Sample records for biotinylated receptor tyrosine

  1. Biotinylated recombinant human erythropoietins: Bioactivity and utility as receptor ligand

    Energy Technology Data Exchange (ETDEWEB)

    Wojchowski, D.M.; Caslake, L. (Pennsylvania State Univ., University Park (USA))


    Recombinant human erythropoietin labeled covalently with biotin at sialic acid moieties has been prepared, and has been shown to possess high biological activity plus utility as a receptor ligand. Initially, the effects on biological activity of covalently attaching biotin to erythropoietin alternatively at carboxylate, amino, or sialic acid groups were compared. Biotinylation of erythropoietin at carboxylate groups using biotin-amidocaproyl hydrazide plus 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide led to substantial biological inactivation, although biotinylated molecules retained detectable activity when prepared at low stoichiometries. Biotinylation at amino groups using sulfosuccinimidyl 6-(biotinamido) hexanoate resulted in a high level of biological inactivation with little, if any, retention of biological activity, regardless of labeling stoichiometries. Biotinylation at sialic acid moieties using periodate and biotinamidocaproyl hydrazide proceeded efficiently (greater than 95% and 80% labeling efficiencies for human urinary and recombinant erythropoietin, respectively) and yielded stably biotinylated erythropoietin molecules possessing comparably high biological activity (ie, 45% of the activity of unmodified hormone). Utility of recombinant biotin-(sialyl)-erythropoietin (in combination with 125I-streptavidin) in the assay of cell surface receptors was demonstrated using two distinct murine erythroleukemia cell lines, Friend 745 and Rauscher Red 1. The densities and affinities of specific hormone binding sites were 116 +/- 4 sites, 3.3 +/- 0.4 nmol/L kd and 164 +/- 5 sites, 2.7 +/- 0.4 nmol/L kd, respectively. It is predicted that the present development of biotin-(sialyl)-erythropoietin as a chemically and biologically stable, bioactive ligand will assist in advancing an understanding of the regulated expression and physicochemistry of the human and murine erythropoietin receptors.

  2. Receptor tyrosine kinases in carcinogenesis. (United States)

    Zhang, Xiao-Ying; Zhang, Pei-Ying


    Receptor tyrosine kinases (RTKs) are cell surface glycoproteins with enzymatic activity involved in the regulation of various important functions. In all-important physiological functions including differentiation, cell-cell interactions, survival, proliferation, metabolism, migration and signaling these receptors are the key players of regulation. Additionally, mutations of RTKs or their overexpression have been described in many human cancers and are being explored as a novel avenue for a new therapeutic approach. Some of the deregulated RTKs observed to be significantly affected in cancers included vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, RTK-like orphan receptor 1 (ROR1) and the platelet-derived growth factor receptor. These deregulated RTKs offer attractive possibilities for the new anticancer therapeutic approach involving specific targeting by monoclonal antibodies as well as kinase. The present review aimed to highlight recent perspectives of RTK ROR1 in cancer.

  3. Multiplex detection of homo- and heterodimerization of g protein-coupled receptors by proximity biotinylation.

    Directory of Open Access Journals (Sweden)

    Elisabeth Steel

    Full Text Available Dimerization of G protein-coupled receptors (GPCRs represents a potential mechanism by which GPCR functions are regulated. Several resonance energy transfer (RET-based methods have revealed GPCR homo- and heterodimerization. However, interpretation of an increase in FRET efficiency could be attributed to either dimerization/oligomerization events or conformational changes within an already dimerized/oligomerized receptor complex. Furthermore, RET-based methods can only measure pairwise dimerization, and cannot easily achieve multiplex detection. In this study, we applied proximity-based biotinylation for detecting receptor dimerization by utilizing a specific enzyme-substrate pair that are fused to GPCRs. The biotin ligase BirA is fused to CXCR4 and site-specifically biotinylates an acceptor peptide (AP in the presence of biotin. As a test case for our newly developed assay, we have characterized the homo-dimerization of chemokine receptor CXCR4 and heterodimerization of CXCR4 with CCR2 or CCR5. The degree of biotinylation varies with the amount of GPCR-AP as well as biotinylation time. Using enzyme/substrate receptor pairs and measuring receptor biotinylation, we demonstrate that CXCR4 can homo-dimerize and hetero-dimerize with CCR2 and CCR5. The effect of CXCL12, agonist for CXCR4, was found to decrease surface biotinylation of CXCR4-AP. This effect is due to a combination of CXCR4 endocytosis and stabilization of CXCR4 homodimers. Finally, when CXCR4-AP, CCR2-AP, and CCR5-AP were expressed together, we observed CXCR4-CXCR4 homodimers and CXCR4-CCR2 and CXCR4-CCR5 heterodimers. The newly developed assay opens new opportunity for multiplex detection for GPCR homo- and heterodimerization within the same cellular context.

  4. Endocytosis of Receptor Tyrosine Kinases (United States)

    Goh, Lai Kuan


    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  5. Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods

    NARCIS (Netherlands)

    M.O. de Jong (Marg); H. Rozemuller (Henk); J.G.J. Bauman (J. G J); J.W.M. Visser (Jan)


    textabstractThe main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that bind

  6. Characterization of receptor proteins using affinity cross-linking with biotinylated ligands. (United States)

    Shinya, Tomonori; Osada, Tomohiko; Desaki, Yoshitake; Hatamoto, Masahiro; Yamanaka, Yuko; Hirano, Hisashi; Takai, Ryota; Che, Fang-Sik; Kaku, Hanae; Shibuya, Naoto


    The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

  7. Complexity of Receptor Tyrosine Kinase Signal Processing (United States)

    Volinsky, Natalia; Kholodenko, Boris N.


    Our knowledge of molecular mechanisms of receptor tyrosine kinase (RTK) signaling advances with ever-increasing pace. Yet our understanding of how the spatiotemporal dynamics of RTK signaling control specific cellular outcomes has lagged behind. Systems-centered experimental and computational approaches can help reveal how overlapping networks of signal transducers downstream of RTKs orchestrate specific cell-fate decisions. We discuss how RTK network regulatory structures, which involve the immediate posttranslational and delayed transcriptional controls by multiple feed forward and feedback loops together with pathway cross talk, adapt cells to the combinatorial variety of external cues and conditions. This intricate network circuitry endows cells with emerging capabilities for RTK signal processing and decoding. We illustrate how mathematical modeling facilitates our understanding of RTK network behaviors by unraveling specific systems properties, including bistability, oscillations, excitable responses, and generation of intricate landscapes of signaling activities. PMID:23906711

  8. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J


    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine-phos...

  9. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation. (United States)

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril


    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  10. BRET biosensor analysis of receptor tyrosine kinase functionality

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    Sana eSiddiqui


    Full Text Available Bioluminescence resonance energy transfer (BRET is an improved version of earlier resonance energy transfer technologies used for the analysis of biomolecular protein interaction. BRET analysis can be applied to many transmembrane receptor classes, however the majority of the early published literature on BRET has focused on G protein-coupled receptor (GPCR research. In contrast, there is limited scientific literature using BRET to investigate receptor tyrosine kinase (RTK activity. This limited investigation is surprising as RTKs often employ dimerization as a key factor in their activation, as well as being important therapeutic targets in medicine, especially in the cases of cancer, diabetes, neurodegenerative and respiratory conditions. In this review, we consider an array of studies pertinent to RTKs and other non-GPCR receptor protein-protein signaling interactions; more specifically we discuss receptor-protein interactions involved in the transmission of signaling communication. We have provided an overview of functional BRET studies associated with the receptor tyrosine kinase (RTK super family involving: neurotrophic receptors (e.g. tropomyosin-related kinase (Trk and p75 neurotrophin receptor (p75NTR; insulinotropic receptors (e.g. insulin receptor (IR and insulin-like growth factor receptor (IGFR and growth factor receptors (e.g. ErbB receptors including the EGFR, the fibroblast growth factor receptor (FGFR, the vascular endothelial growth factor receptor (VEGFR and the c-kit and platelet-derived growth factor receptor (PDGFR. In addition, we review BRET-mediated studies of other tyrosine kinase-associated receptors including cytokine receptors, i.e. leptin receptor (OB-R and the growth hormone receptor (GHR. It is clear even from the relatively sparse experimental RTK BRET evidence that there is tremendous potential for this technological application for the functional investigation of RTK biology.

  11. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors: New Hope for Success in Multiple Sclerosis Therapy. (United States)

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak; Azizi, Gholamreza


    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials.

  12. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D;


    antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  13. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar;


    . The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  14. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  15. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases. (United States)

    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P


    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases.

  16. The role of GH receptor tyrosine phosphorylation in Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, J A; Hansen, L H; Wang, X;


    Stimulation of GH receptors leads to rapid activation of Jak2 kinase and subsequent tyrosine phosphorylation of the GH receptor. Three specific tyrosines located in the C-terminal domain of the GH receptor have been identified as being involved in GH-stimulated transcription of the Spi 2.1 promoter....... Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...

  17. Spatial regulation of receptor tyrosine kinases in development and cancer


    Casaletto, Jessica B.; McClatchey, Andrea I.


    During development and tissue homeostasis, patterns of cellular organization, proliferation, and movement are highly choreographed. Receptor tyrosine kinases (RTKs) play a critical role in establishing these patterns. Individual cells and tissues exhibit tight spatial control of the RTKs that they express, enabling tissue morphogenesis and function while preventing unwarranted cell division and migration that can contribute to tumorigenesis. Indeed, RTKs are deregulated in most human cancers ...

  18. Analysis of receptor tyrosine kinase internalization using flow cytometry. (United States)

    Li, Ning; Hill, Kristen S; Elferink, Lisa A


    The internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization.

  19. EphB4 Receptor Tyrosine Kinase in Prostate Cancer (United States)


    Hassanieh  L,   Ley  EJ,  Scehnet  J,  Kumar  NG,   Hawes  D,  Press  MF,  Weaver  FA,  Gill  PS.  Receptor  tyrosine...J. Pathol. 174 (2009) 1492. [33] T.D. Bartley, R.W. Hunt, A.A. Welcher, W.J. Boyle , V.P. Parker, R.A. Lindberg, H.S. Lu, A.M. Colombero, R.L

  20. Asymmetric Receptor Contact is Required for Tyrosine Autophosphorylation of Fibroblast Growth Factor Receptor in Living Cells

    Energy Technology Data Exchange (ETDEWEB)

    Bae, J.; Boggon, T; Tomé, F; Mandiyan, V; Lax, I; Schlessinge, J


    Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

  1. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Rashna Bhandari; Roy Mathew; K Vijayachandra; Sandhya S Visweswariah


    Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

  2. Asymmetric Tyrosine Kinase Arrangements in Activation or Autophosphorylation of Receptor Tyrosine Kinases

    Energy Technology Data Exchange (ETDEWEB)

    J Bae; J Schlessinger


    Receptor tyrosine kinases (RTKs) play important roles in the control of many cellular processes including cell proliferation, cell adhesion, angiogenesis, and apoptosis. Ligand-induced dimerization of RTKs leads to autophosphorylation and activation of RTKs. Structural studies have shown that while isolated ectodomains of several RTKs form symmetric dimers the isolated cytoplasmic kinase domains of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) form asymmetric dimers during their activation. Binding of one kinase molecule of EGFR to a second kinase molecule asymmetrically leads to stimulation of kinase activity and enhanced autophosphorylation. Furthermore, the structures of the kinase domain of FGFR1 and FGFR2 reveal the formation of asymmetric interfaces in the processes of autophosphorylation at their specific phosphotyrosine (pY) sites. Disruption of asymmetric dimer interface of EGFR leads to reduction in enzymatic activity and drastic reduction of autophosphorylation of FGFRs in ligandstimulated live cells. These studies demonstrate that asymmetric dimer formation is as a common phenomenon critical for activation and autophosphorylation of RTKs.

  3. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D


    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.......-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable...... cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does...

  4. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

    Directory of Open Access Journals (Sweden)

    Mili Jeon


    The respiratory (tracheal system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs, Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr tyrosine kinase (TK. Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

  5. Receptor tyrosine kinases and schistosome reproduction: new targets for chemotherapy

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    Marion eMorel


    Full Text Available Schistosome parasites still represent a serious public health concern and a major economic problem in developing countries. Pathology of schistosomiasis is mainly due to massive egg production by these parasites and to inflammatory responses raised against the eggs which are trapped in host tissues. Tyrosine kinases (TKs are key molecules that control cell differentiation and proliferation and they already represent important targets in cancer therapy. During the recent years, it has been shown that receptor tyrosine kinases (RTK signaling was active in reproductive organs and that it could regulate sexual maturation of schistosomes and egg production. This opens interesting perspectives for the control of transmission and pathogenesis of schistosomiasis based on new therapies targeting schistosome RTKs. This review relates the numerous data showing the major roles of kinase signaling in schistosome reproduction. It describes the conserved and particular features of schistosome RTKs, their implication in gametogenesis and reproduction processes and summarizes recent works indicating that RTKs and their signaling partners are interesting chemotherapeutical targets in new programs of control.

  6. Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells (United States)

    Moody, Terry W.; Berna, Marc J.; Mantey, Samuel; Sancho, Veronica; Ridnour, Lisa; Wink, David A.; Chan, Daniel; Giaccone, Giuseppe; Jensen, Robert T.


    Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr1068 of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transected with NMB receptors. The transactivation of the EGF receptor or the tyrosine phosphorylation of ERK caused by NMB-like peptides was inhibited by AG1478 or gefitinib (tyrosine kinase inhibitors) and NMB receptor antagonist PD168368 but not the GRP receptor antagonist, BW2258U89. The transactivation of the EGF receptor caused by NMB-like peptides was inhibited by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), or transforming growth factor (TGF)α antibody. The transactivation of the EGF receptor and the increase in reactive oxygen species caused by NMB-like peptides was inhibited by N-acetylcysteine (NAC) or Tiron. Gefitinib inhibited the proliferation of NCI-H1299 cells and its sensitivity was increased by the addition of PD168368. The results indicate that the NMB receptor regulates EGF receptor transactivation by a mechanism dependent on Src as well as metalloprotease activation and generation of reactive oxygen species. PMID:20388507

  7. Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications

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    A. Quintanal-Villalonga


    Full Text Available Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as “omics” has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed.

  8. Role of Receptor Tyrosine Kinases and Their Ligands in Glioblastoma

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    Estefanía Carrasco-García


    Full Text Available Glioblastoma multiforme is the most frequent, aggressive and fatal type of brain tumor. Glioblastomas are characterized by their infiltrating nature, high proliferation rate and resistance to chemotherapy and radiation. Recently, oncologic therapy experienced a rapid evolution towards “targeted therapy,” which is the employment of drugs directed against particular targets that play essential roles in proliferation, survival and invasiveness of cancer cells. A number of molecules involved in signal transduction pathways are used as molecular targets for the treatment of various tumors. In fact, inhibitors of these molecules have already entered the clinic or are undergoing clinical trials. Cellular receptors are clear examples of such targets and in the case of glioblastoma multiforme, some of these receptors and their ligands have become relevant. In this review, the importance of glioblastoma multiforme in signaling pathways initiated by extracellular tyrosine kinase receptors such as EGFR, PDGFR and IGF-1R will be discussed. We will describe their ligands, family members, structure, activation mechanism, downstream molecules, as well as the interaction among these pathways. Lastly, we will provide an up-to-date review of the current targeted therapies in cancer, in particular glioblastoma that employ inhibitors of these pathways and their benefits.

  9. Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L


    Many signaling pathways initiated by ligands that activate receptor tyrosine kinases have been shown to involve the binding of SH2 domain-containing proteins to specific phosphorylated tyrosines in the receptor. Although the receptor for growth hormone (GH) does not contain intrinsic tyrosine...

  10. Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma

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    Wen-Bin Ou


    Full Text Available The receptor tyrosine kinases (RTKs epidermal growth factor receptor (EGFR and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3 in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90. Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17demethoxygeldanamycin (17-AAG had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of down-stream signaling intermediates (AKT, mitogen-activated protein kinase, and S6; upregulated the p53, p21, and p27 cell cycle checkpoints; induced G2 phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G1 apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.

  11. A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control. (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Thon, Lutz; Mamat, Uwe; Bellosta, Paola; Basilico, Claudio; Adam, Dieter; Paus, Ralf; Bulfone-Paus, Silvia


    Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a 'promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor alpha (TNFalpha)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFalpha-resistant. IL-15Ralpha and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Ralpha interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Ralpha, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl-/- or IL-15Ralpha-/- mice. Thus, IL-15-induced protection from TNFalpha-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Ralpha and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Ralpha.

  12. Influence of berberine on protein tyrosine kinase of erythrocyte insulin receptors from type 2 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    Xianglei Deng; Xinrong Li; Chenggong Tian


    Objective: Bererine has been used to treat type 2 diabetes mellitus in Chinese traditional medicine because of its hypoglycemic effect. In this report, we compared the intrinsic tyrosine kinase activities of erythrocyte insulin receptors from type 2 diabetes mellitus with or without stimulation by berberine in vitro. Methods: Preparations containing insulin receptors were obtained from soluble human erythrocytes, and the insulin receptors were partially purified by affinity chromatography. The tyrosine kinase activity was measured by the exogenous substrate phosphorylation. Results: Both the membrane tyrosine kinase activity and the purified receptor tyrosine kinase activity from diabetics decreased significantly compared with those of normal individuals (reduced by 67.4 % and 47.2 %, respectively).After incubation with berberine, there is a statistical difference in the activity of membrane tyrosine kinase for diabetic patients (a 150% increase). Bererine had no effect on the tyrosine kinase activity of purified insulin receptors. Conclusion: We concluded from these results that berberine was able to improve the insulin sensitivity by increasing the protein tyrosine kinase activity of membrane-bound insulin receptors from type 2 diabetes mellitus.

  13. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M


    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin to i...

  14. A novel putative tyrosine kinase receptor with oncogenic potential. (United States)

    Janssen, J W; Schulz, A S; Steenvoorden, A C; Schmidberger, M; Strehl, S; Ambros, P F; Bartram, C R


    We have detected transforming activity by a tumorigenicity assay using NIH3T3 cells transfected with DNA from a chronic myeloproliferative disorder patient. Here, we report the cDNA cloning of the corresponding oncogene, designated UFO, in allusion to the as yet unidentified function of its protein. Nucleotide sequence analysis of a 3116bp cDNA clone revealed a 2682-bp-long open reading frame capable of directing the synthesis of a 894 amino acid polypeptide. The predicted UFO protein exhibits characteristic features of a transmembrane receptor with associated tyrosine kinase activity. The UFO proto-oncogene maps to human chromosome 19q13.1 and is transcribed into two 5.0 kb and 3.2 kb mRNAs in human bone marrow and human tumor cell lines. The UFO locus is evolutionarily conserved between vertebrate species. A 4.0 kb mRNA of the murine UFO homolog is expressed in a variety of different mouse tissues. We thus have identified a novel element of the complex signaling network involved in the control of cell proliferation and differentiation.

  15. Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase. (United States)

    Milatovic, Dejan; Jenkins, Jerry W; Hood, Jonathan E; Yu, Yingchun; Rongzhu, Lu; Aschner, Michael


    Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects.

  16. Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. (United States)

    Jayaraman, T; Ondrias, K; Ondriasová, E; Marks, A R


    Tyrosine kinases indirectly raise intracellular calcium concentration ([Ca2+]i) by activating phospholipases that generate inositol 1,4,5-trisphosphate (IP3). IP3 activates the IP3 receptor (IP3R), an intracellular calcium release channel on the endoplasmic reticulum. T cell receptor stimulation triggered a physical association between the nonreceptor protein tyrosine kinase Fyn and the IP3R, which induced tyrosine phosphorylation of the IP3R. Fyn activated an IP3-gated calcium channel in vitro, and tyrosine phosphorylation of the IP3R during T cell activation was reduced in thymocytes from fyn-/- mice. Thus, activation of the IP3R by tyrosine phosphorylation may play a role in regulating [Ca2+]i.

  17. Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation

    Institute of Scientific and Technical Information of China (English)

    Chanan RUBIN; Gal GUR; Yosef YARDEN


    Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cblmediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

  18. Requirement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor for selected GH-stimulated function

    DEFF Research Database (Denmark)

    Lobie, P E; Allevato, G; Norstedt, G;


    We have examined the involvement of tyrosine residues 333 and 338 of the growth hormone (GH) receptor in the cellular response to GH. Stable Chinese hamster ovary (CHO) cell clones expressing a receptor with tyrosine residues at position 333 and 338 of the receptor substituted for phenylalanine...

  19. Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase

    DEFF Research Database (Denmark)

    Wang, X; Uhler, M D; Billestrup, N;


    The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels ...

  20. Cross-membrane signal transduction of receptor tyrosine kinases (RTKs): from systems biology to systems pharmacology. (United States)

    Benson, Neil; van der Graaf, Piet H; Peletier, Lambertus A


    Receptor tyrosine kinases are high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. They straddle the cell wall and play an important role in cross-membrane signalling. We present a two-component systems pharmacology model based on the local physiology and identify characteristic features of its dynamics. We thus present a transparent tool for studying the effects of drug intervention and ways of administration on cross-membrane signalling through these receptors.

  1. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    Directory of Open Access Journals (Sweden)

    Belén Mezquita


    Full Text Available One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT and a truncated isoform of VEGFR-1 (i21-VEGFR-1, which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

  2. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng;


    (RPTPalpha) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPalpha compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPalpha is a novel member of the functional class of genes that control radial......Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha...

  3. DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:... potentialregulators of macrophage inflammatory activities. PubmedID 12472665 Title Macrophage-stim

  4. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors

    DEFF Research Database (Denmark)

    Sorkin, A; Helin, K; Waters, C M


    The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of ...

  5. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L


    Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells...

  6. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J


    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signal...

  7. Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin

    DEFF Research Database (Denmark)

    Yahiro, Kinnosuke; Wada, Akihiro; Yamasaki, Eiki


    Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused...

  8. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.


    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  9. Application of computational approaches to study signalling networks of nuclear and Tyrosine kinase receptors

    Directory of Open Access Journals (Sweden)

    Rebaï Ahmed


    Full Text Available Abstract Background Nuclear receptors (NRs and Receptor tyrosine kinases (RTKs are essential proteins in many cellular processes and sequence variations in their genes have been reported to be involved in many diseases including cancer. Although crosstalk between RTK and NR signalling and their contribution to the development of endocrine regulated cancers have been areas of intense investigation, the direct coupling of their signalling pathways remains elusive. In our understanding of the role and function of nuclear receptors on the cell membrane the interactions between nuclear receptors and tyrosine kinase receptors deserve further attention. Results We constructed a human signalling network containing nuclear receptors and tyrosine kinase receptors that identified a network topology involving eleven highly connected hubs. We further developed an integrated knowledge database, denominated NR-RTK database dedicated to human RTKs and NRs and their vertebrate orthologs and their interactions. These interactions were inferred using computational tools and those supported by literature evidence are indicated. NR-RTK database contains links to other relevant resources and includes data on receptor ligands. It aims to provide a comprehensive interaction map that identifies complex dynamics and potential crosstalk involved. Availability: NR-RTK database is accessible at Conclusions We infer that the NR-RTK interaction network is scale-free topology. We also uncovered the key receptors mediating the signal transduction between these two types of receptors. Furthermore, NR-RTK database is expected to be useful for researchers working on various aspects of the molecular basis of signal transduction by RTKs and NRs. Reviewers This article was reviewed by Professor Paul Harrison (nominated by Dr. Mark Gerstein, Dr. Arcady Mushegian and Dr. Anthony Almudevar.

  10. Tyrosine Kinase Ligand-Receptor Pair Prediction by Using Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Masayuki Yarimizu


    Full Text Available Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs, and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

  11. N-linked glycosylation supports cross-talk between receptor tyrosine kinases and androgen receptor.

    Directory of Open Access Journals (Sweden)

    Harri M Itkonen

    Full Text Available Prostate cancer is the second most common cause of cancer-associated deaths in men and signalling via a transcription factor called androgen receptor (AR is an important driver of the disease. Androgen treatment is known to affect the expression and activity of other oncogenes including receptor tyrosine kinases (RTKs. In this study we report that AR-positive prostate cancer cell-lines express 50% higher levels of enzymes in the hexosamine biosynthesis pathway (HBP than AR-negative prostate cell-lines. HBP produces hexosamines that are used by endoplasmic reticulum and golgi enzymes to glycosylate proteins targeted to plasma-membrane and secretion. Inhibition of O-linked glycosylation by ST045849 or N-linked glycosylation with tunicamycin decreased cell viability by 20%. In addition, tunicamycin inhibited the androgen-induced expression of AR target genes KLK3 and CaMKK2 by 50%. RTKs have been shown to enhance AR activity and we used an antibody array to identify changes in the phosphorylation status of RTKs in response to androgen stimulation. Hormone treatment increased the activity of Insulin like Growth Factor 1-Receptor (IGF-1R ten-fold and this was associated with a concomitant increase in the N-linked glycosylation of the receptor, analyzed by lectin enrichment experiments. Glycosylation is known to be important for the processing and stability of RTKs. Inhibition of N-linked glycosylation resulted in accumulation of IGF-1R pro-receptor with altered mobility as shown by immunoprecipitation. Confocal imaging revealed that androgen induced plasma-membrane localization of IGF-1R was blocked by tunicamycin. In conclusion we have established that the glycosylation of IGF-1R is necessary for the full activation of the receptor in response to androgen treatment and that perturbing this process can break the feedback loop between AR and IGF-1R activation in prostate cells. Achieving similar results selectively in a clinical setting will be an

  12. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E;


    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane......The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  13. PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-d-aspartate receptor subunit NR2A



    Fyn, a member of the Src-family protein-tyrosine kinase (PTK), is implicated in learning and memory that involves N-methyl-d-aspartate (NMDA) receptor function. In this study, we examined how Fyn participates in synaptic plasticity by analyzing the physical and functional interaction between Fyn and NMDA receptors. Results showed that tyrosine phosphorylation of NR2A, one of the NMDA receptor subunits, was reduced in fyn-mutant mice. NR2A was tyrosine-phosphorylated in 293T cells when coexpre...

  14. Elucidation of a four-site allosteric network in fibroblast growth factor receptor tyrosine kinases (United States)

    Chen, Huaibin; Marsiglia, William M; Cho, Min-Kyu; Huang, Zhifeng; Deng, Jingjing; Blais, Steven P; Gai, Weiming; Bhattacharya, Shibani; Neubert, Thomas A; Traaseth, Nathaniel J; Mohammadi, Moosa


    Receptor tyrosine kinase (RTK) signaling is tightly regulated by protein allostery within the intracellular tyrosine kinase domains. Yet the molecular determinants of allosteric connectivity in tyrosine kinase domain are incompletely understood. By means of structural (X-ray and NMR) and functional characterization of pathogenic gain-of-function mutations affecting the FGF receptor (FGFR) tyrosine kinase domain, we elucidated a long-distance allosteric network composed of four interconnected sites termed the ‘molecular brake’, ‘DFG latch’, ‘A-loop plug’, and ‘αC tether’. The first three sites repress the kinase from adopting an active conformation, whereas the αC tether promotes the active conformation. The skewed design of this four-site allosteric network imposes tight autoinhibition and accounts for the incomplete mimicry of the activated conformation by pathogenic mutations targeting a single site. Based on the structural similarity shared among RTKs, we propose that this allosteric model for FGFR kinases is applicable to other RTKs. DOI: PMID:28166054

  15. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

    DEFF Research Database (Denmark)

    Pandey, A.; Blagoev, B.; Kratchmarova, I.;


    We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling mo...

  16. Loss of Bmx Non-Receptor Tyrosine Kinase Prevents Pressure Overload-Induced Cardiac Hypertrophy



    Bmx non-receptor tyrosine kinase has an established role in endothelial and lymphocyte signaling, however its role in the heart is unknown. To determine whether Bmx participates in cardiac growth, we subjected mice deficient in the molecule (Bmx KO mice) to transverse aortic constriction (TAC). In comparison to WT mice, which progressively developed massive hypertrophy following TAC, Bmx KO mice were resistant to TAC-induced cardiac growth at the organ and cell level. Loss of Bmx preserved ca...

  17. Receptor tyrosine kinase signaling regulates replication of the peste des petits ruminants virus. (United States)

    Chaudhary, K; Chaubey, K K; Singh, S V; Kumar, N


    In this study, we found out that blocking the receptor tyrosine kinase (RTK) signaling in Vero cells by tryphostin AG879 impairs the in vitro replication of the peste des petits ruminants virus (PPRV). A reduced virus replication in Trk1-knockdown (siRNA) Vero cells confirmed the essential role of RTK in the virus replication, in particular a specific regulation of viral RNA synthesis. These data represent the first evidence that the RTK signaling regulates replication of a morbillivirus.

  18. Tyrosine Kinase Domain Gene Polymorphism of Epidermal Growth Factor Receptor in Gastric Cancer in Northern Iran

    Directory of Open Access Journals (Sweden)

    Jeivad F


    Full Text Available Background: Gastric cancer is one of the most common diseases of digestive system with a low 5-year survival rate and metastasis is the main cause of death. Multi-factors, such as changes in molecular pathways and deregulation of cells are involved in the disease development. Epidermal growth factor receptor pathway (EGFR which is associated with cell proliferation and survival can influence cancer development. EGFR function is governed by its genetic polymorphism; thus, we aimed to study the tyrosine kinase domain gene mutations of the receptor in patients with gastric cancer.Methods : In this experimental study, 123 subjects (83 patients with gastric cancer and 40 normal subjects were investigated in north of Iran for EGFR gene polymorphisms during 1 year. Genomic DNA was extracted by DNA extraction kit according to the manufacture's protocol. Polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP and silver staining were performed for investigating EGFR gene polymorphisms. Results : The participants included 72 men and 44 women. Gene polymorphism in exon 18 was present in 10% of the study population but SSCP pattern in exon 19 did not show different migrate bands neither in patients nor in normal subjects.Conclusion: It seems that screening for tyrosine kinas gene polymorphism of epidermal growth factor receptor in patients with gastric cancer and use of tyrosine kinas inhibitors could be useful in the prevention of disease progress and improvement of treatment process for a better quality of life in these patients.

  19. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain (United States)

    In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct from one another, and thus while most animal receptor kinases are tyrosin...

  20. Role of receptor tyrosine kinases in gastric cancer: New targets for a selective therapy

    Institute of Scientific and Technical Information of China (English)

    JC Becker; C Müller-Tidow; H Serve; W Domschke; T Pohle


    Receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor family participate in several steps of tumor formation including proliferation and metastatic spread. Several known RTKs are upregulated in gastric cancer being prime targets of a tailored therapy. Only preliminary data exist, however, on the use of the currently clinically available drugs such as trastuzumab,cetuximab, bevacizumab, gefitinib, erlotinib, and imatinib in the setting of gastric cancer. Preclinical data suggest a potential benefit of their use, especially in combination with "conventional" cytostatic therapy. This review summarizes the current knowledge about their use in cancer therapy as well as new approaches and drugs to optimize treatment success.

  1. Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

    Directory of Open Access Journals (Sweden)

    Fernanda Ledda


    Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

  2. Receptor-Tyrosine-Kinase-Targeted Therapies for Head and Neck Cancer

    Directory of Open Access Journals (Sweden)

    Lisa A. Elferink


    Full Text Available Molecular therapeutics for treating epidermal growth factor receptor-(EGFR- expressing cancers are a specific method for treating cancers compared to general cell loss with standard cytotoxic therapeutics. However, the finding that resistance to such therapy is common in clinical trials now dampens the initial enthusiasm over this targeted treatment. Yet an improved molecular understanding of other receptor tyrosine kinases known to be active in cancer has revealed a rich network of cross-talk between receptor pathways with a key finding of common downstream signaling pathways. Such cross talk may represent a key mechanism for resistance to EGFR-directed therapy. Here we review the interplay between EGFR and Met and the type 1 insulin-like growth factor receptor (IGF-1R tyrosine kinases, as well as their contribution to anti-EGFR therapeutic resistance in the context of squamous cell cancer of the head and neck, a tumor known to be primarily driven by EGFR-related oncogenic signals.

  3. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity. (United States)

    Lee, S K; Su, B; Maher, S E; Bothwell, A L


    To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity.

  4. Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

    Directory of Open Access Journals (Sweden)

    Hadas Smadar


    Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

  5. Ligand-based receptor tyrosine kinase partial agonists: New paradigm for cancer drug discovery? (United States)

    Riese, David J.


    Introduction Receptor tyrosine kinases (RTKs) are validated targets for oncology drug discovery and several RTK antagonists have been approved for the treatment of human malignancies. Nonetheless, the discovery and development of RTK antagonists has lagged behind the discovery and development of agents that target G-protein coupled receptors. In part, this is because it has been difficult to discover analogs of naturally-occurring RTK agonists that function as antagonists. Areas covered Here we describe ligands of ErbB receptors that function as partial agonists for these receptors, thereby enabling these ligands to antagonize the activity of full agonists for these receptors. We provide insights into the mechanisms by which these ligands function as antagonists. We discuss how information concerning these mechanisms can be translated into screens for novel small molecule- and antibody-based antagonists of ErbB receptors and how such antagonists hold great potential as targeted cancer chemotherapeutics. Expert opinion While there have been a number of important key findings into this field, the identification of the structural basis of ligand functional specificity is still of the greatest importance. While it is true that, with some notable exceptions, peptide hormones and growth factors have not proven to be good platforms for oncology drug discovery; addressing the fundamental issues of antagonistic partial agonists for receptor tyrosine kinases has the potential to steer oncology drug discovery in new directions. Mechanism based approaches are now emerging to enable the discovery of RTK partial agonists that may antagonize both agonist-dependent and –independent RTK signaling and may hold tremendous promise as targeted cancer chemotherapeutics. PMID:21532939

  6. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen


    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  7. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J;


    The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphor...

  8. Receptor Tyrosine Kinase EphB4 Is a Survival Factor in Breast Cancer


    Kumar, S. Ram; Singh, Jasbir; Xia, Guangbin; Krasnoperov, Valery; Hassanieh, Loubna; Ley, Eric J.; Scehnet, Jeffrey; Kumar, Neil G.; Hawes, Debra; Press, Michael F.; Weaver, Fred A.; Gill, Parkash S.


    EphB4, a member of the largest family of receptor tyrosine kinases, is normally expressed on endothelial and neuronal cells. Although aberrant expression of EphB4 has been reported in several human tumors, including breast cancer, its functional significance is not understood. We report here that EphB4 is expressed in 7 of 12 (58%) human breast cancer specimens and 4 of 4 (100%) breast tumor cell lines examined. Overexpression of EphB4 in breast cancer cells was driven by gene amplification a...

  9. The Receptor Tyrosine Kinase Mertk Regulates Dendritic Cell Production of BAFF


    Gohlke, P.R.; Williams, J. C.; Vilen, B J; Dillon, S.R.; Tisch, R; Matsushima, G.K.


    The MerTK receptor tyrosine kinase is an important negative regulator of dendritic cell function and is required to prevent B cell autoimmunity in vivo. It is not currently known however, if any causal relationship exists between these two aspects of MerTK function. We sought to determine if dendritic cells from mice lacking MerTK (mertk−/− mice) have characteristics that may aid in the development of B cell autoimmunity. Specifically, we found that mertk−/− mice contain an elevated number of...

  10. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor (United States)

    Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie


    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered “off-target” effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  11. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor. (United States)

    Lotz-Jenne, Carina; Lüthi, Urs; Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie


    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo.

  12. Identification and regulation of receptor tyrosine kinases Rse and Mer and their ligand Gas6 in testicular somatic cells. (United States)

    Chan, M C; Mather, J P; McCray, G; Lee, W M


    Receptor tyrosine kinases act to convey extracellular signals to intracellular signaling pathways and ultimately control cell proliferation and differentiation. Rse, Axl, and Mer belong to a newly identified family of cell adhesion molecule-related receptor tyrosine kinase. They bind the vitamin K-dependent protein growth arrest-specific gene 6 (Gas6), which is also structurally related to the anticoagulation factor Protein S. The aim of this study is to investigate the possible role of Rse/Axl/Mer tyrosine kinase receptors and their ligand in regulating testicular functions. Gene expression of Rse, Axl, Mer, and Gas6 in the testis was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis. The results indicated that receptors Rse and Mer and the ligand Gas6 were expressed in the rat endothelial cell line (TR1), mouse Leydig cell line (TM3), rat peritubular myoid cell line (TRM), mouse Sertoli cell line (TM4), and primary rat Sertoli cells. Axl was not expressed in the testicular somatic cells by RT-PCR or Northern blot analysis. The highest level of expression of Gas6 messenger RNA (mRNA) was observed in the Sertoli cells, and its expression was responsive to the addition of forskolin in vitro. The effects of serum, insulin, and transferrin on Gas6 expression by TM4 cells were examined. It was shown that they all exhibited an up-regulating effect on Gas6 expression. The forskolin-stimulated Gas6 expression was accompanied by an increase in tyrosine phosphorylation of the Rse receptor in vitro, suggesting that Gas6 may exhibit an autocrine effect in the Sertoli cells through multiple tyrosine kinase receptors. Our studies so far have demonstrated that tyrosine kinase receptors Rse and Mer and their ligand Gas6 are widely expressed in the testicular somatic cell lines and may play a marked role in promoting testicular cell survival.

  13. Preparation and Characterization of {sup 177}Lu Labeled Antibody against Tyrosine Kinase Receptor Her2

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Hong, Young-Don; Choi, Sun-Ju [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)


    The tyrosine kinase receptor Her2, also known in humans as erbB2, is a member of the epidermal growth factor receptor (EGFR or erbB1) family. The Her2 is highly expressed in many cancer types and over expressed in approximately 30% of all primary breast cancer. Overexpression of Her2 is associated with a poor prognosis. Her2 is a suitable target because it involves an extracellular domain that can be targeted by antibodies produced by B cells. Based on these advantages, we tried to prepare the {sup 177}Lu labeled Her2 antibody. This radioimmunoconjugate could act by not only blocking the Her2 signalling pathway using antibody but also killing the tumour cell using {beta} energy of {sup 177}Lu.

  14. Effects of 4 multitargeted receptor tyrosine kinase inhibitors on regional hemodynamics in conscious, freely moving rats (United States)

    Carter, Joanne J.; Fretwell, Laurice V.; Woolard, Jeanette


    VEGF inhibitors, including receptor tyrosine kinase inhibitors, are used as adjunct therapies in a number of cancer treatments. An emerging issue with these drugs is that most cause hypertension. To gain insight into the physiological mechanisms involved, we evaluated their regional hemodynamic effects in conscious rats. Male Sprague Dawley rats (350–450 g) were chronically implanted with pulsed Doppler flow probes (renal and mesenteric arteries, and the descending abdominal aorta) and catheters (jugular vein, peritoneal cavity, and distal abdominal aorta). Regional hemodynamics were measured over 4 d, before and after daily administration of cediranib (3 and 6 mg/kg, 3 and 6 mg/kg/h for 1 h, i.v.), sorafenib (10 and 20 mg/kg, 10 and 20 mg kg/h for 1 h, i.v.), pazopanib (30 and100 mg/kg, i.p.), or vandetanib (12.5 and 25 mg/kg, i.p.). All drugs evoked significant increases (P phentolamine and propranolol (each 1 mg/kg/h), suggesting a need for new strategies to overcome them.—Carter, J. J., Fretwell, L. V., Woolard, J. Effects of 4 multitargeted receptor tyrosine kinase inhibitors on regional hemodynamics in conscious, freely moving rats. PMID:27986807

  15. Multiple Functions of Let-23, a Caenorhabditis Elegans Receptor Tyrosine Kinase Gene Required for Vulval Induction (United States)

    Aroian, R. V.; Sternberg, P. W.


    The let-23 gene, which encodes a putative tyrosine kinase of the epidermal growth factor (EGF) receptor subfamily, has multiple functions during Caenorhabditis elegans development. We show that let-23 function is required for vulval precursor cells (VPCs) to respond to the signal that induces vulval differentiation: a complete loss of let-23 function results in no induction. However, some let-23 mutations that genetically reduce but do not eliminate let-23 function result in VPCs apparently hypersensitive to inductive signal: as many as five of six VPCs can adopt vulval fates, in contrast to the three that normally do. These results suggest that the let-23 receptor tyrosine kinase controls two opposing pathways, one that stimulates vulval differentiation and another that negatively regulates vulval differentiation. Furthermore, analysis of 16 new let-23 mutations indicates that the let-23 kinase functions in at least five tissues. Since various let-23 mutant phenotypes can be obtained independently, the let-23 gene is likely to have tissue-specific functions. PMID:2071015

  16. [Receptor tyrosine kinase KIT may regulate expression of genes involved in spontaneous regression of neuroblastoma]. (United States)

    Lebedev, T D; Spirin, P V; Suntsova, M V; Ivanova, A V; Buzdin, A A; Prokofjeva, M M; Rubtsov, P M; Prassolov, V S


    Hallmark of neuroblastoma is an ability of this malignant tumor to undergo spontaneous regression or differentiation into benign tumor during any stage of the disease, but it is little known about mechanisms of these phenomena. We studied effect of receptor tyrosine kinase receptor KIT on expression of genes, which may be involved in tumor spontaneous regression. Downregulation of KIT expression by RNA interference in SH-SY5Y cells causes suppression of neurotrophin receptor NGFR expression that may promote the loss of sensibility of cells to nerve growth factors, also it causes upregulation of TrkA receptor expression which can stimulate cell differentiation or apoptosis in NGF dependent manner. Furthermore there is an upregulation of genes which stimulate malignant cell detection by immune system, such as genes of major histocompatibility complex HLA class I HLA-B and HLA-C, and interferon-γ receptors IFNGR1 and IFNGR2 genes. Thus KIT can mediate neuroblastoma cell sensibility to neurotrophins and immune system components--two factors directly contributing to spontaneous regression of neuroblastoma.

