated by Tf-PEI polyplexes in human primary ATCs. Biodistribution of polyplexes in a murine asthmatic model confirmed that Tf-PEI polyplexes can efficiently and selectively deliver siRNA to ATCs. In conclusion, the present work proves the feasibility to target ATCs in asthma via Tf receptor. This strategy could potentially be used to design an efficient siRNA delivery system for asthma therapy. PMID:27001893

  19. Monoclonal Antibody Targeting of Fibroblast Growth Factor Receptor 1c Ameliorates Obesity and Glucose Intolerance via Central Mechanisms

    PubMed Central

    Lelliott, Christopher J.; Ahnmark, Andrea; Admyre, Therese; Ahlstedt, Ingela; Irving, Lorraine; Keyes, Feenagh; Patterson, Laurel; Mumphrey, Michael B.; Bjursell, Mikael; Gorman, Tracy; Bohlooly-Y, Mohammad; Buchanan, Andrew; Harrison, Paula; Vaughan, Tristan; Berthoud, Hans-Rudolf; Lindén, Daniel

    2014-01-01

    We have generated a novel monoclonal antibody targeting human FGFR1c (R1c mAb) that caused profound body weight and body fat loss in diet-induced obese mice due to decreased food intake (with energy expenditure unaltered), in turn improving glucose control. R1c mAb also caused weight loss in leptin-deficient ob/ob mice, leptin receptor-mutant db/db mice, and in mice lacking either the melanocortin 4 receptor or the melanin-concentrating hormone receptor 1. In addition, R1c mAb did not change hypothalamic mRNA expression levels of Agrp, Cart, Pomc, Npy, Crh, Mch, or Orexin, suggesting that R1c mAb could cause food intake inhibition and body weight loss via other mechanisms in the brain. Interestingly, peripherally administered R1c mAb accumulated in the median eminence, adjacent arcuate nucleus and in the circumventricular organs where it activated the early response gene c-Fos. As a plausible mechanism and coinciding with the initiation of food intake suppression, R1c mAb induced hypothalamic expression levels of the cytokines Monocyte chemoattractant protein 1 and 3 and ERK1/2 and p70 S6 kinase 1 activation. PMID:25427253

  20. The Role of Formylated Peptides and Formyl Peptide Receptor 1 in Governing Neutrophil Function during Acute Inflammation

    PubMed Central

    Dorward, David A.; Lucas, Christopher D.; Chapman, Gavin B.; Haslett, Christopher; Dhaliwal, Kevin; Rossi, Adriano G.

    2015-01-01

    Neutrophil migration to sites of inflammation and the subsequent execution of multiple functions are designed to contain and kill invading pathogens. These highly regulated and orchestrated processes are controlled by interactions between numerous receptors and their cognate ligands. Unraveling and identifying those that are central to inflammatory processes may represent novel therapeutic targets for the treatment of neutrophil-dominant inflammatory disorders in which dysregulated neutrophil recruitment, function, and elimination serve to potentiate rather than resolve an initial inflammatory insult. The first G protein–coupled receptor to be described on human neutrophils, formyl peptide receptor 1 (FPR1), is one such receptor that plays a significant role in the execution of these functions through multiple intracellular signaling pathways. Recent work has highlighted important observations with regard to both receptor function and the importance and functional relevance of FPR1 in the pathogenesis of a range of both sterile and infective inflammatory conditions. In this review, we explore the multiple components of neutrophil migration and function in both health and disease, with a focus on the role of FPR1 in these processes. The current understanding of FPR1 structure, function, and signaling is examined, alongside discussion of the potential importance of FPR1 in inflammatory diseases suggesting that FPR1 is a key regulator of the inflammatory environment. PMID:25791526

  1. Functional Genetic Variation of the Cannabinoid Receptor 1 and Cannabis Use Interact on Prefrontal Connectivity and Related Working Memory Behavior

    PubMed Central

    Colizzi, Marco; Fazio, Leonardo; Ferranti, Laura; Porcelli, Annamaria; Masellis, Rita; Marvulli, Daniela; Bonvino, Aurora; Ursini, Gianluca; Blasi, Giuseppe; Bertolino, Alessandro

    2015-01-01

    Cannabinoid signaling is involved in different brain functions and it is mediated by the cannabinoid receptor 1 (CNR1), which is encoded by the CNR1 gene. Previous evidence suggests an association between cognition and cannabis use. The logical interaction between genetically determined cannabinoid signaling and cannabis use has not been determined. Therefore, we investigated whether CNR1 variation predicts CNR1 prefrontal mRNA expression in postmortem prefrontal human tissue. Then, we studied whether functional variation in CNR1 and cannabis exposure interact in modulating prefrontal function and related behavior during working memory processing. Thus, 208 healthy subjects (113 males) were genotyped for the relevant functional SNP and were evaluated for cannabis use by the Cannabis Experience Questionnaire. All individuals performed the 2-back working memory task during functional magnetic resonance imaging. CNR1 rs1406977 was associated with prefrontal mRNA and individuals carrying a G allele had reduced CNR1 prefrontal mRNA levels compared with AA subjects. Moreover, functional connectivity MRI demonstrated that G carriers who were also cannabis users had greater functional connectivity in the left ventrolateral prefrontal cortex and reduced working memory behavioral accuracy during the 2-back task compared with the other groups. Overall, our results indicate that the deleterious effects of cannabis use are more evident on a specific genetic background related to its receptor expression. PMID:25139064

  2. Association of Neurotensin receptor 1 gene polymorphisms with processing speed in healthy Chinese-Han subjects.

    PubMed

    Wang, Man; Ma, Hui; Huang, Ying-lin; Zhu, Gang; Zhao, Jing-ping

    2014-12-01

    Neurotensin modulates dopamine and serotonin transmission in the brain. The study investigated whether genetic polymorphisms in the Neurotensin receptor 1 gene were associated with performance on processing speed and executive function. A total of 129 healthy Chinese-Han volunteers were recruited. Genotyping for three SNPs, including rs6090453, rs6011914, and rs2427422, was analyzed by using a PCR and a restriction fragment length polymorphism analysis. Performances of processing speed and executive function were assessed by using Trail Making Test-A (TMT-A), Wisconsin Card Sorting Test, and Stroop Color-Word Test. We found significant differences in the outcomes of TMT-A score among rs6090453C/G (F(2,126)=4.405, P=0.014) and rs2427422A/G (F(2,126)=7.498, P=0.001) genotypes. Neurotensin receptor 1 SNP polymorphisms were significantly associated with the variance in processing speed performance in a sample of Chinese college students. PMID:25159184

  3. Hyperalgesia and increased neuropathic pain-like response in mice lacking galanin receptor 1 receptors.

    PubMed

    Blakeman, K H; Hao, J-X; Xu, X-J; Jacoby, A S; Shine, J; Crawley, J N; Iismaa, T; Wiesenfeld-Hallin, Z

    2003-01-01

    The neuropeptide galanin may have a role in modulation of nociception, particularly after peripheral nerve injury. The effect of galanin is mediated by at least three subtypes of receptors. In the present study, we assessed the nociceptive sensitivity in mice lacking the galanin receptor 1 gene (Galr1) and the development of neuropathic pain-like behaviours after photochemically induced partial sciatic nerve ischaemic injury. Under basal condition, Galr1 knock-out (Galr1(-/-)) mice had shortened response latency on the hot plate, but not tail flick and paw radiant heat, tests. The mechanical sensitivity was not different between Galr1(-/-) and wild type (Galr1(+/+)) mice, whereas the cold response was moderately enhanced in Galr1(-/-) mice. Both Galr1(-/-) mice and Galr1(+/+) controls developed mechanical and heat hypersensitivity after partial sciatic nerve injury. The duration of such pain-like behaviours was significantly increased in Galr1(-/-). The Galr1(-/-) mice and Galr1(+/+) mice did not differ in their recovery from deficits in toe-spread after sciatic nerve crush. The results provide some evidence for an inhibitory function for the neuropeptide galanin acting on galanin receptor 1 (GALR1) in nociception and neuropathic pain after peripheral nerve injury in mice. PMID:12605908

  4. Central Administration of Galanin Receptor 1 Agonist Boosted Insulin Sensitivity in Adipose Cells of Diabetic Rats

    PubMed Central

    Zhang, Zhenwen; Fang, Penghua; He, Biao; Guo, Lili; Runesson, Johan; Langel, Ülo; Shi, Mingyi; Zhu, Yan; Bo, Ping

    2016-01-01

    Our previous studies testified the beneficial effect of central galanin on insulin sensitivity of type 2 diabetic rats. The aim of the study was further to investigate whether central M617, a galanin receptor 1 agonist, can benefit insulin sensitivity. The effects of intracerebroventricular administration of M617 on insulin sensitivity and insulin signaling were evaluated in adipose tissues of type 2 diabetic rats. The results showed that central injection of M617 significantly increased plasma adiponectin contents, glucose infusion rates in hyperinsulinemic-euglycemic clamp tests, GLUT4 mRNA expression levels, GLUT4 contents in plasma membranes, and total cell membranes of the adipose cells but reduced the plasma C-reactive protein concentration in nondiabetic and diabetic rats. The ratios of GLUT4 contents were higher in plasma membranes to total cell membranes in both nondiabetic and diabetic M617 groups than each control. In addition, the central administration of M617 enhanced the ratios of pAkt/Akt and pAS160/AS160, but not phosphorylative cAMP response element-binding protein (pCREB)/CREB in the adipose cells of nondiabetic and diabetic rats. These results suggest that excitation of central galanin receptor 1 facilitates insulin sensitivity via activation of the Akt/AS160 signaling pathway in the fat cells of type 2 diabetic rats. PMID:27127795

  5. Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages.

    PubMed

    Hossain, Ekhtear; Ota, Akinobu; Karnan, Sivasundaram; Takahashi, Miyuki; Mannan, Shahnewaj B; Konishi, Hiroyuki; Hosokawa, Yoshitaka

    2015-02-01

    There is a growing body of evidence supporting an intimate association of immune activation with the pathogenesis of cardiovascular diseases, including atherosclerosis. Uptake of oxidized low-density lipoprotein (oxLDL) through scavenging receptors promotes the formation of mature lipid-laden macrophages, which subsequently leads to exacerbation of regional inflammation and atherosclerotic plaque formation. In this study, we first examined changes in the mRNA level of the lectin-like oxLDL receptor-1 (LOX-1) in the mouse macrophage cell line RAW264.7 and the human PMA-induced macrophage cell line THP-1 after LPS stimulation. LPS significantly up-regulated LOX-1 mRNA in RAW264.7 cells; LOX-1 cell-surface protein expression was also increased. Flow cytometry and fluorescence microscopy analyses showed that cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with LPS stimulation. The augmented uptake of Dil-oxLDL was almost completely abrogated by treatment with an anti-LOX-1 antibody. Of note, knockdown of Erk1/2 resulted in a significant reduction of LPS-induced LOX-1 up-regulation. Treatment with U0126, a specific inhibitor of MEK, significantly suppressed LPS-induced expression of LOX-1 at both the mRNA and protein levels. Furthermore, LOX-1 promoter activity was significantly augmented by LPS stimulation; this augmentation was prevented by U0126 treatment. Similar results were also observed in human PMA-induced THP-1 macrophages. Taken together, our results indicate that LPS up-regulates LOX-1, at least in part through activation of the Erk1/2 signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of TLR4-mediated aberrant LOX-1 signaling in the pathogenesis of atherosclerosis. PMID:25348362

  6. Antidepressant/anxiolytic potential and adverse effect liabilities of melanin-concentrating hormone receptor 1 antagonists in animal models.

    PubMed

    Chaki, Shigeyuki; Shimazaki, Toshiharu; Nishiguchi, Mariko; Funakoshi, Takeo; Iijima, Michihiko; Ito, Akie; Kanuma, Kosuke; Sekiguchi, Yoshinori

    2015-08-01

    Melanin-concentrating hormone receptor 1 (MCH1 receptor) is known to be involved in the control of mood and stress, in addition to the regulation of feeding. Here, we report further evidence that the blockade of the MCH1 receptor exhibits antidepressant and anxiolytic-like effects in a variety of animal models using TASP0382650 and TASP0489838, newly synthesized MCH1 receptor antagonists, with different scaffolds. Both TASP0382650 and TASP0489838 exhibited high affinities for human MCH1 receptor with IC50 values of 7.13 and 3.80nM, respectively. Both compounds showed potent antagonist activities at the MCH1 receptor, as assessed using MCH-increased [(35)S]GTPγS binding to human MCH1 receptor and an MCH-induced [Ca(2+)]i assay in rat MCH1 receptor expressing cells. In contrast, neither TASP0382650 nor TASP0489838 showed an affinity for the MCH2 receptor, another MCH receptor subtype. The oral administration of TASP0382650 or TASP0489838 significantly reduced the immobility time during the forced swimming test in rats, and reduced hyperemotionality induced by an olfactory bulbectomy, both of which are indicative of an antidepressant-like potential. In the olfactory bulbectomy model, the antidepressant effect of TASP0382650 appeared following a single administration, suggesting a faster onset of action, compared with current medications. Moreover, both TASP0382650 and TASP0489838 exhibited anxiolytic effects in several animal models of anxiety. In contrast, both TASP0382650 and TASP0489838 did not affect spontaneous locomotor activity, motor function, spatial memory during the Morris water maze task, or the convulsion threshold to pentylenetetrazole. These findings provide additional evidence that the blockade of the MCH1 receptor exhibits antidepressant- and anxiolytic activities with no adverse effects in experimental animal models. PMID:26044968

  7. Impacts of Nonsynonymous Single Nucleotide Polymorphisms of Adiponectin Receptor 1 Gene on Corresponding Protein Stability: A Computational Approach

    PubMed Central

    Saleh, Md. Abu; Solayman, Md.; Paul, Sudip; Saha, Moumoni; Khalil, Md. Ibrahim; Gan, Siew Hua

    2016-01-01

    Despite the reported association of adiponectin receptor 1 (ADIPOR1) gene mutations with vulnerability to several human metabolic diseases, there is lack of computational analysis on the functional and structural impacts of single nucleotide polymorphisms (SNPs) of the human ADIPOR1 at protein level. Therefore, sequence- and structure-based computational tools were employed in this study to functionally and structurally characterize the coding nsSNPs of ADIPOR1 gene listed in the dbSNP database. Our in silico analysis by SIFT, nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, and SNPeffect tools identified the nsSNPs with distorting functional impacts, namely, rs765425383 (A348G), rs752071352 (H341Y), rs759555652 (R324L), rs200326086 (L224F), and rs766267373 (L143P) from 74 nsSNPs of ADIPOR1 gene. Finally the aforementioned five deleterious nsSNPs were introduced using Swiss-PDB Viewer package within the X-ray crystal structure of ADIPOR1 protein, and changes in free energy for these mutations were computed. Although increased free energy was observed for all the mutants, the nsSNP H341Y caused the highest energy increase amongst all. RMSD and TM scores predicted that mutants were structurally similar to wild type protein. Our analyses suggested that the aforementioned variants especially H341Y could directly or indirectly destabilize the amino acid interactions and hydrogen bonding networks of ADIPOR1. PMID:27294143

  8. Genetic Inhibition of Fibroblast Growth Factor Receptor 1 in Knee Cartilage Attenuates the Degeneration of Articular Cartilage in Adult Mice

    PubMed Central

    Weng, Tujun; Yi, Lingxian; Huang, Junlan; Luo, Fengtao; Wen, Xuan; Du, Xiaolan; Chen, Qian; Deng, Chuxia; Chen, Di; Chen, Lin