  17. The biological activity of the human epidermal growth factor receptor is positively regulated by its C-terminal tyrosines

    DEFF Research Database (Denmark)

    Helin, K; Velu, T; Martin, P;


    , growth in agar and growth in low serum, mutant receptors display a similar hierarchy of activity. The lower activity is intrinsic in the mutants since they are expressed at similar level as the wild type and bind EGF with similar affinity. Deletion mutants lacking the last 19 or 63 amino acids (Velu et......The epidermal growth factor receptor (EGF-R) C-terminus contains three conserved tyrosines (Y-1068, Y-1148, Y-1173) which are phosphorylated upon EGF activation. To clarify the functional role of these tyrosines, each has been mutated to phenylalanine and studied as single, double and triple...

  18. Interleukin 7 receptor functions by recruiting the tyrosine kinase p59fyn through a segment of its cytoplasmic tail.



    Engagement of the cell surface receptor for interleukin 7 (IL-7R) provokes protein tyrosine phosphorylation, although the receptor lacks a kinase catalytic domain in its cytoplasmic tail. The molecular basis of this response is not known. Here we report that the IL-7R functions by recruiting p59fyn, an intracellular tyrosine kinase of the src family. Treatment of pre-B cells with IL-7 causes an enhancement of the catalytic activity of p59fyn, but not of the related kinase p62yes. IL-7-depende...

  19. Protein tyrosine phosphatase receptor type z negatively regulates oligodendrocyte differentiation and myelination.

    Directory of Open Access Journals (Sweden)

    Kazuya Kuboyama

    Full Text Available BACKGROUND: Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz. METHODOLOGY/PRINCIPAL FINDINGS: We found an early onset of the expression of myelin basic protein (MBP, a major protein of the myelin sheath, and early initiation of myelination in vivo during development of the Ptprz-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from Ptprz-deficient mice than wild-type mice. Furthermore, adult Ptprz-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in Ptprz-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis. CONCLUSIONS/SIGNIFICANCE: Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.

  20. Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling. (United States)

    Kazi, Julhash U; Rönnstrand, Lars


    Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  1. Src-Like adaptor protein (SLAP binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Directory of Open Access Journals (Sweden)

    Julhash U Kazi

    Full Text Available Fms-like tyrosine kinase 3 (Flt3 is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML. Src-like adaptor protein (SLAP is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  2. Small-Molecule Inhibitors of the Receptor Tyrosine Kinases: Promising Tools for Targeted Cancer Therapies

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi


    Full Text Available Chemotherapeutic and cytotoxic drugs are widely used in the treatment of cancer. In spite of the improvements in the life quality of patients, their effectiveness is compromised by several disadvantages. This represents a demand for developing new effective strategies with focusing on tumor cells and minimum side effects. Targeted cancer therapies and personalized medicine have been defined as a new type of emerging treatments. Small molecule inhibitors (SMIs are among the most effective drugs for targeted cancer therapy. The growing number of approved SMIs of receptor tyrosine kinases (RTKs i.e., tyrosine kinase inhibitors (TKIs in the clinical oncology imply the increasing attention and application of these therapeutic tools. Most of the current approved RTK–TKIs in preclinical and clinical settings are multi-targeted inhibitors with several side effects. Only a few specific/selective RTK–TKIs have been developed for the treatment of cancer patients. Specific/selective RTK–TKIs have shown less deleterious effects compared to multi-targeted inhibitors. This review intends to highlight the importance of specific/selective TKIs for future development with less side effects and more manageable agents. This article provides an overview of: (1 the characteristics and function of RTKs and TKIs; (2 the recent advances in the improvement of specific/selective RTK–TKIs in preclinical or clinical settings; and (3 emerging RTKs for targeted cancer therapies by TKIs.

  3. Targeting FMS-related tyrosine kinase receptor 3 with the human immunoglobulin G1 monoclonal antibody IMC-EB10. (United States)

    Youssoufian, Hagop; Rowinsky, Eric K; Tonra, James; Li, Yiwen


    FMS-related tyrosine kinase receptor 3 (FLT3) is a class III receptor tyrosine kinase that holds considerable promise as a therapeutic target in hematologic malignancies. Current efforts directed toward the development of small-molecule tyrosine kinase inhibitors of FLT3 may be limited by off-target toxicities and the development of drug resistance. Target-specific antibodies could overcome these hurdles and provide additional mechanisms to enhance the antitumor efficacy of FLT3 inhibitors. IMC-EB10 is a novel antibody directed against FLT3. The binding of IMC-EB10 to FLT3 results in antiproliferative effects in vitro and in mouse models engrafted with human leukemia cells that harbor wild-type or constitutively activated FLT3. Future clinical trials will test these notions formally and will identify the most appropriate opportunities for this member of a new generation of antileukemic therapies.

  4. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity (United States)

    Chan, Pamela Y.; Carrera Silva, Eugenio A.; De Kouchkovsky, Dimitri; Joannas, Leonel D.; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S.; Herbert, De’Broski R.; Craft, Joseph E.; Flavell, Richard A.; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G.; Torgerson, Dara G.; Ghosh, Sourav; Rothlin, Carla V.


    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded by Tyro3 in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell–specific Pros1 knockouts phenocopied the loss of Tyro3. Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  5. Greater Sensitivity of Blood Pressure Than Renal Toxicity to Tyrosine Kinase Receptor Inhibition With Sunitinib

    DEFF Research Database (Denmark)

    Lankhorst, Stephanie; Baelde, Hans J; Kappers, Mariëtte H W


    of these side effects. Normotensive Wistar Kyoto rats were exposed to 3 different doses of sunitinib or vehicle. After 8 days, rats were euthanized. Telemetrically measured blood pressure rose dose dependently, from 13 to 30 mm Hg. Proteinuria was present at all doses, but a rise in cystatin C occurred only...... histological abnormalities with the low dose. Podocyte number per glomerular circumference did not change. Glomerular nephrin, Neph1, podocin, and endothelin-converting enzyme gene expression were downregulated in a dose-dependent manner. We conclude that the sunitinib-induced rise in blood pressure requires......Hypertension and renal injury are off-target effects of sunitinib, a tyrosine kinase receptor inhibitor used for the treatment of various tumor types. Importantly, these untoward effects are accompanied by activation of the endothelin system. Here, we set up a study to explore the dose dependency...

  6. Non-radioisotopic method for the in vitro measurement of EGF receptor tyrosine kinase

    Institute of Scientific and Technical Information of China (English)


    A non-radioisotopic method was developed for the assay ofepidermal growth factor receptor (EGFR). A peptide with twenty amino acid residues around Tyr 1173, the major phosphorylation site of EGFR, was cloned as a GST fusion protein and used as substrate. Anti-phosphotyrosine monoclonal antibody PY99 was used for the determination of the extent of phosphorylation. Both the specificity and the sensitivity were substantially higher than that of the existing method. Km value of the fusion protein is much lower (10 (mol/L) than that of the synthetic peptide (110 (mol/L). The method can be applied to the measurement of the tyrosine kinase activity of c-erb B2 (Neu/HER2).

  7. The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer. (United States)

    Graham, Douglas K; DeRyckere, Deborah; Davies, Kurtis D; Earp, H Shelton


    The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in multiple haematological and epithelial malignancies. Rather than functioning as oncogenic drivers, their induction in tumour cells predominately promotes survival, chemoresistance and motility. The unique mode of maximal activation of this RTK family requires an extracellular lipid–protein complex. For example, the protein ligand, growth arrest-specific protein 6 (GAS6), binds to phosphatidylserine (PtdSer) that is externalized on apoptotic cell membranes, which activates MERTK on macrophages. This triggers engulfment of apoptotic material and subsequent anti-inflammatory macrophage polarization. In tumours, autocrine and paracrine ligands and apoptotic cells are abundant, which provide a survival signal to the tumour cell and favour an anti-inflammatory, immunosuppressive microenvironment. Thus, TAM kinase inhibition could stimulate antitumour immunity, reduce tumour cell survival, enhance chemosensitivity and diminish metastatic potential.

  8. The C. elegans ROR receptor tyrosine kinase, CAM-1, non-autonomously inhibits the Wnt pathway. (United States)

    Green, Jennifer L; Inoue, Takao; Sternberg, Paul W


    Inhibitors of Wnt signaling promote normal development and prevent cancer by restraining when and where the Wnt pathway is activated. ROR proteins, a class of Wnt-binding receptor tyrosine kinases, inhibit Wnt signaling by an unknown mechanism. To clarify how RORs inhibit the Wnt pathway, we examined the relationship between Wnts and the sole C. elegans ROR homolog, cam-1, during C. elegans vulval development, a Wnt-regulated process. We found that loss and overexpression of cam-1 causes reciprocal defects in Wnt-mediated cell-fate specification. Our molecular and genetic analyses revealed that the CAM-1 extracellular domain (ECD) is sufficient to non-autonomously antagonize multiple Wnts, suggesting that the CAM-1/ROR ECD sequesters Wnts. A sequestration model is supported by our findings that the CAM-1 ECD binds to several Wnts in vitro. These results demonstrate how ROR proteins help to refine the spatial pattern of Wnt activity in a complex multicellular environment.


    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  10. Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates. (United States)

    Brunet, Frédéric G; Volff, Jean-Nicolas; Schartl, Manfred


    The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling.

  11. Extracellular domain determinants of LET-23 (EGF) receptor tyrosine kinase activity in Caenorhabditis elegans. (United States)

    Moghal, Nadeem; Sternberg, Paul W


    Negative regulation of ErbB/EGFR signalling pathways is important for normal development and the prevention of cancer. In a genetic screen to uncover mechanisms that negatively regulate ErbB signalling in Caenorhabditis elegans, we isolated a second-site mutation (sy621) that promotes the activity of a gain-of-function allele (sa62gf) of the let-23 (EGF) receptor tyrosine kinase. We show that activation by the sa62 mutation (C359Y) likely results from a break in the conserved disulphide-bonded eighth module at the junction of CR1 and L2. The sy621 mutation causes a G270E change in the third disulphide-bonded module of CR1, and causes no phenotype on its own, but cooperates with the sa62 mutation to promote receptor activity. Although both sa62 single- and double-mutant receptors can function in the absence of ligand, they can be further activated by ligand. Our results support the current model for ligand-induced dimerization based on the recent crystal structures of HER3 and the EGFR, and provide more evidence for the generation of distinctly activated ErbB family members through mutation.

  12. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

    Directory of Open Access Journals (Sweden)

    Man-Ho eOh


    Full Text Available In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct and thus while most animal receptor kinases are tyrosine kinases the plant receptor kinases are classified as serine/threonine kinases. One of the best studied plant receptor kinases is BRASSINOSTEROID INSENSITIVE 1 (BRI1, which functions in brassinosteroid (BR signaling. Consistent with its classification, BRI1 was shown in early studies to autophosphorylate in vitro exclusively on serine and threonine residues and subsequently numerous specific phosphoserine and phosphothreonine sites were identified. However, several sites of tyrosine autophosphorylation have recently been identified establishing that BRI1 is a dual-specificity kinase. This raises the paradox that BRI1 contains phosphotyrosine but was only observed to autophosphorylate on serine and threonine sites. In the present study, we demonstrate that autophosphorylation on threonine and tyrosine (and presumably serine residues is a post-translational modification, ruling out a co-translational mechanism that could explain the paradox. Moreover, we show that in general, autophosphorylation of the recombinant protein appears to be hierarchal and proceeds in the order: phosphoserine > phosphothreonine > phosphotyrosine. This may explain why tyrosine autophosphorylation was not observed in some studies. Finally, we also show that the juxtamembrane domain of BRI1 is an activator of the kinase domain, and that kinase specificity (serine/threonine versus tyrosine can be affected by residues outside of the kinase domain. This may have implications for identification of signature motifs that distinguish serine/threonine kinases from dual-specificity kinases.

  13. p59fyn tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain.



    Several lines of evidence link the protein tyrosine kinase p59fyn to the T-cell receptor. The molecular basis of this interaction has not been established. Here we show that the tyrosine kinase p59fyn can associate with chimeric proteins that contain the cytoplasmic domains of CD3 epsilon, gamma, zeta (zeta), and eta. Mutational analysis of the zeta cytoplasmic domain demonstrated that the membrane-proximal 41 residues of zeta are sufficient for p59fyn binding and that at least two p59fyn bin...

  14. Role of ErbB family receptor tyrosine kinases in intrahepatic cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)


    Aberrant expression and signaling of epidermal growth factor receptor (ErbB) family receptor tyrosine kinases, most notably that of ErbB2 and ErbB1, have been implicated in the molecular pathogenesis of intrahepatic cholangiocarcinoma. Constitutive overexpression of ErbB2 and/or ErbB1 in malignant cholangiocytes has raised interest in the possibility that agents which selectively target these receptors could potentially be effective in cholangiocarcinoma therapy. However, current experience with such ErbB-directed therapies have at best produced only modest responses in patients with biliary tract cancers. This review provides a comprehensive and critical analysis of both preclinical and clinical studies aimed at assessing the role of altered ErbB2 and/or ErbB1 expression, genetic modifications, and dysregulated signaling on cholangiocarcinoma development and progression. Specific limitations in experimental approaches that have been used to assess human cholangiocarcinoma specimens for ErbB2 and/or ErbB1 overexpression and gene amplification are discussed. In addition, current rodent models of intrahepatic cholangiocarcinogenesis associated with constitutive ErbB2 overexpression are reviewed. Select interactive relationships between ErbB2 or ErbB1 with other relevant molecular signaling pathways associated with intrahepatic cholangiocarcinoma development and progression are also detailed, including those linking ErbB receptors to bile acid, cyclooxygenase-2, interleukin-6/gp130, transmembrane mucins, hepatocyte growth factor/Met, and vascular endothelial growth factor signaling. Lastly, various factors that can limit therapeutic efficacy of ErbB-targeted agents against cholangiocarcinoma are considered.

  15. Bmx tyrosine kinase has a redundant function downstream of angiopoietin and vascular endothelial growth factor receptors in arterial endothelium. (United States)

    Rajantie, I; Ekman, N; Iljin, K; Arighi, E; Gunji, Y; Kaukonen, J; Palotie, A; Dewerchin, M; Carmeliet, P; Alitalo, K


    The Bmx gene, a member of the Tec tyrosine kinase gene family, is known to be expressed in subsets of hematopoietic and endothelial cells. In this study, mice were generated in which the first coding exon of the Bmx gene was replaced with the lacZ reporter gene by a knock-in strategy. The homozygous mice lacking Bmx activity were fertile and had a normal life span without an obvious phenotype. Staining of their tissues using beta-galactosidase substrate to assess the sites of Bmx expression revealed strong signals in the endothelial cells of large arteries and in the endocardium starting between days 10.5 and 12.5 of embryogenesis and continuing in adult mice, while the venular endothelium showed a weak signal only in the superior and inferior venae cavae. Of the five known endothelial receptor tyrosine kinases tested, activated Tie-2 induced tyrosyl phosphorylation of the Bmx protein and both Tie-2 and vascular endothelial growth factor receptor 1 (VEGFR-1) stimulated Bmx tyrosine kinase activity. Thus, the Bmx tyrosine kinase has a redundant role in arterial endothelial signal transduction downstream of the Tie-2 and VEGFR-1 growth factor receptors.

  16. Comparative VEGF receptor tyrosine kinase modeling for the development of highly specific inhibitors of tumor angiogenesis. (United States)

    Schmidt, Ulrike; Ahmed, Jessica; Michalsky, Elke; Hoepfner, Michael; Preissner, Robert


    The Vascular Endothelial Growth Factor receptors (VEGF-Rs) play a significant role in tumor development and tumor angiogenesis and are therefore interesting targets in cancer therapy. Targeting the VEGF-R is of special importance as the feed of the tumor has to be reduced. In general, this can be carried out by inhibiting the tyrosine kinase function of the VEGF-R. Nevertheless, there arise some problems with the specificity of known kinase inhibitors: they bind to the ATP-binding site and inhibit a number of kinases, moreover the so far most specific inhibitors act at least on these three major types of VEGF-Rs: Flt-1, Flk-1/KDR, Flt-4. The goal is a selective VEGF-R-2 (Flk-1/KDR) inhibitor, because this receptor triggers rather unspecific signals from VEGF-A, -C, -D and -E. Here, we describe a protocol starting from an established inhibitor (Vatalanib) with 2D-/3D-searching and property filtering of the in silico screening hits and the "negative docking approach". With this approach we were able to identify a compound, which shows a fourfold higher reduction of the proliferation rate of endothelial cells compared to the reduction effect of the lead structure.

  17. Mice lacking Axl and Mer tyrosine kinase receptors are susceptible to experimental autoimmune orchitis induction. (United States)

    Li, Nan; Liu, Zhenghui; Zhang, Yue; Chen, Qiaoyuan; Liu, Peng; Cheng, C Yan; Lee, Will M; Chen, Yongmei; Han, Daishu


    The mammalian testis is an immunoprivileged organ where male germ cell autoantigens are immunologically ignored. Both systemic immune tolerance to autoantigens and local immunosuppressive milieu contribute to the testicular immune privilege. Testicular immunosuppression has been intensively studied, but information on systemic immune tolerance to autoantigens is lacking. In the present study, we aimed to determine the role of Axl and Mer receptor tyrosine kinases in maintaining the systemic tolerance to male germ cell antigens using the experimental autoimmune orchitis (EAO) model. Axl and Mer double-knockout (Axl(-/-)Mer(-/-)) mice developed evident EAO after a single immunization with germ cell homogenates emulsified with complete Freund's adjuvant. EAO was characterized by the accumulation of macrophages and T lymphocytes in the testis. Damage to the seminiferous epithelium was also observed. EAO induction was associated with pro-inflammatory cytokine upregulation in the testes, impaired permeability of the blood-testis barrier and generation of autoantibodies against germ cell antigens in Axl(-/-)Mer(-/-) mice. Immunization also induced mild EAO in Axl or Mer single-gene-knockout mice. By contrast, a single immunization failed to induce EAO in wild-type mice. The results indicate that Axl and Mer receptors cooperatively regulate the systemic immune tolerance to male germ cell antigens.

  18. The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis. (United States)

    Crosier, P S; Freeman, S A; Orlic, D; Bodine, D M; Crosier, K E


    Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

  19. Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2. (United States)

    Chandler, Kevin Brown; Leon, Deborah R; Meyer, Rosana D; Rahimi, Nader; Costello, Catherine E


    Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of (18)O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.

  20. The c-Met receptor tyrosine kinase inhibitor MP470 radiosensitizes glioblastoma cells

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    Bearss David


    Full Text Available Abstract Purpose Glioblastoma multiforme (GBM is resistant to current cytotoxic therapies, in part because of enhanced DNA repair. Activation of the receptor tyrosine kinase c-Met has been shown to protect cancer cells from DNA damage. We hypothesized that inhibiting c-Met would decrease this protection and thus sensitize resistant tumor cells to the effects of radiation therapy. Materials and methods Eight human GBM cell lines were screened for radiosensitivity to the small-molecule c-Met inhibitor MP470 with colony-count assays. Double-strand (ds DNA breaks was quantified by using antibodies to gamma H2AX. Western blotting demonstrate expression of RAD51, glycogen synthase kinase (GSK-3β, and other proteins. A murine xenograft tumor flank model was used for in vivo radiosensitization studies. Results MP470 reduced c-Met phosphorylation and enhanced radiation-induced cell kill by 0.4 logs in SF767 cells. Cells pretreated with MP470 had more ds DNA damage than cells treated with radiation alone. Mechanistically, MP470 was shown to inhibit dsDNA break repair and increase apoptosis. MP470 influences various survival and DNA repair related proteins such as pAKT, RAD51 and GSK3β. In vivo, the addition of MP470 to radiation resulted in a tumor-growth-delay enhancement ratio of 2.9 over radiation alone and extended survival time. Conclusions GBM is a disease site where radiation is often used to address both macroscopic and microscopic disease. Despite attempts at dose escalation outcomes remain poor. MP470, a potent small-molecule tyrosine kinase inhibitor of c-Met, radiosensitized several GBM cell lines both in vitro and in vivo, and may help to improve outcomes for patients with GBM.

  1. The Plasticity of Oncogene Addiction: Implications for Targeted Therapies Directed to Receptor Tyrosine Kinases

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    Vinochani Pillay


    Full Text Available A common mutation of the epidermal growth factor receptor (EGFR in glioblastoma multiforme (GBM is an extracellular truncation known as the de2-7 EGFR (or EGFRvIII. Hepatocyte growth factor (HGF is the ligand for the receptor tyrosine kinase (RTK c-Met, and this signaling axis is often active in GBM. The expression of the HGF/c-Met axis or de2-7 EGFR independently enhances GBMgrowth and invasiveness, particularly through the phosphatidylinositol-3 kinase/pAkt pathway. Using RTK arrays, we show that expression of de2-7 EGFR in U87MG GBM cells leads to the coactivation of several RTKs, including platelet-derived growth factor receptor β and c-Met. A neutralizing antibody to HGF (AMG102 did not inhibit de2-7 EGFR-mediated activation of c-Met, demonstrating that it is ligand-independent. Therapy for parental U87MG xenografts with AMG 102 resulted in significant inhibition of tumor growth, whereas U87MG.Δ2-7 xenografts were profoundly resistant. Treatment of U87MG.Δ2-7 xenografts with panitumumab, an anti-EGFR antibody, only partially inhibited tumor growth as xenografts rapidly reverted to the HGF/c-Met signaling pathway. Cotreatment with panitumumab and AMG 102 prevented this escape leading to significant tumor inhibition through an apoptotic mechanism, consistent with the induction of oncogenic shock. This observation provides a rationale for using panitumumab and AMG 102 in combination for the treatment of GBM patients. These results illustrate that GBM cells can rapidly change the RTK driving their oncogene addiction if the alternate RTK signals through the same downstream pathway. Consequently, inhibition of a dominant oncogene by targeted therapy can alter the hierarchy of RTKs resulting in rapid therapeutic resistance.

  2. Ror family receptor tyrosine kinases regulate the maintenance of neural progenitor cells in the developing neocortex. (United States)

    Endo, Mitsuharu; Doi, Ryosuke; Nishita, Michiru; Minami, Yasuhiro


    The Ror family receptor tyrosine kinases (RTKs), Ror1 and Ror2, have been shown to play crucial roles in developmental morphogenesis by acting as receptors or co-receptors to mediate Wnt5a-induced signaling. Although Ror1, Ror2 and Wnt5a are expressed in the developing brain, little is known about their roles in the neural development. Here we show that Ror1, Ror2 and their ligand Wnt5a are highly expressed in neocortical neural progenitor cells (NPCs). Small interfering RNA (siRNA)-mediated suppression of Ror1, Ror2 or Wnt5a in cultured NPCs isolated from embryonic neocortex results in the reduction of βIII-tubulin-positive neurons that are produced from NPCs possibly through the generation of T-box brain 2 (Tbr2)-positive intermediate progenitors. BrdU-labeling experiments further reveal that the proportion of proliferative and neurogenic NPCs, which are positive for neural progenitor cell marker (Pax6) but negative for glial cell marker (glial fibrillary acidic protein; GFAP), is reduced within a few days in culture following knockdown of these molecules, suggesting that Ror1, Ror2 and Wnt5a regulate neurogenesis through the maintenance of NPCs. Moreover, we show that Dishevelled 2 (Dvl2) is involved in Wnt5a-Ror1 and Wnt5a-Ror2 signaling in NPCs, and that suppressed expression of Dvl2 indeed reduces the proportion of proliferative and neurogenic NPCs. Interestingly, suppressed expression of either Ror1 or Ror2 in NPCs in the developing neocortex results in the precocious differentiation of NPCs into neurons, and their forced expression results in delayed differentiation. Collectively, these results indicate that Wnt5a-Ror1 and Wnt5a-Ror2 signaling pathways play roles in maintaining proliferative and neurogenic NPCs during neurogenesis of the developing neocortex.

  3. The receptor tyrosine kinase FGFR4 negatively regulates NF-kappaB signaling.

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    Kristine A Drafahl

    Full Text Available BACKGROUND: NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs, including the Fibroblast Growth Factor Receptors (FGFRs are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit, an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling. CONCLUSIONS/SIGNIFICANCE: These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling

  4. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity, and growth regulation by IL-9. (United States)

    Demoulin, J B; Uyttenhove, C; Van Roost, E; DeLestré, B; Donckers, D; Van Snick, J; Renauld, J C


    Interleukin-9 (IL-9), a T-cell-derived cytokine, interacts with a specific receptor associated with the IL-2 receptor gamma chain. In this report, we analyze the functional domains of the human IL-9 receptor transfected into mouse lymphoid cell lines. Three different functions were examined: growth stimulation in factor-dependent pro-B Ba/F3 cells, protection against dexamethasone-induced apoptosis, and Ly-6A2 induction in BW5147 lymphoma cells. The results indicated that a single tyrosine, at position 116 in the cytoplasmic domain, was required for all three activities. In addition, we observed that human IL-9 reduced the proliferation rate of transfected BW5147 cells, an effect also dependent on the same tyrosine. This amino acid was necessary for IL-9-mediated tyrosine phosphorylation of the receptor and for STAT activation but not for IRS-2/4PS activation or for JAK1 phosphorylation, which depended on a domain closer to the plasma membrane. We also showed that JAK1 was constitutively associated with the IL-9 receptor. Activated STAT complexes induced by IL-9 were found to contain STAT1, STAT3, and STAT5 transcription factors. Moreover, sequence homologies between human IL-9 receptor tyrosine 116 and tyrosines (of other receptors activating STAT3 and STAT5 were observed. Taken together, these data indicate that a single tyrosine of the IL-9 receptor, required for activation of three different STAT proteins, is necessary for distinct activities of this cytokine, including proliferative responses.

  5. PDGF-BB-mediated activation of p42(MAPK) is independent of PDGF beta-receptor tyrosine phosphorylation. (United States)

    Cartel, N J; Liu, J; Wang, J; Post, M


    Herein, we investigated the activity of mitogen-activated protein kinase (MAPK), a key component of downstream signaling events, which is activated subsequent to platelet-derived growth factor (PDGF)-BB stimulation. Specifically, p42(MAPK) activity peaked 60 min after addition of PDGF-BB, declined thereafter, and was determined not to be a direct or necessary component of glycosaminoglycan (GAG) synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine phosphorylation inhibition of the PDGF beta-receptor (PDGFR-beta) did not abrogate PDGF-BB-induced p42(MAPK) activation or its threonine or tyrosine phosphorylation. A dominant negative cytoplasmic receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a regulator of MAPKK-MAPK interaction and activation, did not inhibit PDGF-BB-induced p42(MAPK) activation nor did a construct expressing PDGFR-beta with cytoplasmic tyrosines mutated to phenylalanine. However, overexpression of a dominant negative PDGFR-beta lacking the cytoplasmic signaling domain abrogated p42(MAPK) activity. These results suggest that PDGF-BB-mediated activation of p42(MAPK) requires the PDGFR-beta but is independent of its tyrosine phosphorylation.

  6. Receptor-type Protein Tyrosine Phosphatase β Regulates Met Phosphorylation and Function in Head and Neck Squamous Cell Carcinoma

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    Yiru Xu


    Full Text Available Head and neck squamous cell carcinoma (HNSCC is the sixth most common cancer and has a high rate of mortality. Emerging evidence indicates that hepatocyte growth factor receptor (or Met pathway plays a pivotal role in HNSCC metastasis and resistance to chemotherapy. Met function is dependent on tyrosine phosphorylation that is under direct control by receptor-type protein tyrosine phosphatase β (RPTP-β. We report here that RPTP-β expression is significantly downregulated in HNSCC cells derived from metastatic tumors compared to subject-matched cells from primary tumors. Knockdown of endogenous RPTP-β in HNSCC cells from primary tumor potentiated Met tyrosine phosphorylation, downstream mitogen-activated protein (MAP kinase pathway activation, cell migration, and invasion. Conversely, restoration of RPTP-β expression in cells from matched metastatic tumor decreased Met tyrosine phosphorylation and downstream functions. Furthermore, we observed that six of eight HNSCC tumors had reduced levels of RPTP-β protein in comparison with normal oral tissues. Collectively, the results demonstrate the importance of RPTP-β in tumor biology of HNSCC through direct dephosphorylation of Met and regulation of downstream signal transduction pathways. Reduced RPTP-β levels, with or without Met overexpression, could promote Met activation in HNSCC tumors.

  7. Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences

    DEFF Research Database (Denmark)

    Su, J; Yang, L T; Sap, J


    Receptor protein-tyrosine phosphatase RPTPalpha is found associated in vivo with the adaptor protein Grb2. Formation of this complex, which contains no detectable levels of Sos, is known to depend on a C-terminal phosphorylated tyrosine residue (Tyr798) in RPTPalpha and on the Src homology (SH) 2...

  8. Interaction of the growth hormone receptor cytoplasmic domain with the JAK2 tyrosine kinase. (United States)

    Frank, S J; Gilliland, G; Kraft, A S; Arnold, C S


    An early step in GH action involves tyrosine phosphorylation of various cellular proteins. Recently, it has been shown in murine preadipocytes that GH promotes the association of its receptor (the GHR) with and the activation of the JAK2 tyrosine kinase. In this study, we confirmed the human (h) GH-induced association of JAK2 with hGHR in IM-9 cells by coimmunoprecipitation experiments using anti-hGHR serum. We further examined the interaction of JAK2 with the GHR cytoplasmic domain by two lines of investigation. For in vitro studies, we assayed by immunoblotting the ability of cell-derived JAK2 to interact with glutathione S-transferase fusion proteins containing elements of the hGHR cytoplasmic domain. A fusion protein containing the entire hGHR cytoplasmic domain (residues 271-620) specifically associated with JAK2 independent of prior stimulation of cells with hGH. This interaction was not dependent on tyrosine phosphorylation of either partner. Mutational analysis of the hGHR cytoplasmic domain component of the fusions indicated that a membrane-proximal 20-residue region that includes the proline-rich box 1 was necessary for the interaction. This region appeared to cooperate with another region(s), largely in the N-terminal one third of the cytoplasmic domain, to promote full interaction with JAK2. For in vivo reconstitution experiments, wild-type (WT) and mutant rabbit GHRs (rGHRs) along with murine JAK2 were expressed by transient transfection in COS-7 cells. rGHR mutations were confined to the cytoplasmic domain and included C-terminal truncations as well as internal deletions of residues 297-406 and 278-292 (the latter contains box 1). All mutant rGHRs were expressed at the cell surface and bound hGH to a degree similar to the WT rGHR. Receptors were tested for their ability to mediate the hGH-induced immunoprecipitability of JAK2 with phosphotyrosine (APT) antibodies. A rGHR truncated to residue 275 [rGHR-(1-275)], which contains only five cytoplasmic

  9. Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

    DEFF Research Database (Denmark)

    Shen, P; Canoll, P D; Sap, J


    Receptor protein tyrosine phosphatases (RPTPs) comprise a family of proteins that feature intracellular phosphatase domains and an ectodomain with putative ligand-binding motifs. Several RPTPs are expressed in the brain, including RPTP-kappa which participates in homophilic cell-cell interactions...... in vitro [Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, J. Sap, Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region, Mol. Cell. Biol. 13...... processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene...

  10. Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Ming-lei Han; Guo-hua Liu; Jin Guo; Shu-juan Yu; Jing Huang


    Retinal ganglion cell (RGC) degeneration is irreversible in glaucoma and tyrosine kinase receptor B (TrkB)-associated signaling pathways have been implicated in the process. In this study, we attempted to examine whether imipramine, a tricyclic antidepressant, may protect hydrogen peroxide (H2O2)-induced RGC degeneration through the activation of the TrkB pathway in RGC-5 cell lines. RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H2O2. Western blot assay showed that in H2O2-damaged RGC-5 cells, imipramine activated TrkB pathways through extracellular signal-regulated protein kinase/TrkB phosphorylation. TUNEL staining assay also demonstrated that imipramine ameliorated H2O2-induced apoptosis in RGC-5 cells. Finally, TrkB-IgG intervention was able to reverse the protective effect of imipramine on H2O2-induced RGC-5 apoptosis. Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the TrkB signaling pathway.

  11. Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

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    Ming-lei Han


    Full Text Available Retinal ganglion cell (RGC degeneration is irreversible in glaucoma and tyrosine kinase receptor B (TrkB-associated signaling pathways have been implicated in the process. In this study, we attempted to examine whether imipramine, a tricyclic antidepressant, may protect hydrogen peroxide (H 2 O 2 -induced RGC degeneration through the activation of the TrkB pathway in RGC-5 cell lines. RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H 2 O 2 . Western blot assay showed that in H 2 O 2 -damaged RGC-5 cells, imipramine activated TrkB pathways through extracellular signal-regulated protein kinase/TrkB phosphorylation. TUNEL staining assay also demonstrated that imipramine ameliorated H 2 O 2 -induced apoptosis in RGC-5 cells. Finally, TrkB-IgG intervention was able to reverse the protective effect of imipramine on H 2 O 2 -induced RGC-5 apoptosis. Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the TrkB signaling pathway.

  12. Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

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    VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom,Darren L.; Negash, Kitaw; Ono, Michele Y.; Hanrahan, Stephen M.; Taylor,Scott E.; Vanderpoel, Jennifer L.; Slavik, Sarah M.; Morris, Andrew B.; Riese II, David J.


    The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.

  13. Protein tyrosine phosphatase non-receptor type 22 modulates NOD2-induced cytokine release and autophagy.

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    Marianne R Spalinger

    Full Text Available BACKGROUND: Variations within the gene locus encoding protein tyrosine phosphatase non-receptor type 22 (PTPN22 are associated with the risk to develop inflammatory bowel disease (IBD. PTPN22 is involved in the regulation of T- and B-cell receptor signaling, but although it is highly expressed in innate immune cells, its function in other signaling pathways is less clear. Here, we study whether loss of PTPN22 controls muramyl-dipeptide (MDP-induced signaling and effects in immune cells. MATERIAL & METHODS: Stable knockdown of PTPN22 was induced in THP-1 cells by shRNA transduction prior to stimulation with the NOD2 ligand MDP. Cells were analyzed for signaling protein activation and mRNA expression by Western blot and quantitative PCR; cytokine secretion was assessed by ELISA, autophagosome induction by Western blot and immunofluorescence staining. Bone marrow derived dendritic cells (BMDC were obtained from PTPN22 knockout mice or wild-type animals. RESULTS: MDP-treatment induced PTPN22 expression and activity in human and mouse cells. Knockdown of PTPN22 enhanced MDP-induced activation of mitogen-activated protein kinase (MAPK-isoforms p38 and c-Jun N-terminal kinase as well as canonical NF-κB signaling molecules in THP-1 cells and BMDC derived from PTPN22 knockout mice. Loss of PTPN22 enhanced mRNA levels and secretion of interleukin (IL-6, IL-8 and TNF in THP-1 cells and PTPN22 knockout BMDC. Additionally, loss of PTPN22 resulted in increased, MDP-mediated autophagy in human and mouse cells. CONCLUSIONS: Our data demonstrate that PTPN22 controls NOD2 signaling, and loss of PTPN22 renders monocytes more reactive towards bacterial products, what might explain the association of PTPN22 variants with IBD pathogenesis.

  14. Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal tyrosines

    DEFF Research Database (Denmark)

    Helin, K; Beguinot, L


    with receptors in which 1, 2, or all 3 tyrosines were changed to phenylalanines. The triple point mutant EGF-R, expressed in NIH-3T3, exhibited low autophosphorylation in vivo, low biological and reduced kinase activities. Single and double point mutants were down-regulated, as well as wild type EGF-R......The C terminus of the epidermal growth factor receptor (EGF-R) contains three tyrosines (Y1068, Y1148, and Y1173) which correspond to the major autophosphorylation sites. To investigate the role of the tyrosines in internalization and down-regulation of the EGF-R, mutational analysis was performed...... in response to EGF showing a half-life of about 1 h. Degradation of the triple point mutant, however, was impaired and resulted in a half-life of 4 h in the presence of EGF. EGF-dependent down-regulation of surface receptors was decreased in the triple point mutant EGF-R as was internalization and degradation...

  15. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

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    Meehan Maria


    Full Text Available Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA, an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA's primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  16. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria


    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  17. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs). (United States)

    Barrows, Douglas; He, John Z; Parsons, Ramon


    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  18. Receptor tyrosine kinase (c-Kit inhibitors: a potential therapeutic target in cancer cells

    Directory of Open Access Journals (Sweden)

    Abbaspour Babaei M


    Full Text Available Maryam Abbaspour Babaei,1 Behnam Kamalidehghan,2,3 Mohammad Saleem,4–6 Hasniza Zaman Huri,1,7 Fatemeh Ahmadipour1 1Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB, Shahrak-e Pajoohesh, 3Medical Genetics Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 4Department of Urology, 5Department of Laboratory Medicine and Pathology, Masonic Cancer Center, University of Minnesota, 6Section of Molecular Therapeutics & Cancer Health Disparity, The Hormel Institute, Austin, MN, USA; 7Clinical Investigation Centre, University Malaya Medical Centre, Lembah Pantai, Kuala Lumpur, Malaysia Abstract: c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c

  19. Axl receptor tyrosine kinase is a novel target of apigenin for the inhibition of cell proliferation. (United States)

    Kim, Kyung-Chan; Choi, Eun-Ha; Lee, Chuhee


    The Axl receptor tyrosine kinase (RTK), along with Tyro 3 and Mer, belongs to the TAM subfamily that promotes survival, stimulates proliferation and/or inhibits apoptosis. In various types of human cancer, including breast, lung and prostate cancer, Axl expression is increased and correlates with an advanced clinical stage. In this study, we examined whether apigenin has an effect on Axl expression, which in turn can affect cell proliferation. The treatment of the non‑small cell lung cancer (NSCLC) cells, A549 and H460, with apigenin decreased Axl mRNA and protein expression in a dose‑dependent manner. Axl promoter activity was also inhibited by apigenin, indicating that apigenin suppressed Axl expression at the transcriptional level. Upon treatment with apigenin, the viability of both the A549 and H460 cells was gradually decreased and the anti-proliferative effects were further confirmed by the dose‑dependent decrease in the clonogenic ability of the apigenin‑treated cells. Subsequently, we found that the viability and clonogenic ability of the cells treated with apigenin was less or more affected by transfection of the cells with a Axl-expressing plasmid or Axl targeting siRNA, compared to transfection with the empty vector or control siRNA, respectively. In addition, apigenin increased the expression of p21, a cyclin-dependent kinase inhibitor, but reduced the expression of X-linked inhibitor of apoptosis protein (XIAP). These cell cycle arrest and pro-apoptotic effects of apigenin were also attenuated or augmented by the up- or downregulation of Axl expression, respectively, which suggests that Axl is a novel target of apigenin through which it exerts its inhibitory effects on cell proliferation. Taken together, our data indicate that apigenin downregulates Axl expression, which subsequently results in the inhibition of NSCLC cell proliferation through the increase and decrease of p21 and XIAP expression, respectively.

  20. Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients.Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9 and healthy donors (n = 6. IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay.The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05.ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy.

  1. Molecular variation and evolution of the tyrosine kinase domains of insulin receptor IRa and IRb genes in Cyprinidae. (United States)

    Kong, XiangHui; Wang, XuZhen; He, ShunPing


    The insulin receptor (IR) gene plays an important role in regulating cell growth, differentiation and development. In the present study, DNA sequences of insulin receptor genes, IRa and IRb, were amplified and sequenced from 37 representative species of the Cyprinidae and from five outgroup species from non-cyprinid Cypriniformes. Based on coding sequences (CDS) of tyrosine kinase regions of IRa and IRb, molecular evolution and phylogenetic relationships were analyzed to better understand the characteristics of IR gene divergence in the family Cyprinidae. IRa and IRb were clustered into one lineage in the gene tree of the IR gene family, reconstructed using the unweighted pair group method with arithmetic mean (UPGMA). IRa and IRb have evolved into distinct genes after IR gene duplication in Cyprinidae. For each gene, molecular evolution analyses showed that there was no significant difference among different groups in the reconstructed maximum parsimony (MP) tree of Cyprinidae; IRa and IRb have been subjected to similar evolutionary pressure among different lineages. Although the amino acid sequences of IRa and IRb tyrosine kinase regions were highly conserved, our analyses showed that there were clear sequence variations between the tyrosine kinase regions of IRa and IRb proteins. This indicates that IRa and IRb proteins might play different roles in the insulin signaling pathway.

  2. The tyrosine kinase receptor c-met, its cognate ligand HGF and the tyrosine kinase receptor trasducers STAT3, PI3K and RHO in thyroid nodules associated with Hashimoto's thyroiditis: an immunohistochemical characterization

    Directory of Open Access Journals (Sweden)

    R. M. Ruggeri


    Full Text Available Hepatocyte growth factor (HGF exerts proliferative activities in thyrocytes upon binding to its tyrosine kinase receptor c-met and is also expressed in benign thyroid nodules as well as in Hashimoto’s thyroiditis (HT. The simultaneous expression of HGF/c-met and three trasducers of tyrosine kinase receptors (STAT3, PI3K, RHO in both the nodular and extranodular tissues were studied by immunohistochemistry in 50 benign thyroid nodules (NGs, 25 of which associated with HT. The ligand/tyrosine kinase receptor pair HGF/c-met and the two trasducers PI3K and RHO were expressed in NGs, regardless of association with HT, with a higher positive cases percentage in HT-associated NGs compared to not HT-associated NGs (25/25 or 100% vs 7/25 or 28%; P<0.001. HGF, PI3K and RHO expression was only stromal (fibroblasts and endothelial cells, in all 32 reactive NGs, while c-met localization was consistently epithelial (thyrocyes. Immu­noreactions for HGF, c-met, PI3K and RHO were also apparent in the extra-nodular tissue of HT specimens, where HGF and PI3K were expressed not only in stromal cells but also in thyrocyes along with the c-met. Finally, a positive correlation was observed between the proportion of HGF, c-met, PI3K follicular cells and the grade of lymphoid aggregates in HT. In conclusion, HGF, c-met, PI3K are much more frequently and highly expressed in HT compared to NGs, and among all NGs in those present in the context of HT. A paracrine effect of HFG/c-met on nodule development, based on the prevalent stromal expression, may be suggested along with a major role of HGF/c-met and PI3K in HT. Finally, the expression of such molecules in HT may be regulated by lymphoid infiltrate.