    2013-01-01

    Objective Fibroblast growth factor (FGF) family members are involved in the regulation of articular cartilage homeostasis. The aim of this study was to investigate the function of FGF receptor 1 (FGFR-1) in the development of osteoarthritis (OA) and its underlying mechanisms. Methods FGFR-1 was deleted from the articular chondrocytes of adult mice in a cartilage-specific and tamoxifen-inducible manner. Two OA models (aging-associated spontaneous OA, and destabilization-induced OA), as well as an antigen-induced arthritis (AIA) model, were established and tested in Fgfr1-deficient and wild-type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR-1 inhibitor on human articular chondrocytes was examined. Results The gross morphologic features of Fgfr1-deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12-month-old Fgfr1-deficient mice displayed greater aggrecan staining compared to 12-month-old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR-1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP-13) and up-regulation of FGFR-3 in mouse and human articular chondrocytes. Conclusion Disruption of FGFR-1 in adult mouse articular chondrocytes inhibits the progression of cartilage degeneration. Down-regulation of MMP-13 expression and up-regulation of FGFR-3 levels may contribute to the phenotypic changes observed in Fgfr1-deficient mice. PMID:22833219

  9. Impacts of Nonsynonymous Single Nucleotide Polymorphisms of Adiponectin Receptor 1 Gene on Corresponding Protein Stability: A Computational Approach.

    PubMed

    Saleh, Md Abu; Solayman, Md; Paul, Sudip; Saha, Moumoni; Khalil, Md Ibrahim; Gan, Siew Hua

    2016-01-01

    Despite the reported association of adiponectin receptor 1 (ADIPOR1) gene mutations with vulnerability to several human metabolic diseases, there is lack of computational analysis on the functional and structural impacts of single nucleotide polymorphisms (SNPs) of the human ADIPOR1 at protein level. Therefore, sequence- and structure-based computational tools were employed in this study to functionally and structurally characterize the coding nsSNPs of ADIPOR1 gene listed in the dbSNP database. Our in silico analysis by SIFT, nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, and SNPeffect tools identified the nsSNPs with distorting functional impacts, namely, rs765425383 (A348G), rs752071352 (H341Y), rs759555652 (R324L), rs200326086 (L224F), and rs766267373 (L143P) from 74 nsSNPs of ADIPOR1 gene. Finally the aforementioned five deleterious nsSNPs were introduced using Swiss-PDB Viewer package within the X-ray crystal structure of ADIPOR1 protein, and changes in free energy for these mutations were computed. Although increased free energy was observed for all the mutants, the nsSNP H341Y caused the highest energy increase amongst all. RMSD and TM scores predicted that mutants were structurally similar to wild type protein. Our analyses suggested that the aforementioned variants especially H341Y could directly or indirectly destabilize the amino acid interactions and hydrogen bonding networks of ADIPOR1. PMID:27294143

  10. Evaluation of serum transferrin receptor for iron deficiency in women of child-bearing age.

    PubMed

    Lin, Xiao-Ming; Zhang, Juan; Zou, Zhi-Yong; Long, Zhu; Tian, Wei

    2008-11-01

    The objective was to study the evaluation of serum transferrin receptor (sTfR) for Fe deficiency in women of child-bearing age. Primary screening was performed in 942 women ranging in child-bearing age. Serum ferritin (SF), Zn protoporphyrin (ZPP) and Hb were determined. Then the subjects were divided into four groups: normal, Fe store depletion (IDs), Fe-deficiency erythropoiesis and Fe-deficiency anaemia. sTfR was determined and sTfR/SF (sTfR/logSF and log(sTfR/SF)) was calculated. Changes of sTfR in women of different Fe status were observed. A receiver-operating characteristic (ROC) curve was used to evaluate whether sTfR had proper diagnostic efficacy for functional Fe deficiency. The levels of sTfR increased significantly along with the aggravation of Fe deficiency. Increase of STfR/SF along with the aggravation of Fe deficiency was more significant than that of sTfR. STfR had a significant negative correlation with SF and Hb, while it had a significant positive correlation with ZPP. The ROC curve showed that the diagnostic effective rate of sTfR for Fe deficiency could reach 83 %. At this point, the sensitivity was 79 % and the specificity was 63 %. Log(sTfR/SF) could be considered to have the highest effective ratio in detecting IDs, since it reached 99 %. STfR and sTfR/SF could both reflect body Fe-deficiency status specifically. They could be used as reliable indicators for evaluating Fe status and diagnosing Fe deficiency in women of child-bearing age. PMID:18377683

  11. The influence of the synergistic anion on iron chelation by ferric binding protein, a bacterial transferrin.

    PubMed

    Dhungana, Suraj; Taboy, Celine H; Anderson, Damon S; Vaughan, Kevin G; Aisen, Philip; Mietzner, Timothy A; Crumbliss, Alvin L

    2003-04-01

    Although the presence of an exogenous anion is a requirement for tight Fe(3+) binding by the bacterial (Neisseria) transferrin nFbp, the identity of the exogenous anion is not specific in vitro. nFbp was reconstituted as a stable iron containing protein by using a number of different exogenous anions [arsenate, citrate, nitrilotriacetate, pyrophosphate, and oxalate (symbolized by X)] in addition to phosphate, predominantly present in the recombinant form of the protein. Spectroscopic characterization of the Fe(3+)anion interaction in the reconstituted protein was accomplished by UV-visible and EPR spectroscopies. The affinity of the protein for Fe(3+) is anion dependent, as evidenced by the effective Fe(3+) binding constants (K'(eff)) observed, which range from 1 x 10(17) M(-1) to 4 x 10(18) M(-1) at pH 6.5 and 20 degrees C. The redox potentials for Fe(3+)nFbpXFe(2+)nFbpX reduction are also found to depend on the identity of the synergistic anion required for Fe(3+) sequestration. Facile exchange of exogenous anions (Fe(3+)nFbpX + X' --> Fe(3+)nFbpX' + X) is established and provides a pathway for environmental modulation of the iron chelation and redox characteristics of nFbp. The affinity of the iron loaded protein for exogenous anion binding at pH 6.5 was found to decrease in the order phosphate > arsenate approximately pyrophosphate > nitrilotriacetate > citrate approximately oxalate carbonate. Anion influence on the iron primary coordination sphere through iron binding and redox potential modulation may have in vivo application as a mechanism for periplasmic control of iron delivery to the cytosol. PMID:12646708

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

    PubMed

    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. PMID:27305829

  13. Transferrin Receptor-Targeted Lipid Nanoparticles for Delivery of an Antisense Oligodeoxyribonucleotide against Bcl-2

    PubMed Central

    Yang, Xiaojuan; Koh, Chee Guan; Liu, Shujun; Pan, Xiaogang; Santhanam, Ramasamy; Yu, Bo; Peng, Yong; Pang, Jiuxia; Golan, Sharon; Talmon, Yeshayahu; Jin, Yan; Muthusamy, Natarajan; Byrd, John C.; Chan, Kenneth K.; Lee, L. James; Marcucci, Guido; Lee, Robert J.