  3. Deorphanization of the human leukocyte tyrosine kinase (LTK) receptor by a signaling screen of the extracellular proteome (United States)

    Zhang, Hongbing; Pao, Lily I.; Zhou, Aileen; Brace, Arthur D.; Halenbeck, Robert; Hsu, Amy W.; Bray, Thomas L.; Hestir, Kevin; Bosch, Elizabeth; Lee, Ernestine; Wang, Gang; Liu, Haixia; Wong, Brian R.; Kavanaugh, W. Michael; Williams, Lewis T.


    There are many transmembrane receptor-like proteins whose ligands have not been identified. A strategy for finding ligands when little is known about their tissue source is to screen each extracellular protein individually expressed in an array format by using a sensitive functional readout. Taking this approach, we have screened a large collection (3,191 proteins) of extracellular proteins for their ability to activate signaling of an orphan receptor, leukocyte tyrosine kinase (LTK). Only two related secreted factors, FAM150A and FAM150B (family with sequence similarity 150 member A and member B), stimulated LTK phosphorylation. FAM150A binds LTK extracellular domain with high affinity (KD = 28 pM). FAM150A stimulates LTK phosphorylation in a ligand-dependent manner. This strategy provides an efficient approach for identifying functional ligands for other orphan receptors. PMID:25331893

  4. The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response — Biological and Clinical Aspects

    Energy Technology Data Exchange (ETDEWEB)

    Medová, Michaela, E-mail:; Aebersold, Daniel M.; Zimmer, Yitzhak, E-mail: [Department of Radiation Oncology, Inselspital, Bern University Hospital, and University of Bern, 3010 Bern (Switzerland); Department of Clinical Research, University of Bern, DKF, MEM-E807, Murtenstrasse 35, 3010 Bern (Switzerland)


    Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

  5. Tyrosine-based signal mediates LRP6 receptor endocytosis and desensitization of Wnt/β-catenin pathway signaling. (United States)

    Liu, Chia-Chen; Kanekiyo, Takahisa; Roth, Barbara; Bu, Guojun


    Wnt/β-catenin signaling orchestrates a number of critical events including cell growth, differentiation, and cell survival during development. Misregulation of this pathway leads to various human diseases, specifically cancers. Endocytosis and phosphorylation of the LDL receptor-related protein 6 (LRP6), an essential co-receptor for Wnt/β-catenin signaling, play a vital role in mediating Wnt/β-catenin signal transduction. However, its regulatory mechanism is not fully understood. In this study, we define the mechanisms by which LRP6 endocytic trafficking regulates Wnt/β-catenin signaling activation. We show that LRP6 mutant with defective tyrosine-based signal in its cytoplasmic tail has an increased cell surface distribution and decreased endocytosis rate. These changes in LRP6 endocytosis coincide with an increased distribution to caveolae, increased phosphorylation, and enhanced Wnt/β-catenin signaling. We further demonstrate that treatment of Wnt3a ligands or blocking the clathrin-mediated endocytosis of LRP6 leads to a redistribution of wild-type receptor to lipid rafts. The LRP6 tyrosine mutant also exhibited an increase in signaling activation in response to Wnt3a stimulation when compared with wild-type LRP6, and this activation is suppressed when caveolae-mediated endocytosis is blocked. Our results reveal molecular mechanisms by which LRP6 endocytosis routes regulate its phosphorylation and the strength of Wnt/β-catenin signaling, and have implications on how this pathway can be modulated in human diseases.

  6. HER2 Oncogenic Function Escapes EGFR Tyrosine Kinase Inhibitors via Activation of Alternative HER Receptors in Breast Cancer Cells (United States)

    Kong, Anthony; Calleja, Véronique; Leboucher, Pierre; Harris, Adrian; Parker, Peter J.; Larijani, Banafshé


    Background The response rate to EGFR tyrosine kinase inhibitors (TKIs) may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses. Methodology and Principal Findings Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET) which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs) of EGFR, AG1478 and Iressa (Gefitinib) decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment–induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa. Conclusions and Significance These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs) in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients. PMID:18682844

  7. Alteration and potential role of soluble fms-like tyrosine kinase receptor 1 in preeclampsia

    Institute of Scientific and Technical Information of China (English)

    LI Yi; LI Dong-hong; YAO Yuan-qing; ZHAO Hong-xi; ZHANG Chun-li; HUANG Liang


    Objective:To investigate the alteration of serum soluble fms-like tyrosine kinase receptor 1 (sFlt-1), the possible cause in preeclampsia, the relationship between altered sFlt-1 and the development of pregnancy. Methods: Semi-quantitative RT-PCR was carried out to detect the level of sFlt-1 mRNA in placental tissue of 10 preeclampsia (study group) and 10 normotensive pregnancies (control group); ELISA was used to detect the serum level of sFlt-1 in peripheral venous blood and uterine venous blood in control group (n=35) and study group (n=35), and furthermore to detect the volume of peripheral venous blood sFlt-1 in early, middle and later pregnancies. Results: sFlt-1 mRNA of placental tissue was significantly higher in preeclampsia than that in normal pregnancy. The serum level of sFlt-1 of peripheral vein in preeclampsia was higher than that in normal pregnancy ([ 5639.6±3190.5] pg/ml vs[ 2194.4±635.8 ] pg/ml, P<0.01) , so did the serum sFlt-1 of uterine vein in preeclampsia and control group ([7673.3±2295.8] pg/ml vs[ 3056.6±784.6] pg/ml, P<0.01), indicating that the volume of sFlt-1 of uterine vein blood was significantly higher than that of peripheral venous blood (P<0.01). The serum level of sFlt-1 in early, middle and later pregnancy were (31.7±19.6) pg/ml (n=10), (994.0±302.1) pg/ml (n=10), (2194.4±635.8) pg/ml (n=35), respectively, showing that the level of sFlt-1 in peripheral venous blood increasingly elevated with the development of pregnancy (P<0.01). Conclusion: Preeclampsia is associated with increased level of sFlt-1 which might be largely from placenta during pregnancy.

  8. 8-THP-DHI analogs as potent Type I dual TIE-2/VEGF-R2 receptor tyrosine kinase inhibitors. (United States)

    Hudkins, Robert L; Zulli, Allison L; Underiner, Ted L; Angeles, Thelma S; Aimone, Lisa D; Meyer, Sheryl L; Pauletti, Daniel; Chang, Hong; Fedorov, Elena V; Almo, Steven C; Fedorov, Alexander A; Ruggeri, Bruce A


    A novel series of 8-(2-tetrahydropyranyl)-12,13-dihydroindazolo[5,4-a]pyrrolo[3,4-c]carbazoles (THP-DHI) was synthesized and evaluated as dual TIE-2 and VEGF-R2 receptor tyrosine kinase inhibitors. Development of the structure-activity relationships (SAR) with the support of X-ray crystallography led to identification of 7f and 7g as potent, selective dual TIE-2/VEGF-R2 inhibitors with excellent cellular potency and acceptable pharmacokinetic properties. Compounds 7f and 7g were orally active in tumor models with no observed toxicity.

  9. Primary cilia and coordination of receptor tyrosine kinase (RTK) and transforming growth factor β (TGF-β) signaling

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Morthorst, Stine Kjær; Mogensen, Johanne Bay;


    are at the root of a pleiotropic group of diseases and syndromic disorders called ciliopathies. In this review, we present an overview of primary cilia-mediated regulation of receptor tyrosine kinase (RTK) and transforming growth factor β (TGF-β) signaling. Further, we discuss how defects in the coordination...... in their extracellular environment and integrate and transmit signaling information to the cell to regulate various cellular, developmental, and physiological processes. Many different signaling pathways have now been shown to rely on primary cilia to function properly, and mutations that lead to ciliary dysfunction...

  10. Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin

    DEFF Research Database (Denmark)

    Barnea, G; Grumet, M; Milev, P;


    The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically...... labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous...

  11. In vivo Regulation of the Allergic Response by the Interleukin 4 Receptor Alpha Chain Immunoreceptor Tyrosine-based Inhibitory Motif (United States)

    Tachdjian, Raffi; Khatib, Shadi Al; Schwinglshackl, Andreas; Kim, Hong Sook; Chen, Andrew; Blasioli, Julie; Mathias, Clinton; Kim, Hye-Young; Umetsu, Dale T.; Oettgen, Hans C.; Chatila, Talal A.


    Background Signaling by IL-4 and IL-13 via the IL-4 receptor alpha chain (IL-4Rα) plays a critical role in the pathology of allergic diseases. The IL-4Rα is endowed with an immunoreceptor tyrosine-based inhibitory motif (ITIM), centered on tyrosine 709 (Y709) in the cytoplasmic domain, that binds a number of regulatory phosphatases. The function of the ITIM in the in vivo regulation of IL-4R signaling remains unknown. Objective To determine the in vivo function of the IL-4Rα ITIM using mice in which the ITIM was inactivated by mutagenesis of the tyrosine Y709 residue into phenylalanine (F709). Methods F709 ITIM mutant mice were derived by knockin mutagenesis. Activation of intracellular signaling cascades by IL-4 and IL-13 was assessed by intracellular staining of phosphorylated signaling intermediates and by gene expression analysis. In vivo responses to allergic sensitization were assessed using models of allergic airway inflammation. Results The F709 mutation increased STAT6 phosphorylation by IL-4 and, disproportionately, by IL-13. This was associated with exaggerated Th2 polarization, enhanced alternative macrophage activation by IL-13, augmented basal and antigen-induced IgE responses and intensified allergen-induced eosinophilic airway inflammation and hyperreactivity. Conclusions These results point to a physiologic negative regulatory role for the Y709 ITIM in signaling via IL-4Rα, especially by IL-13. PMID:20392476

  12. Carcinoma of an unknown primary: are EGF receptor, Her-2/neu, and c-Kit tyrosine kinases potential targets for therapy?


    Massard, C; Voigt, J-J; Laplanche, A; Culine, S; Lortholary, A; Bugat, R; Theodore, C.; Priou, F; Kaminsky, M-C; Lesimple, T; Pivot, X; B. Coudert; Douillard, J-Y; Merrouche, Y; Fizazi, K


    Carcinomas of an unknown primary site (CUP) are heterogeneous tumours with a median survival of only 8 months. Tyrosine kinase inhibitors are promising new drugs. The aim of this study was to determine the expression of EGF-receptor, Her-2/neu, and c-Kit tyrosine kinases in CUP. Paraffin-embedded specimens were obtained from 54 patients with a CUP who were included in the GEFCAPI 01 randomised phase II trial. Immunohistochemistry was performed using the Dako autostainer with antibodies direct...

  13. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J;


    CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...

  14. Antidepressant drug exposure is associated with mRNA levels of tyrosine receptor kinase B in major depressive disorder. (United States)

    Bayer, T A; Schramm, M; Feldmann, N; Knable, M B; Falkai, P


    1. Recent studies have provided support for the notion that the high affinity neurotrophin receptor tyrosine receptor kinase B (trk B) may be involved in the treatment of depression. 2. Using a quantitative RT-PCR approach trk B mRNA levels were determined in brain material from cerebellum, temporal cortex, and frontal cortex of control specimen and patients with major depressive disorder, schizophrenia and bipolar disorder (15 subjects each). 3. Interestingly, elevated trk B mRNA levels were found in cerebellum (3.6-fold) in patients with major depressive disorder, reaching statistical significance (p=0.03). 4. The major depressive disorder-on drugs group differed from controls (p=0.006) in the cerebellum. 5. Since only patients with major depressive disorder received antidepressants, elevated trk B mRNA levels are possibly related to drug treatment.

  15. Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila. (United States)

    Tsarouhas, Vasilios; Yao, Liqun; Samakovlis, Christos


    Metazoans have evolved efficient mechanisms for epidermal repair and survival following injury. Several cellular responses and key signaling molecules that are involved in wound healing have been identified in Drosophila, but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair are poorly understood. The Ret-like receptor tyrosine kinase (RTK) Stitcher (Stit, also known as Cad96Ca) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce restoration of the extracellular barrier. Here, we describe the immediate downstream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src family tyrosine kinases bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src family kinases (SFKs) control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier-repair genes. Our analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode for Src kinases and their central functions during epidermal wound healing in vivo.

  16. Coexpression of receptor-tyrosine-kinases in gastric adenocarcinoma-a rationale for a molecular targeting strategy?

    Institute of Scientific and Technical Information of China (English)

    Daniel Drescher; Thomas Wehler; Andreas Teufel; Kerstin Herzer; Thomas Fischer; Martin R Berger; Theodor Junginger; Peter R Galle; Carl C Schimanski; Markus Moehler; Ines Gockel; Kirsten Frerichs; Annett Müller; Friedrich Dünschede; Thomas Borschitz; Stefan Biesterfeld; Martin Holtmann


    AIM: To define the (co-)expression pattern of target receptor-tyrosine-kinases (RTK) in human gastric adenocarcinoma.METHODS: The (co-)expression pattern of VEGFR1-3,PDGFRα/β and EGFR1 was analyzed by RT-PCR in 51 human gastric adenocarcinomas. In addition, IHC staining was applied for confirmation of expression and analysis of RTK Iocalisation.RESULTS: The majority of samples revealed a VEGFR1(98%), VEGFR2 (80%), VEGFR3 (67%), PDGFRα(82%) and PDGFRβ (82%) expression, whereas only 62% exhibited an EGFR1 expression. 78% of cancers expressed at least four out of six RTKs. While VEGFR1-3and PDGFRα revealed a predominantly cytoplasmatic staining in tumor cells, accompanied by an additional nuclear staining for VEGFR3, EGFR1 was almost exclusively detected on the membrane of tumor cells.PDGFRβ was restricted to stromal pericytes, which also depicted a PDGFRα expression.CONCLUSION: Our results reveal a high rate of receptor-tyrosine-kinases coexpression in gastric adenocarcinoma and might therefore encourage an application of multiple-target RTK-inhibitors within a combination therapy.

  17. RTK SLAP down: the emerging role of Src-like adaptor protein as a key player in receptor tyrosine kinase signaling. (United States)

    Wybenga-Groot, Leanne E; McGlade, C Jane


    SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

  18. Proliferation of Ewing sarcoma cell lines is suppressed by the receptor tyrosine kinase inhibitors gefitinib and vandetanib

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    Åman Pierre


    Full Text Available Abstract Background Tyrosine kinase inhibitors (TKIs have gained much attention in recent years as targeted agents for the treatment of a wide range of human cancers. We have investigated the effect of the TKIs gefitinib and vandetanib on tumor cell lines derived from Ewing sarcoma, a highly malignant tumor affecting bone and soft tissue in children and young adults. Gefitinib is an inhibitor of epidermal growth factor receptor tyrosine kinase activity (EGFR and vandetanib selectively targets vascular endothelial growth factor receptor-2 (VEGFR-2 with additional activity against VEGFR-3, EGFR and RET kinase receptors. Results Two Ewing sarcoma cell lines investigated showed high levels of nuclear EGFR expression as well as moderate expression in plasma membrane and cytoplasm. When treated with concentrations of 5 μM and more of either gefitinib or vandetanib, we observed a significant decrease in cell proliferation. However, there were no detectable changes in p44/42 MAPK and Akt-1 phosphorylation, or in the expression of cyclin D1 or c-Myc following gefitinib or vandetanib treatment. Conclusion We conclude that Ewing sarcoma tumor cell proliferation is not highly sensitive to inhibition of EGFR signaling alone or the simultaneous inhibition of VEGFR receptors, EGFR and RET kinase. Decreased tumor cell proliferation could be achieved with gefitinib and vandetanib, but only at higher doses where non-specific effects of the compounds may be overriding. As Ewing tumor cells do not seem to depend on EGFR and VEGFR pathways for survival, other key factors in the cellular signaling of Ewing sarcoma should be targeted in order to obtain a potent therapeutic response.

  19. Involvement of receptor tyrosine kinase Tyro3 in amyloidogenic APP processing and β-amyloid deposition in Alzheimer's disease models.

    Directory of Open Access Journals (Sweden)

    Yan Zheng

    Full Text Available Alzheimer's disease (AD is the most common progressive neurodegenerative disease known to humankind. It is characterized by brain atrophy, extracellular amyloid plaques, and intracellular neurofibril tangles. β-Amyloid cascade is considered the major causative player in AD. Up until now, the mechanisms underlying the process of Aβ generation and accumulation in the brain have not been well understood. Tyro3 receptor belongs to the TAM receptor subfamily of receptor protein tyrosine kinases (RPTKs. It is specifically expressed in the neurons of the neocortex and hippocampus. In this study, we established a cell model stably expressing APPswe mutants and producing Aβ. We found that overexpression of Tyro3 receptor in the cell model significantly decreased Aβ generation and also down-regulated the expression of β-site amyloid precursor protein cleaving enzyme (BACE1. However, the effects of Tyro3 were inhibited by its natural ligand, Gas6, in a concentration-dependent manner. In order to confirm the role of Tyro3 in the progression of AD development, we generated an AD transgenic mouse model accompanied by Tyro3 knockdown. We observed a significant increase in the number of amyloid plaques in the hippocampus in the mouse model. More plaque-associated clusters of astroglia were also detected. The present study may help researchers determine the role of Tyro3 receptor in the neuropathology of AD.

  20. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1 into Diverse Memory T-Cell Populations.

    Directory of Open Access Journals (Sweden)

    Drew C Deniger

    Full Text Available T cells modified with chimeric antigen receptors (CARs targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1 is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28 or CD137 (designated ROR1RCD137 and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC, which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

  1. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations (United States)

    Deniger, Drew C.; Yu, Jianqiang; Huls, M. Helen; Figliola, Matthew J.; Mi, Tiejuan; Maiti, Sourindra N.; Widhopf, George F.; Hurton, Lenka V.; Thokala, Radhika; Singh, Harjeet; Olivares, Simon; Champlin, Richard E.; Wierda, William G.; Kipps, Thomas J.; Cooper, Laurence J. N.


    T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire. PMID:26030772

  2. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site. (United States)

    Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W


    Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

  3. IL-4 function can be transferred to the IL-2 receptor by tyrosine containing sequences found in the IL-4 receptor alpha chain. (United States)

    Wang, H Y; Paul, W E; Keegan, A D


    IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL-2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences.

  4. Regeneration of human epidermis on acellular dermis is impeded by small-molecule inhibitors of EGF receptor tyrosine kinase. (United States)

    Forsberg, Sofi; Ostman, Arne; Rollman, Ola


    The family of human epidermal growth factor receptors (EGFR, HER2-4) exerts key functions in normal and malignant epithelial cells. Both EGFR and HER2 are valuable targets for anti-cancer drugs by interfering with ligand binding, receptor dimerization, or tyrosine kinase activity. A similar therapeutic strategy has been advocated for chronic psoriasis since plaque lesions overexpress EGFR and its ligands. Our aim was to characterize EGFR/HER2 protein expression in skin cultures and to evaluate the effects of tyrosine kinase inhibitors on epidermal outgrowth, morphology, and EGFR activation. Human skin explants were established on cell-free dermis and cultured at the air-liquid interface. The impact of small-molecule HER inhibitors on outgrowth was assayed by fluorescence-based image analysis and histometry. Effects of a dual EGFR/HER2 kinase inhibitor, PKI166, on neoepidermis were studied by immunohistochemistry and Western blot. Receptor immunostaining showed in vivo-like distributions with highest EGFR intensity in the proliferative layers whereas HER2 was mainly expressed by suprabasal keratinocytes. Reepithelialization was associated with EGFR autophosphorylation irrespective of exogenous ligand stimulation. PKI166 inhibited neoepidermal EGFR activation, keratinocyte proliferation, and outgrowth from normal and psoriatic skin explants. The rate of epidermalization in presence of other HER inhibitors varied suggesting that drug specificity, potency, and reversibility determine the dynamic outcome. Overall, agents predominantly targeting EGFR kinase were more efficient inhibitors of epidermal regeneration than an HER2-selective drug. The study illustrates the usefulness of a dynamic skin model and emphasizes the potential of HER-directed approaches to control epidermal growth in hyperproliferative skin disorders.

  5. A "turn-on" fluorescent receptor for detecting tyrosine phosphopeptide using the surface imprinting procedure and the epitope approach. (United States)

    Li, Dong-Yan; Qin, Ya-Ping; Li, Hong-Yu; He, Xi-Wen; Li, Wen-You; Zhang, Yu-Kui


    A new strategy for the manufacture of a turn-on fluorescent molecularly imprinted polymer (CdTe/SiO2/MIP) receptor for detecting tyrosine phosphopeptide (pTyr peptide) was proposed. The receptor was prepared by the surface imprinting procedure and the epitope approach with silica-capped CdTe quantum dots (QDs) as core substrate and fluorescent signal, phenylphosphonic acid (PPA) as the dummy template, 1-[3-(trimethoxysilyl) propyl] urea as the functional monomer, and octyltrimethoxysilane as the cross-linker. The synthetic CdTe/SiO2/MIP was able to selectively capture the template PPA and corresponding target pTyr peptide with fluorescence enhancement via the special interaction between them and the recognition cavities. The receptor exhibited the linear fluorescence enhancement to pTyr peptide in the range of 0.5-35μM, and the detection limit was 0.37μM. The precision for five replicate detections of pTyr peptide at 20μM was 2.60% (relative standard deviation). Combining the fluorescence property of the CdTe QDs with the merits of the surface imprinting technique and the epitope approach, the receptor not only owned high recognition site accessibility and good binding affinities for target pTyr peptide, but also improved the fluorescence selectivity of the CdTe QDs, as well revealed the feasibility of fabrication of a turn-on fluorescence probe using the surface imprinting procedure and the epitope approach.

  6. Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

    Energy Technology Data Exchange (ETDEWEB)

    Primo M. E.; Jakoncic J.; Noguera M.E.; Risso V.A.; Sosa L.; Sica M.P.; Solimena M.; Poskus E. and Ermacora M.


    ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  7. Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain.

    Directory of Open Access Journals (Sweden)

    María E Primo

    Full Text Available ICA512 (or IA-2 is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512 and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  8. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain. (United States)

    Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J


    We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

  9. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of); Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon [Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Cho, Dong-Hyung, E-mail: [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of)


    Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  10. Replication of biotinylated human immunodeficiency viruses. (United States)

    Belshan, Michael; Matthews, John M; Madson, Christian J


    Previous work demonstrated recently the adaptation of the Escherichia coli biotin ligase BirA - biotin acceptor sequence (BAS) labeling system to produce human immunodeficiency virus type 1 viruses with biotinylated integrase (NLXIN(B)) and matrix (NLXMA(B)) proteins (Belshan et al., 2009). This report describes the construction of an HIV permissive cell line stably expressing BirA (SupT1.BirA). Consistent with the results in the previous report, NLXMA(B) replicated similar to wild-type levels and expressed biotinylated Gag and MA proteins in the SupT1.BirA cells, whereas the replication of NLXIN(B) was reduced severely. Three additional HIV type 2 (HIV-2) viruses were constructed with the BAS inserted into the vpx and vpr accessory genes. Two BAS insertions were made into the C-terminal half of the Vpx, including one internal insertion, and one at the N-terminus of Vpr. All three viruses were replication competent in the SupT1.BirA cells and their target proteins biotinylated efficiently and incorporated into virions. These results demonstrate the potential utility of the biotinylation system to label and capture HIV protein complexes in the context of replicating virus.

  11. Dopamine D1-dependent trafficking of striatal N-methyl-D-aspartate glutamate receptors requires Fyn protein tyrosine kinase but not DARPP-32. (United States)

    Dunah, Anthone W; Sirianni, Ana C; Fienberg, Allen A; Bastia, Elena; Schwarzschild, Michael A; Standaert, David G


    Interactions between dopaminergic and glutamatergic systems in the striatum are thought to underlie both the symptoms and adverse effects of treatment of Parkinson's disease. We have previously reported that activation of the dopamine D1 receptor triggers a rapid redistribution of striatal N-methyl-d-aspartate (NMDA) receptors between intracellular and postsynaptic sub-cellular compartments. To unravel the signaling pathways underlying this trafficking, we studied mice with targeted disruptions of either the gene that encodes the dopamine- and cAMP-regulated phosphoprotein (DARPP-32), a potent and selective inhibitor of protein phosphatase-1, or the protein tyrosine kinase Fyn. In striatal tissue from DARPP-32-depleted mice, basal tyrosine and serine phosphorylation of striatal NMDA receptor subunits NR1, NR2A, and NR2B was normal, and activation of dopamine D1 receptors with the agonist SKF-82958 [(+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-benzazepine] produced redistribution of NMDA receptors from vesicular compartments (P3 and LP2) to synaptosomal membranes (LP1). In the Fyn knockout mice, basal tyrosine phosphorylation of NR2A and NR2B was drastically reduced, whereas serine phosphorylation of these NMDA subunits was unchanged. In the Fyn knockout mice, the dopamine D1 receptor agonist failed to induce subcellular redistribution of NMDA receptors. In addition, Fyn-depleted mice lesioned with 6-hydroxydopamine also failed to exhibit l-DOPA-induced behavioral sensitization, but this may be caused, at least in part, by resistance of these mice to the neurotoxic lesion. These findings suggest a novel mechanism for the trafficking of striatal NMDA receptors by signaling pathways that are independent of DARPP-32 but require Fyn protein tyrosine kinase. Strategies that prevent NMDA receptor subcellular redistribution through inhibition of Fyn kinase may prove useful in the treatment of Parkinson's disease.

  12. The role of oestrogen receptor {alpha} in human thyroid cancer: contributions from coregulatory proteins and the tyrosine kinase receptor HER2.

    LENUS (Irish Health Repository)

    Kavanagh, Dara O


    Epidemiological, clinical, and molecular studies suggest a role for oestrogen in thyroid cancer. How oestrogen mediates its effects and the consequence of it on clinical outcome has not been fully elucidated. The participation of coregulatory proteins in modulating oestrogen receptor (ER) function and input of crosstalk with the tyrosine kinase receptor HER2 was investigated. Oestrogen induced cell proliferation in the follicular thyroid cancer (FTC)-133 cells, but not in the anaplastic 8305C cell line. Knockdown of the coactivator steroid receptor coactivator (SRC)-1 inhibited FTC-133 basal, but not oestrogen induced, cell proliferation. Oestrogen also increased protein expression of SRC-1 and the ER target gene cyclin D1 in the FTC-133 cell line. ERalpha, ERbeta, the coregulatory proteins SRC-1 and nuclear corepressor (NCoR), and the tyrosine kinase receptor HER2 were localised by immunohistochemistry and immnofluorescence in paraffin-embedded tissue from thyroid tumour patients (n=111). ERalpha was colocalised with both SRC-1 and NCoR to the nuclei of the tumour epithelial cells. Expression of ERalpha and NCoR was found predominantly in non-anaplastic tumours and was significantly associated with well-differentiated tumours and reduced incidence of disease recurrence. In non-anaplastic tumours, HER2 was significantly associated with SRC-1, and these proteins were associated with poorly differentiated tumours, capsular invasion and disease recurrence. Totally, 87% of anaplastic tumours were positive for SRC-1. Kaplan-Meier estimates of disease-free survival indicated that in thyroid cancer, SRC-1 strongly correlates with reduced disease-free survival (P<0.001), whereas NCoR predicted increased survival (P<0.001). These data suggest opposing roles for the coregulators SRC-1 and NCoR in thyroid tumour progression.

  13. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Energy Technology Data Exchange (ETDEWEB)

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)


    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  14. Growth-Factor-Driven Rescue to Receptor Tyrosine Kinase (RTK) Inhibitors through Akt and Erk Phosphorylation in Pediatric Low Grade Astrocytoma and Ependymoma

    NARCIS (Netherlands)

    Sie, Mariska; den Dunnen, Wilfred F. A.; Lourens, Harm Jan; Meeuwsen-de Boer, Tiny G. J.; Scherpen, Frank J. G.; Zomerman, Walderik W.; Kampen, Kim R.; Hoving, Eelco W.; de Bont, Eveline S. J. M.


    Up to now, several clinical studies have been started investigating the relevance of receptor tyrosine kinase (RTK) inhibitors upon progression free survival in various pediatric brain tumors. However, single targeted kinase inhibition failed, possibly due to tumor resistance mechanisms. The present

  15. Combination therapy with gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, gemcitabine and cisplatin in patients with advanced solid tumors.

    NARCIS (Netherlands)

    Giaccone, G.; Gonzalez-Larriba, JL; Oosterom, van A.T.; Alfonso, R; Smit, E.F.; Martens, M.; Peters, G.J.; Vijgh, van der WJ; Smith, R; Averbuch, S; Fandi, A


    BACKGROUND: The aim of this study was to investigate the tolerability, pharmacokinetic interaction and antitumor activity of gefitinib ("Iressa", ZD1839), an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor, combined with gemcitabine and cisplatin in chemotherapy-n

  16. Three generations of epidermal growth factor receptor tyrosine kinase inhibitors developed to revolutionize the therapy of lung cancer

    Directory of Open Access Journals (Sweden)

    Zhang H


    Full Text Available Haijun Zhang Department of Oncology, Zhongda Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China Abstract: Lung cancer, ~80%–85% of which is non-small-cell lung cancer (NSCLC, is the leading cause of cancer-related mortality worldwide. Sensitizing mutations in epidermal growth factor receptor (EGFR gene (EGFRm+, such as exon 19 deletions and exon 21 L858R point mutations, are the most important drivers in NSCLC patients. In this respect, small-molecule EGFR tyrosine kinase inhibitors (TKIs have been designed and developed, which launched the era of targeted, personalized and precise medicine for lung cancer. Patients with EGFRm+ could achieve good responses to the treatment with the first-generation EGFR TKIs, such as erlotinib and gefitinib. However, most patients develop acquired drug resistance mostly driven by the T790M mutation occurring within exon 20. Although the second-generation EGFR TKIs, such as afatinib, dacomitinib and neratinib, demonstrated promising activity against T790M in preclinical models, they have failed to overcome resistance in patients due to dose-limiting toxicity. Recently, the third-generation EGFR TKIs have shown to be effective against cell lines and murine models harboring T790M mutations while sparing wild-type EGFR, which represents a promising breakthrough approach in overcoming T790M-mediated resistance in NSCLC patients. This article provides a comprehensive review of the therapy revolution for NSCLC with three generations of EGFR TKIs. Keywords: lung cancer, epidermal growth factor receptor, tyrosine kinase inhibitors, T790M mutation

  17. Inhibition of corneal neovascularization with new Tyrosine Kinase Inhibitors targeting vascular endothelial growth factor receptors: Sunitinib malate and Sorafenib

    Directory of Open Access Journals (Sweden)

    Delnia Arshadi


    Full Text Available Corneal neovascularization (NV is a significant, sight-threatening, complication of many ocular surface disorders. Presence of new vessels in cornea can compromise clarity and thus vision. The data supporting a causal role for vascular endothelial growth factor (VEGF in corneal NV are extensive. Inhibition of VEGF remains as a main strategy for treating corneal NV. There is a growing body of evidence that corneal NV can be reduced by using anti-VEGF agents. Sunitinib malate and Sorafenib are new orally bio-available anti-angiogenic agents undergoing tests of efficacy in the treatment of various types of cancers. The main mechanism of these drugs is inhibiting angiogenesis by diminishing signaling through VEGF receptor1 (VEGFR1, VEGFR2, and platelet-derived growth factor receptors. Since VEGF exerts its angiogenic effects through tyrosine kinase receptors in cornea, any mechanisms which reduce VEGF signaling may inhibit corneal NV or at least attenuate it. Based on this fact we herein hypothesize that Sunitinib malate and Sorafenib can be prepared in topical form and be used in corneal neovascularization states. These approaches offer new hope for the successful treatment of corneal NV. Further investigations in animal models are needed to place these two drugs alongside corneal NV therapeutics.

  18. Protein Tyrosine Phosphatase PTPRS Is an Inhibitory Receptor on Human and Murine Plasmacytoid Dendritic Cells

    NARCIS (Netherlands)

    Bunin, A.; Sisirak, V.; Ghosh, H.S.; Grajkowska, L.T.; Hou, Z.E.; Miron, M.; Yang, C.; Ceribelli, M.; Uetani, N.; Chaperot, L.; Plumas, J.; Hendriks, W.J.; Tremblay, M.L.; Hacker, H.; Staudt, L.M.; Green, P.H.; Bhagat, G.; Reizis, B.


    Plasmacytoid dendritic cells (pDCs) are primary producers of type I interferon (IFN) in response to viruses. The IFN-producing capacity of pDCs is regulated by specific inhibitory receptors, yet none of the known receptors are conserved in evolution. We report that within the human immune system, re

  19. Protein tyrosine phosphatase receptor type O regulates development and function of the sensory nervous system. (United States)

    Gonzalez-Brito, Manuel R; Bixby, John L


    The roles of protein tyrosine phosphatases (PTPs) in differentiation and axon targeting by dorsal root ganglion (DRG) neurons are essentially unknown. The type III transmembrane PTP, PTPRO, is expressed in DRG neurons, and is implicated in the guidance of motor and retinal axons. We examined the role of PTPRO in DRG development and function using PTPRO(-/-) mice. The number of peptidergic nociceptive neurons in the DRG of PTPRO(-/-) mice was significantly decreased, while the total number of sensory neurons appeared unchanged. In addition, spinal pathfinding by both peptidergic and proprioceptive neurons was abnormal in PTPRO(-/-) mice. Lastly, PTPRO(-/-) mice performed abnormally on tests of thermal pain and sensorimotor coordination, suggesting that both nociception and proprioception were perturbed. Our data indicate that PTPRO is required for peptidergic differentiation and process outgrowth of sensory neurons, as well as mature sensory function, and provide the first evidence that RPTPs regulate DRG development.

  20. Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3.


    King, C. R.; Borrello, I; Bellot, F; Comoglio, P; Schlessinger, J


    The epidermal growth factor receptor (EGF-R) and the erbB-2 proto-oncogene product protein are closely related by their structural homology and their shared enzymatic activity as autophosphorylating tyrosine kinases. We show that in mammary tumor cells (SK-BR-3) EGF causes a rapid increase in tyrosine phosphorylation of the erbB-2 protein. Phosphorylation of erbB-2 does not occur in cells lacking the EGF-R (MDA-MB-453). Phosphorylation of erbB-2 in SK-BR-3 cells is blocked if EGF is prevented...

  1. Aversive odorant causing appetite decrease downregulates tyrosine decarboxylase gene expression in the olfactory receptor neuron of the blowfly, Phormia regina (United States)

    Ishida, Yuko; Ozaki, Mamiko


    In the blowfly Phormia regina, exposure to d-limonene for 5 days during feeding inhibits proboscis extension reflex behavior due to decreasing tyramine (TA) titer in the brain. TA is synthesized by tyrosine decarboxylase (Tdc) and catalyzed into octopamine (OA) by TA ß-hydroxylase (Tbh). To address the mechanisms of TA titer regulation in the blowfly, we cloned Tdc and Tbh cDNAs from P. regina (PregTdc and PregTbh). The deduced amino acid sequences of both proteins showed high identity to those of the corresponding proteins from Drosophila melanogaster at the amino acid level. PregTdc was expressed in the antenna, labellum, and tarsus whereas PregTbh was expressed in the head, indicating that TA is mainly synthesized in the sensory organs whereas OA is primarily synthesized in the brain. d-Limonene exposure significantly decreased PregTdc expression in the antenna but not in the labellum and the tarsus, indicating that PregTdc expressed in the antenna is responsible for decreasing TA titer. PregTdc-like immunoreactive material was localized in the thin-walled sensillum. In contrast, the OA/TA receptor (PregOAR/TAR) was localized to the thick-walled sensillum. The results indicated that d-limonene inhibits PregTdc expression in the olfactory receptor neurons in the thin-walled sensilla, likely resulting in reduced TA levels in the receptor neurons in the antenna. TA may be transferred from the receptor neuron to the specific synaptic junction in the antennal lobe of the brain through the projection neurons and play a role in conveying the aversive odorant information to the projection and local neurons.

  2. Exploring minimal biotinylation conditions for biosensor analysis using capture chips. (United States)

    Papalia, Giuseppe; Myszka, David


    Using Biacore's new regenerateable streptavidin capture (CAP) sensor chips, we investigated a number of biotinylation conditions for target ligands. We explored standard amine as well as the less commonly used carboxyl biotinylation methods. We illustrate the time scales required for efficient biotinylation as well as the hazards of overbiotinylation. We evaluated a range of desalting methods, including spin columns, dialysis membranes, and filters. Finally, we tested the effects of common buffer components, such as Tris and glycerol, on the biotinylation process. Together, our results serve as a general guide of the steps to consider when minimally biotinylating a target ligand.

  3. Neuroprotection of GluR5-containing kainate receptor activation against ischemic brain injury through decreasing tyrosine phosphorylation of N-methyl-D-aspartate receptors mediated by Src kinase. (United States)

    Xu, Jie; Liu, Yong; Zhang, Guang-Yi


    Previous studies indicate that cerebral ischemia breaks the dynamic balance between excitatory and inhibitory inputs. The neural excitotoxicity induced by ionotropic glutamate receptors gain the upper hand during ischemia-reperfusion. In this paper, we investigate whether GluR5 (glutamate receptor 5)-containing kainate receptor activation could lead to a neuroprotective effect against ischemic brain injury and the related mechanism. The results showed that (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective GluR5 agonist, could suppress Src tyrosine phosphorylation and interactions among N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A), postsynaptic density protein 95 (PSD-95), and Src and then decrease NMDA receptor activation through attenuating tyrosine phosphorylation of NR2A and NR2B. More importantly, ATPA had a neuroprotective effect against ischemia-reperfusion-induced neuronal cell death in vivo. However, four separate drugs were found to abolish the effects of ATPA. These were selective GluR5 antagonist NS3763; GluR5 antisense oligodeoxynucleotides; CdCl(2), a broad spectrum blocker of voltage-gated calcium channels; and bicuculline, an antagonist of gamma-aminobutyric acid A (GABA(A)) receptor. GABA(A) receptor agonist muscimol could attenuate Src activation and interactions among NR2A, PSD-95 and Src, resulting the suppression of NMDA receptor tyrosine phosphorylation. Moreover, patch clamp recording proved that the activated GABA(A) receptor could inhibit NMDA receptor-mediated whole-cell currents. Taken together, the results suggest that during ischemia-reperfusion, activated GluR5 may facilitate Ca(2+)-dependent GABA release from interneurons. The released GABA can activate postsynaptic GABA(A) receptors, which then attenuates NMDA receptor tyrosine phosphorylation through inhibiting Src activation and disassembling the signaling module NR2A-PSD-95-Src. The final result of this process is that the pyramidal

  4. Coarse-grained molecular simulation of epidermal growth factor receptor protein tyrosine kinase multi-site self-phosphorylation.

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    John G Koland


    Full Text Available Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR, the intrinsic protein tyrosine kinase (PTK activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites in either of the two C-terminal (CT domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in

  5. Ligand-induced tyrosine phosphorylation of cysteinyl leukotriene receptor 1 triggers internalization and signaling in intestinal epithelial cells.

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    Ladan Parhamifar

    Full Text Available BACKGROUND: Leukotriene D(4 (LTD(4 belongs to the bioactive lipid group known as eicosanoids and has implications in pathological processes such as inflammation and cancer. Leukotriene D(4 exerts its effects mainly through two different G-protein-coupled receptors, CysLT(1 and CysLT(2. The high affinity LTD(4 receptor CysLT(1R exhibits tumor-promoting properties by triggering cell proliferation, survival, and migration in intestinal epithelial cells. In addition, increased expression and nuclear localization of CysLT(1R correlates with a poorer prognosis for patients with colon cancer. METHODOLOGY/PRINCIPAL FINDINGS: Using a proximity ligation assay and immunoprecipitation, this study showed that endogenous CysLT(1R formed heterodimers with its counter-receptor CysLT(2R under basal conditions and that LTD(4 triggers reduced dimerization of CysLTRs in intestinal epithelial cells. This effect was dependent upon a parallel LTD(4-induced increase in CysLT(1R tyrosine phosphorylation. Leukotriene D(4 also led to elevated internalization of CysLT(1Rs from the plasma membrane and a simultaneous increase at the nucleus. Using sucrose, a clathrin endocytic inhibitor, dominant-negative constructs, and siRNA against arrestin-3, we suggest that a clathrin-, arrestin-3, and Rab-5-dependent process mediated the internalization of CysLT(1R. Altering the CysLT(1R internalization process at either the clathrin or the arrestin-3 stage led to disruption of LTD(4-induced Erk1/2 activation and up-regulation of COX-2 mRNA levels. CONCLUSIONS/SIGNIFICANCE: Our data suggests that upon ligand activation, CysLT(1R is tyrosine-phosphorylated and released from heterodimers with CysLT(2R and, subsequently, internalizes from the plasma membrane to the nuclear membrane in a clathrin-, arrestin-3-, and Rab-5-dependent manner, thus, enabling Erk1/2 signaling and downstream transcription of the COX-2 gene.