    2013-01-01

    Antisense oligonucleotide G3139-mediated down-regulation of Bcl-2 is a potential strategy for overcoming chemoresistance in leukemia. However, the limited efficacy shown in recent clinical trials calls attention to the need for further development of novel and more efficient delivery systems. In order to address this issue, transferrin receptor (TfR)-targeted, protamine-containing lipid nanoparticles (Tf-LNs) were synthesized as delivery vehicles for G3139. The LNs were produced by an ethanol dilution method and lipid-conjugated Tf ligand was then incorporated by a post-insertion method. The resulting Tf-LNs had a mean particle diameter of ~ 90 nm and G3139 loading efficiency of 90.4%. Antisense delivery efficiency of Tf-LNs was evaluated in K562, MV4-11 and Raji leukemia cell lines. The results showed that Tf-LNs were more effective than non-targeted LNs and free G3139 (p <0.05) in decreasing Bcl-2 expression (by up to 62% at the mRNA level in K562 cells) and in inducing caspase-dependent apoptosis. In addition, Bcl-2 down-regulation and apoptosis induced by Tf-LN G3139 were shown to be blocked by excess free Tf and thus were TfR-dependent. Cell lines with higher TfR expression also showed greater Bcl-2 down-regulation. Furthermore, upregulation of TfR expression in leukemia cells by iron chelator deferoxamine resulted in a further increase in antisense effect (up to 79% Bcl-2 reduction in K562 at the mRNA level) and in caspase-dependent apoptosis (by ~ 3-fold) by Tf-LN. Tf-LN mediated delivery combined with TfR up-regulation by deferoxamine appears to be a potentially promising strategy for enhancing the delivery efficiency and therapeutic efficacy of antisense oligonucleotides. PMID:19183107

  14. Transferrin-Targeted Nanoparticles Containing Zoledronic Acid as a Potential Tool to Inhibit Glioblastoma Growth.

    PubMed

    Salzano, G; Zappavigna, S; Luce, A; D'Onofrio, N; Balestrieri, M L; Grimaldi, A; Lusa, S; Ingrosso, D; Artuso, S; Porru, M; Leonetti, C; Caraglia, M; De Rosa, G

    2016-04-01

    The treatment of glioblastoma (GBM) is a challenge for the biomedical research since cures remain elusive. Its current therapy, consisted on surgery, radiotherapy, and concomitant chemotherapy with temozolomide (TMZ), is often uneffective. Here, we proposed the use of zoledronic acid (ZOL) as a potential agent for the treatment of GBM. Our group previously developed self-assembling nanoparticles, also named PLCaPZ NPs, to use ZOL in the treatment of prostate cancer. Here, we updated the previously developed nanoparticles (NPs) by designing transferrin (Tf)-targeted self-assembling NPs, also named Tf-PLCaPZ NPs, to use ZOL in the treatment of brain tumors, e.g., GBM. The efficacy of Tf-PLCaPZ NPs was evaluated in different GBM cell lines and in an animal model of GBM, in comparison with PLCaPZ NPs and free ZOL. Tf-PLCaPZ NPs were characterized by a narrow size distribution and a high incorporation efficiency of ZOL. Moreover, the presence of Tf significantly reduced the hemolytic activity of the formulation. In vitro, in LN229 cells, a significant uptake and cell growth inhibition after treatment with Tf-PLCaPZ NPs was achieved. Moreover, the sequential therapy of TMZ and Tf-PLCaPZ NPs lead to a superior therapeutic activity compared to their single administration. The results obtained in mice xenografted with U373MG, revealed a significant anticancer activity of Tf-PLCaPZ NPs, while the tumors remained unaffected with free TMZ. These promising results introduce a novel type of easy-to-obtain NPs for the delivery of ZOL in the treatment of GBM tumors. PMID:27301207

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Programmable cellular retention of nanoparticles by replacing the synergistic anion of transferrin.

    PubMed

    Wu, Li-Chen; Chu, Li-Wei; Lo, Leu-Wei; Liao, Yen-Chen; Wang, Yu-Chao; Yang, Chung-Shi

    2013-01-22

    The ability to program the intracellular retention of nanoparticles (NPs) would increase their applicability for imaging and therapeutic applications. To date, there has been no efficient method developed to control the fate of NPs once they enter cells. Existing approaches to manipulate the intracellular retention of NPs are mostly "passive" and particle size-dependent. Different sized particles hold distinct cellular responses. The adverse effect of particle size may limit the utility of nanodelivery systems. Therefore, the development of tunable/"active" NP intracellular retention systems with fixed particle sizes remains a considerable challenge. By replacing the synergistic anions of transferrin (Tf) immobilized on quantum dots (Tf-QDs, ca. 25 nm), we have examined the feasibility of this concept. Substitution of synergistic anions of Tf from carbonate (holo-Tf) to oxalate (oxa-Tf) significantly increased the intracellular accumulation of the oxa-Tf-QDs as a result of (i) a delay in cellular removal triggered by oxalate (oxa-Tf)-induced endosomal Tf iron-release retardation and (ii) enhanced recycling of Tf-QD/TfR (Tf receptor) complexes from early endosomes to the plasma membrane. This accumulation extended the intracellular NP retention interval. The half-maximum fluorescence intensity of the oxa-Tf-QDs in vivo was 4 times higher than that of the holo-Tf-QDs. Programming of the intracellular NP retention time was accomplished through manipulation of the ratio of holo- and oxa-Tfs on the surfaces of the QDs. Using this simple and efficient approach, it was possible to readily achieve a desirable intracellular retention interval for the NPs. PMID:23194060

  17. The Invasion and Reproductive Toxicity of QDs-Transferrin Bioconjugates on Preantral Follicle in vitro

    PubMed Central

    Xu, Gaixia; Lin, Suxia; Law, Wing-Cheung; Roy, Indrajit; Lin, Xiaotan; Mei, Shujiang; Ma, Hanwu; Chen, Siping; Niu, Hanben; Wang, Xiaomei

    2012-01-01

    The toxicity of QD has been extensively studied over the past decade. However, the potential toxicity of QDs impedes its use for clinical research. In this work, we established a preantral follicle in vitro culture system to investigate the effects of QD-Transferrin (QDs-Tf) bioconjugates on follicle development and oocyte maturation. The preantral follicles were cultured and exposed to CdTe/ZnTe QDs-Tf bioconjugates with various concentrations and the reproductive toxicity was assessed at different time points post-treatment. The invasion of QDs-Tf for oocytes was verified by laser scanning confocal microscope. Steroid production was evaluated by immunoassay. C-band Giemsa staining was performed to observe the chromosome abnormality of oocytes. The results showed that the QDs-Tf bioconjugates could permeate into granulosa cells and theca cells, but not into oocyte. There are no obvious changes of oocyte diameter, the mucification of cumulus-oocyte-complexes and the occurrence of aneulpoidy as compared with the control group. However, delay in the antrum formation and decrease in the ratio of oocytes with first polar body were observed in QDs-Tf-treated groups. The matured oocytes with first polar body decreased significantly by ~16% (from 79.6±10 % to 63±2.9 %) when the concentration of QDs-Tf bioconjugates exceeded 2.89 nmol·L-1 (P < 0.05). Our results implied that the CdTe/ZnTe QDs-Tf bioconjugates were reproductive toxic for follicle development, and thus also revealed that this in vitro culture system of preantral follicle is a highly sensitive tool for study on the reproductive toxicity of nanoparticles. PMID:22916073

  18. Miiuy croaker transferrin gene and evidence for positive selection events reveal different evolutionary patterns.

    PubMed

    Sun, Yueyan; Zhu, Zhihuang; Wang, Rixin; Sun, Yuena; Xu, Tianjun

    2012-01-01

    Transferrin (TF) is a protein that plays a central role in iron metabolism. This protein is associated with the innate immune system, which is responsible for disease defense responses after bacterial infection. The clear link between TF and the immune defense mechanism has led researchers to consider TF as a candidate gene for disease resistance. In this study, the Miichthys miiuy (miiuy croaker) TF gene (MIMI-TF) was cloned and characterized. The gene structure consisted of a coding region of 2070 nucleotides divided into 17 exons, as well as a non-coding region that included 16 introns and spans 6757 nucleotides. The deduced MIMI-TF protein consisted of 689 amino acids that comprised a signal peptide and two lobes (N- and C-lobes). MIMI-TF expression was significantly up-regulated after infection with Vibrio anguillarum. A series of model tests implemented in the CODEML program showed that TF underwent a complex evolutionary process. Branch-site models revealed that vertebrate TF was vastly different from that of invertebrates, and that the TF of the ancestors of aquatic and terrestrial organisms underwent different selection pressures. The site models detected 10 positively selected sites in extant TF genes. One site was located in the cleft between the N1 and N2 domains and was expected to affect the capability of TF to bind to or release iron indirectly. In addition, eight sites were found near the TF exterior. Two of these sites, which could have evolved from the competition for iron between pathogenic bacteria and TF, were located in potential pathogen-binding domains. Our results could be used to further investigate the function of TF and the selective mechanisms involved. PMID:22957037

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

    PubMed

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

    2015-05-01

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

  20. The human endogenous retrovirus K(HML-2) has a broad envelope-mediated cellular tropism and is prone to inhibition at a post-entry, pre-integration step.

    PubMed

    Kramer, Philipp; Lausch, Veronika; Volkwein, Alexander; Hanke, Kirsten; Hohn, Oliver; Bannert, Norbert

    2016-01-01

    The HERV-K(HML-2) family is the most recent addition to the collection of human endogenous retroviruses. It comprises proviruses that encode functional proteins that can assemble into replication defective particles carrying the envelope protein. Using a reconstituted HERV-K113 envelope sequence, we have analyzed its ability to mediate entry into a set of 33 cell lines from 10 species. Of these, 30 were permissive, demonstrating an amphotropism consistent with a broad expression of receptor protein(s). In an initial effort to identify a receptor for HERV-K(HML-2) we investigated whether transferrin receptor 1 and hyaluronidase 2, known cellular receptors of the closely related betaretroviruses mouse mammary tumor virus (MMTV) and Jaagsiekte sheep retrovirus (JSRV), could facilitate HERV-K(HML-2) entry. However, neither of these proteins could serve as a receptor for HERV-K(HML-2). Moreover, during attempts to further characterize the tropism of HERV-K(HML-2), we identified a cellular activity that inhibits infection at a post-entry, pre-integration step. PMID:26517399

  1. A Novel Isoquinoline Derivative Anticancer Agent and Its Targeted Delivery to Tumor Cells Using Transferrin-Conjugated Liposomes

    PubMed Central

    Yang, Xuewei; Yang, Shuang; Chai, Hongyu; Yang, Zhaogang; Lee, Robert J.; Liao, Weiwei; Teng, Lesheng

    2015-01-01

    We have screened 11 isoquinoline derivatives and α-methylene-γ-butyrolactones using the 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay in HeLa and HEK-293T cells. Compound 2 was identified as potential anticancer agent. To further improve its therapeutic potential, this agent was incorporated into transferrin (Tf)-conjugated liposomes (LPs) for targeted delivery to tumor cells. We have demonstrated Tf-LP-Compound 2 have superior antitumor activity compared to non-targeted controls and the free drug. These data show Tf-LP-Compound 2 to be a promising agent that warrants further evaluation. PMID:26309138

  2. Function of G-Protein-Coupled Estrogen Receptor-1 in Reproductive System Tumors

    PubMed Central

    Qian, Hongyan; Xuan, Jingxiu; Liu, Yuan; Shi, Guixiu

    2016-01-01

    The G-protein-coupled estrogen receptor-1 (GPER-1), also known as GPR30, is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types. The progress of estrogen-related cancer is promoted by GPER-1 activation through mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC) signaling pathways. However, this promoting effect of GPER-1 is nonclassic estrogen receptor (ER) dependent manner. In addition, clinical evidences revealed that GPER-1 is associated with estrogen resistance in estrogen-related cancer patients. These give a hint that GPER-1 may be a novel therapeutic target for the estrogen-related cancers. However, preclinical studies also found that GPER-1 activation of its special agonist G-1 inhibits cancer cell proliferation. This review aims to summarize the characteristics and complex functions of GPER-1 in cancers. PMID:27314054

  3. Synthesis and biological evaluation of phenoxyacetic acid derivatives as novel free fatty acid receptor 1 agonists.

    PubMed

    Wang, Xuekun; Zhao, Tianxiao; Yang, Baowei; Li, Zheng; Cui, Jian; Dai, Yuxuan; Qiu, Qianqian; Qiang, Hao; Huang, Wenlong; Qian, Hai

    2015-01-01

    Free fatty acid receptor 1 (FFA1) is a new potential drug target for the treatment of type 2 diabetes because of its role in amplifying glucose-stimulated insulin secretion in pancreatic β-cell. In the present studies, we identified phenoxyacetic acid derivative (18b) as a potent FFA1 agonist (EC50=62.3 nM) based on the structure of phenylpropanoic acid derivative 4p. Moreover, compound 18b could significantly improve oral glucose tolerance in ICR mice and dose-dependently reduced glucose levels in type 2 diabetic C57BL/6 mice without observation of hypoglycemic side effect. Additionally, compound 18b exhibited acceptable PK profiles. In summary, compound 18b with ideal PK profiles exhibited good activity in vitro and in vivo, and might be a promising drug candidate for the treatment of diabetes mellitus. PMID:25481394

  4. Trace amine-associated receptor 1: a promising target for the treatment of psychostimulant addiction

    PubMed Central

    Jing, Li; Li, Jun-Xu

    2015-01-01

    Abuse of and addiction to psychostimulants remains a challenging clinical issue, yet no effective pharmacotherapy is available. Trace amine associated receptor 1 (TAAR 1) is increasingly recognized as a novel drug target that participates in the modulation of drug abuse. This review analyzed existing preclinical evidence from electrophysiological, biochemical to behavioral aspects regarding the functional interactions between TAAR 1 and dopaminergic system. TAAR 1 knockout mice demonstrate increased sensitivity to dopaminergic activation while TAAR 1 agonists reduce the neurochemical effects of cocaine and amphetamines, attenuate abuse- and addiction-related behavioral effects of cocaine and methamphetamine. It is concluded that TAAR 1 activation functionally modulate the dopaminergic activity and TAAR 1 agonists appear to be promising pharmacotherapies against psychostimulant addiction. PMID:26092759

  5. Pancreatitis Secondary to Anti-Programmed Death Receptor 1 Immunotherapy Diagnosed by FDG PET/CT.

    PubMed

    Alabed, Yazan Z; Aghayev, Ayaz; Sakellis, Christopher; Van den Abbeele, Annick D

    2015-11-01

    A 57-year-old man with metastatic melanoma developed colitis secondary to ipilimumab, a known immune-related adverse event (irAE). The patient then received pembrolizumab immunotherapy, an anti-programmed-death-receptor-1 (PD-1) antibody. Restaging FDG PET/CT study following 3 cycles of therapy demonstrated diffuse increased FDG uptake throughout the body of the pancreas associated with fat stranding in the peripancreatic region, suggestive of pembrolizumab-induced pancreatitis. Although the patient was clinically asymptomatic, diagnosis was biochemically confirmed with elevated amylase and lipase levels. In the era of immunotherapy, it will be critical to recognize irAEs early to allow prompt initiation of appropriate therapy and reduce the risk of long-term sequelae. PMID:26284765

  6. Engineering superactive granulocyte macrophage colony-stimulating factor transferrin fusion proteins as orally-delivered candidate agents for treating neurodegenerative disease.