  6. A receptor tyrosine kinase, UFO/Axl, and other genes isolated by a modified differential display PCR are overexpressed in metastatic prostatic carcinoma cell line DU145. (United States)

    Jacob, A N; Kalapurakal, J; Davidson, W R; Kandpal, G; Dunson, N; Prashar, Y; Kandpal, R P


    We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.

  7. An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment

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    Tiziana Grafone


    Full Text Available Hematopoiesis, the process by which the hematopoietic stem cells and progenitors differentiate into blood cells of various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Despite the many controls that regulate hematopoiesis, mutations in the regulatory genes capable of promoting leukemogenesis may occur. The FLT3 gene encodes a tyrosine kinase receptor that plays a key role in controlling survival, proliferation and differentiation of hematopoietic cells. Mutations in this gene are critical in causing a deregulation of the delicate balance between cell proliferation and differentiation. In this review, we provide an update on the structure, synthesis and activation of the FLT3 receptor and the subsequent activation of multiple downstream signaling pathways. We also review activating FLT3 mutations that are frequently identified in acute myeloid leukemia, cause activation of more complex downstream signaling pathways and promote leukemogenesis. Finally, FLT3 has emerged as an important target for molecular therapy. We, therefore, report on some recent therapies directed against it.

  8. Reduced Proteolytic Shedding of Receptor Tyrosine Kinases is a Post-Translational Mechanism of Kinase Inhibitor Resistance (United States)

    Miller, Miles A.; Oudin, Madeleine J.; Sullivan, Ryan J.; Wang, Stephanie J.; Meyer, Aaron S.; Im, Hyungsoon; Frederick, Dennie T.; Tadros, Jenny; Griffith, Linda G.; Lee, Hakho; Weissleder, Ralph; Flaherty, Keith T.; Gertler, Frank B.; Lauffenburger, Douglas A.


    Kinase inhibitor resistance often involves upregulation of poorly understood “bypass” signaling pathways. Here, we show that extracellular proteomic adaptation is one path to bypass signaling and drug resistance. Proteolytic shedding of surface receptors, which can provide negative feedback on signaling activity, is blocked by kinase inhibitor treatment and enhances bypass signaling. In particular, MEK inhibition broadly decreases shedding of multiple receptor tyrosine kinases (RTKs) including HER4, MET, and most prominently AXL, an ADAM10 and ADAM17 substrate, thus increasing surface RTK levels and mitogenic signaling. Progression-free survival of melanoma patients treated with clinical BRAF/MEK inhibitors inversely correlates with RTK shedding reduction following treatment, as measured non-invasively in blood plasma. Disrupting protease inhibition by neutralizing TIMP1 improves MAPK inhibitor efficacy, and combined MAPK/AXL inhibition synergistically reduces tumor growth and metastasis in xenograft models. Altogether, extracellular proteomic rewiring through reduced RTK shedding represents a surprising mechanism for bypass signaling in cancer drug resistance. PMID:26984351

  9. Met receptor tyrosine kinase signaling induces secretion of the angiogenic chemokine interleukin-8/CXCL8 in pancreatic cancer.

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    Kristen S Hill

    Full Text Available At diagnosis, the majority of pancreatic cancer patients present with advanced disease when curative resection is no longer feasible and current therapeutic treatments are largely ineffective. An improved understanding of molecular targets for effective intervention of pancreatic cancer is thus urgent. The Met receptor tyrosine kinase is one candidate implicated in pancreatic cancer. Notably, Met is over expressed in up to 80% of invasive pancreatic cancers but not in normal ductal cells correlating with poor overall patient survival and increased recurrence rates following surgical resection. However the functional role of Met signaling in pancreatic cancer remains poorly understood. Here we used RNA interference to directly examine the pathobiological importance of increased Met signaling for pancreatic cancer. We show that Met knockdown in pancreatic tumor cells results in decreased cell survival, cell invasion, and migration on collagen I in vitro. Using an orthotopic model for pancreatic cancer, we provide in vivo evidence that Met knockdown reduced tumor burden correlating with decreased cell survival and tumor angiogenesis, with minimal effect on cell growth. Notably, we report that Met signaling regulates the secretion of the pro-angiogenic chemokine interleukin-8/CXCL8. Our data showing that the interleukin-8 receptors CXCR1 and CXCR2 are not expressed on pancreatic tumor cells, suggests a paracrine mechanism by which Met signaling regulates interleukin-8 secretion to remodel the tumor microenvironment, a novel finding that could have important clinical implications for improving the effectiveness of treatments for pancreatic cancer.

  10. Oncogenic activation of the Met receptor tyrosine kinase fusion protein, Tpr-Met, involves exclusion from the endocytic degradative pathway. (United States)

    Mak, H H L; Peschard, P; Lin, T; Naujokas, M A; Zuo, D; Park, M


    Multiple mechanisms of dysregulation of receptor tyrosine kinases (RTKs) are observed in human cancers. In addition to gain-of-function, loss of negative regulation also contributes to oncogenic activation of RTKs. Negative regulation of many RTKs involves their internalization and degradation in the lysosome, a process regulated through ubiquitination. RTK oncoproteins activated following chromosomal translocation, are no longer transmembrane proteins, and are predicted to escape lysosomal degradation. To test this, we used the Tpr-Met oncogene, generated following chromosomal translocation of the hepatocyte growth factor receptor (Met). Unlike Met, Tpr-Met is localized in the cytoplasm and also lacks the binding site for Cbl ubiquitin ligases. We determined whether subcellular localization of Tpr-Met, and/or loss of its Cbl-binding site, is important for oncogenic activity. Presence of a Cbl-binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter their transforming activity. In contrast, plasma membrane targeting allows Tpr-Met to enter the endocytic pathway, and Tpr-Met transforming activity as well as protein stability are decreased in a Cbl-dependent manner. We show that transformation by Tpr-Met is in part dependent on its ability to escape normal downregulatory mechanisms. This provides a paradigm for many RTK oncoproteins activated following chromosomal translocation.

  11. Neurotrophic tyrosine kinase receptor 1 is a direct transcriptional and epigenetic target of IL-13 involved in allergic inflammation. (United States)

    Rochman, M; Kartashov, A V; Caldwell, J M; Collins, M H; Stucke, E M; Kc, K; Sherrill, J D; Herren, J; Barski, A; Rothenberg, M E


    Although interleukin (IL)-13 and neurotrophins are functionally important for the pathogenesis of immune responses, the interaction of these pathways has not been explored. Herein, by interrogating IL-13-induced responses in human epithelial cells we show that neurotrophic tyrosine kinase receptor, type 1 (NTRK1), a cognate, high-affinity receptor for nerve growth factor (NGF), is an early transcriptional IL-13 target. Induction of NTRK1 was accompanied by accumulation of activating epigenetic marks in the promoter; transcriptional and epigenetic changes were signal transducer and activator of transcription 6 dependent. Using eosinophilic esophagitis as a model for human allergic inflammation, we found that NTRK1 was increased in inflamed tissue and dynamically expressed as a function of disease activity and that the downstream mediator of NTRK1 signaling early growth response 1 protein was elevated in allergic inflammatory tissue compared with control tissue. Unlike NTRK1, its ligand NGF was constitutively expressed in control and disease states, indicating that IL-13-stimulated NTRK1 induction is a limiting factor in pathway activation. In epithelial cells, NGF and IL-13 synergistically induced several target genes, including chemokine (C-C motif) ligand 26 (eotaxin-3). In summary, we have demonstrated that IL-13 confers epithelial cell responsiveness to NGF by regulating NTRK1 levels by a transcriptional and epigenetic mechanism and that this process likely contributes to allergic inflammation.

  12. DSD-1-Proteoglycan/Phosphacan and receptor protein tyrosine phosphatase-beta isoforms during development and regeneration of neural tissues. (United States)

    Faissner, Andreas; Heck, Nicolas; Dobbertin, Alexandre; Garwood, Jeremy


    Interactions between neurons and glial cells play important roles in regulating key events of development and regeneration of the CNS. Thus, migrating neurons are partly guided by radial glia to their target, and glial scaffolds direct the growth and directional choice of advancing axons, e.g., at the midline. In the adult, reactive astrocytes and myelin components play a pivotal role in the inhibition of regeneration. The past years have shown that astrocytic functions are mediated on the molecular level by extracellular matrix components, which include various glycoproteins and proteoglycans. One important, developmentally regulated chondroitin sulfate proteoglycan is DSD-1-PG/phosphacan, a glial derived proteoglycan which represents a splice variant of the receptor protein tyrosine phosphatase (RPTP)-beta (also known as PTP-zeta). Current evidence suggests that this proteoglycan influences axon growth in development and regeneration, displaying inhibitory or stimulatory effects dependent on the mode of presentation, and the neuronal lineage. These effects seem to be mediated by neuronal receptors of the Ig-CAM superfamily.

  13. ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia. (United States)

    Shankar, Deepa B; Li, Junling; Tapang, Paul; Owen McCall, J; Pease, Lori J; Dai, Yujia; Wei, Ru-Qi; Albert, Daniel H; Bouska, Jennifer J; Osterling, Donald J; Guo, Jun; Marcotte, Patrick A; Johnson, Eric F; Soni, Niru; Hartandi, Kresna; Michaelides, Michael R; Davidsen, Steven K; Priceman, Saul J; Chang, Jenny C; Rhodes, Katrin; Shah, Neil; Moore, Theodore B; Sakamoto, Kathleen M; Glaser, Keith B


    In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3-internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G(0)/G(1) phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)-FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

  14. Motor activity affects adult skeletal muscle re-innervation acting via tyrosine kinase receptors. (United States)

    Sartini, Stefano; Bartolini, Fanny; Ambrogini, Patrizia; Betti, Michele; Ciuffoli, Stefano; Lattanzi, Davide; Di Palma, Michael; Cuppini, Riccardo


    Recently, muscle expression of brain-derived neurotrophic factor (BDNF) mRNA and protein under activity control has been reported. BDNF is a neurotrophin known to be involved in axon sprouting in the CNS. Hence, we set out to study the effect of chronic treadmill mid-intensity running on adult rat muscle re-innervation, and to explore the involvement of BDNF and tropomyosin-related kinase (Trk) receptors. After nerve crush, muscle re-innervation was evaluated using intracellular recordings, tension recordings, immunostaining and Western blot analyses. An enhanced muscle multiple innervation was found in running rats that was fully reversed to control values blocking Trk receptors or interrupting the running activity. An increase in muscle multiple innervation was also found in sedentary rats treated with a selective TrkB receptor agonist. The expression of TrkB receptors by intramuscular axons was demonstrated, and increased muscle expression of BDNF was found in running animals. The increase in muscle multiple innervation was consistent with the faster muscle re-innervation that we found in running animals. We conclude that, when regenerating axons contact muscle cells, muscle activity progressively increases modulating BDNF and possibly other growth factors, which in turn, acting via Trk receptors, induce axon sprouting to re-innervate skeletal muscle.

  15. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

    DEFF Research Database (Denmark)

    Zhang, Qian; Wang, HongYi; Kantekure, Kanchan;


    Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression via......RNA and protein. Stimulation of the ICOS receptor with anti-ICOS antibody or ICOS ligand-expressing B cells markedly enhanced proliferation of the ALK(+) TCL cells. These results demonstrate that NPM-ALK, acting through STAT3 as the gene transcriptional activator, induces the expression of ICOS, a cell growth...

  16. The Role of Epidermal Growth Factor Receptor Mutations and Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in the Treatment of Lung Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Shih-Chieh [Department of Internal Medicine, National Yang-Ming University Hospital, Yilan 260, Taiwan (China); Chang, Cheng-Yu [Department of Chest Medicine, Far Eastern Memorial Hospital, Taipei 220, Taiwan (China); Shih, Jin-Yuan, E-mail: [Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei 100, Taiwan (China)


    Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC) cases comprise approximately 85% of the lung cancer cases. Before the era of target therapy, platinum-based doublet chemotherapy only led to a median survival of 8–9 months and a one-year survival of 30%–40% in patients with advanced NSCLC. In July 2002, gefitinib, a small-molecule epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), was approved for the treatment of patients with advanced NSCLC in Japan. After the widespread use of gefitinib in the treatment of NSCLC, there have been many new studies regarding the association between the clinical anticancer efficacy of gefitinib and the somatic EGFR mutation status in patients with NSCLC. This article summarizes the role of EGFR mutations in lung cancer and the use of EGFR antagonists in the treatment of lung cancer and its associated adverse effects.

  17. Inhibition of tyrosine kinase receptor Tie2 reverts HCV-induced hepatic stellate cell activation.

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    Samuel Martín-Vílchez

    Full Text Available BACKGROUND: Hepatitis C virus (HCV infection is a major cause of chronic liver disease (CLD and is frequently linked to intrahepatic microvascular disorders. Activation of hepatic stellate cells (HSC is a central event in liver damage, due to their contribution to hepatic renewal and to the development of fibrosis and hepatocarcinoma. During the progression of CLDs, HSC attempt to restore injured tissue by stimulating repair processes, such as fibrosis and angiogenesis. Because HSC express the key vascular receptor Tie2, among other angiogenic receptors and mediators, we analyzed its involvement in the development of CLD. METHODS: Tie2 expression was monitored in HSC cultures that were exposed to media from HCV-expressing cells (replicons. The effects of Tie2 blockade on HSC activation by either neutralizing antibody or specific signaling inhibitors were also examined. RESULTS: Media from HCV-replicons enhanced HSC activation and invasion and upregulated Tie2 expression. Notably, the blockade of Tie2 receptor (by a specific neutralizing antibody or signaling (by selective AKT and MAPK inhibitors significantly reduced alpha-smooth muscle actin (α-SMA expression and the invasive potential of HCV-conditioned HSC. CONCLUSIONS: These findings ascribe a novel profibrogenic function to Tie2 receptor in the progression of chronic hepatitis C, highlighting the significance of its dysregulation in the evolution of CLDs and its potential as a novel therapeutic target.

  18. Negative Suppressors of Oncogenic Activation of the Met Receptor Tyrosine Kinase (United States)


    We thank members of the Park lab, Dr. Sergio Grinstein and Dr. Stephane Laporte for their helpful comments on the manuscript. We would like to...of Hrs couples receptor sorting to vesicle formation. Journal of cell science 116: 4169-4179 Veracini L, Franco M, Boureux A, Simon V, Roche S

  19. Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

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    Kathleen Busman-Sahay

    Full Text Available Following antigen recognition, B cell receptor (BCR-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2 is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs. Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

  20. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS. (United States)

    Zhang, Qian; Wang, Hongyi; Kantekure, Kanchan; Paterson, Jennifer C; Liu, Xiaobin; Schaffer, Andras; Paulos, Chrystal; Milone, Michael C; Odum, Niels; Turner, Suzanne; Marafioti, Teresa; Wasik, Mariusz A


    Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression via STAT3, which triggers the transcriptional activity of the ICOS gene promoter. In addition, STAT3 suppresses the expression of miR-219 that, in turn, selectively inhibits ICOS expression. ALK(+) TCL cell lines display extensive DNA methylation of the CpG island located within intron 1, the putative enhancer region, of the ICOS gene, whereas cutaneous T-cell lymphoma cell lines, which strongly express ICOS, show no methylation of the island. Treatment of the ALK(+) TCL cell lines with DNA methyltransferase inhibitor reversed the CpG island methylation and augmented the expression of ICOS mRNA and protein. Stimulation of the ICOS receptor with anti-ICOS antibody or ICOS ligand-expressing B cells markedly enhanced proliferation of the ALK(+) TCL cells. These results demonstrate that NPM-ALK, acting through STAT3 as the gene transcriptional activator, induces the expression of ICOS, a cell growth promoting receptor. These data also show that the DNA methylation status of the intronic CpG island affects transcriptional activity of the ICOS gene and, consequently, modulates the concentration of the expressed ICOS protein.

  1. Effect of propofol on brain-derived neurotrophic factor and tyrosine kinase receptor B in the hippocampus of aged rats with chronic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Gang Chen; Qiang Fu; Jiangbei Cao; Weidong Mi


    We intraperitoneally injected 10 and 50 mg/kg of propofol for 7 consecutive days to treat a rat model of chronic cerebral ischemia. A low-dose of propofol promoted the expression of brain-derived neurotrophic factor, tyrosine kinase receptor B, phosphorylated cAMP response element binding protein, and cAMP in the hippocampus of aged rats with chronic cerebral ischemia, but a high-dose of propofol inhibited their expression. Results indicated that the protective effect of propofol against cerebral ischemia in aged rats is related to changes in the expression of brain-derived neurotrophic factor and tyrosine kinase receptor B in the hippocampus, and that the cAMP-cAMP responsive element binding protein pathway is involved in the regulatory effect of propofol on brain-derived neurotrophic factor expression.

  2. Study of Mutation in Tyrosine Protein Kinase of Insulin Receptor Gene in Patients with Polycystic Ovarian Syndrome

    Institute of Scientific and Technical Information of China (English)

    Min LI; Hong-yu QIU; Yong-yu SUN; Hong-fa LI; Yong-li CHU


    Objective To explore the molecular mechanism of insulin resistance in the patients with polycystic ovarian syndrome (PCOS)Methods Polymerase chain reaction, silver staining-single strand conformation polymorphism(PCR-SSCP) and DNA direct sequencing were used to detect the mutation of insulin receptor(INSR) gene in exon 17~21 with the abdominal wall adipose tissue from 31 patients with PCOS (PCOS Group) and 30 patients with pure hysteromyoma in reproductive lift (Control Group).Results Twenty-two variant SSCP patterns in exon 17 of INSR gene were detected. Direct sequence analysis of exon 17 showed that homozygous nonsense mutation was two alleles single nucleotide polymorphism(SNP) at the codon 1058 (CAC→CAT). Exons 18~21 were not detected with any significantly mutation. The INSR gene His1058C→T substitution collecting rate and insulin resistance were significantly higher in the PCOS group than in the control group (P=0.0293, P<0.05, P<0.01).Conclusion It is suggested that the SNP in codon 1058 of the INSR gene might be related with the insulin resistance in PCOS patients, which has hereditary tendency. And the missense mutation,nonsense mutation and frameshift mutation at exons 18~21 in tyrosine protein kinase region of INSR gene for PCOS patients were not frequently observed.

  3. Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling. (United States)

    Friedman, Adam A; Tucker, George; Singh, Rohit; Yan, Dong; Vinayagam, Arunachalam; Hu, Yanhui; Binari, Richard; Hong, Pengyu; Sun, Xiaoyun; Porto, Maura; Pacifico, Svetlana; Murali, Thilakam; Finley, Russell L; Asara, John M; Berger, Bonnie; Perrimon, Norbert


    Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification-mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type- and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine-protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

  4. Bisubstrate analog probes for the insulin receptor protein tyrosine kinase: molecular yardsticks for analyzing catalytic mechanism and inhibitor design. (United States)

    Hines, Aliya C; Parang, Keykavous; Kohanski, Ronald A; Hubbard, Stevan R; Cole, Philip A


    Bisubstrate analogs have the potential to provide enhanced specificity for protein kinase inhibition and tools to understand catalytic mechanism. Previous efforts led to the design of a peptide-ATP conjugate bisubstrate analog utilizing aminophenylalanine in place of tyrosine and a thioacetyl linker to the gamma-phosphate of ATP which was a potent inhibitor of the insulin receptor kinase (IRK). In this study, we have examined the contributions of various electrostatic and structural elements in the bisubstrate analog to IRK binding affinity. Three types of changes (seven specific analogs in all) were introduced: a Tyr isostere of the previous aminophenylalanine moiety, modifications of the spacer between the adenine and the peptide, and deletions and substitutions within the peptide moiety. These studies allowed a direct evaluation of the hydrogen bond strength between the anilino nitrogen of the bisubstrate analog and the enzyme catalytic base Asp and showed that it contributes 2.5 kcal/mol of binding energy, in good agreement with previous predictions. Modifications of the linker length resulted in weakened inhibitory affinity, consistent with the geometric requirements of an enzyme-catalyzed dissociative transition state. Alterations in the peptide motif generally led to diminished inhibitory potency, and only some of these effects could be rationalized based on prior kinetic and structural studies. Taken together, these results suggest that a combination of mechanism-based design and empirical synthetic manipulation will be necessary in producing optimized protein kinase bisubstrate analog inhibitors.

  5. Expression and function of the protein tyrosine phosphatase receptor J (PTPRJ) in normal mammary epithelial cells and breast tumors. (United States)

    Smart, Chanel E; Askarian Amiri, Marjan E; Wronski, Ania; Dinger, Marcel E; Crawford, Joanna; Ovchinnikov, Dmitry A; Vargas, Ana Cristina; Reid, Lynne; Simpson, Peter T; Song, Sarah; Wiesner, Christiane; French, Juliet D; Dave, Richa K; da Silva, Leonard; Purdon, Amy; Andrew, Megan; Mattick, John S; Lakhani, Sunil R; Brown, Melissa A; Kellie, Stuart


    The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

  6. Loss of Protein Tyrosine Phosphatase Receptor J Expression Predicts an Aggressive Clinical Course in Patients with Esophageal Squamous Cell Carcinoma. (United States)

    Qiao, Dongfeng; Li, Ming; Pu, Juan; Wang, Wanwei; Zhu, Weiguo; Liu, Haiyan


    Protein Tyrosine Phosphatase Receptor J (PTPRJ) has been reported to be a tumor suppressor in various human cancers. The aim of this study was to investigate the clinical significance of PTPRJ in ESCC patients and its effects on biological behaviors of ESCC cells. PTPRJ expression, at mRNA and protein levels, were respectively detected by quantitative real-time PCR, western blot and immunohistochemistry, based on 106 newly diagnosed ESCC patients. The associations between PTPRJ expression and clinicopathological characteristics of ESCC patients were statistically analyzed. Then, the effects of PTPRJ in migration and invasion were determined by wound healing and transwell assays based on ESCC cell line transfected with siRNA or expression vector of PTPRJ. Expression of PTPRJ at mRNA and protein levels were both significantly lower in ESCC tissues than those in normal esophageal mucosa. Immunohistochemistry showed that PTPRJ protein was localized in the cytoplasm of cancer cells in ESCC tissues. In addition, PTPRJ downregulation was found to be closely correlated with advanced tumor stage (P = 0.01) and poor differentiation (P = 0.03). Moreover, knockdown of PTPRJ in KYSE510 cells could significantly promote cell migration and invasion (both P ESCC patients. PTPRJ may function as a tumor suppressor and play an important role in the regulation of ESCC cell motility, suggesting its potentials as a therapeutic agent for human ESCC.

  7. Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE. (United States)

    Orme, Jacob J; Du, Yong; Vanarsa, Kamala; Mayeux, Jessica; Li, Li; Mutwally, Azza; Arriens, Cristina; Min, Soyoun; Hutcheson, Jack; Davis, Laurie S; Chong, Benjamin F; Satterthwaite, Anne B; Wu, Tianfu; Mohan, Chandra


    Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.

  8. EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE.

    Directory of Open Access Journals (Sweden)

    Kathryn M Munro

    Full Text Available The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE, axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

  9. TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations (United States)

    Castoldi, Raffaella; Schanzer, Jürgen; Panke, Christian; Jucknischke, Ute; Neubert, Natalie J.; Croasdale, Rebecca; Scheuer, Werner; Auer, Johannes; Klein, Christian; Niederfellner, Gerhard; Kobold, Sebastian; Sustmann, Claudio


    Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development. PMID:27578890

  10. Tyrosine kinase of insulin-like growth factor receptor as target for novel treatment and prevention strategies of colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Michael H(o)pfner; Andreas P Sutter; Alexander Huether; Viola Baradari; Hans Scherübl


    AIM: To investigate the antineoplastic potency of the novel insulin-like growth factor 1 receptor (IGF-1R) tyrosine kinase inhibitor (TKI) NVP-AEW541 in cell lines and primary cell cultures of human colorectal cancer (CRC).METHODS: Cells of primary colorectal carcinomas were from 8 patients. Immunostaining and crystal violet staining were used for analysis of growth factor receptor protein expression and detection of cell number changes,respectively. Cytotoxicity was determined by measuring the release of the cytoplasmic enzyme lactate dehydrogenase (LDH). The proportion of apoptotic cells was determined by quantifying the percentage of sub-G1(hypodiploid) cells. Cell cycle status reflected by the DNA content of the nuclei was detected by flow cytometry.RESULTS: NVP-AEW541 dose-dependently inhibited the proliferation of colorectal carcinoma cell lines and primary cell cultures by inducing apoptosis and cell cycle arrest. Apoptosis was characterized by caspase-3 activation and nuclear degradation. Cell cycle was arrested at the G1/S checkpoint. The NVP-AEW541-mediated cell cycle-related signaling involved the inactivation of Akt and extracellular signal-regulated kinase (ERK) 1/2, the upregulation of the cyclin-dependent kinase inhibitors p21Waf1/CIP1 and p27Kip1, and the downregulation of the cell cycle promoter cyclin D1. Moreover, BAX was upregulated during NVP-AEW541-induced apoptosis, whereas Bcl-2 was downregulated. Measurement of LDH release showed that the antineoplastic effect of NVP-AEW541 was not due to general cytotoxicity of the compound.However, augmented antineoplastic effects were observed in combination treatments of NVP-AEW541 with either 5-FU, or the EGFR-antibody cetuximab, or the HMG-CoA-reductase inhibitor fluvastatin.CONCLUSION: IGF-1R-TK inhibition is a promising novel approach for either mono- or combination treatment strategies of colorectal carcinoma and even for CRC chemoprevention.

  11. Association study of neurotrophic tyrosine kinase receptor type 2 (NTRK2) and childhood-onset mood disorders. (United States)

    Adams, Jennifer H; Wigg, Karen G; King, Nicole; Burcescu, Irina; Vetró, Agnes; Kiss, Eniko; Baji, Ildikó; George, Charles J; Kennedy, James L; Kovacs, Maria; Barr, Cathy L


    Childhood-onset mood disorders (COMD) are often familial, and twin studies of COMD provide compelling evidence that genetic factors are involved. Deficits in neural plasticity have been suggested to underlie the development of depression. The receptor tropomyosin related kinase B (TrkB) and its ligand, brain derived neurotrophic factor (BDNF), play essential roles in neural plasticity, and mRNA expression of both of these genes has been shown to be influenced by stress and chronic antidepressant treatment. In addition, TrkB knock-out mice display inappropriate stress coping mechanisms. Having previously shown that BDNF is associated with COMD, in this study we investigated the gene encoding TrkB, neurotrophic tyrosine kinase, receptor, type 2 (NTRK2) as a susceptibility factor in COMD. We tested for association of NTRK2 with COMD in two independent samples: (a) a case-control sample matched on ethnicity and gender, consisting of 120 cases who met DSM III/IV criteria for major depressive or dysthymic disorder before age 14 or bipolar I/II before the age of 18, and controls, and (b) a family based control sample of 113 families collected in Hungary, identified by a proband between the age of 7 and 14 who met DSM IV criteria for major depressive disorder or bipolar I/II disorder. There was no evidence for an allelic or genotypic association of three polymorphisms of NTRK2 with COMD in the case-control sample. Also, in the family based sample, using the transmission disequilibrium test (TDT), we did not identify any evidence of allelic association for each marker individually or when haplotypes were analyzed. Based on these results, using these three polymorphisms, we do not find support for NTRK2 as a susceptibility gene for COMD.

  12. Activation of nonreceptor tyrosine kinase Bmx/Etk mediated by phosphoinositide 3-kinase, epidermal growth factor receptor, and ErbB3 in prostate cancer cells. (United States)

    Jiang, Xinnong; Borgesi, Robert A; McKnight, Nicole C; Kaur, Ramneet; Carpenter, Christopher L; Balk, Steven P


    Pathways activated downstream of constitutively active phosphatidylinositol (PI) 3-kinase in PTEN-deficient prostate cancer (PCa) cells are possible therapeutic targets. We found that the nonreceptor Tec family tyrosine kinase Bmx/Etk was activated by tyrosine phosphorylation downstream of Src and PI 3-kinase in PTEN-deficient LNCaP and PC3 PCa cells and that Bmx down-regulation by short interfering RNA markedly inhibited LNCaP cell growth. Bmx also associated with ErbB3 in LNCaP cells, and heregulin-beta1 enhanced this interaction and further stimulated Bmx activity. Epidermal growth factor (EGF) similarly stimulated an interaction between Bmx and EGF receptor and rapidly increased Bmx kinase activity. Bmx stimulation in response to heregulin-beta1 and EGF was Src-dependent, and heregulin-beta1 stimulation of Bmx was also PI 3-kinase-dependent. In contrast, the rapid tyrosine phosphorylation and activation of Bmx in response to EGF was PI 3-kinase-independent. Taken together, these results demonstrate that Bmx is a critical downstream target of the constitutively active PI 3-kinase in PTEN-deficient PCa cells and further show that Bmx is recruited by the EGF receptor and ErbB3 and activated in response to their respective ligands. Therefore, Bmx may be a valuable therapeutic target in PCa and other epithelial malignancies in which PI 3-kinase or EGF receptor family pathways are activated.

  13. Overexpression of the receptor tyrosine kinase EphA4 in human gastric cancers

    Institute of Scientific and Technical Information of China (English)

    Mariko Oki; Hiroyuki Yamamoto; Hiroaki Taniguchi; Yasushi Adachi; Kohzoh Imai; Yasuhisa Shinomura


    AIM: To clarify the expression and role of Ephrin receptor A4 (EphA4) in gastric cancer in relation to clinicopathological characteristics and the expression of fibroblast growth factor receptor 1 (RGFR1) and ephrin ligands.METHODS: Eleven gastric carcinoma cell lines,24 paired surgical fresh specimens of gastric adenocarcinoma and adjacent nontumor tissue,74 conventional formalin-fixed,paraffin-embedded tumor specimens,and 55 specimens spotted on tissue microarray (TMA)were analyzed.Reverse transcription-PCR (RT-PCR),real-time RT-PCR,immunohistochemistry,and cell growth assays were performed.RESULTS: Overexpression of EphA4 mRNA expression was observed in 8 (73%) of 11 gastric cancer cell lines and 10 (42%) of 24 gastric cancer tissues.Overexpression of EphA4,analyzed by immunohistochemistry,was observed in 62 (48%) of 129 gastric cancer tissues.EphA4 overexpression,at the protein level,was significantly associated with depth of invasion and recurrence.EphA4 overexpression was also correlated with FGFR1 overexpression.Patients with EphA4-positive cancer had significantly shorter overall survival periods than did those with EphA4-negative cancer (P= 0.0008).The mRNAs for ephrin ligands were coexpressed in various combinations in gastric cancer cell lines and cancer tissues.Downregulation of EphA4 expression by siRNA in EphA4-overexpressing gastric cancer cell lines resulted in a significant decrease in cell growth.CONCLUSION: Our results suggest that overexpression of EphA4 plays a role in gastric cancer.

  14. Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.



    mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutati...

  15. Nonreceptor protein tyrosine and lipid phosphatases in type I fc(epsilon) receptor-mediated activation of mast cells and basophils. (United States)

    Heneberg, Petr; Dráber, Petr


    Protein tyrosine and lipid phosphorylations are early and critical events in type 1 Fc(epsilon) receptor (Fc(epsilon)RI)-mediated activation of mast cells and basophils. Tyrosine phosphorylation of Fc(epsilon)RI subunits as well as other signal transduction molecules reflects the balance between the action of protein tyrosine kinases and phosphatases. Similarly, the phosphate content of inositol phospholipids, involved in the recruitment of signalling molecules to the plasma membrane and the generation of secondary messengers, is the net result of the opposing effects of phosphoinositide kinases and lipid phosphatases. This review summarizes the current understanding of the structural and functional aspects of nonreceptor protein tyrosine phosphatases (SHP-1, SHP-2, HePTP, PTP20, PRL1, PRL2, PTP-MEG1 and PTP-MEG2) and lipid phosphatases (SHIP and SHIP2) in the activation of mast cells and basophils after Fc(epsilon)RI aggregation. New approaches towards a deeper understanding of the role of phosphatases in mast cell physiology are also discussed.

  16. Tyrosine kinase inhibitors for epidermal growth factor receptor gene mutation-positive non-small cell lung cancers: an update for recent advances in therapeutics. (United States)

    Chung, Clement


    The presence of activating gene mutations in the epidermal growth factor receptor of non-small cell lung cancer patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations are in-frame deletions in exon 19 (LREA deletions) and substitution in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, exon 19 deletion and exon 21 L858R gene substitution are present in about 10% of Caucasian patients and 20-40% of Asian patients with non-small cell lung cancer. T790M gene mutation at exon 20 is associated with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline epidermal growth factor receptor tyrosine kinase inhibitor therapy with afatinib is associated with improved progression-free survival compared to chemotherapy regardless of race. Moreover, guidelines now suggest EGFR gene mutation testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component, regardless of characteristics such as smoking status, gender or race. The success of targeted therapies in non-small cell lung cancer patients has changed the treatment paradigm in metastatic non-small cell lung cancer. However, despite a durable response of greater than a year, resistance to epidermal growth factor receptor tyrosine kinase inhibitors inevitably occurs. This mini-review describes the clinically relevant EGFR gene mutations and the efficacy/toxicity of small molecule epidermal growth factor receptor tyrosine kinase

  17. c-MET receptor tyrosine kinase as a molecular target in advanced hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Granito A


    Full Text Available Alessandro Granito,1 Elena Guidetti,1 Laura Gramantieri2,3 1Dipartimento di Scienze Mediche e Chirurgiche Università di Bologna, Bologna, Italy; 2Dipartimento dell'Apparato Digerente, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 3Centro di Ricerca Biomedica Applicata (CRBA, Azienda Ospedaliero-Universitaria Policlinico S Orsola-Malpighi e Università di Bologna, Bologna, Italy Abstract: c-MET is the membrane receptor for hepatocyte growth factor (HGF, also known as scatter factor or tumor cytotoxic factor, a mitogenic growth factor for hepatocytes. HGF is mainly produced by cells of mesenchymal origin and it mainly acts on neighboring epidermal and endothelial cells, regulating epithelial growth and morphogenesis. HGF/MET signaling has been identified among the drivers of tumorigenesis in human cancers. As such, c-MET is a recognized druggable target, and against it, targeted agents are currently under clinical investigation. c-MET overexpression is a common event in a wide range of human malignancies, including gastric, lung, breast, ovary, colon, kidney, thyroid, and liver carcinomas. Despite c-MET overexpression being reported by a large majority of studies, no evidence for a c-MET oncogenic addiction exists in hepatocellular carcinoma (HCC. In particular, c-MET amplification is a rare event, accounting for 4%–5% of cases while no mutation has been identified in c-MET oncogene in HCC. Thus, the selection of patient subgroups more likely to benefit from c-MET inhibition is challenging. Notwithstanding, c-MET overexpression was reported to be associated with increased metastatic potential and poor prognosis in patients with HCC, providing a rationale for its therapeutic inhibition. Here we summarize the role of activated HGF/MET signaling in HCC, its prognostic relevance, and the implications for therapeutic approaches in HCC. Keywords: hepatocellular carcinoma, c-MET, clinical trials

  18. Midgut-enriched receptor protein tyrosine phosphatase PTP52F is required for Drosophila development during larva-pupa transition. (United States)

    Santhanam, Abirami; Liang, Suh-Yuen; Chen, Dong-Yuan; Chen, Guang-Chao; Meng, Tzu-Ching


    To date our understanding of Drosophila receptor protein tyrosine phosphatases (R-PTPs) in the regulation of signal transduction is limited. Of the seven R-PTPs identified in flies, six are involved in the axon guidance that occurs during embryogenesis. However, whether and how R-PTPs may control key steps of Drosophila development is not clear. In this study we investigated the potential role of Drosophila R-PTPs in developmental processes outside the neuronal system and beyond the embryogenesis stage. Through systematic data mining of available microarray databases, we found the mRNA level of PTP52F to be highly enriched in the midgut of flies at the larva-pupa transition. This finding was confirmed by gut tissue staining with a specific antibody. The unique spatiotemporal expression of PTP52F suggests that it is possibly involved in regulating metamorphosis during the transformation from larva to pupa. To test this hypothesis, we employed RNA interference to examine the defects of transgenic flies. We found that ablation of endogenous PTP52F led to high lethality characterized by the pharate adult phenotype, occurring due to post pupal eclosion failure. These results show that PTP52F plays an indispensable role during the larva-pupa transition. We also found that PTP52F could be reclassified as a member of the subtype R3 PTPs instead of as an unclassified R-PTP without a human ortholog, as suggested previously. Together, these findings suggest that Drosophila R-PTPs may control metamorphosis and other biological processes beyond our current knowledge.

  19. Dopaminergic Receptors and Tyrosine Hydroxylase Expression in Peripheral Blood Mononuclear Cells: A Distinct Pattern in Central Obesity (United States)

    Leite, Fernanda; Lima, Margarida; Marino, Franca; Cosentino, Marco; Ribeiro, Laura


    Background Dopamine (DA) may be involved in central obesity (CO), an inflammatory condition, through its role in the central nervous system and in periphery, where it may affect immune cell function through five different DA receptors (DR). Whether dopaminergic pathways in peripheral immune cells are implicated in the inflammatory condition linked to CO is however unknown. Methods In a cohort of blood donors with and without CO, categorized by waist circumference (WC) (CO: WC ≥0.80 m in women and ≥0.94 m in men), we studied the expression of DR and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, in peripheral blood mononuclear cells (PBMCs) and their relation with anthropometric and metabolic/endocrine and inflammatory parameters. DR D1-5 and TH expression was assessed by semi quantitative real-time PCR. As inflammatory markers we investigated the immunophenotype of monocyte subsets by flow cytometry, staining for CD14, CD16, CD11b and CD36. Results CO individuals showed higher plasma levels of leptin and higher inflammatory pattern of monocytes compared with non-CO. PBMC expression of DR D2, DR D4 and DR D5 as well as of TH were lower in CO in comparison with non-CO. DR D2, and DR D5 expression correlated with lower WC and weight, and with lower inflammatory pattern of monocytes, and TH expression correlated with lower WC. DR D4 expression correlated with lower plasma levels of glycosylated hemoglobin, and DR D2 expression correlated with lower CO. Conclusions Results show that CO is associated with peripheral inflammation and downregulation of dopaminergic pathways in PBMCs, possibly suggesting DR expressed on immune cells as pharmacological targets in obesity for better metabolic outcome. PMID:26808524

  20. The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling. (United States)

    Hikasa, Hiroki; Shibata, Mikihito; Hiratani, Ichiro; Taira, Masanori


    The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.

  1. Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases. (United States)

    Johnston, J A; Wang, L M; Hanson, E P; Sun, X J; White, M F; Oakes, S A; Pierce, J H; O'Shea, J J


    The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.

  2. Immunohistochemical and Western blot analysis of two protein tyrosine phosphatase receptors, R and Z1, in colorectal carcinoma, colon adenoma and normal colon tissues. (United States)

    Woźniak, Marta; Gamian, Elżbieta; Łaczmańska, Izabela; Sąsiadek, Maria M; Duś-Szachniewicz, Kamila; Ziółkowski, Piotr


    Two classes of proteins, namely tyrosine kinases (PTK) and phosphatases (PTP), play an important role in cell proliferation and differentiation, thus leading to an acceleration or inhibition of tumour growth. The role of the above proteins in colorectal carcinoma (CRC) growth is a well-known event. In this study we carried out immunohistochemical and Western blot analysis of colorectal carcinoma, adenoma and normal colon tissue in relation to two protein tyrosine phosphatase receptors, R and Z1. Twenty-five cases of CRC were analyzed and the results were compared with similar data obtained in non-malignant tissues. High expression of both PTP receptors was observed in all examined cases of CRC, adenoma and normal colon tissue in this study. These results are not in line with recently published data, showing that genetic coding for PTPRR and PTPRZ1 were hypermethylated in CRC's. We presume that the protein tyrosine phosphatase overexpression in colorectal carcinoma is not enough to protect from the progression of disease.

  3. Toxicity profile of epidermal growth factor receptor tyrosine kinase inhibitors in patients with epidermal growth factor receptor gene mutation-positive lung cancer (United States)

    Takeda, Masayuki; Nakagawa, Kazuhiko


    Recent progress in the research on the molecular biology of lung cancer revealed that the clinical response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is associated with the presence of activating EGFR mutations. Three EGFR-TKIs, namely afatinib, erlotinib and gefitinib, are currently available for the treatment of patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC). Due to the dearth of published phase III trials prospectively evaluating the effects of one EGFR-TKI in comparison with another in such patients, the decision-making regarding which agent to recommend to any given patient lies with the treating physician. Given the potential long-term exposure of such patients to EGFR-TKIs, the toxicological properties of these drugs in such patients may differ from those observed in unselected patients. The aim of the present study was to provide an overview of the key adverse events (rash, diarrhea, hepatotoxicity and interstitial lung disease) reported for EGFR-TKIs in clinical trials including patients with advanced NSCLC. PMID:28123721

  4. The Role of Epidermal Growth Factor Receptor Mutations and Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in the Treatment of Lung Cancer

    Directory of Open Access Journals (Sweden)

    Shih-Chieh Chang


    Full Text Available Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC cases comprise approximately 85% of the lung cancer cases. Before the era of target therapy, platinum-based doublet chemotherapy only led to a median survival of 8–9 months and a one-year survival of 30%–40% in patients with advanced NSCLC. In July 2002, gefitinib, a small-molecule epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI, was approved for the treatment of patients with advanced NSCLC in Japan. After the widespread use of gefitinib in the treatment of NSCLC, there have been many new studies regarding the association between the clinical anticancer efficacy of gefitinib and the somatic EGFR mutation status in patients with NSCLC. This article summarizes the role of EGFR mutations in lung cancer and the use of EGFR antagonists in the treatment of lung cancer and its associated adverse effects.

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

    DEFF Research Database (Denmark)

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


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

  6. Preliminary measurement results of biotinylated BSA detection of a low cost optical cavity based biosensor using differential detection (United States)

    Cowles, Peter; Joy, Cody; Bujana, Antonio; Rho, DongGee; Kim, Seunghyun


    We report an optical cavity based biosensor using a novel differential detection method for point-of-care applications. Two laser diodes allow for multiplexing capability along with the ability to enhance the responsivity using differential detection. The laser wavelengths are chosen so that the optical intensities of two lasers change monotonically with opposite slopes upon the adsorption of desired biomarkers. The cavity width, PMMA thickness, and silver thickness have been optimized to achieve a large change in scaled differential value. We chose biotinylated BSA detection with Avidin as a receptor molecule to demonstrate the proposed design. Avidin is attached directly to the PMMA layer by physisorption. Then, biotinylated BSA is introduced to the sample and the intensities of the laser diodes are measured by a sCMOS camera. A change in the scaled differential value will correlate to the binding of biotinylated BSA. In this presentation, we will discuss simulation results, fabrication procedures, and preliminary measurement results.

  7. Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Wei-Lan Yeh

    Full Text Available Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD. In contrast to the Selective Estrogen Receptor Modulators (SERMs such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK, a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

  8. Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

    Directory of Open Access Journals (Sweden)

    Sundler Roger


    Full Text Available Abstract Background Yeast and bacteria elicit arachidonate release in macrophages, leading to the formation of leukotrienes and prostaglandins, important mediators of inflammation. Receptors recognising various microbes have been identified, but the signalling pathways are not entirely understood. Cytosolic phospholipase A2 is a major down-stream target and this enzyme is regulated by both phosphorylation and an increase in intracellular Ca2+. Potential signal components are MAP kinases, phosphatidylinositol 3-kinase and phospholipase Cγ2. The latter can undergo tyrosine phosphorylation, and Src family kinases might carry out this phosphorylation. Btk, a Tec family kinase, could also be important. Our aim was to further elucidate the role of Src family kinases and Btk. Methods Arachidonate release from murine peritoneal macrophages was measured by prior radiolabeling. Furthermore, immunoprecipitation and Western blotting were used to monitor changes in activity/phosphorylation of intermediate signal components. To determine the role of Src family kinases two different inhibitors with broad specificity (PP2 and the Src kinase inhibitor 1, SKI-1 were used as well as the Btk inhibitor LFM-A13. Results Arachidonate release initiated by either Staphylococcus aureus or yeast-derived zymosan beads was shown to depend on members of the Src kinase family as well as Btk. Src kinases were found to act upstream of Btk, phosphatidylinositol 3-kinase, phospholipase Cγ2 and the MAP kinases ERK and p38, thereby affecting all branches of the signalling investigated. In contrast, Btk was not involved in the activation of the MAP-kinases. Since the cytosolic phospholipase A2 in macrophages is regulated by both phosphorylation (via ERK and p38 and an increase in intracellular Ca2+, we propose that members of the Src kinase family are involved in both types of regulation, while the role of Btk may be restricted to the latter type. Conclusion Arachidonate release

  9. Research Progress on Resistance Mechanisms of Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Yuan LI


    Full Text Available With a greater understanding of tumor biology, novel molecular-targeted strategies that block cancer progression pathways have been evaluated as a new therapeutic approach for treating non-small cell lung cancer (NSCLC. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs, such as gefitinib and erlotinib, show favorable response to EGFR mutant lung cancer in some populations of NSCLC patients. However, the efficacy of EGFR-TKIs is limited by either primary (de novo or acquired resistance after therapy. This review will focus on recently identified mechanisms of primary and acquired resistance to EGFR TKIs and strategies currently being employed to overcome resistance.

  10. Decreased signaling competence as a result of receptor overexpression: overexpression of CD4 reduces its ability to activate p56lck tyrosine kinase and to regulate T-cell antigen receptor expression in immature CD4+CD8+ thymocytes.


    Nakayama, T.; Wiest, D L; Abraham, K.M.; Munitz, T I; Perlmutter, R M; Singer, A


    Thymic selection of the developing T-cell repertoire occurs in immature CD4+CD8+ thymocytes, with the fate of individual thymocytes determined by the specificity of T-cell antigen receptor they express. However, T-cell antigen receptor expression in immature CD4+CD8+ thymocytes is actively down-regulated in CD4+CD8+ thymocytes by CD4-mediated tyrosine kinase signals that are generated in the thymus as a result of CD4 engagement by intrathymic ligands. In the present study we have examined the...

  11. Tyrosine phosphorylation of transcriptional coactivator WW-domain binding protein 2 regulates estrogen receptor α function in breast cancer via the Wnt pathway. (United States)

    Lim, Shen Kiat; Orhant-Prioux, Magali; Toy, Weiyi; Tan, Kah Yap; Lim, Yoon Pin


    WW-binding protein 2 (WBP2) has been demonstrated in different studies to be a tyrosine kinase substrate, to activate estrogen receptor α (ERα)/progesterone receptor (PR) transcription, and to play a role in breast cancer. However, the role of WBP2 tyrosine phosphorylation in regulating ERα function and breast cancer biology is unknown. Here, we established WBP2 as a tyrosine phosphorylation target of estrogen signaling via EGFR crosstalk. Using dominant-negative, constitutively active mutants, RNAi, and pharmacological studies, we demonstrated that phosphorylation of WBP2 at Tyr192 and Tyr231 could be regulated by c-Src and c-Yes kinases. We further showed that abrogating WBP2 phosphorylation impaired >60% of ERα reporter activity, putatively by blocking nuclear entry of WBP2 and its interaction with ERα. Compared to vector control, overexpression of WBP2 and its phospho-mimic mutant in MCF7 cells resulted in larger tumors in mice, induced loss of cell-cell adhesion, and enhanced cell proliferation, anchorage-independent growth, migration, and invasion in both estrogen-dependent and -independent manners, events of which could be substantially abolished by overexpression of the phosphorylation-defective mutant. Hormone independence of cells expressing WBP2 phospho-mimic mutant was associated with heightened ERα and Wnt reporter activities. Wnt/β-catenin inhibitor FH535 blocked phospho-WBP2-mediated cancer cell growth more pronouncedly than tamoxifen and fulvestrant, in part by reducing the expression of ERα. Wnt pathway is likely to be a critical component in WBP2-mediated breast cancer biology.

  12. Different Biotinylation Strategies for Competitive Immunoassay of Estradiol

    Institute of Scientific and Technical Information of China (English)

    ZHAO,Jin-Fu(赵金富); WANG,Yong-Cheng(王永成); LI,Yuan-Zong(李元宗); CHANG,Wen-Bao(常文保)


    Study on biotinylation strategies for competitive immunoassay of estradiol (E2) was carried out. Two types of competitive enzyme immunoassay (EIA) with Biotin-Avidin amplification system were established and optimized.The E2-Biotin conjugate was used as a tracer in one assay, and biotinylated antibody was used as a tracer in the other. In both of EIAs, horseradish peroxidase-labelled Avidin (Avidin-HRP) was used with a spectrometric determination of enzyme activity. The precision, sensitivity and specificity were measured and compared. The results showed that although both were satisfactory in specificity, the EIA with hapten-Biotin showed to be superior to the EIA with biotinylated antibody in sensitivity and precision. The limit of detection of serum E2 was 8 and 50 pg/mL with E2-Biotin and biotinylated antibody as tracer, respectively.

  13. The Caenorhabditis elegans matrix non-peptidase MNP-1 is required for neuronal cell migration and interacts with the Ror receptor tyrosine kinase CAM-1. (United States)

    Craft, Teresa R; Forrester, Wayne C


    Directed cell migration is critical for metazoan development. During Caenorhabditis elegans development many neuronal, muscle and other cell types migrate. Multiple classes of proteins have been implicated in cell migration including secreted guidance cues, receptors for guidance cues and intracellular proteins that respond to cues to polarize cells and produce the forces that move them. In addition, cell surface and secreted proteases have been identified that may clear the migratory route and process guidance cues. We report here that mnp-1 is required for neuronal cell and growth cone migrations. MNP-1 is expressed by migrating cells and functions cell autonomously for cell migrations. We also find a genetic interaction between mnp-1 and cam-1, which encodes a Ror receptor tyrosine kinase required for some of the same cell migrations.

  14. Tyrosine phosphorylation of the N-Methyl-D-Aspartate receptor 2B subunit in spinal cord contributes to remifentanil-induced postoperative hyperalgesia: the preventive effect of ketamine

    Directory of Open Access Journals (Sweden)

    Cui Songqin


    Full Text Available Abstract Background Experimental and clinical studies showed that intraoperative infusionof remifentanil has been associated with postoperative hyperalgesia. Previous reports suggested that spinal N-methyl-D-aspartate (NMDA receptors may contribute to the development and maintenance of opioid-induced hyperalgesia. In the present study, we used a rat model of postoperative pain to investigate the role of tyrosine phosphorylation of NMDA receptor 2B (NR2B subunit in spinal cord in the postoperative hyperalgesia induced by remifentanil and the intervention of pretreatment with ketamine. Results Intraoperative infusion of remifentanil (0.04 mg/kg, subcutaneous significantly enhanced mechanical allodynia and thermal hyperalgesia induced by the plantar incision during the postoperative period (each lasting between 2 h and 48 h, which was attenuated by pretreatment with ketamine (10 mg/kg, subcutaneous. Correlated with the pain behavior changes, immunocytochemical and western blotting experiments in our study revealed that there was a marked increase in NR2B phosphorylation at Tyr1472 in the superficial dorsal horn after intraoperative infusion of remifentanil, which was attenuated by pretreatment with ketamine. Conclusions This study provides direct evidence that tyrosine phosphorylation of the NR2B at Tyr1472 in spinal dosal horn contributes to postoperative hyperalgesia induced by remifentanil and supports the potential therapeutic value of ketamine for improving postoperative hyperalgesia induced by remifentanil.

  15. The human epidermal growth factor receptor (EGFR gene in European patients with advanced colorectal cancer harbors infrequent mutations in its tyrosine kinase domain

    Directory of Open Access Journals (Sweden)

    Delvenne Philippe


    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR, a member of the ErbB family of receptors, is a transmembrane tyrosine kinase (TK activated by the binding of extracellular ligands of the EGF-family and involved in triggering the MAPK signaling pathway, which leads to cell proliferation. Mutations in the EGFR tyrosine kinase domain are frequent in non-small-cell lung cancer (NSCLC. However, to date, only very few, mainly non-European, studies have reported rare EGFR mutations in colorectal cancer (CRC. Methods We screened 236 clinical tumor samples from European patients with advanced CRC by direct DNA sequencing to detect potential, as yet unknown mutations, in the EGFR gene exons 18 to 21, mainly covering the EGFR TK catalytic domain. Results EGFR sequences showed somatic missense mutations in exons 18 and 20 at a frequency of 2.1% and 0.4% respectively. Somatic SNPs were also found in exons 20 and 21 at a frequency of about 3.1% and 0.4% respectively. Of these mutations, four have not yet been described elsewhere. Conclusions These mutation frequencies are higher than in a similarly sized population characterized by Barber and colleagues, but still too low to account for a major role played by the EGFR gene in CRC.

  16. Recent advancements in small molecule inhibitors of insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase as anticancer agents. (United States)

    Negi, Arvind; Ramarao, P; Kumar, Raj


    Advancements in understanding of the genetics, genomics, biochemistry and the pharmacology of cancer inhuman, have driven the current cancer chemotherapy to intently focus on development of target-based approaches rather than conventional approaches. From among the various targets identified, validated and inhibited at different hallmarks of cancer, protein tyrosine kinases (PTKs) have been exploited the most. Insulin receptors (IRs), insulin like growth factor receptors (IGF-1R) and their hybrid receptors belong to tyrosine kinase receptor (TKR) family, constitute a structural homology among them and generate a growth promoting IGF system on binding with either insulin, IGF-1 or IGF-2. The system induces the mitogenic effects through a torrent of cell signals produced as a result of cross talk with other growth promoting peptides and steroidal hormones, ultimately resulting in hijacking apoptosis and increasing cell proliferation and cell survival in cancer cells. Various strategies such as anti-IGF-1R antibodies, IGF-1 mimetic peptides, antisense strategies, IGF-1R specific peptide aptamers, targeted degradation of IGF-1R and expression of dominant negative IGF-1R mutants have been explored to inhibit the IGF-1R signaling. However, targeting IGF-1R with small molecules has gained considerable attention in last few years due to their ease of synthesis, ease of optimization of absorption,distribution, metabolism, excretion and toxicity (ADMET) parameters, oral route of administration, lesser side effects and cost effectiveness. The present review provides a broad overview and discusses the highlights on discoveries, SAR studies and binding interactions of small molecules with either IGF-1R active or allosteric sites reported till date.

  17. Overexpression of Tyro3 receptor tyrosine kinase leads to the acquisition of taxol resistance in ovarian cancer cells. (United States)

    Lee, Chuhee


    The majority of patients with ovarian cancer are diagnosed at the advanced stages (III/IV) and their 5-year-survival rate is relatively low. One of the major causes of the poor prognosis of ovarian cancer is the development of resistance to first-line chemotherapy, including platinum and taxol. Therefore, improvements in current understanding of chemoresistance is required for the successful treatment of ovarian cancer. In the present study, taxol-resistant ovarian cancer cells, SKOV3/TR, were established by exposing parental SKOV3 cells to increasing concentrations of taxol. . Briefly, cells were treated with 1.5 nM (for 4 weeks), 3 nM (for 4 weeks), 6 nM (for 5 weeks), 12 nM (for 5 weeks) and 24 nM taxol (for 8 weeks) over 6 months. The SKOV3/TR cells were found to be smaller in size and rounder in shape compared with their parental cells. Cell viability and colony formation assays demonstrated an increase in the population doubling time of the SKOV3/TR cells, indicating a reduction in the proliferative capacity of these cells. Reverse transcription-polymerase chain reaction and western blot analysis revealed that, among the TAM receptor tyrosine kinases (RTKs), the mRNA and protein expression levels of Tyro3 RTK were increased, while those of Axl and Mer RTK were decreased in the SKOV3/TR cells. In addition, restoration of the level of Tyro3 by transfecting Tyro3-specific small interfering RNA into the SKOV3/TR cells reduced the proliferative capacity of the cells, indicating that upregulation of the expression of Tyro3 in SKOV3/TR cells may promote survival in the presence of taxol, which eventually resulted in the acquisition of resistance upon taxol treatment. The present study subsequently found that, in the SKOV3/TR cells, the level of intracellular reactive oxygen species (ROS) was elevated, and antioxidant treatment with N-acetyl cysteine (NAC) exerted more profound antiproliferative effects compared with the parental cells. The western blot analysis

  18. Helicobacter pylori VacA, acting through receptor protein tyrosine phosphatase α, is crucial for CagA phosphorylation in human duodenum carcinoma cell line AZ-521 (United States)

    Yahiro, Kinnosuke; Yamasaki, Eiki; Kurazono, Hisao; Akada, Junko; Yamaoka, Yoshio; Niidome, Takuro; Hatakeyama, Masanori; Suzuki, Hidekazu; Yamamoto, Taro; Moss, Joel; Isomoto, Hajime; Hirayama, Toshiya


    ABSTRACT Helicobacter pylori, a major cause of gastroduodenal diseases, produces vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA), which seem to be involved in virulence. VacA exhibits pleiotropic actions in gastroduodenal disorders via its specific receptors. Recently, we found that VacA induced the phosphorylation of cellular Src kinase (Src) at Tyr418 in AZ-521 cells. Silencing of receptor protein tyrosine phosphatase (RPTP)α, a VacA receptor, reduced VacA-induced Src phosphorylation. Src is responsible for tyrosine phosphorylation of CagA at its Glu-Pro-Ile-Tyr-Ala (EPIYA) variant C (EPIYA-C) motif in Helicobacter pylori-infected gastric epithelial cells, resulting in binding of CagA to SHP-2 phosphatase. Challenging AZ-521 cells with wild-type H. pylori induced phosphorylation of CagA, but this did not occur when challenged with a vacA gene-disrupted mutant strain. CagA phosphorylation was observed in cells infected with a vacA gene-disrupted mutant strain after addition of purified VacA, suggesting that VacA is required for H. pylori-induced CagA phosphorylation. Following siRNA-mediated RPTPα knockdown in AZ-521 cells, infection with wild-type H. pylori and treatment with VacA did not induce CagA phosphorylation. Taken together, these results support our conclusion that VacA mediates CagA phosphorylation through RPTPα in AZ-521 cells. These data indicate the possibility that Src phosphorylation induced by VacA is mediated through RPTPα, resulting in activation of Src, leading to CagA phosphorylation at Tyr972 in AZ-521 cells. PMID:27935824

  19. Cellular maturation defects in Bruton's tyrosine kinase-deficient immature B cells are amplified by premature B cell receptor expression and reduced by receptor editing

    NARCIS (Netherlands)

    S. Middendorp; R.W. Hendriks (Rudi)


    textabstractIn the mouse, Bruton's tyrosine kinase (Btk) is essential for efficient developmental progression of CD43(+)CD2(-) large cycling into CD43(-)CD2(+) small resting pre-B cells in the bone marrow and of IgM(high) transitional type 2 B cells into IgM(low) mature B cells in

  20. Binding assays with streptavidin-functionalized superparamagnetic nanoparticles and biotinylated analytes using fluxgate magnetorelaxometry

    Energy Technology Data Exchange (ETDEWEB)

    Heim, Erik [TU Braunschweig, Institut fuer Elektrische Messtechnik und Grundlagen der Elektrotechnik, Hans-Sommer-Str. 66, 38106 Braunschweig (Germany)], E-mail:; Ludwig, Frank; Schilling, Meinhard [TU Braunschweig, Institut fuer Elektrische Messtechnik und Grundlagen der Elektrotechnik, Hans-Sommer-Str. 66, 38106 Braunschweig (Germany)


    Binding assays based on the magnetorelaxation of superparamagnetic nanoparticles as markers are presented utilizing a differential fluxgate system. As ligand and receptor, streptavidin and biotin, respectively, are used. Superparamagnetic nanoparticles are functionalized with streptavidin and bound to two types of biotinylated analytes: agarose beads and bovine serum (BSA) proteins. The size difference of the two analytes causes a different progress of the reaction. As a consequence, the analysis of the relaxation signal is carried out dissimilarly for the two analytes. In addition, we studied the reaction kinetics of the two kinds of analytes with the fluxgate system.

  1. Role of protein tyrosine phosphatase non-receptor type 7 in the regulation of TNF-α production in RAW 264.7 macrophages.

    Directory of Open Access Journals (Sweden)

    Huiyun Seo

    Full Text Available Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7, a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs. Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2 and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.

  2. Role of protein tyrosine phosphatase non-receptor type 7 in the regulation of TNF-α production in RAW 264.7 macrophages. (United States)

    Seo, Huiyun; Lee, In-Seon; Park, Jae Eun; Park, Sung Goo; Lee, Do Hee; Park, Byoung Chul; Cho, Sayeon


    Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7), a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs). Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS) that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA) analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2) and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.

  3. Development of epidermal growth factor receptor tyrosine kinase inhibitors against EGFR T790M. Mutation in non small-cell lung carcinoma

    Directory of Open Access Journals (Sweden)

    Wang Yuli


    Full Text Available Individualized therapies targeting epidermal growth factor receptor (EGFR mutations show promises for the treatment of non small-cell lung carcinoma (NSCLC. However, disease progression almost invariably occurs 1 year after tyrosine kinase inhibitor (TKI treatment. The most prominent mechanism of acquired resistance involves the secondary EGFR mutation, namely EGFR T790M, which accounts for 50%–60% of resistant tumors. A large amount of studies have focused on the development of effective strategies to treat TKI-resistant EGFR T790M mutation in lung tumors. Novel generations of EGFR inhibitors are producing encouraging results in patients with acquired resistance against EGFR T790M mutation. This review will summarize the novel inhibitors, which might overcome resistance against EGFR T790M mutation.

  4. Research progress in the use of combinations of platinum-based chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors

    Institute of Scientific and Technical Information of China (English)

    Chi Pan; Suzhan Zhang; Jianjin Huang


    In the past decade, the advent of the epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has dramatically influenced the therapeutic strategies for treating lung cancer, but with tumor progression and drug resistance, patients will ultimately develop reduced sensitivity to EGFR-TKIs. How can we delay the emergence of drug resistance? What is the next strategy after drug resistance? How to reasonably combine platinum-based chemotherapy and EGFR-TKIs? These questions are currently the focus of lung cancer research. Clinical studies have reported that platinum-based chemotherapy can increase the sensitivity to EGFR-TKIs. However, results of pre-clinical and clinical studies have been inconsistent. The mechanisms of platinum chemotherapy and EGFR-TKIs are still unknown due to the lack of systematic research. Therefore, systematic studies are required to show the mechanisms of EGFR-TKIs and chemotherapy agents and define the markers sensitive to their combinations when given concurrently or sequentially.

  5. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways

    DEFF Research Database (Denmark)

    Pandey, A; Fernandez, M M; Steen, H;


    In an effort to clone novel tyrosine-phosphorylated substrates of the epidermal growth factor receptor, we have initiated an approach coupling affinity purification using anti-phosphotyrosine antibodies to mass spectrometry-based identification. Here, we report the identification of a signaling m...

  6. Epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutation testing in adults with locally advanced or metastatic non-small cell lung cancer : a systematic review and cost-effectiveness analysis

    NARCIS (Netherlands)

    Westwood, Marie; Joore, Manuela; Whiting, Penny; van Asselt, Thea; Ramaekers, Bram; Armstrong, Nigel; Misso, Kate; Severens, Johan; Kleijnen, Jos


    BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. Some epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutations make tumours responsive to treatment with EGFR-TK inhibitors (EGFR-TKIs) but less responsive to treatment with standard chemotherapy. Patie

  7. Epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutation testing in adults with locally advanced or metastatic non-small cell lung cancer: A systematic review and cost-effectiveness analysis

    NARCIS (Netherlands)

    M. Westwood (Marie); M.A. Joore (Manuela); P. Whiting (Penny); T. van Asselt (Thea); B.L.T. Ramaekers (Bram); N. Armstrong (Nigel); K. Misso (Kate); J.L. Severens (Hans); J. Kleijnen (Jos)


    markdownabstract__Abstract__ Background: Non-small cell lung cancer (NSCLC) is the most common form of lung cancer. Some epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutations make tumours responsive to treatment with EGFR-TK inhibitors (EGFR-TKIs) but less responsive to treatment wit

  8. 6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Barden, E-mail: [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); VanderLaan, Paul A. [Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); Sukhatme, Vikas P. [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States)


    Highlights: •Expression of 6PGD positively correlates with advancing stage of lung carcinoma. •Knockdown of 6PGD by shRNA potently inhibits c-Met tyrosine phosphorylation. •Exogenous HGF fails to restore c-Met phosphorylation in cells with 6PGD knocked down. •6PGD knockdown results in inhibition of cell migration in vitro. •Constitutively active TPR-cMet significantly restores migration of cells without 6PGD. -- Abstract: 6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functional role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.

  9. A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase. (United States)

    Hassan, Yousef I; Zempleni, Janos


    Holocarboxylase synthetase catalyzes the covalent binding of biotin to histones in humans and other eukaryotes. Eleven biotinylation sites have been identified in histones H2A, H3, and H4. K12-biotinylated histone H4 is enriched in heterochromatin, repeat regions, and plays a role in gene repression. About 30% of the histone H4 molecules are biotinylated at K12 in histone H4 in human fibroblast telomeres. The abundance of biotinylated histones at distinct genomic loci depends on biotin availability. Decreased histone biotinylation decreases life span and stress resistance in Drosophila. Low enrichment of biotinylated histones at transposable elements impairs repression of these elements.

  10. Relative expression of the p75 neurotrophin receptor, tyrosine receptor kinase A, and insulin receptor in SH-SY5Y neuroblastoma cells and hippocampi from Alzheimer's disease patients. (United States)

    Ito, Shingo; Ménard, Michel; Atkinson, Trevor; Brown, Leslie; Whitfield, James; Chakravarthy, Balu


    We have previously shown in SH-SY5Y human neuroblastoma cells that the expressions of basal (75 kDa) and high molecular weight (HMW; 85 kDa) isoforms of the p75 neurotrophic receptor (p75NTR) are stimulated by amyloid-β peptide1-42 oligomers (AβOs) via the insulin-like growth factor-1 receptor (IGF-1R). On the other hand, it is known that AβOs inhibit insulin receptor (IR) signaling. The purpose of the present study was to determine the involvement of IR signaling in the regulation of p75 neurotrophin receptor (p75NTR) protein isoform expression in cultured SH-SY5Y cells and in hippocampi from late-stage human Alzheimer's disease (AD) brains. Interestingly, insulin induced the expression of basal and HMW p75NTR isoforms in SH-SY5Y cells, suggesting the presence of cross-talk between the IR and IGF-1R for the regulation of p75NTR expression. Reducing IR signaling with an IR kinase inhibitor (AG 1024) or IR-targeted siRNAs increased HMW p75NTR expression and reduced tyrosine receptor kinase-A (Trk-A) expression as well as postsynaptic density protein 95 (PSD95) expression in SH-SY5Y cells. Both basal and HMW p75NTR isoforms were increased in the hippocampi of post-mortem late-stage human AD brains (relative to non-AD brains), and the protein expression of HMW p75NTR was negatively associated with Trk-A expression, PSD95 expression, and IR expression. Thus, increased p75NTR expression, specifically an increased p75NTR-to-Trk-A ratio, is likely to play a role in synaptic loss and neuronal cell death in late-stage AD. Collectively, these findings suggest that increased expression of the p75NTR due to IR signaling inhibition by AβOs might be involved in the pathology of AD.

  11. Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHEN Yang; YANG Su-juan; FU Yao; WANG Jia-peng; ZHAO Zhi-zhuang; FU Xue-qi


    A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPa.

  12. Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

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    Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Wang, Shu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Dong, Xin; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China)


    Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury.

  13. Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

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    Yuan Jian


    Full Text Available Abstract Background The downstream of tyrosine kinase/docking protein (Dok adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk receptor family, which has three members (TrkA, TrkB and TrkC, are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST precipitation assays and coimmunoprecipitation (Co-IP experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3 stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

  14. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases. (United States)

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun


    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment.

  15. N-Stearoyl-L-Tyrosine Inhibits the Senescence of Neural Stem/Progenitor Cells Induced by Aβ1–42 via the CB2 Receptor

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    Wen-Qing Li


    Full Text Available Alzheimer’s disease, one of the neurodegenerative diseases, shows the progressive senescence of neural progenitor/stem cells. N-Stearoyl-L-tyrosine (NsTyr showed neuroprotective effect against chronic brain ischemia in previous reports. In the present study, we find the antisenescent effects of NsTyr-2K in NSPCs induced by Aβ1–42 in vitro. Cell viability of NSPCs was evaluated by CCK8 assay; SA-β-gal staining was used to evaluate senescence of NSPCs. CB receptors were detected by immunohistochemistry in NSPCs. AM251 or AM630 was used to offset the anti-senescence effects afforded by NsTyr-2K. The positive rate of SA-β-gal staining was significantly increased in NSPCs after incubation with Aβ1–42 for 9 days. CB receptors were found on the surface of NSPCs. The expression level of CB1 receptors was significantly decreased in NSPCs after incubation with Aβ1–42. This phenomenon was reversed dose-dependently by NsTyr-2K. NsTyr-2K attenuated Aβ1–42 induced NSPCs senescence dose-dependently, and its antisenescence effect was completely abolished by AM630. Aβ1–42 dose-dependently increased the prosenescence molecules p16 and Rb. Their expression was inhibited by NsTyr-2K dose-dependently and blocked by AM630 in NSPCs. These results suggest that NsTyr-2K can alleviate the senescence of NSPCs induced by Aβ1–42 via CB2 receptor.

  16. Involvement of β3A Subunit of Adaptor Protein-3 in Intracellular Trafficking of Receptor-like Protein Tyrosine Phosphatase PCP-2

    Institute of Scientific and Technical Information of China (English)

    Hui DONG; Hong YUAN; Weirong JIN; Yan SHEN; Xiaojing XU; Hongyang WANG


    PCP-2 is a human receptor-like protein tyrosine phosphatase and a member of the MAM domain family cloned in human pancreatic adenocarcinoma cells. Previous studies showed that PCP-2 directly interacted with β-catenin through the juxtamembrane domain, dephosphorylated β-catenin and played an important role in the regulation of cell adhesion. Recent study showed that PCP-2 was also involved in the repression of β-catenin-induced transcriptional activity. Here we describe the interactions of PCP-2 with the β3A subunit of adaptor protein (AP)-3 and sorting nexin (SNX) 3. These protein complexes were detected using the yeast two-hybrid assay with the juxtamembrane and membrane-proximal catalytic domain of PCP-2 as "bait". Both AP-3 and SNX3 are molecules involved in intracellular trafficking of membrane receptors. The association between the β3A subunit of AP-3 and PCP-2 was further confirmed in mammalian cells. Our results suggested a possible mechanism of intracellular trafficking of PCP-2 mediated by AP-3 and SNX3 which might participate in the regulation of PCP-2 functions.

  17. Simple methods for the 3' biotinylation of RNA. (United States)

    Moritz, Bodo; Wahle, Elmar


    Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3' ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3'-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3' ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of 29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme's proofreading activity, can cope with more extended 3' heterogeneities.

  18. Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fc epsilon receptors. (United States)

    Amoui, M; Dráberová, L; Tolar, P; Dráber, P


    Activation of rat mast cells through the receptor with high affinity for IgE (Fc epsilonRI) requires a complex set of interactions involving transmembrane subunits of the Fc epsilonRI and two classes of nonreceptor protein tyrosine kinase (PTK). the Src family PTK p53/p56(lyn) (Lyn) and the Syk/ZAP-family PTK p72(syk) (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated Fc epsilonRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of Fc epsilonRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of Fc epsilonRI beta or gamma subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of Fc epsilonRI beta or gamma subunits were used, suggesting that Fc epsilonRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in Fc epsilonRI signaling.

  19. Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available The receptor tyrosine kinase (RTK ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL. There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8 induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC and antibody dependent cellular cytotoxicity (ADCC. The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375 including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

  20. Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells

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    Gui Anping


    Full Text Available Abstract Background Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling. Methods We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS, and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling. Results Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045, were also observed in RBE cells. Conclusion In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.

  1. Activation of platelet-activating factor receptor and pleiotropic effects on tyrosine phospho-EGFR/Src/FAK/paxillin in ovarian cancer. (United States)

    Aponte, Margarita; Jiang, Wei; Lakkis, Montaha; Li, Ming-Jiang; Edwards, Dale; Albitar, Lina; Vitonis, Allison; Mok, Samuel C; Cramer, Daniel W; Ye, Bin


    Among the proinflammatory mediators, platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a major primary and secondary messenger involved in intracellular and extracellular communication. Evidence suggests that PAF plays a significant role in oncogenic transformation, tumor growth, angiogenesis, and metastasis. However, PAF, with its receptor (PAFR) and their downstream signaling targets, has not been thoroughly studied in cancer. Here, we characterized the PAFR expression pattern in 4 normal human ovarian surface epithelial (HOSE) cell lines, 13 ovarian cancer cell lines, paraffin blocks (n = 84), and tissue microarrays (n = 230) from patients with ovarian cancer. Overexpression of PAFR was found in most nonmucinous types of ovarian cancer but not in HOSE and mucinous cancer cells. Correspondingly, PAF significantly induced cell proliferation and invasion only in PAFR-positive cells (i.e., OVCA429 and OVCA432), but not in PAFR-negative ovarian cells (HOSE and mucinous RMUG-L). The dependency of cell proliferation and invasion on PAFR was further confirmed using PAFR-specific small interfering RNA gene silencing probes, antibodies against PAFR and PAFR antagonist, ginkgolide B. Using quantitative multiplex phospho-antibody array technology, we found that tyrosine phosphorylation of EGFR/Src/FAK/paxillin was coordinately activated by PAF treatment, which was correlated with the activation of phosphatidylinositol 3-kinase and cyclin D1 as markers for cell proliferation, as well as matrix metalloproteinase 2 and 9 for invasion. Specific tyrosine Src inhibitor (PP2) reversibly blocked PAF-activated cancer cell proliferation and invasion. We suggest that PAFR is an essential upstream target of Src and other signal pathways to control the PAF-mediated cancer progression.

  2. A multiscale modeling approach to investigate molecular mechanisms of pseudokinase activation and drug resistance in the HER3/ErbB3 receptor tyrosine kinase signaling network. (United States)

    Telesco, Shannon E; Shih, Andrew J; Jia, Fei; Radhakrishnan, Ravi


    Multiscale modeling provides a powerful and quantitative platform for investigating the complexity inherent in intracellular signaling pathways and rationalizing the effects of molecular perturbations on downstream signaling events and ultimately, on the cell phenotype. Here we describe the application of a multiscale modeling scheme to the HER3/ErbB3 receptor tyrosine kinase (RTK) signaling network, which regulates critical cellular processes including proliferation, migration and differentiation. The HER3 kinase is a topic of current interest and investigation, as it has been implicated in mechanisms of resistance to tyrosine kinase inhibition (TKI) of EGFR and HER2 in the treatment of many human malignancies. Moreover, the commonly regarded status of HER3 as a catalytically inactive 'pseudokinase' has recently been challenged by our previous study, which demonstrated robust residual kinase activity for HER3. Through our multiscale model, we investigate the most significant molecular interactions that contribute to potential mechanisms of HER3 activity and the physiological relevance of this activity to mechanisms of drug resistance in an ErbB-driven tumor cell in silico. The results of our molecular-scale simulations support the characterization of HER3 as a weakly active kinase that, in contrast to its fully-active ErbB family members, depends upon a unique hydrophobic interface to coordinate the alignment of specific catalytic residues required for its activity. Translating our molecular simulation results of the uniquely active behavior of the HER3 kinase into a physiologically relevant environment, our HER3 signaling model demonstrates that even a weak level of HER3 activity may be sufficient to induce AKT signaling and TKI resistance in the context of an ErbB signaling-dependent tumor cell, and therefore therapeutic targeting of HER3 may represent a superior treatment strategy for specific ErbB-driven cancers.

  3. Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP. (United States)

    Chiodi, Valentina; Mallozzi, Cinzia; Ferrante, Antonella; Chen, Jiang F; Lombroso, Paul J; Di Stasi, Anna Maria Michela; Popoli, Patrizia; Domenici, Maria Rosaria


    The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine.

  4. Mapping of the receptor protein-tyrosine kinase 10 to human chromosome 1q21-q23 and mouse chromosome 1H1-5 by fluorescence in situ hybridization

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    Edelhoff, S.; Disteche, C.M. [Univ. of Washington School of Medicine, Seattle, WA (United States); Lai, C. [Scripps Research Inst., LaJolla, CA (United States)


    Receptor protein-tyrosine kinases (PTKs) play a critical role in the transduction of signals important to cell growth, differentiation, and survival. Mutations affecting the expression of receptor PTK genes have been associated with a number of vertebrate and invertebrate developmental abnormalities, and the aberrant regulation of tyrosine phosphorylation is implicated in a variety of neoplasias. One estimate suggests that approximately 100 receptor PTK genes exist in the mammalian genome, about half of which have been identified. The tyro-10 receptor protein-tyrosine kinase, first identified in a PCR-based survey for novel tyrosine kinases in the rat nervous system, defines a new subfamily of PTKs. It exhibits a catalytic domain most closely related to those found in the trk PTK receptor subfamily, which transduces signals for nerve growth factor and the related molecules brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 (NT-3 and NT-4). Trk and the related PTK receptors trkB and trkC play a critical role in the neurotrophin-dependent survival of subsets of sensory and motor neurons. The predicted tyro-10 extracellular region is, however, distinct from that of the trk subfamily and is unique except for a domain shared with the blood coagulation factors V and VIII, thought to be involved in phospholipid binding. Although tyro-10 RNA is most abundant in heart and skeletal muscle in the adult rat, it is expressed in a wide variety of tissues, including the developing and mature brain. Tyro-10 appears identical to the murine TKT sequence reported by Karn et al. and exhibits a high degree of similarity with the CaK, DDR, and Nep PTKs. A ligand for tyro-10 has not yet been identified. 10 refs., 1 fig.

  5. Biotinyl-methyl 4-(amidomethyl) benzoate is a competitive inhibitor of human biotinidase (United States)

    Posttranslational modification of histones by biotinylation can be catalyzed by both biotinidase (BTD) and holocarboxylase synthetase (HCS). Biotinylation of histones is an important epigenetic mechanism to regulate gene expression, DNA repair, and chromatin remodeling. The role of BTD in histone ...

  6. Effects of tenuigenin on the expression of brain-derived neurotrophic factor and its receptor tyrosine protein kinase B in the hippocampus of Alzheimer's disease model rats

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    Wei-rong CHEN


    Full Text Available Objective To investigate the effects of tenuigenin (TEN on expression of brain-derived neurotrophic factor (BDNF, and its receptor tyrosine protein kinase B (TrkB in the hippocampal CA1 region of Alzheimer's disease (AD model rats.  Methods Sixty male Wistar rats were divided randomly into 4 groups: the control group, the model group, 12.50 mg/ml TEN group and 37.50 mg/ml TEN group. AD model rats were made by injecting ibotenic acid into Meynert basal nuclei of aging rats induced by D-galactose. The expressions of BDNF and its receptor TrkB in the hippocampal CA1 region were measured by immunohistochemistry method.  Results The positive expressions of BDNF and TrkB were pale brown and mainly in neuronal cell membrane of the hippocampal CA1 region measured by immunohistochemistry method. The average absorbance values of BDNF and its receptor TrkB in the control group were 0.47 ± 0.02 and 0.46 ± 0.05, while in the model group were 0.30 ± 0.02 and 0.21 ± 0.07 which were significantly lower than that of the control group (P = 0.000, for all. The average absorbance values of BDNF and its receptor TrkB in 12.50 mg/ml TEN group were 0.35 ± 0.05 and 0.32 ± 0.07, which were significantly higher than that of the model group (P = 0.000, for all and 37.50 mg/ml TEN group were 0.43 ± 0.05 and 0.37 ± 0.03, which were significantly higher than that of the model group (P = 0.000, for all. The average absorbance values of BDNF and its receptor TrkB in 37.50 mg/ml TEN group increased significantly than that in 12.50 mg/ml TEN group (P = 0.000.  Conclusions TEN can dose-dependently increase BDNF and its receptor TrkB expression in the hippocampal CA1 region of Alzheimer's disease model rats, which may partly explain the beneficial effects of TEN on cognitive function. doi: 10.3969/j.issn.1672-6731.2014.05.011

  7. Identification of the tyrosine-protein phosphatase non-receptor type 2 as a rheumatoid arthritis susceptibility locus in europeans.

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    Joanna E Cobb

    Full Text Available OBJECTIVES: Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA. However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA. METHODS: A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data. RESULTS: Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10(-9. CONCLUSIONS: This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene.

  8. Identification of the Tyrosine-Protein Phosphatase Non-Receptor Type 2 as a Rheumatoid Arthritis Susceptibility Locus in Europeans (United States)

    Cobb, Joanna E.; Plant, Darren; Flynn, Edward; Tadjeddine, Meriem; Dieudé, Philippe; Cornélis, François; Ärlestig, Lisbeth; Dahlqvist, Solbritt Rantapää; Goulielmos, George; Boumpas, Dimitrios T.; Sidiropoulos, Prodromos; Krintel, Sophine B.; Ørnbjerg, Lykke M.; Hetland, Merete L.; Klareskog, Lars; Haeupl, Thomas; Filer, Andrew; Buckley, Christopher D.; Raza, Karim; Witte, Torsten; Schmidt, Reinhold E.; FitzGerald, Oliver; Veale, Douglas; Eyre, Stephen; Worthington, Jane


    Objectives Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA). However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA. Methods A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data. Results Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10−9). Conclusions This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase) with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene. PMID:23840476

  9. Impact of protein tyrosine kinase 6 (PTK6) on human epidermal growth factor receptor (HER) signalling in breast cancer. (United States)

    Ludyga, Natalie; Anastasov, Nataša; Gonzalez-Vasconcellos, Iria; Ram, Manuela; Höfler, Heinz; Aubele, Michaela


    PTK6, also known as Brk, is highly expressed in over 80% of breast cancers. In the last decade several substrates and interaction partners were identified localising PTK6 downstream of HER receptors. PTK6 seems to be involved in progression of breast tumours, in particular in HER receptor signalling. Here, we show the down-regulation effects of PTK6 in the T47D, BT474 and JIMT-1 breast cancer cell lines. PTK6 knockdown leads to a decreased phosphorylation of HER2, PTEN, MAPK (ERK), p38 MAPK, STAT3 and to a reduced expression of cyclin E. Our findings show that silencing PTK6 impairs the downstream targets of HER receptors and consequently the activation of signalling molecules. Furthermore, lower levels of PTK6 result in reduced migration of T47D and JIMT-1 breast cancer cells. Due to decreased migration, the PTK6 RNA interference might contribute to reduced metastasis and malignant potential of breast cancer cells. Since PTK6 plays an important role in HER receptor signal transduction, its down-regulation might be suitable for future therapy approaches in breast cancer.