    PubMed

    Heinzelman, Pete; Priebe, Molly C

    2015-01-01

    Intravenously injected granulocyte macrophage colony-stimulating factor (GM-CSF) has shown efficacy in Alzheimer's Disease (AD) and Parkinson's Disease (PD) animal studies and is undergoing clinical evaluation. The likely need for dosing of GM-CSF to patients over months or years motivates pursuit of avenues for delivering GM-CSF to circulation via oral administration. Flow cytometric screening of 37 yeast-displayed GM-CSF saturation mutant libraries revealed residues P12, H15, R23, R24, and K72 as key determinants of GM-CSF's CD116 and CD131 GM-CSF receptor (GM-CSFR) subunit binding affinity. Screening combinatorial GM-CSF libraries mutated at positions P12, H15, and R23 yielded variants with increased affinities toward both CD116 and CD131. Genetic fusion of GM-CSF to human transferrin (Trf), a strategy that enables oral delivery of other biopharmaceuticals in animals, yielded bioactive wild type and variant cytokines upon secretion from cultured Human Embryonic Kidney cells. Surface plasmon resonance (SPR) measurements showed that all evaluated variants possess decreases in CD116 and CD131 binding KD values of up to 2.5-fold relative to wild type. Improved affinity led to increased in vitro bioactivity; the most bioactive variant, P12D/H15L/R23L, had a leukocyte proliferation assay EC50 value 3.5-fold lower than the wild type GM-CSF/Trf fusion. These outcomes are important first steps toward our goal of developing GM-CSF/Trf fusions as orally available AD and PD therapeutics. PMID:25737095

  7. Conjugation of an anti transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells.

    PubMed

    Daniels, Tracy R; Ng, Patrick P; Delgado, Tracie; Lynch, Maureen R; Schiller, Gary; Helguera, Gustavo; Penichet, Manuel L

    2007-11-01

    We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition, anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling, leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6), a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells. PMID:18025284

  8. Structural Model of the Cytosolic Domain of the Plant Ethylene Receptor 1 (ETR1)

    PubMed Central

    Mayerhofer, Hubert; Panneerselvam, Saravanan; Kaljunen, Heidi; Tuukkanen, Anne; Mertens, Haydyn D. T.; Mueller-Dieckmann, Jochen

    2015-01-01

    Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants. PMID:25451923

  9. A ruthenium(ii) based photosensitizer and transferrin complexes enhance photo-physical properties, cell uptake, and photodynamic therapy safety and efficacy.

    PubMed

    Kaspler, Pavel; Lazic, Savo; Forward, Sarah; Arenas, Yaxal; Mandel, Arkady; Lilge, Lothar

    2016-04-01

    Metal-based photosensitizers are of interest as their absorption and chemical binding properties can be modified via the use of different ligands. Ru(2+) based photosensitizers are known to be effective photodynamic therapy (PDT) agents against bacteria, whereas use for oncological indications in vivo has not been demonstrated with the same level of evidence. We present data showing that premixing the Ru(2+)-complex TLD1433 with transferrin increases the molar extinction coefficient, including longer activation wavelengths, reduces photobleaching rates, and reduces the toxicity of the complex improving overall PDT efficacy. As the transferrin receptor is upregulated in most malignancies, premixing the Ru(2+) complex with transferrin converts the active pharmaceutical ingredient TLD1433 into a drug of potentially considerable clinical utility. PMID:26947517

  10. Impairment of blastogenic response of splenic lymphocytes from iron-deficient mice. In vitro repletion by hemin, transferrin, and ferric chloride.

    PubMed

    Kuvibidila, S R; Nauss, K M; Baliga, S B; Suskind, R M

    1983-04-01

    Splenic lymphocytes from iron deficient C57BL/6 mice gave smaller proliferative responses to T and B cell mitogens than those from either the control of pair-fed mice. The addition of hemin to the culture medium partially restored the responses to Con A and phytohemagglutinin but not to bacterial lipopolysaccharide in unfractionated spleen cells and enriched T cell fractions. The responses of lymphocytes from the control and pair-fed mice were either unchanged or decreased. Hemin restored the blastogenic response to Con A more efficiently than to phytohemagglutinin. The blastogenic responses were increased linearly with increasing doses of hemin. Ferric chloride and iron saturated mouse transferrin did not restore the response to either Con A or lipopolysaccharide. However, both transferrin and ferric chloride partially restored the response to phytohemagglutinin. The possible mechanism of selective restoration of blastogenesis by hemin, transferrin, and ferric chloride in iron-deficient T lymphocytes is discussed. PMID:6601454

  11. Role of non-transferrin bound iron in iron overload and liver dysfunction in long term survivors of acute leukaemia and bone marrow transplantation.

    PubMed Central

    Harrison, P; Neilson, J R; Marwah, S S; Madden, L; Bareford, D; Milligan, D W

    1996-01-01

    AIMS: To determine whether nontransferrin bound iron is present in the serum of long term survivors of acute leukaemia and bone marrow transplantation who have liver dysfunction as indicated by consistently raised serum aspartate aminotransferase (AST) activities. METHODS: Thirty eight patients, who were at least three years from the end of treatment, were studied. Serum samples were analysed for hepatitis C, hepatitis B, AST, ferritin, and non-transferrin bound iron. A bleomycin based assay was used to detect non-transferrin bound iron. Patient and blood bank records were examined to determine the number of units of transfused blood received by each patient. RESULTS: Ten patients had consistently raised serum AST activities. Of these, two had evidence of hepatitis C infection, one had chronic hepatitis B infection and one had chronic graft versus host disease affecting the liver. None of these four patients had detectable non-transferrin bound iron. The remaining six patients had no obvious reason for raised AST activities, but four had non-transferrin bound iron detectable in their serum as compared with only two out of 28 patients with normal AST activities. Patients with abnormal AST activities had higher serum ferritin concentrations than those with normal AST, though serum ferritin was raised in 21 of 28 patients without liver dysfunction. CONCLUSION: Non-transferrin bound iron may be found in this group of patients, suggesting that iron overload is the cause of the observed liver dysfunction. Non-transferrin bound iron may also be a more specific indicator of iron overload than the serum ferritin concentrations. PMID:8943756

  12. Inhibition of metabotropic glutamate receptor 1 suppresses tumor growth and angiogenesis in experimental non-small cell lung cancer.

    PubMed

    Xia, Hui; Zhao, Ying-Nan; Yu, Chang-Hai; Zhao, Yun-Long; Liu, Yang

    2016-07-15

    Metabotropic glutamate receptor 1 (mGlu1 receptor) is expressed in many cancer cell types as compared to normal counterparts underscoring its potential role in tumor behavior. The aim of present study was to test the role of mGlu1 receptor in experimental non-small cell lung cancer (NSCLC). First, protein expression of mGlu1 receptor was higher in human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes, when compared to normal bronchial epithelial cells. Inhibition of mGlu1 receptor by BAY36-7620 (an mGlu1 receptor-specific inhibitor) inhibited tumor growth and prolonged survival of mice with tumors of A549 or H1299. Treatment with BAY36-7620 suppressed AKT phosphorylation in A549 tumors and pre-treatment with BAY36-7620 blocked the L-quisqualate (a potent mGlu1 receptor agonist)-induced AKT phosphorylation in A549 cells. Treatment with BAY36-7620 reduced cellular proliferation of A549 cells. Treatment with BAY36-7620 enhanced cleaved PARP levels and reduced protein expression of bcl-2, HIF-1α, and VEGF. In contrast, treatment with L-quisqualate reduced cleaved PARP levels and enhanced protein expression of bcl-2, HIF-1α, VEGF, and IL-8, which was reversed by co-incubation with MK2206 (an AKT inhibitor). Pre-treatment with BAY36-7620 blocked the VEGF-induced AKT phosphorylation in HUVECs. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was reversed by co-incubation with MK2206. Furthermore, mGlu1 receptor knockdown suppressed tumor growth and prolonged survival of mice with tumors of A549 or H1299. Collectively, inhibition of mGlu1 receptor suppressed tumor growth and angiogenesis in experimental NSCLC. PMID:27132814

  13. Fibroblast Growth Factor Receptor 1 Overexpression Is Associated with Poor Survival in Patients with Resected Muscle Invasive Urothelial Carcinoma

    PubMed Central

    Lim, Seungtaek; Koh, Myoung Ju; Jeong, Hyeon Joo; Cho, Nam Hoon; Choi, Young Deuk; Cho, Do Yeun; Lee, Hoi Young

    2016-01-01

    Purpose To examine the usefulness of various receptor tyrosine kinase expressions as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients. Materials and Methods We retrospectively analyzed the data of 98 patients with muscle invasive UC who underwent radical cystectomy between 2005 and 2010 in Yonsei Cancer Center. Using formalin fixed paraffin embedded tissues of primary tumors, immunohistochemical staining was done for human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 3 (FGFR3). Results There were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, significantly shorter median time to recurrence (TTR) (12.9 months vs. 49.0 months; p=0.008) and overall survival (OS) (22.3 months vs. 52.7 months; p=0.006) was found in patients with FGFR1 overexpression. By contrast, there was no difference in TTR or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% confidence interval: 1.27–3.90, p=0.006) in multivariate analysis. Conclusion Our result showed that FGFR1 expression, but not FGFR3, is an adverse prognostic factor in muscle invasive UC patients after radical cystectomy. FGFR1 might be feasible for prognosis prediction and a potential therapeutic target after thorough validation in muscle invasive UC. PMID:27189274

  14. Neuropeptide S receptor 1 (NPSR1) activates cancer-related pathways and is widely expressed in neuroendocrine tumors.