  10. Ligand-induced tyrosine phosphorylation of cysteinyl leukotriene receptor 1 triggers internalization and signaling in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Sime, Wondossen; Yudina, Yuliana;


    Leukotriene D(4) (LTD(4)) belongs to the bioactive lipid group known as eicosanoids and has implications in pathological processes such as inflammation and cancer. Leukotriene D(4) exerts its effects mainly through two different G-protein-coupled receptors, CysLT(1) and CysLT(2). The high affinity...

  11. In vitro and in vivo inhibition of epidermal growth factor receptor-tyrosine kinase pathway by photodynamic therapy. (United States)

    Ahmad, N; Kalka, K; Mukhtar, H


    PDT, a new therapeutic procedure for the management of many malignant conditions including skin cancer, involves the administration of a photosensitizing compound followed by illumination of the lesion with visible light. We earlier showed an involvement of: (i) WAF1/p21-cyclins (D1 and E)-cdk (2 and 6) network; and (ii) Rb/E2F-DP machinery during silicon phthalocyanine (Pc4)-PDT-mediated cell cycle dysregulation and apoptosis of human epidermoid carcinoma (A431) cells. Here, we investigated the involvement of EGFR-pathway during antiproliferative responses of Pc4-PDT in A431 cells and during ablation of murine skin papillomas. Pc4-PDT of A431 cells was found to result in a time-dependent down-modulation of the protein expression and phosphorylation of EGFR and Shc (an immediate downstream molecule in EGFR-pathway), during progressive increase in apoptotic response. To establish the relevance of these in vitro findings to in vivo situations, we subjected chemically- as well as ultraviolet B radiation-induced squamous papillomas in SENCAR and SKH-1 hairless mice, respectively, to Pc4-PDT, and assessed its effect on EGFR-pathway during ablation of these tumors. Pc4-PDT was found to result in a time-dependent: (i) inhibition of protein expressions of EGFR; and (ii) tyrosine phosphorylation of EGFR and Shc; and (iii) induction of apoptosis, during the regression of these tumors. These data suggest the involvement of EGFR-pathway during the antiproliferative effects of PDT. It is tempting to speculate that inhibitors of EGFR could enhance the therapeutic efficacy of PDT.

  12. Non-peptide fibrinogen receptor antagonists. 2. Optimization of a tyrosine template as a mimic for Arg-Gly-Asp. (United States)

    Egbertson, M S; Chang, C T; Duggan, M E; Gould, R J; Halczenko, W; Hartman, G D; Laswell, W L; Lynch, J J; Lynch, R J; Manno, P D


    Inhibitors of platelet-fibrinogen binding offer an opportunity to interrupt the final, common pathway for platelet aggregation. Small molecule inhibitors of the platelet fibrinogen receptor GPIIb/IIIa were prepared and evaluated for their ability to prevent platelet aggregation. Compound 23m (L-700,462/MK-383) inhibited in vitro platelet aggregation with an IC50 of 9 nM and demonstrated a selectivity of > 24,000-fold between platelet and human umbilical vein endothelial cell fibrinogen receptors. Dose-dependent inhibition of ex vivo platelet aggregation induced by ADP was achieved with i.v. infusions of 0.1-10 micrograms/kg/min of 23m in anesthetized dogs, with 10 micrograms/kg/min completely inhibiting platelet aggregation during the entire 6 h infusion protocol. Platelet aggregatability returned rapidly after the termination of the 23m infusions. These features suggest that 23m may be useful in the treatment of arterial occlusive disorders.

  13. MD-2 interacts with Lyn kinase and is tyrosine phosphorylated following LPS-induced activation of the Toll-like receptor 4 signaling pathway (United States)

    Gray, Pearl; Dagvadorj, Jargalsaikhan; Michelsen, Kathrin S.; Brikos, Constantinos; Rentsendorj, Altan; Town, Terrence; Crother, Timothy R.; Arditi, Moshe


    Stimulation with LPS induces tyrosine phosphorylation of numerous proteins involved in the TLR signaling pathway. In this study, we demonstrate that MD-2 is also tyrosine phosphorylated following LPS stimulation. LPS-induced tyrosine phosphorylation of MD-2 is specific, it is blocked by the tyrosine kinase inhibitor, Herbimycin A, and by an inhibitor of endocytosis, Cytochalsin-D, suggesting that MD-2 phosphorylation occurs during trafficking of MD2 and not on cell surface. Furthermore, we identify two possible phospho-accepting tyrosine residues at positions 22 and 131. Mutant proteins in which these tyrosines were changed to phenylalanine have reduced phosphorylation and significantly diminished ability to activate NF-κB in response to LPS. In addition, MD2 co-precipitates and colocalizes with Lyn kinase, most likely in ER. A Lyn-binding peptide inhibitor abolished MD2 tyrosine phosphorylation, suggesting that Lyn is a likely candidate to be the kinase required for MD-2 tyrosine phophorylation. Our study demonstrates that tyrosine phosphorylation of MD-2 is important for signaling following exposure to LPS and underscores the importance of this event in mediating an efficient and prompt immune response. PMID:21918188

  14. Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Galpha12/13. (United States)

    Mao, J; Xie, W; Yuan, H; Simon, M I; Mano, H; Wu, D


    A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Moreover, expression of Galpha12 and Galpha13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 did not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed.

  15. Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma. (United States)

    Patwardhan, Parag P; Ivy, Kathryn S; Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K


    Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

  16. A comparative study of Pointed and Yan expression reveals new complexity to the transcriptional networks downstream of receptor tyrosine kinase signaling. (United States)

    Boisclair Lachance, Jean-François; Peláez, Nicolás; Cassidy, Justin J; Webber, Jemma L; Rebay, Ilaria; Carthew, Richard W


    The biochemical regulatory network downstream of receptor tyrosine kinase (RTK) signaling is controlled by two opposing ETS family members: the transcriptional activator Pointed (Pnt) and the transcriptional repressor Yan. A bistable switch model has been invoked to explain how pathway activation can drive differentiation by shifting the system from a high-Yan/low-Pnt activity state to a low-Yan/high-Pnt activity state. Although the model explains yan and pnt loss-of-function phenotypes in several different cell types, how Yan and Pointed protein expression dynamics contribute to these and other developmental transitions remains poorly understood. Toward this goal we have used a functional GFP-tagged Pnt transgene (Pnt-GFP) to perform a comparative study of Yan and Pnt protein expression throughout Drosophila development. Consistent with the prevailing model of the Pnt-Yan network, we found numerous instances where Pnt-GFP and Yan adopt a mutually exclusive pattern of expression. However we also observed many examples of co-expression. While some co-expression occurred in cells where RTK signaling is presumed low, other co-expression occurred in cells with high RTK signaling. The instances of co-expressed Yan and Pnt-GFP in tissues with high RTK signaling cannot be explained by the current model, and thus they provide important contexts for future investigation of how context-specific differences in RTK signaling, network topology, or responsiveness to other signaling inputs, affect the transcriptional response.

  17. Deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in twins with a Rett syndrome-like phenotype. (United States)

    Williamson, Sarah L; Ellaway, Carolyn J; Peters, Greg B; Pelka, Gregory J; Tam, Patrick P L; Christodoulou, John


    Rett syndrome (RTT), a neurodevelopmental disorder that predominantly affects females, is primarily caused by variants in MECP2. Variants in other genes such as CDKL5 and FOXG1 are usually associated with individuals who manifest distinct phenotypes that may overlap with RTT. Individuals with phenotypes suggestive of RTT are typically screened for variants in MECP2 and then subsequently the other genes dependent on the specific phenotype. Even with this screening strategy, there are individuals in whom no causative variant can be identified, suggesting that there are other novel genes that contribute to the RTT phenotype. Here we report a de novo deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in identical twins with a RTT-like phenotype. We also demonstrate the reduced expression of Ptpn4 in a Mecp2 null mouse model of RTT, as well as the activation of the PTPN4 promoter by MeCP2. Our findings suggest that PTPN4 should be considered for addition to the growing list of genes that warrant screening in individuals with a RTT-like phenotype.

  18. Conditional downregulation of brain- derived neurotrophic factor and tyrosine kinase receptor B blocks epileptogenesis in the human temporal lobe epilepsy hippocampus

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    Hou Xiaohua


    Full Text Available Backgroud : Brain-derived neurotrophic factor (BDNF has been implicated as a potential therapeutic target in temporal lobe epilepsy (TLE. However, whether BDNF exerts an epileptogenic or antiepileptogenic function remains controversial. Materials and Methods : BDNF/tyrosine kinase receptor B (trkB expression levels were comparatively assessed in the hippocampal tissue of TLE patients with (HS group and without hippocampal sclerosis (non-HS group as well as from non-epileptic controls. Results : Immunohistochemistry and immunoblot analysis revealed a marked increase in BDNF/trkB expression in the dentate gyrus and CA3 regions of HS and non-HS groups. The lack of any differences in expression levels was observed between HS and non-HS patients. Meanwhile, treatment with VPA (Valproic acid, anti-epileptic drug resulted in a significant down-regulation of BDNF/trkB protein expression in sclerotic and non-sclerotic hippocampus (P < 0.001. In contrast, no marked change was noticed in VPA-untreated and OA-treated groups (sodium octanoate. Conclusion : These results suggest that the up-regulation of BDNF/trkB pathway might be at least in part responsible for the epileptogenesis.

  19. Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases. (United States)

    Booij, Tijmen H; Klop, Maarten J D; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S


    3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting.

  20. Fibroblast growth factor receptor 3 interacts with and activates TGFβ-activated kinase 1 tyrosine phosphorylation and NFκB signaling in multiple myeloma and bladder cancer.

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    Lisa Salazar

    Full Text Available Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1. Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3 tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

  1. Kinome-wide shRNA Screen Identifies the Receptor Tyrosine Kinase AXL as a Key Regulator for Mesenchymal Glioblastoma Stem-like Cells

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    Peng Cheng


    Full Text Available Glioblastoma is a highly lethal cancer for which novel therapeutics are urgently needed. Two distinct subtypes of glioblastoma stem-like cells (GSCs were recently identified: mesenchymal (MES and proneural (PN. To identify mechanisms to target the more aggressive MES GSCs, we combined transcriptomic expression analysis and kinome-wide short hairpin RNA screening of MES and PN GSCs. In comparison to PN GSCs, we found significant upregulation and phosphorylation of the receptor tyrosine kinase AXL in MES GSCs. Knockdown of AXL significantly decreased MES GSC self-renewal capacity in vitro and inhibited the growth of glioblastoma patient-derived xenografts. Moreover, inhibition of AXL with shRNA or pharmacologic inhibitors also increased cell death significantly more in MES GSCs. Clinically, AXL expression was elevated in the MES GBM subtype and significantly correlated with poor prognosis in multiple cancers. In conclusion, we identified AXL as a potential molecular target for novel approaches to treat glioblastoma and other solid cancers.

  2. Kinase activation of the non-receptor tyrosine kinase Etk/BMX alone is sufficient to transactivate STAT-mediated gene expression in salivary and lung epithelial cells. (United States)

    Wen, X; Lin, H H; Shih, H M; Kung, H J; Ann, D K


    Etk/BMX is a non-receptor protein tyrosine kinase that requires a functional phosphatidylinositol 3-kinase via the pleckstrin homology domain to be activated by cytokine. In the present study, a conditionally active form of Etk was constructed by fusing the hormone-binding domain of estrogen receptor (ER) to an amino terminus truncated form of Etk, PHDelta1-68Etk, to generate DeltaEtk:ER. In stably transfected Pa-4DeltaEtk:ER cells, the activity of DeltaEtk:ER was stimulated within minutes by the treatment of DeltaEtk:ER stimulant, estradiol, and sustained for greater than 24 h. A robust induction in the phosphorylation of signal transducers and activators of transcription (STAT) proteins, including STAT1, STAT3, and STAT5, was accompanied with DeltaEtk:ER activation. Moreover, the conditionally activated Etk stimulated STAT1- and STAT5-dependent reporter activities by approximately 160- and approximately 15-fold, respectively, however, elicited only a modest STAT3-mediated reporter activation. Qualitatively comparable results were obtained in lung A549 cells, indicating that DeltaEtk:ER inducible system could function in an analogous fashion in different epithelial cells. Furthermore, we demonstrated that Etk activation alone augmented cyclin D1 promoter/enhancer activity via its STAT5 response element in both Pa-4DeltaEtk:ER and A549 cells. Altogether, these findings support the notion that the activation of Etk kinase is sufficient to transactivate STAT-mediated gene expression. Hence, our inducible DeltaEtk:ER system represents a novel approach to investigate the biochemical events following Etk activation and to evaluate the contribution by kinase activation of Etk alone or in conjunction with other signaling pathway(s) to the ultimate biological responses.

  3. Association between Single-Nucleotide Polymorphisms of the Tyrosine Kinase Receptor B (TrkB and Post-Stroke Depression in China.

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    Zhiming Zhou

    Full Text Available Polymorphisms of the brain-derived neurotrophic factor (BDNF have been investigated as candidate genes for post-stroke depression (PSD, and its receptor, neurotrophic tyrosine kinase receptor B (TrkB, has been associated with depression. However, no further data have yet reported the association between PSD and polymorphisms in TrkB. This study aims to investigate whether a relationship exists between TrkB polymorphisms and PSD.A total of 312 depression patients (PSD patients and 472 non-depression patient controls (NPSD patients were recruited. All patients were evaluated using the Hamilton Rating Scale for Depression (HAMD to determine depression severity, and PSD patients were diagnosed in accordance with DSM-V criteria. Three single-nucleotide polymorphisms (SNPs, namely, rs1187323, rs1212171, and rs1778929, in the TrkB gene were genotyped by high-resolution melt analysis.The SNP rs1778929 was significantly more associated with incident PSD in participants with the TT genotype than in those with CC (OR 0.482, 95% CI: 0.313-0.744. In terms of rs1187323, stroke was significantly more associated with incident depression in participants with the AC genotype than in those with AA (OR 0.500, 95% CI: 0.368-0.680. The minor allele (T of rs1778929 (P = 0.024, OR = 0.725, 95% CI = 0.590-0.890 and the minor allele (C of rs1187323 (P = 0.000, OR = 0.598, 95% CI = 0.466-0.767 were found to be significantly associated with PSD. Neither genotype nor allele frequencies of rs1212171 showed statistically significant differences between PSD and NPSD patients.The results suggest that rs1778929 and rs1187323 in the TrkB gene are significantly associated with post-stroke depression in the Chinese population. Further studies are necessary to confirm our findings.

  4. Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression. (United States)

    Kotani, H; Ebi, H; Kitai, H; Nanjo, S; Kita, K; Huynh, T G; Ooi, A; Faber, A C; Mino-Kenudson, M; Yano, S


    Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR

  5. Effects of ketoconazole or rifampin on the pharmacokinetics of tivozanib hydrochloride, a vascular endothelial growth factor receptor tyrosine kinase inhibitor. (United States)

    Cotreau, Monette M; Siebers, Nicholas M; Miller, James; Strahs, Andrew L; Slichenmyer, William


    The vascular endothelial growth factor (VEGF) pathway is associated with the promotion of endothelial cell proliferation, migration, and survival necessary for angiogenesis. VEGF and its three receptor isoforms are often overexpressed in many human solid tumors. Tivozanib is a potent, selective inhibitor of VEGF receptors 1, 2, and 3, with a long half-life. The purpose of these studies was to evaluate the effect of ketoconazole, a potent inhibitor of CYP3A4, and rifampin, a potent inducer of CYP3A4, on the pharmacokinetics of tivozanib. Two phase I, open-label, 2-period, single-sequence studies evaluated the effect of steady-state ketoconazole (NCT01363778) or rifampin (NCT01363804) on the pharmacokinetic profile, safety, and tolerability of a single oral 1.5-mg dose of tivozanib. Tivozanib was well tolerated in both studies. Steady-state ketoconazole did not cause a clinically significant change in the pharmacokinetics of a single dose of tivozanib; therefore, dosing of tivozanib with a CYP3A4 pathway inhibitor should not cause a clinically significant change in serum tivozanib levels. However, coadministration of tivozanib with rifampin caused a significant decrease in the area under the curve from 0 to infinity and half-life and an increase in clearance of tivozanib, which suggest increased clearance via the enhanced CYP3A4-mediated metabolism of tivozanib.

  6. Spatial memory training modifies the expression of brain-derived neurotrophic factor tyrosine kinase receptors in young and aged rats. (United States)

    Silhol, M; Arancibia, S; Maurice, T; Tapia-Arancibia, L


    Aging leads to alterations in the function of the hippocampus, a brain structure largely involved in learning processes. This study aimed at examining the basal levels and the impact of a learning-associated task on brain-derived neurotrophic factor (BDNF), on BDNF full-length catalytic receptor (TrkB.FL) and on the truncated forms (TrkB.T1 and TrkB.T2) receptor expression (mRNA and protein) in the hippocampus of young (2-month-old) and aged (24-month-old) Wistar rats. Spatial memory was evaluated using a water-maze procedure involving visible and invisible platform location learning. Aged rats showed higher latencies during the first two training days but rapidly exhibited learning performances similar to patterns observed with young rats. Real-time PCR measurements showed that aged rats had significantly higher levels of trkB.FL mRNAs than young rats under basal conditions. In situ hybridization analysis indicated that the highest level of trkB.FL mRNA (mRNA encoding for TrkB.FL receptor) was noted in the dentate gyrus, and in the CA2 and CA3 hippocampal layers. In contrast, there was no marked difference in trkB.T1 signal in any hippocampal region. Training induced a significant reduction in trkB.FL mRNA levels solely in aged rats. In contrast, in young and aged rats, trkB.T2 mRNA levels were significantly increased after training. Measurements of proteins revealed that learning significantly increased TrkB.FL content in aged rats. Untrained aged rats presented higher levels of BDNF and brain-derived neurotrophic factor precursor (proBDNF) proteins than young rats. Training strongly increased precursor BDNF metabolism in young and aged rats, resulting in increased levels of proBDNF in the two groups but in old rats the mature BDNF level did not change. This study shows that Wistar rats present age-related differences in the levels of BDNF and TrkB isoforms and that spatial learning differentially modifies some of these parameters in the hippocampus.

  7. Tyrosylprotein sulfotransferase-1 and tyrosine sulfation of chemokine receptor 4 are induced by Epstein-Barr virus encoded latent membrane protein 1 and associated with the metastatic potential of human nasopharyngeal carcinoma.

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    Juan Xu

    Full Text Available The latent membrane protein 1 (LMP1, which is encoded by the Epstein-Barr virus (EBV, is an important oncogenic protein that is closely related to carcinogenesis and metastasis of nasopharyngeal carcinoma (NPC, a prevalent cancer in China. We previously reported that the expression of the functional chemokine receptor CXCR4 is associated with human NPC metastasis. In this study, we show that LMP1 induces tyrosine sulfation of CXCR4 through tyrosylprotein sulfotransferase-1 (TPST-1, an enzyme that is responsible for catalysis of tyrosine sulfation in vivo, which is likely to contribute to the highly metastatic character of NPC. LMP1 could induce tyrosine sulfation of CXCR4 and its associated cell motility and invasiveness in a NPC cell culture model. In contrast, the expression of TPST-1 small interfering RNA reversed LMP1-induced tyrosine sulfation of CXCR4. LMP1 conveys signals through the epidermal growth factor receptor (EGFR pathway, and EGFR-targeted siRNA inhibited the induction of TPST-1 by LMP1. We used a ChIP assay to show that EGFR could bind to the TPST-1 promoter in vivo under the control of LMP1. A reporter gene assay indicated that the activity of the TPST-1 promoter could be suppressed by deleting the binding site between EGFR and TPST-1. Finally, in human NPC tissues, the expression of TPST-1 and LMP1 was directly correlated and clinically, the expression of TPST-1 was associated with metastasis. These results suggest the up-regulation of TPST-1 and tyrosine sulfation of CXCR4 by LMP1 might be a potential mechanism contributing to NPC metastasis.

  8. Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors

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    Rüegg Markus A


    Full Text Available Abstract Background Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ, agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs. Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs and protein tyrosine kinases (PTKs such as those of the Src-family may be essential in stabilizing clusters of AChRs. Results We have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src-/-;fyn-/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src-/-;fyn-/- myotubes contained elevated levels of SHP-2 protein. Conclusion Our data

  9. Phase Ⅰ trial of icotinib, a novel epidermal growth factor receptor tyrosine kinase inhibitor, in Chinese patients with non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    WANG Han-ping; XIAO Yi; ZHANG Li; WANG Yin-xiang; TAN Fen-lai; XIA Ying; REN Guan-jun; HU Pei; JIANG Ji; WANG Meng-zhao


    Background The preclinical experiments and studies of congener drugs show icotinib, a new epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, can specifically bind to the tyrosine kinase domain of the EGFR, block the EGFR related signal, thereby inhibit the growth of tumor cell. The objective of this study was to investigate the safety, tolerability and dose-related biologic effects of icotinib in patients with non-small cell lung cancer (NSCLC) in a Chinese patient population.Methods This was an open-label, phase Ⅰ, dose escalation, safety/tolerability trial of oral icotinib (100 to 400 mg), administered twice per day for 28-continuous-day cycles until disease progression or undue toxiclty.Results Forty patients with stage ⅢB (15%) or Ⅳ (85%) NSCLC were included in the study. They had mainly adenocarcinoma (85%), with a performance status (PS) of 0 (45%) or 1 (55%) and less than half the patients (45%) had histories of smoking and all were pretreated by at least one regimen of chemotherapy. Patients were assigned to three dose levels of 150 mg b.i.d, 200 mg b.i.d, or 125 mg t.i.d. The follow-up periods ranged from 5 to 80 weeks. Adverse events were found in 35% patients, most of which were mild and reversible. The adverse events mainly occurred in the first 4 weeks and included rash (25%), diarrhea, nausea and abdominal distention. One definite interstitial lung disease (ILD) was found in a patient in the dose of 200 mg b.i.d. According to an 8-week assessment, one (2.5%) patient receiving 150 mg gained complete response (CR) that persisted for 44 weeks, seven (17.50%) patients had partial remission (PR), and 18 (45%) patients had stable disease (SD). The objective response including CR+PR was 20%. The median time of progression-free survival for the 40 patients was 20 weeks (range: 12 to 32 weeks). The response was not affected by pathological type, history of smoking, or numbers of previous therapeutic regimens. No relationship between dose

  10. Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer

    DEFF Research Database (Denmark)

    Mereiter, Stefan; Magalhães, Ana; Adamczyk, Barbara


    gastric carcinoma cells transfected with the sialyltransferase ST3GAL4 were established as a model overexpressing sialylated terminal glycans. We have evaluated at the structural level the glycome and the sialoproteome of this gastric cancer cell line applying liquid chromatography and mass spectrometry...... known to be key players in malignancy. Further analysis of RON confirmed its modification with SLe(X) and the concomitant activation. SLe(X) and RON co-expression was validated in gastric tumors. CONCLUSION: The overexpression of ST3GAL4 interferes with the overall glycophenotype of cancer cells...... affecting a multitude of key proteins involved in malignancy. Aberrant glycosylation of the RON receptor was shown as an alternative mechanism of oncogenic activation. GENERAL SIGNIFICANCE: This study provides novel targets and points to an integrative tumor glycomic/proteomic-profiling for gastric cancer...

  11. Cetuximab resistance in squamous carcinomas of the upper aerodigestive tract is driven by receptor tyrosine kinase plasticity

    DEFF Research Database (Denmark)

    Kjær, Ida; Lindsted, Trine; Fröhlich, Camilla;


    Squamous cell carcinomas (SCC) arising in upper parts of the aero-digestive tract (UAT) are among the leading causes of death worldwide. The epidermal growth factor receptor (EGFR) has been found to play an essential role in driving the malignancy of SCCUAT, but despite this, clinical results using...... a range of different EGFR- targeted agents have been disappointing. Cetuximab is currently the only EGFR-targeted agent approved by the FDA for treatment of SCCUAT. However, intrinsic and acquired cetuximab resistance is a major problem for effective therapy. Thus, a better understanding of the mechanisms...... by continuous selective pressure in vitro and in vivo. Our results show that resistant clones maintain partial dependency on EGFR and that RTK plasticity mediated by HER3 and IGF1R plays an essential role. A multi-target mAb mixture against EGFR, HER3, and IGF1R was able to overcome cetuximab resistance...

  12. Interaction Potential of the Multitargeted Receptor Tyrosine Kinase Inhibitor Dovitinib with Drug Transporters and Drug Metabolising Enzymes Assessed in Vitro

    Directory of Open Access Journals (Sweden)

    Johanna Weiss


    Full Text Available Dovitinib (TKI-258 is under development for the treatment of diverse cancer entities. No published information on its pharmacokinetic drug interaction potential is available. Thus, we assessed its interaction with important drug metabolising enzymes and drug transporters and its efficacy in multidrug resistant cells in vitro. P-glycoprotein (P-gp, MDR1, ABCB1 inhibition was evaluated by calcein assay, inhibition of breast cancer resistance protein (BCRP, ABCG2 by pheophorbide A efflux, and inhibition of organic anion transporting polypeptides (OATPs by 8-fluorescein-cAMP uptake. Inhibition of cytochrome P450 3A4, 2C19, and 2D6 was assessed by using commercial kits. Induction of transporters and enzymes was quantified by real-time RT-PCR. Possible aryl hydrocarbon receptor (AhR activating properties were assessed by a reporter gene assay. Substrate characteristics were evaluated by growth inhibition assays in cells over-expressing P-gp or BCRP. Dovitinib weakly inhibited CYP2C19, CYP3A4, P-gp and OATPs. The strongest inhibition was observed for BCRP (IC50 = 10.3 ± 4.5 μM. Among the genes investigated, dovitinib only induced mRNA expression of CYP1A1, CYP1A2, ABCC3 (coding for multidrug resistance-associated protein 3, and ABCG2 and suppressed mRNA expression of some transporters and drug metabolising enzymes. AhR reporter gene assay demonstrated that dovitinib is an activator of this nuclear receptor. Dovitinib retained its efficacy in cell lines over-expressing P-gp or BCRP. Our analysis indicates that dovitinib will most likely retain its efficacy in tumours over-expressing P-gp or BCRP and gives first evidence that dovitinib might act as a perpetrator drug in pharmacokinetic drug–drug interactions.

  13. Tyrosine kinase discoidin domain receptors DDR1 and DDR2 are coordinately deregulated in triple-negative breast cancer. (United States)

    Toy, Kathy A; Valiathan, Rajeshwari R; Núñez, Fernando; Kidwell, Kelley M; Gonzalez, Maria E; Fridman, Rafael; Kleer, Celina G


    Receptor kinases Discoidin Domain Receptors (DDRs) 1 and 2 are emerging as new therapeutic targets in breast cancer (BC). However, the expression of DDR proteins during BC progression and their association with BC subtypes remain poorly defined. Herein we report the first comprehensive immunohistochemical analyses of DDR protein expression in a wide range of breast tissues. DDR1 and DDR2 expression was investigated by immunohistochemistry in 218 samples of normal breast (n = 10), ductal carcinoma in situ (DCIS, n = 10), and invasive carcinomas (n = 198), arrayed in tissue microarrays with comprehensive clinical and follow-up information. Staining was evaluated for cell type, subcellular localization, percentage and intensity (scores 1-4), and association with disease subtype and outcome. In normal epithelium and DCIS, DDR1 was highly expressed, while DDR2 was negative in normal epithelium, and in DCIS it localized to cells at the epithelial-stromal interface. Of the 198 invasive carcinomas, DDR1 was high in 87 (44 %) and low in 103 (52 %), and DDR2 was high in 110 (56 %) and low in 87 (44 %). High DDR2 was associated with high tumor grade (P = 0.002), triple-negative subtype (TNBC) (P DDR2(High) profile significantly associated with TNBC, compared to luminal tumors (P = 0.012), and with worse overall survival. In conclusion, DDR2 upregulation occurs in DCIS, before stromal invasion, and may reflect epithelial-stromal cross-talk. A DDR1(Low)/DDR2(High) protein profile is associated with TNBC and may identify invasive carcinomas with worse prognosis.

  14. Oligomerization of epidermal growth factor receptors (EGFR) on A431 cells studied by time-resolved fluorescence imaging microscopy: a stereochemical model for tyrosine kinase receptor activation

    NARCIS (Netherlands)

    Th.W.J. Gadella; T.M. Jovin


    The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance en- ergy transfer: donor photobleaching fluorescence reso- nance energy transfer (pbFRET) microscopy and

  15. Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study

    Directory of Open Access Journals (Sweden)

    Akahoshi Eiichi


    Full Text Available Abstract Background Dioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR. This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR. Methods N2a-Rβ cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA. Results Induction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE, though AHRE-III was not identical to XRE, the conventional AhR-binding motif. Conclusion Our results suggest TCDD directly regulate the dopamine system by TH gene

  16. Ginsenoside-Rg{sub 1} induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a

    Energy Technology Data Exchange (ETDEWEB)

    Kwok, Hoi-Hin [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Chan, Lai-Sheung [Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Poon, Po-Ying [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Yue, Patrick Ying-Kit [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Wong, Ricky Ngok-Shun, E-mail: [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China)


    Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. Ginsenoside-Rg{sub 1} (Rg{sub 1}), one of the most abundant active components of ginseng, has been demonstrated as an angiogenesis-stimulating compound in different models. There is increasing evidence implicating microRNAs (miRNAs), a group of non-coding RNAs, as important regulators of angiogenesis, but the role of microRNAs in Rg{sub 1}-induced angiogenesis has not been fully explored. In this report, we found that stimulating endothelial cells with Rg{sub 1} could reduce miR-23a expression. In silico experiments predicted hepatocyte growth factor receptor (MET), a well-established mediator of angiogenesis, as the target of miR-23a. Transfection of the miR-23a precursor or inhibitor oligonucleotides validated the inverse relationship of miR-23a and MET expression. Luciferase reporter assays further confirmed the interaction between miR-23a and the MET mRNA 3′-UTR. Intriguingly, ginsenoside-Rg{sub 1} was found to increase MET protein expression in a time-dependent manner. We further demonstrated that ginsenoside-Rg{sub 1}-induced angiogenic activities were indeed mediated through the down-regulation of miR-23a and subsequent up-regulation of MET protein expression, as confirmed by gain- and loss-of-function angiogenic experiments. In summary, our results demonstrated that ginsenoside-Rg{sub 1} could induce angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a. This study has broadened our understanding of the non-genomic effects of ginsenoside-Rg{sub 1,} and provided molecular evidence that warrant further development of natural compound as novel angiogenesis-promoting therapy. - Highlights: • Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. • Ginsenoside-Rg{sub 1} (Rg{sub 1}) has been demonstrated as an angiogenesis-stimulating compound. • We found that Rg{sub 1} induces angiogenesis by

  17. Pigment pattern formation in the guppy, Poecilia reticulata, involves the Kita and Csf1ra receptor tyrosine kinases. (United States)

    Kottler, Verena A; Fadeev, Andrey; Weigel, Detlef; Dreyer, Christine


    Males of the guppy (Poecilia reticulata) vary tremendously in their ornamental patterns, which are thought to have evolved in response to a complex interplay between natural and sexual selection. Although the selection pressures acting on the color patterns of the guppy have been extensively studied, little is known about the genes that control their ontogeny. Over 50 years ago, two autosomal color loci, blue and golden, were described, both of which play a decisive role in the formation of the guppy color pattern. Orange pigmentation is absent in the skin of guppies with a lesion in blue, suggesting a defect in xanthophore development. In golden mutants, the development of the melanophore pattern during embryogenesis and after birth is affected. Here, we show that blue and golden correspond to guppy orthologs of colony-stimulating factor 1 receptor a (csf1ra; previously called fms) and kita. Most excitingly, we found that both genes are required for the development of the black ornaments of guppy males, which in the case of csf1ra might be mediated by xanthophore-melanophore interactions. Furthermore, we provide evidence that two temporally and genetically distinct melanophore populations contribute to the adult camouflage pattern expressed in both sexes: one early appearing and kita-dependent and the other late-developing and kita-independent. The identification of csf1ra and kita mutants provides the first molecular insights into pigment pattern formation in this important model species for ecological and evolutionary genetics.

  18. Antitumor activity of F90,an epidermal growth factor receptor tyrosine kinase inhibitor,on glioblastoma cell line SHG-44

    Institute of Scientific and Technical Information of China (English)

    LIU Fang-jun; GUI Song-bai; LI Chu-zhong; SUN Ze-lin; ZHANG Ya-zhuo


    Background Over-expression of epidermal growth factor receptor (EGFR) is thought to be related to cell proliferation,invasion,metastasis,resistance to chemoradiotherapy and poor prognosis of various human cancers.Forty percent to fifty percent of glioblastoma multiforme (GBM) possess deregulated EGFR,which may contribute to the aggressive and refractory course of GBM.Therefore,blockade of EGFR signal transduction may be a promising treatment strategy for GBM.Methods MIT assay,cell growth curve assay and tumor xenograft model were used to evaluate the antitumor activity of F90 against SHG-44 in vitro and in vivo.Western blot assay was applied to evaluate the expression of p-EGFR,p-ERK1,p-JNK,p-P38,Bcl2 and P53 proteins.Results F90 inhibited the cell proliferation in a dose-dependent manner in vitro.The growth of SHG-44 tumor xenografts was suppressed by F90 at a high dose level (100 of EGFR and activated downstream signaling proteins,such as ERK1,JNK and P38,were found to be depressed after incubation with F90 for 48 hours in vitro.Down-regulated Bcl2 protein and up-regulated P53 protein were also observed.Conclueions The results demonstrate that F90 is effective in inhibiting the proliferation of SHG-44 cells in vitro and tumor growth in vivo,suggesting that F90 may be a new therapeutic option for treatment of GBM.

  19. The MET Receptor Tyrosine Kinase Confers Repair of Murine Pancreatic Acinar Cells following Acute and Chronic Injury (United States)

    Gaziova, Ivana; Jackson, Daniel; Boor, Paul J.; Carter, Dwayne; Cruz-Monserrate, Zobeida; Elferink, Cornelis J.; Joshi, Aditya D.; Kaphalia, Bhupendra; Logsdon, Craig D.; Pereira de Castro, Karen; Soong, Lynn; Tao, Xinrong; Qiu, Suimin; Elferink, Lisa A.


    Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury. PMID:27798657

  20. Molecular mechanisms of the synergy between cysteinyl-leukotrienes and receptor tyrosine kinase growth factors on human bronchial fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Hajime Yoshisue


    Full Text Available We have reported that cysteinyl-leukotrienes (cys-LTs synergise not only with epidermal growth factor (EGF but also with platelet-derived growth factor (PDGF and fibroblast growth factor (FGF to induce mitogenesis in human bronchial fibroblasts. We now describe the molecular mechanisms underlying this synergism. Mitogenesis was assessed by incorporation of [3H]thymidine into DNA and changes in protein phosphorylation by Western blotting. Surprisingly, no CysLT receptor antagonists (MK-571, montelukast, BAY u9773 prevented the synergistic mitogenesis. LTD4 did not cause phosphorylation of EGFR nor did it augment EGF-induced phosphorylation of EGFR, and the synergy between LTD4 and EGF was not blocked by the metalloproteinase inhibitor GM6001 or by an HB-EGF neutralising antibody. The EGFR-selective kinase inhibitor, AG1478, suppressed the synergy by LTD4 and EGF, but had no effect on the synergy with PDGF and FGF. While inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C (PKC prevented the synergy, these drugs also inhibited mitogenesis elicited by EGF alone. In contrast, pertussis toxin (PTX efficiently inhibited the potentiating effect of LTD4 on EGF-induced mitogenesis, as well as that provoked by PDGF or FGF, but had no effect on mitogenesis elicited by the growth factors alone. Whereas LTD4 alone did not augment phosphorylation of extracellular signal-regulated kinase (Erk-1/2 and Akt, it increased phosphorylation of PKC in a Gi-dependent manner. Addition of LTD4 prolonged the duration of EGF-induced phosphorylation of Erk-1/2 and Akt, both of which were sensitive to PTX. The effect of cys-LTs involves a PTX-sensitive and PKC-mediated intracellular pathway leading to sustained growth factor-dependent phosphorylation of Erk-1/2 and Akt.

  1. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Minyong Kang


    Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

  2. Cooperation of tyrosine kinase receptor TrkB and epidermal growth factor receptor signaling enhances migration and dispersal of lung tumor cells.

    Directory of Open Access Journals (Sweden)

    Rudolf Götz

    Full Text Available TrkB mediates the effects of brain-derived neurotrophic factor (BDNF in neuronal and nonnneuronal cells. Based on recent reports that TrkB can also be transactivated through epidermal growth-factor receptor (EGFR signaling and thus regulates migration of early neurons, we investigated the role of TrkB in migration of lung tumor cells. Early metastasis remains a major challenge in the clinical management of non-small cell lung cancer (NSCLC. TrkB receptor signaling is associated with metastasis and poor patient prognosis in NSCLC. Expression of this receptor in A549 cells and in another adenocarcinoma cell line, NCI-H441, promoted enhanced migratory capacity in wound healing assays in the presence of the TrkB ligand BDNF. Furthermore, TrkB expression in A549 cells potentiated the stimulatory effect of EGF in wound healing and in Boyden chamber migration experiments. Consistent with a potential loss of cell polarity upon TrkB expression, cell dispersal and de-clustering was induced in A549 cells independently of exogeneous BDNF. Morphological transformation involved extensive cytoskeletal changes, reduced E-cadherin expression and suppression of E-cadherin expression on the cell surface in TrkB expressing tumor cells. This function depended on MEK and Akt kinase activity but was independent of Src. These data indicate that TrkB expression in lung adenoma cells is an early step in tumor cell dissemination, and thus could represent a target for therapy development.

  3. ZEB1 Mediates Acquired Resistance to the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

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    Takeshi Yoshida

    Full Text Available Epithelial-mesenchymal transition (EMT is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs in non-small cell lung cancer (NSCLC. The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8, a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin, ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.

  4. Characterization of the activation of protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1 by hypoxia inducible factor-2 alpha.

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    Victoria Wang

    Full Text Available BACKGROUND: Hypoxia inducible factors (HIFs are the principal means by which cells upregulate genes in response to hypoxia and certain other stresses. There are two major HIFs, HIF-1 and HIF-2. We previously found that certain genes are preferentially activated by HIF-2. One was protein tyrosine phosphatase, receptor-type, Z polypeptide 1 (PTPRZ1. PTPRZ1 is overexpressed in a number of tumors and has been implicated in glioblastoma pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To understand the preferential activation of PTPRZ1 by HIF-2, we studied the PTPRZ1 promoter in HEK293T cells and Hep3B cells. Through deletion and mutational analysis, we identified the principal hypoxia response element. This element bound to both HIF-1 and HIF-2. We further identified a role for ELK1, an E26 transformation-specific (Ets factor that can bind to HIF-2alpha but not HIF-1alpha, in the HIF-2 responsiveness. Knock-down experiments using siRNA to ELK1 decreased HIF-2 activation by over 50%. Also, a deletion mutation of one of the two Ets binding motifs located near the principal hypoxia response element similarly decreased activation of the PTPRZ1 promoter by HIF-2. Finally, chromatin immunoprecipitation assays showed binding of HIF and ELK1 to the PTPRZ1 promoter region. CONCLUSIONS/SIGNIFICANCE: These results identify HIF-binding and Ets-binding motifs on the PTPRZ1 promoter and provide evidence that preferential activation of PTPRZ1 by HIF-2 results at least in part from cooperative binding of HIF-2 and ELK1 to nearby sites on the PTPRZ1 promoter region. These results may have implications in tumor pathogenesis and in understanding neurobiology, and may help inform the development of novel tumor therapy.

  5. SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Buchanan, Sean G.; Hendle, Jorg; Lee, Patrick S.; Smith, Christopher R.; Bounaud, Pierre-Yves; Jessen, Katti A.; Tang, Crystal M.; Huser, Nanni H.; Felce, Jeremy D.; Froning, Karen J.; Peterman, Marshall C.; Aubol, Brandon E.; Gessert, Steve F.; Sauder, J. Michael; Schwinn, Kenneth D.; Russell, Marijane; Rooney, Isabelle A.; Adams, Jason; Leon, Barbara C.; Do, Tuan H.; Blaney, Jeff M.; Sprengeler, Paul A.; Thompson, Devon A.; Smyth, Lydia; Pelletier, Laura A.; Atwell, Shane; Holme, Kevin; Wasserman, Stephen R.; Emtage, Spencer; Burley, Stephen K.; Reich, Siegfried H.; (Ventana); (SGX); (Genentech); (Amira)


    The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC{sub 50} of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.

  6. Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during Experimental Autoimmune Encephalomyelitis

    Directory of Open Access Journals (Sweden)

    Prieto Anne L


    Full Text Available Abstract Background Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6 are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis. Methods WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays. Results Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+ were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals. Conclusions These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data

  7. The rat ErbB2 tyrosine kinase receptor produced in plants is immunogenic in mice and confers protective immunity against ErbB2(+) mammary cancer. (United States)

    Matić, Slavica; Quaglino, Elena; Arata, Lucia; Riccardo, Federica; Pegoraro, Mattia; Vallino, Marta; Cavallo, Federica; Noris, Emanuela


    The rat ErbB2 (rErbB2) protein is a 185-kDa glycoprotein belonging to the epidermal growth factor-related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2-pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ-HT expression vector as 6X His tag fusions. All rErbB2 variants (72-74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2(+) mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.