    PubMed

    Pulkkinen, V; Ezer, S; Sundman, L; Hagström, J; Remes, S; Söderhäll, C; Greco, D; Dario, G; Haglund, C; Kere, J; Arola, J

    2014-08-01

    Neuroendocrine tumors (NETs) arise from disseminated neuroendocrine cells and express general and specific neuroendocrine markers. Neuropeptide S receptor 1 (NPSR1) is expressed in neuroendocrine cells and its ligand neuropeptide S (NPS) affects cell proliferation. Our aim was to study whether NPS/NPSR1 could be used as a biomarker for neuroendocrine neoplasms and to identify the gene pathways affected by NPS/NPSR1. We collected a cohort of NETs comprised of 91 samples from endocrine glands, digestive tract, skin, and lung. Tumor type was validated by immunostaining of chromogranin-A and synaptophysin expression and tumor grade was analyzed by Ki-67 proliferation index. NPS and NPSR1 expression was quantified by immunohistochemistry using polyclonal antibodies against NPS and monoclonal antibodies against the amino-terminus and carboxy-terminus of NPSR1 isoform A (NPSR1-A). The effects of NPS on downstream signaling were studied in a human SH-SY5Y neuroblastoma cell line which overexpresses NPSR1-A and is of neuroendocrine origin. NPSR1 and NPS were expressed in most NET tissues, with the exception of adrenal pheochromocytomas in which NPS/NPSR1 immunoreactivity was very low. Transcriptome analysis of NPSR1-A overexpressing cells revealed that mitogen-activated protein kinase (MAPK) pathways, circadian activity, focal adhesion, transforming growth factor beta, and cytokine-cytokine interactions were the most altered gene pathways after NPS stimulation. Our results show that NETs are a source of NPS and NPSR1, and that NPS affects cancer-related pathways. PMID:24915894

  15. Blockage of protease-activated receptor 1 ameliorates heat-stress induced intestinal high permeability and bacterial translocation.

    PubMed

    Xu, Qiu-lin; Guo, Xiao-hua; Liu, Jing-xian; Chen, Bin; Liu, Zhi-feng; Su, Lei

    2015-04-01

    Accumulated evidences indicate intestinal lesions play an important role in the pathogenesis of heatstroke. However, the underlying mechanisms by which heat stress causes intestinal barrier dysfunction and bacterial translocation remain unclear. In this study, we investigated the role of protease-activated receptor 1 (PAR1) in heat stress-induced intestinal hyper-permeability and bacterial translocation. Intestinal permeability in heat stressed mouse was evaluated by determining plasma endotoxin concentration and urinal lactulose/mannitol (L/M) ratio with gastric administration of L/M solution. Venous blood, liver, spleen and mesenteric lymph node tissues were collected for bacterial load test. Real time PCR was used to determine ileum PAR1 mRNA expression. In vitro study, permeability was assessed by determining trans-epithelial electrical resistance (TEER) in human intestinal Caco-2 cell line. RWJ-58259, a selective antagonist of PAR1, was used both in vivo and in vitro studies. The results showed that heat stress could increase ileum PAR1 mRNA level, urinal L/M ratio, plasma endotoxin concentration and bacterial load in the blood, spleen and mesenteric lymph nodes. Blocking PAR1 with RWJ-58259 (10 mg/kg) pretreatment could significantly reduce heat stress-induced above changes, but have no role to PAR1 mRNA level. In Caco-2 cells, heat stress-induced high permeability could also be reduced by RWJ-58259 (5-20 µmol/L). In summary, our results demonstrated that PAR1 signaling pathway may play an important role in the heat stress-induced elevation of intestinal permeability, bacterial translocation and the occurrence of endotoxemia. PMID:25492552

  16. The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase.

    PubMed

    Cao, Hong; Schroeder, Barbara; Chen, Jing; Schott, Micah B; McNiven, Mark A

    2016-08-01

    Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments. PMID:27226592

  17. Luminescence turn-on/off sensing of biological iron by carbon dots in transferrin.

    PubMed

    Bhattacharya, Arpan; Chatterjee, Surajit; Khorwal, Vijaykant; Mukherjee, Tushar Kanti

    2016-02-21

    Iron is a key nutrient as well as a potential toxin for almost all living organisms. In mammalian cells, serum transferrin (Tf) is responsible for iron transport and its iron overload/deficiency causes various diseases. Therefore, closely regulated iron homeostasis is extremely essential for cellular metabolism. In the present article we report the pH-dependent luminescence turn-on/off sensing of bound Fe(3+) ions of serum Tf by carbon dots (CDs) with the help of photoluminescence (PL) spectroscopy, FTIR spectroscopy, dynamic light scattering (DLS), circular dichroism (CD) and PL imaging techniques. At physiological pH (7.4), the intrinsic luminescence of CDs gets quenched in the presence of Tf as a consequence of ground-state association, which is driven by favorable electrostatic interactions between negatively charged CDs (-25.45 ± 1.23 mV) and positively charged Fe(3+) ions of Tf. The estimated detection limit of Tf by CDs at physiological pH is found to be 1.82 μM (signal-to-noise ratio of 3), which is much lower than the in vivo plasma concentration of Tf (∼25-35 μM). Various thermodynamic parameters have been evaluated by using the van't Hoff equation. Importantly, the secondary structure of Tf remains unaltered upon association with CDs. However, at pH 3.5, no such luminescence quenching of CDs has been observed in the presence of Tf due to the lack of ground-state interactions between positively charged (+17.63 ± 0.84 mV) CDs and Tf. Furthermore, the results from UV-Vis and far-UV CD measurements revealed a significant conformational change of Tf at pH 3.5 relative to pH 7.4, which triggers the subsequent release of bound iron from Tf. PL microscopy of individual CD revealed significant luminescence quenching at the single particle level, which further supports the non-emissive ground-state complexation at pH 7.4. Our present results show that these chemically synthesized water-dispersed CDs have the ability to selectively sense the bound iron from

  18. Synthesis and characterization of tumor-targeted copolymer nanocarrier modified by transferrin

    PubMed Central

    Liu, Ran; Wang, Yonglu; Li, Xueming; Bao, Wen; Xia, Guohua; Chen, Wei; Cheng, Jian; Xu, Yuanlong; Guo, Liting; Chen, Baoan

    2015-01-01

    To increase the encapsulation of hydrophilic antitumor agent daunorubicin (DNR) and multidrug resistance reversal agent tetrandrine (Tet) in the drug delivery system of nano-particles (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 4.27%±0.13% for Tet. Notably, the accumulated release of DNR and Tet could be sustained over 1 week, and the Tf content was 2.18%±0.04%. In cell viability tests, DNR/Tet–PLGA–PLL–PEG–Tf-NPs could inhibit the proliferation of K562/ADR cells in a dose-dependent manner, and the half maximal inhibitory concentration value (total drug) of DNR/Tet–PLGA–PLL–PEG–Tf-NPs was lower than that of DNR