  8. A hallmark of immunoreceptor, the tyrosine-based inhibitory motif ITIM, is present in the G protein-coupled receptor OX1R for orexins and drives apoptosis: a novel mechanism. (United States)

    Voisin, Thierry; El Firar, Aadil; Rouyer-Fessard, Christiane; Gratio, Valérie; Laburthe, Marc


    Orexins acting at the G protein-coupled receptor (GPCR) OX1R have recently been shown to promote dramatic apoptosis in cancer cells. We report here that orexin-induced apoptosis is driven by an immunoreceptor tyrosine-based inhibitory motif (ITIM) (IIY(358)NFL) present in the OX1R. This effect is mediated by SHP-2 phosphatase recruitment via a mechanism that requires Gq protein but is independent of phospholipase C activation. This is based on the following observations: 1) mutation of Y(358) into F abolished orexin-induced tyrosine phosphorylation in ITIM, orexin-induced apoptosis, and uncoupled OX1R from Gq protein in transfected Chinese hamster ovary (CHO) cells; 2) orexin-induced apoptosis in CHO cells expressing recombinant OX1R and in colon cancer cells expressing the native receptor was abolished by treatment with the tyrosine phosphatase inhibitor PAO and by transfection with a dominant-negative mutant of SHP-2; 3) orexins were unable to promote apoptosis in fibroblast cells invalidated for the G alpha q subunit and transfected with OX1R cDNA, whereas they promoted apoptosis in cells equipped with G alpha q and OX1R; and 4) the phospholipase C inhibitor U-73122 blocked orexin-stimulated inositol phosphate formation, whereas it had no effect on orexin-induced apoptosis in CHO cells expressing OX1R. These data unravel a novel mechanism, whereby ITIM-expressing GPCRs may trigger apoptosis.

  9. Influence of ginsenoside on expression of brain-derived neurotrophic factor and receptor tyrosine kinase B in the medial septum of aged rats

    Institute of Scientific and Technical Information of China (English)

    Liang Zeng; Haihua Zhao; Yongli Lü; Wenbo Dai


    BACKGROUND: It has been shown that ginsenoside, the effective component of ginseng, can enhance expression of choline acetyl transferase, as well as brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (TrkB), in cholinergic neurons of the basal forebrain.OBJECTIVE: To qualitatively and quantitatively verify the influence of ginsenoside on expression of BDNF and its receptor, TrkB, in the medial septum of aged rats, and to provide a molecular basis for clinical application.DESIGN, TIME AND SETTING: A contrast study, which was performed in the Department of Anatomy, China Medical University, and the Department of Anatomy, Shenyang Medical College between December 2005 and May 2007.MATERIALS: Thirty-five, healthy, female, Sprague Dawley rats were selected for this study. Ginsenoside (81% purity) was provided by Jilin Ji'an Wantai Chinese Medicine Factory; anti-BDNF antibody, anti-TrkB antibody, and their kits were provided by Wuhan Boster Company.METHODS: A total of 35 rats were divided into three groups: young (four months old), aging (26 months old), and ginsenoside. Rats in the ginsenoside group were administered ginsenoside (25mg/kg/d) between 17 months and 26 months.MAIN OUTCOME MEASURES: Immunohistochemistry and in situ hybridization were used to measure expression of BDNF and TrkB in the medial septum of aged rats, and the detected results were expressed as gray values.RESULTS: ①Qualitative detection: using microscopy, degenerative neurons were visible in the medial septum in the aging group. However, neuronal morphology in the ginsenoside group was similar to neurons in the young group.②Quantitative detection: the mean gray value of BDNF-positive and TrkB-positive products in the aging group were significantly higher than in the young group (t=3.346,4.169, P<0.01); however, the mean gray value in the ginsenoside group was significantly lower than in the aging group (t=2.432,2.651, P<0.01).CONCLUSION: Ginsenoside can increase

  10. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

    DEFF Research Database (Denmark)

    Jiang, Y P; Wang, H; D'Eustachio, P;


    We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology......, separated from the transmembrane segment by an uncharacteristically large juxta-membrane region. The extracellular domain of the R-PTP-kappa precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of about 150 amino acids...... with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (S. Takagi, T. Hsrata, K. Agata, M. Mochii, G. Eguchi, and H. Fujisawa, Neuron 7:295-307, 1991). Antibodies directed against the intra- and extracellular domains reveal that the R-PTP-kappa precursor protein undergoes proteolytic...

  11. Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

    Directory of Open Access Journals (Sweden)

    Chun Wu


    Full Text Available Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA.

  12. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay


    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya


    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-ba...

  13. Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation.

    Directory of Open Access Journals (Sweden)

    Patrice N Mimche

    Full Text Available The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC and bone-marrow derived DCs (BMDC express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.

  14. Targeting the epidermal growth factor receptor in non-small cell lung cancer cells: the effect of combining RNA interference with tyrosine kinase inhibitors or cetuximab

    Directory of Open Access Journals (Sweden)

    Chen Gang


    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is a validated therapeutic target in non-small cell lung cancer (NSCLC. However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. In the current study we have examined, in different NSCLC cell lines, the combined effect of RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling using different receptor tyrosine kinase inhibitors (TKIs or a monoclonal antibody cetuximab. Methods NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975 were transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of EGFR mRNA expression was measured by real-time quantitative RT-PCR. The down-regulation of EGFR protein expression was measured by western blot, and the proliferation, viability, caspase3/7 activity, and apoptotic morphology were monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The combined effect of EGFR siRNA and different drugs was evaluated using a combination index. Results EGFR-specific siRNA strongly inhibited EGFR protein expression almost equally in all cell lines and inhibited cell growth and induced cell apoptosis in all NSCLC cell lines studied, albeit with a different magnitude. The effects on growth obtained with siRNA was strikingly different from the effects obtained with TKIs. The effects of siRNA probably correlate with the overall oncogenic significance of the receptor, which is only partly inhibited by the TKIs. The cells which showed weak response to TKIs, such as the H1975 cell line containing the T790M resistance mutation, were found to be responsive to siRNA knockdown of EGFR, as were cell lines with downstream TKI resistance mutations. The cell line HCC827, harboring an exon 19 deletion mutation, was more than 10-fold

  15. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology. (United States)

    Challa, Anil Kumar; Chatti, Kiranam


    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.

  16. Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein.

    Directory of Open Access Journals (Sweden)

    Gerrit Volkmann

    Full Text Available BACKGROUND: Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. METHODOLOGY: A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. CONCLUSIONS: We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.

  17. Skin problems and EGFR-tyrosine kinase inhibitor. (United States)

    Kozuki, Toshiyuki


    Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities.

  18. Increased risk of severe infections in cancer patients treated with vascular endothelial growth factor receptor tyrosine kinase inhibitors: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Ma Q


    Full Text Available Qing Ma, Li-Yan Gu, Yao-Yao Ren, Li-Li Zeng, Ting Gong, Dian-Sheng Zhong Department of Oncology, The General Hospital of Tianjin Medical University, Tianjin, People’s Republic of China Background: Vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs have been widely used in a variety of solid malignancies. Concerns have arisen regarding the risk of severe infections (≥grade 3 with use of these drugs, but the contribution of VEGFR-TKIs to infections is still unknown.Methods: The databases of PubMed and abstracts presented at oncology conferences’ proceedings were searched for relevant studies from January 2000 to December 2014. Summary incidences, Peto odds ratio (Peto OR, and 95% confidence intervals (CIs were calculated by using either random-effects or fixed-effects models according to the heterogeneity of included studies.Results: A total of 16,488 patients from 27 randomized controlled trials were included. The risk of developing severe (Peto OR 1.69, 95% CI: 1.45–1.96, P<0.001 and fatal infections (Peto OR 1.78, 95% CI: 1.13–2.81, P=0.013 was significantly increased in patients treated with VEGFR-TKIs when compared to controls. Exploratory subgroup analysis showed no effect of tumor types, phase of trials, or agent used on the Peto OR of severe infections. When stratified according to specific infectious events, the risks of high-grade febrile neutropenia, pneumonia, fever, and sepsis were increased compared with controls, with Peto ORs of 1.57 (95% CI: 1.30–1.88, P<0.001, 1.79 (95% CI: 1.29–2.49, P<0.001, 5.35 (95% CI: 1.47–19.51, P=0.011, and 3.68 (95% CI: 1.51–8.99, P=0.004, respectively. Additionally, VEGFR-TKIs significantly increased the risk of fatal sepsis (OR 3.66, 95% CI: 1.47–9.13, P=0.005 but not fatal pneumonia (OR 1.34, 95% CI: 0.80–2.25, P=0.26.Conclusion: The use of VEGFR-TKIs significantly increases the risk of developing severe and fatal infectious events in cancer

  19. Early life stress and post-weaning high fat diet alter tyrosine hydroxylase regulation and AT1 receptor expression in the adrenal gland in a sex dependent manner. (United States)

    Bobrovskaya, Larisa; Maniam, Jayanthi; Ong, Lin Kooi; Dunkley, Peter R; Morris, Margaret J


    Previous studies have shown that early life stress induced by maternal separation or non-handling can lead to behavioural deficits in rats and that these deficits can be alleviated by providing palatable cafeteria high-fat diet (HFD). In these studies we investigated the effects of maternal separation or non-handling and HFD on tyrosine hydroxylase (TH) protein and TH phosphorylation at Ser40 (pSer40TH) and the expression of angiotensin II receptor type 1 (AT1R) protein in the adrenal gland as markers of sympatho-adrenomedullary activation. After littering, Sprague-Dawley rats were assigned to short maternal separation, S15 (15 min), prolonged maternal separation, S180 (180 min) daily from postnatal days 2-14 or were non-handled (NH) until weaning. Siblings were exposed to HFD or chow from day 21 until 19 weeks when adrenals were harvested. Maternal separation and non-handling had no effects on adrenal TH protein in both sexes. We found an effect of HFD only in the females; HFD significantly increased TH levels in NH rats and pSer40TH in S180 rats (relative to corresponding chow-fed groups), but had no effect on AT1R expression in any group. In contrast, in male rats HFD had no effect on TH protein levels, but significantly increased pSer40TH across all treatment groups. There was no effect of HFD on AT1R expression in male rats; however, maternal separation (for 15 or 180 min) caused significant increases in AT1R expression (relative to NH group regardless of diet). This is the first study to report that early life stress and diet modulate TH protein, pSer40TH and AT1R protein levels in the adrenal gland in a sex dependent manner. These results are interpreted in respect to the potential adverse effects that these changes in the adrenal gland may have in males and females in adult life.

  20. Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses

    Directory of Open Access Journals (Sweden)

    Wang HY


    Full Text Available Hsian-Yu Wang,1,2 Min-Kung Hsu,3,4 Kai-Hsuan Wang,1 Ching-Ping Tseng,2,4 Feng-Chi Chen,3,4 John T-A Hsu1,4 1Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 2Institute of Molecular Medicine and Bioengineering, National Chiao Tung University (NCTU, Hsinchu, 3Division of Biostatistics and Bioinformatics, Institute of Population Health Sciences, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 4Department of Biological Science and Technology, National Chiao Tung University (NCTU, Hsinchu, Taiwan, Republic of China Background: Epidermal growth factor receptor (EGFR tyrosine kinase inhibitors (TKIs, such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs.Results: Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits

  1. Synthesis of receptor tyrosine kinase inhibitors linifanib%受体酪氨酸激酶抑制剂linifanib的合成

    Institute of Scientific and Technical Information of China (English)

    王志锋; 李亮; 周辛波; 郑志兵; 李松


    目的 合成受体酪氨酸激酶抑制剂linifanib.方法 以2-氟-5-甲基苯胺为起始原料,经3步反应制得中间体N-(4-硼酸频哪醇酯苯基)-N'-(2-氟-5-甲基苯基)脲(5);以2,6-二氯苯腈为起始原料,经关环反应制得另一中间体3-氨基-4-氯-吲唑(7);中间体5和7经Suzuki偶联反应得到目标化合物linifanib.结果与结论 目标化合物和中间体的结构经1H-NMR、MS谱确证,总收率为11.4%.%The synthesis of linifanib,a receptor tyrosine kinase inhibitor,has been accomplished via 5 steps according to the literature. Linifanib was synthesized through Suzuki coupling reaction between the intermediate 1 -(2-fluoro-5-methylphenyl) -3-( 4-( 4,4,5,5-teramethyl-1, 3,2-dioxaborolan-2-yl) phenyl) urea (5) and 4-chloro-1H-indazol-3-amine(7). The intermediate 5 was obtained by a palladium-catalyzed coupling reaction from bis (pinacolato) diboron and 1 -(4-bromophenyl) -3-( 2-fluoro-5-methyl phenyl) urea (4), which was synthesized from 2-fluoro-5-methylaniline by a carbamate bond formation and amine-ester exchange reaction. The other intermediate 7 was synthesized from 2,6-dichlorobenzonitrile by a substitution and cyclization reaction. The structure of the target compound and the intermediates were confirmed by MS and 'H-NMR. The total yield of linifanib from 2-fluoro-5-methylaniline is 11. 4%. Compared with the method reported in the literature, the new synthesis method showed some advantages, such as inexpensive and readily available starting materials, a simplified and easier synthesis process and with much lower synthesis cost of linifanib.

  2. Regulation of T cell receptor expression in immature CD4+CD8+ thymocytes by p56lck tyrosine kinase: basis for differential signaling by CD4 and CD8 in immature thymocytes expressing both coreceptor molecules



    Signals transduced through the T cell antigen receptor (TCR) are modulated by the src family tyrosine kinase p56lck (lck), which associates in mature T cells with the coreceptor molecules CD4 and CD8. Here we describe a novel function of lck in immature CD4+CD8+ thymocytes, that of regulating TCR expression. Activation of lck in immature CD4+CD8+ thymocytes by intrathymic engagement of CD4 maintains low TCR expression by causing most TCR components to be retained and degraded within the endop...

  3. Synthesis of N11-anchoring biotinylated artemisinin derivatives and their preliminary biological assessment

    Institute of Scientific and Technical Information of China (English)


    Unique endoperoxide moiety of artemisinin and its derivatives has been considered the functionality exhibiting highly potent antimalarial and anticancer activities.To investigate the mechanisms of their biological actions,development of suitable molecular probes including biotinylated derivatives is of extreme significance.The synthesis and preliminary biological assessment of four new biotinylated artemisinin derivatives have been reported in this work.

  4. Potent quinoxaline-based inhibitors of PDGF receptor tyrosine kinase activity. Part 2: the synthesis and biological activities of RPR127963 an orally bioavailable inhibitor. (United States)

    He, Wei; Myers, Michael R; Hanney, Barbara; Spada, Alfred P; Bilder, Glenda; Galzcinski, Helen; Amin, Dilip; Needle, Saul; Page, Ken; Jayyosi, Zaid; Perrone, Mark H


    RPR127963 demonstrates an excellent pharmacokinetic profile in several species and was found to be efficacious in the prevention of restenosis in a Yucatan mini-pig model upon oral administration of 1-5 mg/kg. The in vitro selectivity profile and SAR of the highly optimized PDGF-R tyrosine kinase inhibitor are highlighted.

  5. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.

    NARCIS (Netherlands)

    E. de Boer (Ernie); P. Rodriguez (Patrick); E. Bonte (Edgar); J. Krijgsveld (Jeroen); E. Katsantoni (Eleni); A.J.R. Heck (Albert); F.G. Grosveld (Frank); J. Strouboulis (John)


    textabstractProteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated temp

  6. Ligand-independent activation of the arylhydrocarbon receptor by ETK (Bmx) tyrosine kinase helps MCF10AT1 breast cancer cells to survive in an apoptosis-inducing environment. (United States)

    Fujisawa, Yasuko; Li, Wen; Wu, Dalei; Wong, Patrick; Vogel, Christoph; Dong, Bin; Kung, Hsing-Jien; Matsumura, Fumio


    It has been reported that the arylhydrocarbon receptor (AHR) is overexpressed in certain types of breast tumors. However, so far no concrete evidence has been provided yet as to why and how the overexpressed AHR in those cancer cells is functionally activated without exogenous ligands. Here we show that the AHR was functionally activated when estrogen receptor-negative, AHR overexpressing MCF10AT1 human breast cancer cells (designated P20E) were subjected to serum starvation. Transfection of cells with ETK-KQ, a plasmid for kinase-dead epithelial and endothelial tyrosine kinase (ETK), attenuated this AHR activation. Artificial over-expression of ETK in P20E cells through transfection with wild-type ETK plasmid (ETK-wt) caused up-regulation of cytochrome P4501a1 (CYP1A1; a marker of functional activation of AHR). Furthermore, ablation of ETK expression by a specific antisense oligonucleotide or AG879, a specific inhibitor of ETK kinase suppressed activation of AHR induced by omeprazole, a strong ligand-independent activator of AHR. Activation of ETK in those cells conferred them resistance to UVB- as well as doxorubicin-induced apoptosis, both of which were reversed by ETK-KQ. Together, these findings support our conclusion that ETK is the tyrosine kinase responsible for the functional activation of the AHR in these mammary epithelial cells.

  7. Staphylococcal enterotoxins modulate interleukin 2 receptor expression and ligand-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription (Stat proteins)

    DEFF Research Database (Denmark)

    Nielsen, M; Svejgaard, A; Röpke, C;


    Staphylococcal enterotoxins (SE) stimulate T cells expressing the appropriate variable region beta chain of (V beta) T-cell receptors and have been implicated in the pathogenesis of several autoimmune diseases. Depending on costimulatory signals, SE induce either proliferation or anergy in T cells...

  8. Early radiographic response to epidermal growth factor receptor-tyrosine kinase inhibitor in non-small cell lung cancer patients with epidermal growth factor receptor mutations: A prospective study

    Directory of Open Access Journals (Sweden)

    John WC Chang


    Full Text Available Background: The time schedules for response evaluation of epidermal growth factor receptor-tyrosine kinase Inhibitor (EGFR-TKI in non-small cell lung cancer (NSCLC patients are still ill-defined. Methods: Stage IIIB/IV patients with histologically proven NSCLC were enrolled in this study if the tumor cells bore EGFR mutations other than T790M. Eligible patients were treated with either 250 mg of gefitinib or 150 mg of erlotinib once daily. The early response rate [computed tomography (CT scan on Day 14], definitive response rate determined on Day 56, progression-free survival (PFS, overall survival (OS, and toxicity profile were assessed prospectively. Results: Thirty-nine patients were enrolled in this study. A total of 29 patients (29/39, 74.4% achieved partial response (PR. Twenty-one patients (21/39, 53.8% had early radiological response on Day 14. The early radiological response rate in patients with PR was 72.4% (21/29. Only eight patients without a PR on early CT still ended with PR. Among the 29 patients with PR, the PFS (8.1 months and OS (18.3 months of the 21 patients with early CT response were shorter than those of the 8 patients without early CT response (11.9 and 24.0 months for PFS and OS, respectively. But the survival differences were statistically non-significant. Conclusions: A very high percentage (72.4%, 21/29 of NSCLC patients with EGFR mutations with PR demonstrates early radiological response to EGFR-TKIs, which would advocate early radiological examination for EGFR-TKI therapy in NSCLC patients.

  9. Research Progress of Small-molecule Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors%小分子表皮生长因子受体酪氨酸激酶抑制剂的研究进展

    Institute of Scientific and Technical Information of China (English)

    李莹; 田驰; 王磊; 谢蒙蒙; 程泽能


    Epidermal growth factor receptor (EGFR), a receptor on membrane surface, is over expressed in non-small cell lung cancer with tyrosine kinase activity. It plays an important role in proliferation, metastasis, metabolism, differentiation and survival of cells. Currently, the development of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has become a hot topic in the field of tumor therapy. According to the reversibility of binding with EGFR, the EGFR-TKIs can be classified into reversible and irreversible EGFR-TKIs. Reversible EGFR-TKIs include gefitinib, erlotinib and some others which are available in the market. Irreversible EGFR-TKIs involve afatinib and some novel antineoplastics which are still in clinical trials, including AZD9291, CO-1686 and HM61713. In this work, some latest researches about the development of EGFR-TKIs were reviewed for provide some information to non-small cell lung cancer therapies in clinic.%表皮生长因子受体是一种具有酪氨酸激酶活性的膜受体,在细胞的增殖、迁移、代谢、分化和存活中发挥重要作用。目前,表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitors,EGFR-TKIs)的开发已成为肿瘤治疗领域的热点。EGFR-TKIs 分为可逆性 EGFR-TKIs 和不可逆性 EGFR-TKIs,可逆性 EGFR-TKIs 主要包括吉非替尼和厄洛替尼等已上市的药物,不可逆性 EGFR-TKIs主要包括阿法替尼和一些正处于临床研究中的药物,如 AZD9291、CO-1686和 HM61713等。本文对 EGFR-TKIs 的研究进展进行了综述,为非小细胞肺癌临床治疗的药物选择提供参考。

  10. Proximity Utilizing Biotinylation of Nuclear Proteins in vivo

    Directory of Open Access Journals (Sweden)

    Arman Kulyyassov


    Full Text Available Introduction. The human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 10,000 proteins can be produced by the cell at any given time (the cellular “proteome”. It has been estimated that over 80% of proteins do not operate alone, but in complexes. These protein-protein interactions (PPI are regulated by several mechanisms. For example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination or metal-binding can lead to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo.Methods. The biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP was fused to an interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry.Results. Using several experimental systems (BirA.A + BAP.B, we showed that the biotinylation is interaction/proximity dependent. Here, A and B are the next nuclear proteins used in the experiments – 3 paralogues of heterochromatin protein HP1a (CBX5, HP1b (CBX1, HP1g (CBX3, wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA, ubiquitin Ub, SUMO-2/3, different types and isoforms of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP (Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation and is designed to purify and study the protein

  11. Increased activity of the Vesicular Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor TI-VAMP/VAMP7 by Tyrosine Phosphorylation in the Longin Domain* (United States)

    Burgo, Andrea; Casano, Alessandra M.; Kuster, Aurelia; Arold, Stefan T.; Wang, Guan; Nola, Sébastien; Verraes, Agathe; Dingli, Florent; Loew, Damarys; Galli, Thierry


    Vesicular (v)- and target (t)-SNAREs play essential roles in intracellular membrane fusion through the formation of cytoplasmic α-helical bundles. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. The molecular mechanism leading to Longin v-SNARE activation is largely unknown. Here we find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. In search of a potential downstream mechanism, we found that TI-VAMP is phosphorylated in vitro by c-Src kinase on tyrosine 45 of the Longin domain. Accordingly, a mutation of tyrosine 45 into glutamate, but not phenylalanine, activates both t-SNARE binding and exocytosis. Activation of TI-VAMP-mediated exocytosis thus relies on tyrosine phosphorylation. PMID:23471971

  12. Increased activity of the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor TI-VAMP/VAMP7 by tyrosine phosphorylation in the Longin domain. (United States)

    Burgo, Andrea; Casano, Alessandra M; Kuster, Aurelia; Arold, Stefan T; Wang, Guan; Nola, Sébastien; Verraes, Agathe; Dingli, Florent; Loew, Damarys; Galli, Thierry


    Vesicular (v)- and target (t)-SNAREs play essential roles in intracellular membrane fusion through the formation of cytoplasmic α-helical bundles. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. The molecular mechanism leading to Longin v-SNARE activation is largely unknown. Here we find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. In search of a potential downstream mechanism, we found that TI-VAMP is phosphorylated in vitro by c-Src kinase on tyrosine 45 of the Longin domain. Accordingly, a mutation of tyrosine 45 into glutamate, but not phenylalanine, activates both t-SNARE binding and exocytosis. Activation of TI-VAMP-mediated exocytosis thus relies on tyrosine phosphorylation.

  13. Biotinylation: a novel posttranslational modification linking cell autonomous circadian clocks with metabolism. (United States)

    He, Lan; Hamm, J Austin; Reddy, Alex; Sams, David; Peliciari-Garcia, Rodrigo A; McGinnis, Graham R; Bailey, Shannon M; Chow, Chi-Wing; Rowe, Glenn C; Chatham, John C; Young, Martin E


    Circadian clocks are critical modulators of metabolism. However, mechanistic links between cell autonomous clocks and metabolic processes remain largely unknown. Here, we report that expression of the biotin transporter slc5a6 gene is decreased in hearts of two distinct genetic mouse models of cardiomyocyte-specific circadian clock disruption [i.e., cardiomyocyte-specific CLOCK mutant (CCM) and cardiomyocyte-specific BMAL1 knockout (CBK) mice]. Biotinylation is an obligate posttranslational modification for five mammalian carboxylases: acetyl-CoA carboxylase α (ACCα), ACCβ, pyruvate carboxylase (PC), methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PCC). We therefore hypothesized that the cardiomyocyte circadian clock impacts metabolism through biotinylation. Consistent with decreased slc5a6 expression, biotinylation of all carboxylases is significantly decreased (10-46%) in CCM and CBK hearts. In association with decreased biotinylated ACC, oleate oxidation rates are increased in both CCM and CBK hearts. Consistent with decreased biotinylated MCC, leucine oxidation rates are significantly decreased in both CCM and CBK hearts, whereas rates of protein synthesis are increased. Importantly, feeding CBK mice with a biotin-enriched diet for 6 wk normalized myocardial 1) ACC biotinylation and oleate oxidation rates; 2) PCC/MCC biotinylation (and partially restored leucine oxidation rates); and 3) net protein synthesis rates. Furthermore, data suggest that the RRAGD/mTOR/4E-BP1 signaling axis is chronically activated in CBK and CCM hearts. Finally we report that the hepatocyte circadian clock also regulates both slc5a6 expression and protein biotinylation in the liver. Collectively, these findings suggest that biotinylation is a novel mechanism by which cell autonomous circadian clocks influence metabolic pathways.

  14. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    Directory of Open Access Journals (Sweden)

    Yu-Ren Liou

    Full Text Available Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs conjugated with antibodies (i.e., targeted biotin-MBs. Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs, which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+ and MDA-MB-453 cells (CD44-, which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+ is a commonly used cancer

  15. Detection of active matriptase using a biotinylated chloromethyl ketone peptide.

    Directory of Open Access Journals (Sweden)

    Sine Godiksen

    Full Text Available Matriptase is a member of the family of type II transmembrane serine proteases that is essential for development and maintenance of several epithelial tissues. Matriptase is synthesized as a single-chain zymogen precursor that is processed into a two-chain disulfide-linked form dependent on its own catalytic activity leading to the hypothesis that matriptase functions at the pinnacle of several protease induced signal cascades. Matriptase is usually found in either its zymogen form or in a complex with its cognate inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1, whereas the active non-inhibited form has been difficult to detect. In this study, we have developed an assay to detect enzymatically active non-inhibitor-complexed matriptase by using a biotinylated peptide substrate-based chloromethyl ketone (CMK inhibitor. Covalently CMK peptide-bound matriptase is detected by streptavidin pull-down and subsequent analysis by Western blotting. This study presents a novel assay for detection of enzymatically active matriptase in living human and murine cells. The assay can be applied to a variety of cell systems and species.

  16. Biotinylated dextran amine anterograde tracing of the canine corticospinal tract

    Institute of Scientific and Technical Information of China (English)

    Xiao Han; Guangming Lv; Huiqun Wu; Dafeng Ji; Zhou Sun; Yaofu Li; Lemin Tang


    In this study, biotinylated dextran amine (BDA) was microinjected into the left cortical motor area of the canine brain. Fluorescence microscopy results showed that a large amount of BDA-labeled pyramidal cells were visible in the left cortical motor area after injection. In the left medulla oblongata, the BDA-labeled corticospinal tract was evenly distributed, with green fluorescence that had a clear boundary with the surrounding tissue. The BDA-positive corticospinal tract entered into the right lateral funiculus of the spinal cord and descended into the posterior part of the right lateral funiculus, close to the posterior horn, from cervical to sacral segments. There was a small amount of green fluorescence in the sacral segment. The distribution of BDA labeling in the canine central nervous system was consistent with the course of the corticospinal tract. Fluorescence labeling for BDA gradually diminished with time after injection. Our findings indicate that the BDA anterograde tracing technique can be used to visualize the localization and trajectory of the corticospinal tract in the canine central nervous system.

  17. Tyrosine Kinase Inhibition: An Approach to Drug Development (United States)

    Levitzki, Alexander; Gazit, Aviv


    Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

  18. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan. (United States)

    Moraz, Marie-Laurence; Pythoud, Christelle; Turk, Rolf; Rothenberger, Sylvia; Pasquato, Antonella; Campbell, Kevin P; Kunz, Stefan


    The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.

  19. Tyrosine Kinase Display of Prostate Cancer Cells (United States)


    Swanson, P.E., Ratliff, T.L., Vollmer, R.T., Day, M.L. (1995) HGF and its receptor c-met in prostatic carcinoma. Am. J. Pathol. 147:386-396. 4. Levitzki , A...alpha and erbB-3 receptor in human prostatic adenocarcinoma. Br. J. Urol. 79: 212-216. 78. Levitzki , A. and A. Gazit. 1995. Tyrosine kinase inhibition

  20. Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib

    DEFF Research Database (Denmark)

    Sorensen, Boe S; Wu, Lin; Wei, Wen;


    BACKGROUND: The feasibility of monitoring epidermal growth factor receptor (EGFR) mutations in plasma DNA from patients with advanced non-small cell lung cancer (NSCLC) during treatment with erlotinib and its relation to disease progression was investigated. METHODS: The amount of EGFR-mutant DNA...... was tested in plasma DNA from patients with advanced NSCLC with allele-specific polymerase chain reaction assays. Blood samples from 23 patients with adenocarcinoma of NSCLC that carried tyrosine kinase inhibitor-sensitizing EGFR mutations were taken immediately before treatment with erlotinib. Additional...... blood samples were taken at timed intervals until erlotinib treatment was withdrawn. RESULTS: The amount of plasma DNA with sensitizing EGFR mutations was found to be reduced after the first cycle of erlotinib treatment in 22 of 23 patients (96%). No patients presented with the resistant T790M mutation...

  1. Antenatal betamethasone produces protracted changes in anxiety-like behaviors and in the expression of microtubule-associated protein 2, brain-derived neurotrophic factor and the tyrosine kinase B receptor in the rat cerebellar cortex. (United States)

    Pascual, Rodrigo; Valencia, Martina; Bustamante, Carlos


    Using classic Golgi staining methods, we previously showed that the administration of synthetic glucocorticoid betamethasone in equivalent doses to those given in cases of human premature birth generates long-term alterations in Purkinje cell dendritic development in the cerebellar cortex. In the present study, we evaluated whether betamethasone alters the immunohistochemical expression of proteins that participate in cerebellar Purkinje cell dendritic development and maintenance, including microtubule-associated protein 2 (MAP2), brain-derived neurotrophic factor (BDNF) and the tyrosine kinase B receptor (TrkB), which are located predominantly in the cerebellar molecular layer where Purkinje cell dendritogenesis occurs. Consistent with our previous Golgi stain studies, we observed that animals prenatally exposed to a single course of betamethasone showed long-term alterations in the expression of MAP2, BDNF and TrkB. Additionally, these protracted molecular changes were accompanied by anxiety-like behaviors in the elevated plus maze and marble burying tests.

  2. The role of histone H4 biotinylation in the structure of nucleosomes.

    Directory of Open Access Journals (Sweden)

    Nina A Filenko

    Full Text Available BACKGROUND: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4 is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p<0.001 difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. CONCLUSIONS/SIGNIFICANCE: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation.

  3. A phase I dose escalation study of BIBW 2992, an irreversible dual inhibitor of epidermal growth factor receptor I (EGFR) and 2 (HER2) tyrosine kinase in a 2-week on, 2-week off schedule in patients with advanced solid tumours

    NARCIS (Netherlands)

    Eskens, Falm; Mom, C. H.; Planting, A. S. T.; Gietema, J. A.; Amelsberg, A.; Huisman, H.; van Doorn, L.; Burger, H.; Stopfer, P.; Verweij, J.; de Vries, Ege


    To assess tolerability, pharmacokinetics ( PK), pharmacodynamics ( PD) and clinical activity of the dual epidermal growth factor receptor ( EGFR) 1 and 2 ( HER2) tyrosine kinase inhibitor BIBW 2992. An escalating schedule of once-daily ( OD) BIBW 2992 for 14 days followed by 14 days off medication w

  4. A phase I dose escalation study of BIBW 2992, an irreversible dual inhibitor of epidermal growth factor receptor 1 (EGFR) and 2 (HER2) tyrosine kinase in a 2-week on, 2-week off schedule in patients with advanced solid tumours

    NARCIS (Netherlands)

    F.A.L.M. Eskens (Ferry); C.H. Mom (Constantijne); A.S.Th. Planting (André); J.A. Gietema (Jourik); A. Amelsberg; H. Huisman (Henkjan); L. van Doorn (Leni); H. Burger (Herman); P. Stopfer; J. Verweij (Jaap); E. de Vries (Esther)


    textabstractTo assess tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and clinical activity of the dual epidermal growth factor receptor (EGFR) 1 and 2 (HER2) tyrosine kinase inhibitor BIBW 2992. An escalating schedule of once-daily (OD) BIBW 2992 for 14 days followed by 14 days off medic

  5. Protein-tyrosine phosphatase 1B (PTP1B) is a novel regulator of central brain-derived neurotrophic factor and tropomyosin receptor kinase B (TrkB) signaling. (United States)

    Ozek, Ceren; Kanoski, Scott E; Zhang, Zhong-Yin; Grill, Harvey J; Bence, Kendra K


    Neuronal protein-tyrosine phosphatase 1B (PTP1B) deficiency in mice results in enhanced leptin signaling and protection from diet-induced obesity; however, whether additional signaling pathways in the brain contribute to the metabolic effects of PTP1B deficiency remains unclear. Here, we show that the tropomyosin receptor kinase B (TrkB) receptor is a direct PTP1B substrate and implicate PTP1B in the regulation of the central brain-derived neurotrophic factor (BDNF) signaling. PTP1B interacts with activated TrkB receptor in mouse brain and human SH-SY5Y neuroblastoma cells. PTP1B overexpression reduces TrkB phosphorylation and activation of downstream signaling pathways, whereas PTP1B inhibition augments TrkB signaling. Notably, brains of Ptpn1(-/-) mice exhibit enhanced TrkB phosphorylation, and Ptpn1(-/-) mice are hypersensitive to central BDNF-induced increase in core temperature. Taken together, our findings demonstrate that PTP1B is a novel physiological regulator of TrkB and that enhanced BDNF/TrkB signaling may contribute to the beneficial metabolic effects of PTP1B deficiency.

  6. Tyrosine phosphorylation-dependent activation of phosphatidylinositide 3-kinase occurs upstream of Ca^(2+)-signalling induced by Fcy receptor cross-linking in human neutrophils

    NARCIS (Netherlands)

    Vossebeld, Paula J. M.; Homburg, Christa H. E.; Schweizer, R.C.; Ibarrola, Iñaki; Kessler, Jan; Koenderman, L.; Roos, Dirk; Verhoeven, Arthur J.


    The effect of wortmannin on IgG-receptor (FcyR)-mediated stimulation of human neutrophils was investigated. The Ca^(2+) influx induced by clustering of both Fcy receptors was inhibited by wortmannin, as was the release of Ca^(2+) from intracellular stores. Wortmannin also inhibited, with the same ef

  7. Effect of treatment with a colloidal oatmeal lotion on the acneform eruption induced by epidermal growth factor receptor and multiple tyrosine-kinase inhibitors. (United States)

    Alexandrescu, D T; Vaillant, J G; Dasanu, C A


    Current treatment modalities for epidermal growth factor (EGFR)-positive cancers have recently included the use of antibodies and small-molecule tyrosine-kinase inhibitors (TKI). A significant limiting step in the use of these agents is dermatological toxicity, frequently in the form of an acneiform eruption. Present management modalities for this toxicity are largely ineffective. Colloidal oatmeal lotion demonstrates multiple anti-inflammatory properties with known effects on arachidonic acid, cytosolic phospholipase A2 and tumour necrosis factor-alpha pathways, along with an excellent side-effect profile. Treatment with colloidal oatmeal was applied to 11 patients with a rash induced by cetuximab, erlotinib, panitumumab and sorafenib. Of the 10 assessable patients, 6 had complete response and 4 partial response, giving a response rate of 100% with no associated toxicities. Treatment with colloidal oatmeal lotion is efficient in controlling the rash associated with EGFR and multiple TKI, and allows continuation of the antineoplastic treatment.

  8. Third-generation epidermal growth factor receptor-tyrosine kinase inhibitors:a clinical update%第三代表皮生长因子受体-酪氨酸激酶抑制剂研究进展

    Institute of Scientific and Technical Information of China (English)

    王沁雪; 曾晓宁; 刘亚南; 赵欣; 黄茂


    肺癌是中国最常见且病死率最高的恶性肿瘤之一。近年来随着分子靶向药物的发展,尤其是表皮生长因子受体(epidermal growth factor receptor,EGFR)-酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)的研发及应用,部分存在基因敏感型突变的非小细胞肺癌患者已获得了良好效果。现已进入临床应用的第一代可逆性 TKIs 如吉非替尼、厄洛替尼、埃克替尼用药后易继发T790M 突变而导致耐药,第二代不可逆性泛 ErbB 家族抑制剂阿法替尼等仍未能克服此缺陷。现同时针对 EGFR 基因敏感型突变及 T790M 耐药突变的第三代不可逆性 TKIs———AZD9291、CO1686、HM61713已进入临床研究并取得部分可喜结果,或可成为获得性耐药肺癌患者治疗的理想选择。%Lung cancer is one of the most common malignant tumors with higher mortality in China.Patients with epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer have achieved favorable therapeutic effect since the development of molecular targeted therapy,especially the application of EGFR-tyrosine kinase inhibitors (TKIs).However,patients receiving the first-generation EGFR-TKIs such as gefitinib,erlotinib and icotinib would yield progression of the disease due to acquired resistance mostly caused by T790M mutation.Since the second-generation TKIs,which serve as irreversible pan-ErbB receptors inhibitors such as afatinib,play a limited role in these patients,the third-generation TKIs targeted both EGFR and T790M mutation,including AZD9291,CO1 686 and HM61 713 have achieved the predictive results from clinical trials,may provide optimal options for patients with acquired resistance.

  9. The Tyrosine Kinome Dictates Breast Cancer Heterogeneity and Therapeutic Responsiveness. (United States)

    Ha, Jacqueline R; Siegel, Peter M; Ursini-Siegel, Josie


    Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility, and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression, from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of tyrosine phosphorylation networks in breast cancer pathology. However, a significant degree of redundancy exists within the tyrosine kinome. Numerous receptor and cytoplasmic tyrosine kinases converge on a core set of signaling regulators, including adaptor proteins and tyrosine phosphatases, to amplify pro-tumorigenic signal transduction pathways. Mutational activation, amplification, or overexpression of one or more components of the tyrosine kinome represents key contributing events responsible for the tumor heterogeneity that is observed in breast cancers. It is this molecular heterogeneity that has become the most significant barrier to durable clinical responses due to the development of therapeutic resistance. This review focuses on recent literature that supports a prominent role for specific components of the tyrosine kinome in the emergence of unique breast cancer subtypes and in shaping breast cancer plasticity, sensitivity to targeted therapies, and the eventual emergence of acquired resistance. J. Cell. Biochem. 117: 1971-1990, 2016. © 2016 Wiley Periodicals, Inc.

  10. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice



    Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demon...

  11. Development of an immunoFET biosensor for the detection of biotinylated PCR product

    Directory of Open Access Journals (Sweden)

    Wannaporn Muangsuwan


    Full Text Available ImmunoFET (IMFET biosensor is a simple platform for the detection of biotinylated products of polymerase chain reaction (PCR. Construction of the IMFET biosensor started with adsorption of 1.5 mg/mL of protein A (PA onto the insulated gate surface of ISFET for 90 min. Next, the immobilized 1/500 dilution of anti-biotin antibody was adsorbed onto the PA layer for 60 min. The IMFET biosensor was subsequently ready for detection of the biotinylated amplicon. The IMFET biosensor showed highly specific binding to the biotinylated PCR product of the phaE gene of Haloquadratum walsbyi DSM 16854. The phaE gene is a biomarker of polyhydroxyalkanoate (PHA producers that contain PHA synthase class III. The lowest amount of DNA template of H. walsbyi DSM 16854 that the IMFET biosensor could detect was 125 fg. The IMFET biosensor has a lower amount of detection compared with a DNA lateral flow biosensor from our previous study. The degree of linearity of the biosensor signal was influenced by the concentration of the biotinylated amplicon. The IMFET biosensor also has a short response time (approximately 30 times to detect the phaE amplicon compared to an agarose gel electrophoresis. The IMFET biosensor is a promising tool for the detection of the biotinylated PCR product, and it can be integrated into a micro total analysis system (μTAS.

  12. Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in histone H3. (United States)

    Kobza, Keyna; Sarath, Gautam; Zempleni, Janos


    BirA ligase is a prokaryotic ortholog of holocarboxylase synthetase (HCS) that can biotinylate proteins. This study tested the hypothesis that BirA ligase catalyzes the biotinylation of eukaryotic histones. If so, this would mean that recombinant BirA ligase is a useful surrogate for HCS in studies of histone biotinylation. The biological activity of recombinant BirA ligase was confirmed by enzymatic biotinylation of p67. In particular, it was found that BirA ligase biotinylated both calf thymus histone H1 and human bulk histone extracts. Incubation of recombinant BirA ligase with H3-based synthetic peptides showed that lysines 4, 9, 18, and 23 in histone H3 are the targets for the biotinylation by BirA ligase. Modification of the peptides (e.g., serine phosphorylation) affected the subsequent biotinylation by BirA ligase, suggesting crosstalk between modifications. In conclusion, this study suggests that prokaryotic BirA ligase is a promiscuous enzyme and biotinylates eukaryotic histones. Moreover the biotinylation of histones by BirA ligase is consistent with the proposed role of human HCS in chromatin.