  19. Synthesis and characterization of tumor-targeted copolymer nanocarrier modified by transferrin.

    PubMed

    Liu, Ran; Wang, Yonglu; Li, Xueming; Bao, Wen; Xia, Guohua; Chen, Wei; Cheng, Jian; Xu, Yuanlong; Guo, Liting; Chen, Baoan

    2015-01-01

    To increase the encapsulation of hydrophilic antitumor agent daunorubicin (DNR) and multidrug resistance reversal agent tetrandrine (Tet) in the drug delivery system of nano-particles (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 4.27%±0.13% for Tet. Notably, the accumulated release of DNR and Tet could be sustained over 1 week, and the Tf content was 2.18%±0.04%. In cell viability tests, DNR/Tet-PLGA-PLL-PEG-Tf-NPs could inhibit the proliferation of K562/ADR cells in a dose-dependent manner, and the half maximal inhibitory concentration value (total drug) of DNR/Tet-PLGA-PLL-PEG-Tf-NPs was lower than that of DNR, a mixture of DNR and Tet, and DNR

  20. Examination of the mechanism of action of nitrogen monoxide on iron uptake from transferrin.

    PubMed

    Watts, R N; Richardson, D R

    2000-08-01

    Nitrogen monoxide (NO) exerts many of its functions by binding to iron (Fe) in the active sites of a number of key proteins. Previously we have shown that NO produced by NO-generating agents decreased cellular Fe uptake from transferrin (Tf). However, the mechanism of this effect was not elucidated. In this study we examined the possible mechanisms whereby NO could interfere with Fe uptake. Our experiments demonstrate that NO produced by the NO generator S-nitroso-N-acetylpenicillamine was slightly more effective than the Fe chelator deferoxamine at reducing iron 59 uptake from 59Fe-labeled Tf by LMTK- fibroblasts. Other NO generators including S-nitrosoglutathione (GSNO) and spermine-NONOate also decreased 59Fe uptake from 59Fe-labeled Tf. In contrast, precursors of these compounds that do not release NO had no effect. When the RAW264.7 macrophage cell line was activated to produce NO by incubation with lipopolysaccharide or lipopolysaccharide and interferon-gamma, a decrease in 59Fe uptake from 59Fe-labeled Tf was also observed. Experiments with electron paramagnetic resonance spectroscopy and ultraviolet-Vis spectrophotometry demonstrated that NO did not prevent Fe uptake by binding to the Fe-ligating sites of Tf, suggesting that it acted more distally. Because the uptake of Fe is an energy-dependent process, and since NO inhibits mitochondrial respiration, cellular adenosine triphosphate (ATP) was estimated after incubation with GSNO. In the presence of D-glucose (D-G), GSNO reduced ATP levels by 35% as compared with the control, while in the absence of D-G, GSNO reduced ATP by 72%. When the same experiments were performed with D-fructose (D-F), which cannot be efficiently metabolized by fibroblasts, no "rescue" effect was observed on ATP levels. The addition of D-G to GSNO prevented the decrease in 59Fe uptake from 59Fe-labeled Tf while D-F did not, in good correlation with their effects on ATP levels. These results suggest that D-G acts as a salvage

  1. Evaluation of recombinant transferrin-binding protein B variants from Neisseria meningitidis for their ability to induce cross-reactive and bactericidal antibodies against a genetically diverse collection of serogroup B strains.

    PubMed Central

    Rokbi, B; Mignon, M; Maitre-Wilmotte, G; Lissolo, L; Danve, B; Caugant, D A; Quentin-Millet, M J

    1997-01-01

    Transferrin-binding protein B (TbpB) is a surface-exposed protein, variable among strains of Neisseria meningitidis, that has been considered as a vaccine candidate. To define a TbpB molecule that would give rise to broadly cross-reactive antibodies with TbpB of many strains, specific antisera were produced against three recombinant TbpB variants from strain M982: one corresponding to the full-length TbpB; one in which stretches of amino acids located in the central part of the molecule, described as hypervariable, have been deleted; and one corresponding to the N-terminal half of the molecule, described as the human transferrin binding domain. The reactivity of these antisera against 58 serogroup B strains with a 2.1-kb tbpB gene representing different genotypes, serotypes, and subtypes and different geographic origins was tested on intact meningococcal cells. In parallel, the bactericidal activity of the antisera was evaluated against 15 of the 58 strains studied. Of the 58 strains, 56 (98%) reacted with the antiserum specific for the N-terminal half of TbpB M982; this antiserum was bactericidal against 9 of 15 strains (60%). On the other hand, 43 of 58 strains reacted with the antiserum raised to full-length TbpB while 12 of 15 (80%) were killed with this antiserum. The antiserum specific to TbpB deleted of its central domain gave intermediate results, with 53 of 58 strains (91.3%) recognized and 10 of 15 (66.6%) killed. These results indicate that the N-terminal half of TbpB was sufficient to induce cross-reactive antibodies reacting with the protein on meningococcal cells but that the presence of the C-terminal half of the protein is necessary for the induction of cross-bactericidal antibodies. PMID:8975892

  2. A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

    PubMed

    Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco

    2016-01-01

    Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification). PMID:27279446

  3. Dihydroartemisinin and transferrin dual-dressed nano-graphene oxide for a pH-triggered chemotherapy.

    PubMed

    Liu, Lijuan; Wei, Yanchun; Zhai, Shaodong; Chen, Qun; Xing, Da

    2015-09-01

    Dihydroartemisinin (DHA) is a unique anti-malarial drug isolated from the plant Artemisia annua. Recently, it has been studied as an alternative modality for cancer therapy, utilizing its reactive oxygen species (ROS) yielding mechanism from interacting with Ferrous ion (Fe (II)). In this work, a novel nanodrug (DHA-GO-Tf) is constructed based on nanoscale Graphene oxide (GO) dual-dressed with DHA and Transferrin (Tf). Tf dually functions as a pilot for the nanoparticle to target tumor cell with over expressed Transferrin receptor (TfR) and a ferric ion carrier. Upon tumor cellular endocytosis, Ferric ion (Fe(III)) is released from the Tf, triggered by the low pH in the lysosomes of the tumor cell. The intracellular Fe (III) is reduced to Fe (II) and interacts with DHA to increase its cytotoxicity. The potential of this alternative anti-tumor modality is demonstrated both in vitro and in vivo. Comparing with DHA alone, the nanodrug DHA-GO-Tf resulted in a significantly enhanced tumor delivery specificity and cytotoxicity, and achieved a complete tumor cure in mice with minimal side-effects. PMID:26022978

  4. Self-assembled IR780-loaded transferrin nanoparticles as an imaging, targeting and PDT/PTT agent for cancer therapy.

    PubMed

    Wang, Kaikai; Zhang, Yifan; Wang, Juan; Yuan, Ahu; Sun, Minjie; Wu, Jinhui; Hu, Yiqiao

    2016-01-01

    Combination of photothermal and photodynamic therapy (PTT/PDT) offer unique advantages over PDT alone. However, to achieve synergetic PDT/PTT effect, one generally needs two lasers with different wavelengths. Near-infrared dye IR-780 could be used as photosensitizer both for PTT and PDT, but its lipophilicity limits its practical use and in vivo efficiency. Herein, a simple multifunctional IR780-loaded nanoplatform based on transferrin was developed for targeted imaging and phototherapy of cancer compatible with a single-NIR-laser irradiation. The self-assembled transferrin-IR780 nanoparticles (Tf-IR780 NPs) exhibited narrow size distribution, good photo-stability, and encouraging photothermal performance with enhanced generation of ROS under laser irradiation. Following intravenous injection, Tf-IR780 NPs had a high tumor-to-background ratio in CT26 tumor-bearing mice. Treatment with Tf-IR780 NPs resulted in significant tumor suppression. Overall, the Tf-IR780 NPs show notable targeting and theranostic potential in cancer therapy. PMID:27263444

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

    PubMed Central

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

    2015-01-01