  13. K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase. (United States)

    Kobza, Keyna; Camporeale, Gabriela; Rueckert, Brian; Kueh, Alice; Griffin, Jacob B; Sarath, Gautam; Zempleni, Janos


    Histones are modified post-translationally, e.g. by methylation of lysine and arginine residues, and by phosphorylation of serine residues. These modifications regulate processes such as gene expression, DNA repair, and mitosis and meiosis. Recently, evidence has been provided that histones are also modified by covalent binding of the vitamin biotin. The aims of this study were to identify biotinylation sites in histone H3, and to investigate the crosstalk among histone biotinylation, methylation and phosphorylation. Synthetic peptides based on the sequence of human histone H3 were used as substrates for enzymatic biotinylation by biotinidase; biotin in peptides was probed using streptavidin peroxidase. These studies provided evidence that K4, K9 and K18 in histone H3 are good targets for biotinylation; K14 and K23 are relatively poor targets. Antibodies were generated to histone H3, biotinylated either at K4, K9 or K18. These antibodies localized to nuclei in human placental cells in immunocytochemistry and immunoblotting experiments, suggesting that lysines in histone H3 are biotinylated in vivo. Dimethylation of R2, R8 and R17 increased biotinylation of K4, K9 and K18, respectively, by biotinidase; phosphorylation of S10 abolished biotinylation of K9. These observations are consistent with crosstalk between biotinylation of histones and other known modifications of histones. We speculate that this crosstalk provides a link to known roles for biotin in gene expression and cell proliferation.

  14. The expression of a novel receptor-type tyrosine phosphatase suggests a role in morphogenesis and plasticity of the nervous system

    DEFF Research Database (Denmark)

    Canoll, P D; Barnea, G; Levy, J B


    . In the adult, high levels of RPTP-beta are seen in regions of the brain where there is continued neurogenesis and neurite outgrowth. The spatial and temporal patterns of RPTP-beta expression suggest that this receptor phosphatase plays a role in morphogenesis and plasticity of the nervous system....

  15. Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA. (United States)

    Rispens, Theo; Ooijevaar-de Heer, Pleuni


    Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protein.

  16. Transcriptome analysis reveals an unexpected role of a collagen tyrosine kinase receptor gene, Ddr2, as a regulator of ovarian function. (United States)

    Matsumura, Hirokazu; Kano, Kiyoshi; Marín de Evsikova, Caralina; Young, James A; Nishina, Patsy M; Naggert, Jürgen K; Naito, Kunihiko


    Mice homozygous for the smallie (slie) mutation lack a collagen receptor, discoidin domain receptor 2 (DDR2), and are dwarfed and infertile due to peripheral dysregulation of the endocrine system of unknown etiology. We used a systems biology approach to identify biological networks affected by Ddr2(slie/slie) mutation in ovaries using microarray analysis and validate findings using molecular, cellular, and functional biological assays. Transcriptome analysis indicated several altered gene categories in Ddr2(slie/slie) mutants, including gonadal development, ovulation, antiapoptosis, and steroid hormones. Subsequent biological experiments confirmed the transcriptome analysis predictions. For instance, a significant increase of TUNEL-positive follicles was found in Ddr2(slie/slie) mutants vs. wild type, which confirm the transcriptome prediction for decreased chromatin maintenance and antiapoptosis. Decreases in gene expression were confirmed by RT-PCR and/or qPCR; luteinizing hormone receptor and prostaglandin type E and F receptors in Ddr2(slie/slie) mutants, compared with wild type, confirm hormonal signaling pathways involved in ovulation. Furthermore, deficiencies in immunohistochemistry for DDR2 and luteinizing hormone receptor in the somatic cells, but not the oocytes, of Ddr2(slie/slie) mutant ovaries suggest against an intrinsic defect in germ cells. Indeed, Ddr2(slie/slie) mutants ovulated significantly fewer oocytes; their oocytes were competent to complete meiosis and fertilization in vitro. Taken together, our convergent data signify DDR2 as a novel critical player in ovarian function, which acts upon classical endocrine pathways in somatic, rather than germline, cells.

  17. Absorption of CH330331, a novel 4-anilinoquinazoline inhibitor of epidermal growth factor receptor tyrosine kinase: comparative studies using in vitro, in situ and in vivo models. (United States)

    Sun, Haiyan; Bi, Huichang; Huang, Min; Liu, Dong; Qin, Zhenyu


    CH330331 is a prototype of a new class of synthetic small molecule tyrosine kinase inhibitors (TKIs). In vitro Caco-2 cell monolayers, the in situ single-pass rat intestinal perfusion (SPIP) technique with mesenteric vein cannulated and an in vivo animal model were employed to investigate its permeability and transepithelial transport mechanisms. The Caco-2 model showed that the transport of CH330331 across the monolayers from the apical (AP) to basolateral (BL) side was 6- to 10-fold higher than that from the BL to AP side. The apparent permeability coefficient (P(app) ) values of CH330331 at 5-20 µg/ml from the AP to BL and from BL to AP side were 5.30-2.21 × 10(-6)  cm/s, with a decrease in P(app) values from the AP to BL side at increased CH330331 concentrations. In the perfused rat intestinal model, a concentration dependent change in permeability was detected where P(blood) at 5 µg/ml (1.66 ± 0.69 × 10(-6)  cm/s) and 10 µg/ml (1.80 ± 0.45 × 10(-6)  cm/s) was significantly different from P(blood) at 20 µg/ml (0.98 ± 0.31 × 10(-6)  cm/s, prat. All the results confirmed that the transepithelial transport of CH330331 was rapid and saturable, which might involve an active mechanism. The oral bioavailability of CH330331 was relatively high in vivo.

  18. Salinomycin co-treatment enhances tamoxifen cytotoxicity in luminal A breast tumor cells by facilitating lysosomal degradation of receptor tyrosine kinases. (United States)

    Sommer, Ann-Katrin; Hermawan, Adam; Mickler, Frauke Martina; Ljepoja, Bojan; Knyazev, Pjotr; Bräuchle, Christoph; Ullrich, Axel; Wagner, Ernst; Roidl, Andreas


    Luminal A breast cancer is the most common breast cancer subtype which is usually treated with selective estrogen receptor modulators (SERMS) like tamoxifen. Nevertheless, one third of estrogen receptor positive breast cancer patients initially do not respond to endocrine therapy and about 40% of luminal A breast tumors recur in five years. In this study, we investigated an alternative treatment approach by combining tamoxifen and salinomycin in luminal A breast cancer cell lines. We have found that salinomycin induces an additional cytotoxic effect by inhibiting the ligand independent activation of ERα. Thereby salinomycin increases the intracellular calcium level. This leads to a premature fusion of endosomes with lysosomes and thus to the degradation of Egfr family members. Since this process is essential for luminal A breast cancer cells to circumvent tamoxifen treatment, the combination of both drugs induces cytotoxicity in tamoxifen sensitive as well as resistant luminal A breast cancer cell lines.

  19. New 2',6'-dimethyl-L-tyrosine (Dmt) opioid peptidomimetics based on the Aba-Gly scaffold. Development of unique mu-opioid receptor ligands. (United States)

    Ballet, Steven; Salvadori, Severo; Trapella, Claudio; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H; Negri, Lucia; Giannini, Elisa; Lattanzi, Roberta; Tourwé, Dirk; Balboni, Gianfranco


    The Aba-Gly scaffold, incorporated into Dmt-Tic ligands (H-Dmt-Tic-Gly-NH-CH2-Ph, H-Dmt-Tic-Gly-NH-Ph, H-Dmt-Tic-NH-CH2-Bid), exhibited mixed micro/delta or delta opioid receptor activities with micro agonism. Substitution of Tic by Aba-Gly coupled to -NH-CH2-Ph (1), -NH-Ph (2), or -Bid (Bid=1H-benzimidazole-2-yl) (3) shifted affinity (Ki(micro)=0.46, 1.48, and 19.9 nM, respectively), selectivity, and bioactivity to micro-opioid receptors. These compounds represent templates for a new class of lead opioid agonists that are easily synthesized and suitable for therapeutic pain relief.

  20. ABL Tyrosine Kinase Stimulates PUMA Protein Expression


    Oon, Chet K


    ABL is an ubiquitously expressed non-receptor tyrosine kinase involved in multiple cellular functions including programmed cell death. Upon DNA damage, ABL has been shown to upregulate PUMA, p53 upregulated modulator of apoptosis, and causes downstream mitochondrial intrinsic apoptotic events. However, the mechanism by which ABL regulates PUMA expression remains unknown. We have shown that ABL does not change PUMA protein subcellular localization through immunofluorescence. Through protein an...

  1. Tyrosine phosphorylation of NEDD4 activates its ubiquitin ligase activity. (United States)

    Persaud, Avinash; Alberts, Philipp; Mari, Sara; Tong, Jiefei; Murchie, Ryan; Maspero, Elena; Safi, Frozan; Moran, Michael F; Polo, Simona; Rotin, Daniela


    Ligand binding to the receptor tyrosine kinase fibroblast growth factor (FGF) receptor 1 (FGFR1) causes dimerization and activation by transphosphorylation of tyrosine residues in the kinase domain. FGFR1 is ubiquitylated by the E3 ligase NEDD4 (also known as NEDD4-1), which promotes FGFR1 internalization and degradation. Although phosphorylation of FGFR1 is required for NEDD4-dependent endocytosis, NEDD4 directly binds to a nonphosphorylated region of FGFR1. We found that activation of FGFR1 led to activation of c-Src kinase-dependent tyrosine phosphorylation of NEDD4, enhancing the ubiquitin ligase activity of NEDD4. Using mass spectrometry, we identified several FGF-dependent phosphorylated tyrosines in NEDD4, including Tyr(43) in the C2 domain and Tyr(585) in the HECT domain. Mutating these tyrosines to phenylalanine to prevent phosphorylation inhibited FGF-dependent NEDD4 activity and FGFR1 endocytosis and enhanced cell proliferation. Mutating the tyrosines to glutamic acid to mimic phosphorylation enhanced NEDD4 activity. Moreover, the NEDD4 C2 domain bound the HECT domain, and the presence of phosphomimetic mutations inhibited this interaction, suggesting that phosphorylation of NEDD4 relieves an inhibitory intra- or intermolecular interaction. Accordingly, activation of FGFR1 was not required for activation of NEDD4 that lacked its C2 domain. Activation of c-Src by epidermal growth factor (EGF) also promoted tyrosine phosphorylation and enhanced the activity of NEDD4. Thus, we identified a feedback mechanism by which receptor tyrosine kinases promote catalytic activation of NEDD4 and that may represent a mechanism of receptor crosstalk.

  2. Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in eukaryotic histone H3 (United States)

    BirA ligase, a prokaryotic ortholog of human holocarboxylase synthetase (HCS), is known to biotinylate proteins. Here, we tested the hypothesis that BirA ligase may also catalyze biotinylation of eukaryotic histones. If so, this would render recombinant BirA ligase a useful surrogate for HCS in stud...

  3. Effects of the activated mitogen-activated protein kinase pathway via the c-ros receptor tyrosine kinase on the T47D breast cancer cell line following alcohol exposure. (United States)

    Lee, Hyung Tae; Kim, Se Kye; Choi, Mi Ran; Park, Ji Hyun; Jung, Kyoung Hwa; Chai, Young Gyu


    Compared to other cancers affecting women, breast cancer is significantly associated with alcohol consumption. However, the principles underlying the carcinogenesis of alcohol-induced breast cancer and the related metastatic mechanisms have yet to be established. To observe the effect of alcohol on the growth regulation in breast cancer cells, we identified differentially expressed proteins in alcohol-exposed human breast cancer T47D cells using gel-based proteomics analysis. The expression of c-ros receptor tyrosine kinase (ROS1) was increased and activated by autophosphorylation, thereby activating mitogen- and stress-activated protein kinase 1 (MSK1) through the mitogen‑activated protein kinase (MAPK) pathway; activated MSK1, in turn, phosphorylated histone 3 serine 10 (H3S10p) residues in the nucleus. The increase in H3S10 phosphorylation consequently increased the level of expression of immediate-early gene such as c-fos. This study demonstrated that when breast cancer cells are exposed to alcohol, phosphorylated ROS1 activates MSK1 via Erk1/2 in the MAPK pathway, which then induces modifications to histone residues that regulate gene expression by 14-3-3 protein recruitment, leading to a lack of control of breast cancer cell proliferation.

  4. Targeted exome sequencing for the identification of a protective variant against Internet gaming disorder at rs2229910 of neurotrophic tyrosine kinase receptor, type 3 (NTRK3): A pilot study. (United States)

    Kim, Jeong-Yu; Jeong, Jo-Eun; Rhee, Je-Keun; Cho, Hyun; Chun, Ji-Won; Kim, Tae-Min; Choi, Sam-Wook; Choi, Jung-Seok; Kim, Dai-Jin


    Background and aims Internet gaming disorder (IGD) has gained recognition as a potential new diagnosis in the fifth revision of the Diagnostic and Statistical Manual of Mental Disorders, but genetic evidence supporting this disorder remains scarce. Methods In this study, targeted exome sequencing was conducted in 30 IGD patients and 30 control subjects with a focus on genes linked to various neurotransmitters associated with substance and non-substance addictions, depression, and attention deficit hyperactivity disorder. Results rs2229910 of neurotrophic tyrosine kinase receptor, type 3 (NTRK3) was the only single nucleotide polymorphism (SNP) that exhibited a significantly different minor allele frequency in IGD subjects compared to controls (p = .01932), suggesting that this SNP has a protective effect against IGD (odds ratio = 0.1541). The presence of this potentially protective allele was also associated with less time spent on Internet gaming and lower scores on the Young's Internet Addiction Test and Korean Internet Addiction Proneness Scale for Adults. Conclusions The results of this first targeted exome sequencing study of IGD subjects indicate that rs2229910 of NTRK3 is a genetic variant that is significantly related to IGD. These findings may have significant implications for future research investigating the genetics of IGD and other behavioral addictions.

  5. Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi. (United States)

    Aizen, Joseph; Chandler, Jennifer C; Fitzgibbon, Quinn P; Sagi, Amir; Battaglene, Stephen C; Elizur, Abigail; Ventura, Tomer


    In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans.

  6. The transcriptional response of neurotrophins and their tyrosine kinase receptors in lumbar sensorimotor circuits to spinal cord contusion is affected by injury severity and survival time.

    Directory of Open Access Journals (Sweden)

    M Tyler Hougland


    Full Text Available Traumatic spinal cord injury (SCI results in changes to the anatomical, neurochemical, and physiological properties of cells in the central and peripheral nervous system. Neurotrophins, acting by binding to their cognate Trk receptors on target cell membranes, contribute to modulation of anatomical, neurochemical, and physiological properties of neurons in sensorimotor circuits in both the intact and injured spinal cord. Neurotrophin signaling is associated with many post-SCI changes including maladaptive plasticity leading to pain and autonomic dysreflexia, but also therapeutic approaches such as training-induced locomotor improvement. Here we characterize expression of mRNA for neurotrophins and Trk receptors in lumbar dorsal root ganglia (DRG and spinal cord after two different severities of mid-thoracic injury and at 6 and 12 weeks post-SCI. There was complex regulation that differed with tissue, injury severity, and survival time, including reversals of regulation between 6 and 12 weeks, and the data suggest that natural regulation of neurotrophins in the spinal cord may continue for months after birth. Our assessments determined that a coordination of gene expression emerged at the 12 week post-SCI time point and bioinformatic analyses address possible mechanisms. These data can inform studies meant to determine the role of the neurotrophin signaling system in post-SCI function and plasticity, and studies using this signaling system as a therapeutic approach.

  7. Biotinylated chitosan-based SPIONs with potential in blood-contacting applications

    Energy Technology Data Exchange (ETDEWEB)

    Balan, Vera [Technical University ' Gh.Asachi' , Faculty of Chemical Engineering and Environmental Protection (Romania); Petrache, Ivona Andreea [' Gr.T.Popa' University of Medicine and Pharmacy, Department of Biomedical Sciences, Faculty of Medical Bioengineering (Romania); Popa, Marcel Ionel [Technical University ' Gh.Asachi' , Faculty of Chemical Engineering and Environmental Protection (Romania); Butnaru, Maria [' Gr.T.Popa' University of Medicine and Pharmacy, Department of Biomedical Sciences, Faculty of Medical Bioengineering (Romania); Barbu, Eugen; Tsibouklis, John [University of Portsmouth, School of Pharmacy and Biomedical Sciences (United Kingdom); Verestiuc, Liliana, E-mail: [' Gr.T.Popa' University of Medicine and Pharmacy, Department of Biomedical Sciences, Faculty of Medical Bioengineering (Romania)


    Haemocompatible biotinylated superparamagnetic nanoparticles (size range 300-700 nm) have been obtained by coating magnetite through ionic gelation with a mixture of chitosan and sodium tripolyphosphate, followed by subsequent functionalisation with biotin. The evaluations of their magnetic properties together with haemocompatibility tests have shown that these nanoparticles exhibit the prerequisite behaviour for use in magnetic field-assisted separations within biological systems.

  8. Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Santos P.R.P.


    Full Text Available Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 µg/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite


    NARCIS (Netherlands)



    Objective: To develop a method to detect acrosome-reacted spermatozoa on human zonae pellucidae using only commercially available reagents and without need for sperm fixation. Design: Sperm head labeling with biotinylated soybean trypsin inhibitor (SBTI-biotin) was compared with results of a known m

  10. Magnetic molecularly imprinted polymer for the isolation and detection of biotin and biotinylated biomolecules. (United States)

    Ben Aissa, A; Herrera-Chacon, A; Pupin, R R; Sotomayor, M D P T; Pividori, M I


    Magnetic separation based on biologically-modified magnetic particles is a preconcentration procedure commonly integrated in magneto actuated platforms for the detection of a huge range of targets. However, the main drawback of this material is the low stability and high cost. In this work, a novel hybrid molecularly-imprinted polymer with magnetic properties is presented with affinity towards biotin and biotinylated biomolecules. During the synthesis of the magneto core-shell particles, biotin was used as a template. The characterization of this material by microscopy techniques including SEM, TEM and confocal microscopy is presented. The application of the magnetic-MIPs for the detection of biotin and biotinylated DNA in magneto-actuated platforms is also described for the first time. The magnetic-MIP showed a significant immobilization capacity of biotinylated molecules, giving rise to a cheaper and a robust method (it is not required to be stored at 4°C) with high binding capacity for the separation and purification under magnetic actuation of a wide range of biotinylated molecules, and their downstream application including determination of their specific targets.

  11. Characterisation of adiponectin multimers and the IGF axis in humans with a heterozygote mutation in the tyrosine kinase domain of the insulin receptor gene

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning; Flyvbjerg, Allan;


    Objective: Low levels of adiponectin, IGF-binding protein (IGFBP)-1, and IGFBP-2, and high levels of leptin correlate with several indices of insulin resistance and risk of type 2 diabetes. However, in insulin receptoropathies plasma adiponectin is paradoxically increased despite severe insulin r...... in insulin receptor deficient individuals.......Objective: Low levels of adiponectin, IGF-binding protein (IGFBP)-1, and IGFBP-2, and high levels of leptin correlate with several indices of insulin resistance and risk of type 2 diabetes. However, in insulin receptoropathies plasma adiponectin is paradoxically increased despite severe insulin...... resistance, whereas the IGF-axis is sparsely described. Here, we aimed to characterize the multimeric distribution of adiponectin and the IGF-axis in humans with a heterozygous INSR mutation (Arg1174Gln).Methods: Blood samples obtained in six Arg1174Gln-carriers and 10 lean, healthy controls before and after...

  12. Protein tyrosine phosphorylation in streptomycetes. (United States)

    Waters, B; Vujaklija, D; Gold, M R; Davies, J


    Using phosphotyrosine-specific antibodies, we demonstrate that in several Streptomyces spp. a variety of proteins are phosphorylated on tyrosine residues. Tyrosine phosphorylation was found in a number of Streptomyces species including Streptomyces lividans, Streptomyces hygroscopicus and Streptomyces lavendulae. Each species exhibited a unique pattern of protein tyrosine phosphorylation. Moreover, the patterns of tyrosine phosphorylation varied during the growth phase and were also influenced by culture conditions. We suggest that metabolic shifts during the complex growth cycle of these filamentous bacteria, and possibly secondary metabolic pathways, may be controlled by the action of protein tyrosine kinases and phosphatases, as has been demonstrated in signal transduction pathways in eukaryotic organisms.

  13. Tyrosine Kinase Inhibitors and Diabetes: A Novel Treatment Paradigm? (United States)

    Fountas, Athanasios; Diamantopoulos, Leonidas-Nikolaos; Tsatsoulis, Agathocles


    Deregulation of protein tyrosine kinase (PTK) activity is implicated in various proliferative conditions. Multi-target tyrosine kinase inhibitors (TKIs) are increasingly used for the treatment of different malignancies. Recently, several clinical cases of the reversal of both type 1 and 2 diabetes mellitus (T1DM, T2DM) during TKI administration have been reported. Experimental in vivo and in vitro studies have elucidated some of the mechanisms behind this effect. For example, inhibition of Abelson tyrosine kinase (c-Abl) results in β cell survival and enhanced insulin secretion, while platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) inhibition leads to improvement in insulin sensitivity. In addition, inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) reduces the degree of islet cell inflammation (insulitis). Therefore, targeting several PTKs may provide a novel approach for correcting the pathophysiologic disturbances of diabetes.

  14. Novel CoQ10 antidiabetic mechanisms underlie its positive effect: modulation of insulin and adiponectine receptors, Tyrosine kinase, PI3K, glucose transporters, sRAGE and visfatin in insulin resistant/diabetic rats.

    Directory of Open Access Journals (Sweden)

    Mohamed M Amin

    Full Text Available As a nutritional supplement, coenzyme Q10 (CoQ10 was tested previously in several models of diabetes and/or insulin resistance (IR; however, its exact mechanisms have not been profoundly explicated. Hence, the objective of this work is to verify some of the possible mechanisms that underlie its therapeutic efficacy. Moreover, the study aimed to assess the potential modulatory effect of CoQ10 on the antidiabetic action of glimebiride. An insulin resistance/type 2 diabetic model was adopted, in which rats were fed high fat/high fructose diet (HFFD for 6 weeks followed by a single sub-diabetogenic dose of streptozotocin (35 mg/kg, i.p.. At the end of the 7(th week animals were treated with CoQ10 (20 mg/kg, p.o and/or glimebiride (0.5 mg/kg, p.o for 2 weeks. CoQ10 alone opposed the HFFD effect and increased the hepatic/muscular content/activity of tyrosine kinase (TK, phosphatidylinositol kinase (PI3K, and adiponectin receptors. Conversely, it decreased the content/activity of insulin receptor isoforms, myeloperoxidase and glucose transporters (GLUT4; 2. Besides, it lowered significantly the serum levels of glucose, insulin, fructosamine and HOMA index, improved the serum lipid panel and elevated the levels of glutathione, sRAGE and adiponectin. On the other hand, CoQ10 lowered the serum levels of malondialdehyde, visfatin, ALT and AST. Surprisingly, CoQ10 effect surpassed that of glimepiride in almost all the assessed parameters, except for glucose, fructosamine, TK, PI3K, and GLUT4. Combining CoQ10 with glimepiride enhanced the effect of the latter on the aforementioned parameters.These results provided a new insight into the possible mechanisms by which CoQ10 improves insulin sensitivity and adjusts type 2 diabetic disorder. These mechanisms involve modulation of insulin and adiponectin receptors, as well as TK, PI3K, glucose transporters, besides improving lipid profile, redox system, sRAGE, and adipocytokines. The study also points to the

  15. Effect of FK506 and cyclosporine A on the expression of BDNF, tyrosine kinase B and p75 neurotrophin receptors in astrocytes exposed to simulated ischemia in vitro. (United States)

    Gabryel, Bozena; Bernacki, Jacek


    We investigated whether the immunosuppressive drugs, FK506 and cyclosporine A, increase BDNF protein and/or mRNA expression in ischemic astrocytes and if an increase could be related to changes in the nuclear expression of p-CREB, p-Erk1/2 and p-Akt. The influence of these immunosuppressants on protein and mRNA levels of TrkB and p75(NTR) receptors was also examined. On day 21, cultures of rat astrocytes were subjected to ischemic conditions simulated in vitro (combined oxygen glucose deprivation, OGD) for 8h and exposed to FK506 (10-1000nM) and cyclosporine A (0.25-10microM). FK506 and cyclosporine A (at 1000nM and 0.25microM, respectively) stimulated the expression and release of BDNF in cultured rat cerebral cortical astrocytes exposed to OGD. The immunosuppressants at these doses simultaneously increased p-CREB and p-Erk1/2 expression in the nuclear fraction of astrocytes. The results RT-PCR and Western blot analysis provided further evidence of a modulating influence of the drugs on the expression of trkB and p75(NTR) genes and their protein products in ischemic astrocytes.

  16. Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

    DEFF Research Database (Denmark)

    Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno;


    The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 ...

  17. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice (United States)

    de Boer, Ernie; Rodriguez, Patrick; Bonte, Edgar; Krijgsveld, Jeroen; Katsantoni, Eleni; Heck, Albert; Grosveld, Frank; Strouboulis, John


    Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

  18. In vivo biotinylation of recombinant beta-glucosidase enables simultaneous purification and immobilization on streptavidin coated magnetic particles

    DEFF Research Database (Denmark)

    Alftrén, Johan; Ottow, Kim Ekelund; Hobley, Timothy John


    Beta-glucosidase from Bacillus licheniformis was in vivo biotinylated in Escherichia coli and subsequently immobilized directly from cell lysate on streptavidin coated magnetic particles. In vivo biotinylation was mediated by fusing the Biotin Acceptor Peptide to the C-terminal of beta......-glucosidase and co-expressing the BirA biotin ligase. The approach enabled simultaneous purification and immobilization of the enzyme from crude cell lysate on magnetic particles because of the high affinity and strong interaction between biotin and streptavidin. After immobilization of the biotinylated beta...

  19. Efficacy of Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors for Advanced Squamous Cell Lung Carcinoma Patients with Sensitive EGFR Mutations%EGFR-TKI治疗EGFR敏感突变的晚期肺鳞癌的疗效分析

    Institute of Scientific and Technical Information of China (English)

    刘咏梅; 赵倩; 唐源; 张衍; 李艳莹; 王永生; 卢铀


    目的 探讨表皮生长因子酪氨酸激酶抑制剂(Epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)治疗EGFR敏感突变的晚期肺鳞癌患者的疗效.方法 收集20例四川大学华西医院经病理确诊、EGFR检测敏感突变、并接受EGFR-TKI治疗的Ⅳ期或术后复发转移肺鳞癌患者,分析其与EGFR-TKI的疗效关系.结果 20例EGFR敏感突变的晚期鳞癌患者接受EGFR-TKI治疗,随访资料完整.10例19-del(+),8例L858R(+),1例同时存在外显子21(L858R)点突变和外显子20(T790M)突变,1例外显子18(G719X)突变.其中部分缓解(PR)9例,疾病稳定(SD)7例,疾病进展(PD)4例.客观缓解率(ORR) 45%,疾病控制率80%,中位无进展生存期(mPFS)为5.0月,中位生存期(mOS)为14.7月.结论 EGFR-TKI对部分EGFR敏感突变的鳞癌患者有一定疗效.在临床工作中,应重视这部分患者的EGFR基因检测,以便明确获益的患者.

  20. Postnatal development of brain-derived neurotrophic factor (BDNF) and tyrosine protein kinase B (TrkB) receptor immunoreactivity in multiple brain stem respiratory-related nuclei of the rat. (United States)

    Liu, Qiuli; Wong-Riley, Margaret T T


    Previously, we found a transient imbalance between suppressed excitation and enhanced inhibition in the respiratory network of the rat around postnatal days (P) 12-13, a critical period when the hypoxic ventilatory response is at its weakest. The mechanism underlying the imbalance is poorly understood. Brain-derived neurotrophic factor (BDNF) and its tyrosine protein kinase B (TrkB) receptors are known to potentiate glutamatergic and attenuate gamma-aminobutyric acid (GABA)ergic neurotransmission, and BDNF is essential for respiratory development. We hypothesized that the excitation-inhibition imbalance during the critical period stemmed from a reduced expression of BDNF and TrkB at that time within respiratory-related nuclei of the brain stem. An in-depth, semiquantitative immunohistochemical study was undertaken in seven respiratory-related brain stem nuclei and one nonrespiratory nucleus in P0-21 rats. The results indicate that the expressions of BDNF and TrkB: 1) in the pre-Bötzinger complex, nucleus ambiguus, commissural and ventrolateral subnuclei of solitary tract nucleus, and retrotrapezoid nucleus/parafacial respiratory group were significantly reduced at P12, but returned to P11 levels by P14; 2) in the lateral paragigantocellular nucleus and parapyramidal region were increased from P0 to P7, but were strikingly reduced at P10 and plateaued thereafter; and 3) in the nonrespiratory cuneate nucleus showed a gentle plateau throughout the first 3 postnatal weeks, with only a slight decline of BDNF expression after P11. Thus, the significant downregulation of both BDNF and TrkB in respiratory-related nuclei during the critical period may form the basis of, or at least contribute to, the inhibitory-excitatory imbalance within the respiratory network during this time.

  1. Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin transactivates aryl hydrocarbon receptor-responsive element III in the tyrosine hydroxylase immunoreactive neurons of the mouse midbrain. (United States)

    Tanida, Takashi; Tasaka, Ken; Akahoshi, Eiichi; Ishihara-Sugano, Mitsuko; Saito, Michiko; Kawata, Shigehisa; Danjo, Megumi; Tokumoto, Junko; Mantani, Youhei; Nagahara, Daichi; Tabuchi, Yoshiaki; Yokoyama, Toshifumi; Kitagawa, Hiroshi; Kawata, Mitsuhiro; Hoshi, Nobuhiko


    Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000 ng kg⁻¹ body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg⁻¹ TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD).

  2. Roles of the tyrosine isomers meta-tyrosine and ortho-tyrosine in oxidative stress. (United States)

    Ipson, Brett R; Fisher, Alfred L


    The damage to cellular components by reactive oxygen species, termed oxidative stress, both increases with age and likely contributes to age-related diseases including Alzheimer's disease, atherosclerosis, diabetes, and cataract formation. In the setting of oxidative stress, hydroxyl radicals can oxidize the benzyl ring of the amino acid phenylalanine, which then produces the abnormal tyrosine isomers meta-tyrosine or ortho-tyrosine. While elevations in m-tyrosine and o-tyrosine concentrations have been used as a biological marker of oxidative stress, there is emerging evidence from bacterial, plant, and mammalian studies demonstrating that these isomers, particularly m-tyrosine, directly produce adverse effects to cells and tissues. These new findings suggest that the abnormal tyrosine isomers could in fact represent mediators of the effects of oxidative stress. Consequently the accumulation of m- and o-tyrosine may disrupt cellular homeostasis and contribute to disease pathogenesis, and as result, effective defenses against oxidative stress can encompass not only the elimination of reactive oxygen species but also the metabolism and ultimately the removal of the abnormal tyrosine isomers from the cellular amino acid pool. Future research in this area is needed to clarify the biologic mechanisms by which the tyrosine isomers damage cells and disrupt the function of tissues and organs and to identify the metabolic pathways involved in removing the accumulated isomers after exposure to oxidative stress.

  3. Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing. (United States)

    Chen, Zhiyao; Liu, Yunlong; Duan, Wenbang; Ye, Hui; Wu, Haiping; Li, Jinheng; Zhou, Guohua


    Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.

  4. Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

    NARCIS (Netherlands)

    S. Middendorp; G.M. Dingjan (Gemma); A. Maas (Alex); K. Dahlenborg; R.W. Hendriks (Rudi)


    textabstractThe Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, an

  5. Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of small molecule tyrosine kinase inhibitors and cytotoxic drugs (United States)

    Puvanenthiran, Soozana; Essapen, Sharadah; Seddon, Alan M.; Modjtahedi, Helmout


    Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 μM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2

  6. Biofunctionalization of polyelectrolyte microcapsules with biotinylated polyethylene glycol-grafted liposomes. (United States)

    Gao, Jie; Reibetanz, Uta; Venkatraman, Subbu; Neu, Björn


    Hollow polyelectrolyte microcapsules (PEMC) are prepared using layer-by-layer self-assembly of polyelectrolytes on melamine formaldehyde templates, followed by template dissolution, and subsequent coating with biotinylated polyethylene glycol-grafted liposomes. These potential site-specific carrier systems show a high specificity for NeutrAvidin binding and a strong resistance against unspecific protein binding. It is concluded that this design with NeutrAvidin as the outermost layer of such capsules provides an ideal platform for the biofunctionalization of PEMC as drug delivery systems or as artificial cell-like structures for biomimetic studies.

  7. A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations. (United States)

    Hörmann, Katrin; Stukalov, Alexey; Müller, André C; Heinz, Leonhard X; Superti-Furga, Giulio; Colinge, Jacques; Bennett, Keiryn L


    Plasma membrane (PM) proteins contribute to the identity of a cell, mediate contact and communication, and account for more than two-thirds of known drug targets.1-8 In the past years, several protocols for the proteomic profiling of PM proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach.9-11 We compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation, and surface coating with silica beads to isolate PM proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of PM proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by a competitive biotin elution strategy achieving an average PM annotated protein fraction of 54% (347 proteins). Computational analysis using additional databases and prediction tools revealed that in total over 90% of the purified proteins were associated with the PM, mostly as interactors. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. Glycosylphosphatidylinositol (GPI)-anchored proteins were depleted in PM purifications from cells deficient in the GPI transamidase component PIGS, and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications.

  8. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. (United States)

    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya


    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

  9. Tyrosine Sulfation of Statherin

    Directory of Open Access Journals (Sweden)

    C. Kasinathan, N. Gandhi, P. Ramaprasad, P. Sundaram, N. Ramasubbu


    Full Text Available Tyrosylprotein sulfotransferase (TPST, responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96. In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl2. Increase in the level of total sulfation was observed with increasing statherin concentration. The Km value of tyrosylprotein sulfotransferase for statherin was 40 μM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed 35S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.

  10. Interferon signaling is dependent on specific tyrosines located within the intracellular domain of IFNAR2c. Expression of IFNAR2c tyrosine mutants in U5A cells. (United States)

    Wagner, T Charis; Velichko, Sharlene; Vogel, David; Rani, M R Sandhya; Leung, Stewart; Ransohoff, Richard M; Stark, George R; Perez, H Daniel; Croze, Ed


    Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

  11. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko


    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  12. Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation. (United States)

    Kikawa, Keith D; Vidale, Derika R; Van Etten, Robert L; Kinch, Michael S


    Intracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2.

  13. Protein-tyrosine phosphatases in zebrafish gastrulation

    NARCIS (Netherlands)

    van Eekelen, M.J.L.


    Protein tyrosine phosphorylation plays a key role in relaying external stimuli and signals into the cell towards the appropriate responses. This process is mediated by protein-tyrosine kinases adding a phosphor group to a tyrosine residue and protein-tyrosine phosphatases removing a phosphor group f

  14. Rapid plasmid library screening using RecA-coated biotinylated probes. (United States)

    Rigas, B; Welcher, A A; Ward, D C; Weissman, S M


    A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.

  15. Area density of streptavidin can be evaluated by the number density of biotinylated microbubbles (United States)

    Yokoi, Yasuhiro; Yoshida, Kenji; Otsuki, Yuta; Watanabe, Yoshiaki


    Targeted microbubbles (TMBs) that specifically accumulate on target sites via biochemical bonds have been studied for using ultrasound diagnoses and therapies (e.g., ultrasound molecular imaging) in the research field. To understand the specific interactions between TMBs and their target molecules, a biosensor system with a quartz crystal microbalance (QCM) was constructed. In this system, TMBs become absorbed on their target molecule, which was fixed to the QCM surface via a self-assembled monolayer. Our previous studies showed that the system allowed the evaluation of the interaction between biotinylated MBs and the target molecule, streptavidin, by monitoring changes in the resonant frequency of QCM [Muramoto et al., Ultrasound Med. Biol., 40(5), 1027-1033 (2014)]. This paper investigates how the amount of streptavidin relates to the amount of absorbed biotinylated MBs. The amount of streptavidin on the QCM surface was evaluated by measuring the difference in its resonant frequency before and after the fixation of streptavidin. After which, the amount of absorbed MBs was also evaluated by measuring the frequency shift during the interaction between MBs and the target molecule. Our results showed a weak correlation between the amounts of bound MB and the density of streptavidin (correlation coefficient, r = 0.44), suggesting that the area density of target molecule can be evaluated by estimating the number density of TMBs.

  16. Detection of HCV-RNA by Reverse Transcription Polymerase Chain Reaction Using Biotinylated and Radioiodinated Primers

    Energy Technology Data Exchange (ETDEWEB)

    Ryu, Jin Sook; Moon, Dae Hyuk; Cheon, Jun Hong; Chung, Yoon Young; Park, Hung Dong; Chung, Young Hwa; Lee, Young Sang [Asan Medical Center, University of Ulsan, Seoul (Korea, Republic of)


    This study was performed to evaluate the clinical applicability of the reverse transcription polymerase chain reaction (RT-PCR) kit of HCV-RNA using biotinylated and radioiodinated primers. Study subjects were 118 patients with positive anti-HCV. HCV-RNA in patients serum was extracted by guanidium thiocyanate method. After first amplification, the product was reamplified by primers labelled with biotin and I-125. The final amplification product was detected by counting the radioactivity after incubation in avidin coated tubes. In 51 samples, the test was repeated for evaluation of reproducibility. This new method was also compared with conventional RT-PCR methods in 34 samples from patients with chronic liver disease. The results were as follows, 1) HCV-RNA was positive in 85(97%)of 88 patients with chronic liver disease, and in 23 (73%) of 30 patients with normal liver function. 2) In comparison with conventional method, HCV-RNA was detected in 32(94%) of 34 patients with new method, whereas in 27(79% ) of the same group with conventional method 3) Repeated test with new method in 52 samples demonstrated 82% of concordant result. In conclusion, new method with biotinylated and radioiodinated primers was more sensitive than conventional method. However, great care must be taken for quality control because there were considerable interassay variation and possibility of false positivity and false negativity.

  17. TrkB和BDNF在胃癌组织中的表达及意义%Expression and significance of tyrosine kinase receptor B and brain-derived neurotrophic factor in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    刘天卿; 任海军; 温爽; 张众


    目的 观察酪氨酸激酶受体B(TrkB)及其配体脑源性神经营养因子(BDNF)在胃癌组织、癌旁组织和正常胃黏膜组织中的表达情况,探讨TrkB和BDNF与胃癌临床病理参数的关系.方法 采用免疫组织化学sP法检测64例原发性胃癌组织、癌旁组织,34例有淋巴结转移患者中对应的阳性淋巴结和20例正常胃黏膜组织中TrkB和BDNF的表达,分析其与临床病理特征的关系.结果 胃癌组织中TrkB和BDNF阳性表达率分别为60.9%、59.4%,而癌旁组织和正常胃黏膜组织中无一例表达.TrkB和BDNF表达与患者性别、年龄、肿瘤分化程度等无关,而与浸润深度、淋巴结转移和TNM分期有关.浸润至胃壁全层、有淋巴结转移和TNM分期Ⅲ~Ⅳ期的TrkB和BDNF阳性表达率明显高于未浸润至胃壁全层、无淋巴结转移和TNM分期Ⅰ~Ⅱ期(P0.05).结论 TrkB和BDNF表达与胃癌发生、发展密切相关,二者的表达增强提示肿瘤可能已发生局部侵袭和远处转移.%Objective To investigate the expressions of tyrosine kinase receptor (Trk)B and brain-derived neurotrophic factor (BDNF) protein in human gastric careinoma and compare them with those in epithelial cells of normal mucous, in order to evaluate their clinicopathological significance. Method The expressions of TrkB and BDNF protein in tumor tissues, matched with para-tumor mueosal tissues from 64 cases with gastric carcinoma and normal mucous of 20 cases were observed immunohistochemically and related to some of the clinicopathological parameters. Results The positive rates of TrkB and BDNF protein in tumor tissues were 60.9% and 59.4% respectively, but there was negative in matched para-tumor mueosal tissues and normal mucosal tissues. TrkB and BDNF protein expressions were related to invasive depth,lymph node metastasis and TNM stage of cancer, but not to sex, age and degrees of cancerous differentiation.The positive rates of TrkB and BDNF protein in cases with

  18. Schwann cells express erythropoietin receptor and represent a major target for Epo in peripheral nerve injury. (United States)

    Li, Xiaoqing; Gonias, Steven L; Campana, W Marie


    Erythropoietin (Epo) expresses potent neuroprotective activity in the peripheral nervous system; however, the underlying mechanism remains incompletely understood. In this study, we demonstrate that Epo is upregulated in sciatic nerve after chronic constriction injury (CCI) and crush injury in rats, largely due to local Schwann cell production. In uninjured and injured nerves, Schwann cells also express Epo receptor (EpoR), and its expression is increased during Wallerian degeneration. CCI increased the number of Schwann cells at the injury site and the number was further increased by exogenously administered recombinant human Epo (rhEpo). To explore the activity of Epo in Schwann cells, primary cultures were established. These cells expressed cell-surface Epo receptors, with masses of 71 and 62 kDa, as determined by surface protein biotinylation and affinity precipitation. The 71-kDa species was rapidly but transiently tyrosine-phosphorylated in response to rhEpo. ERK/MAP kinase was also activated in rhEpo-treated Schwann cells; this response was blocked by pharmacologic antagonism of JAK-2. RhEpo promoted Schwann cell proliferation, as determined by BrdU incorporation. Cell proliferation was ERK/MAP kinase-dependent. These results support a model in which Schwann cells are a major target for Epo in injured peripheral nerves, perhaps within the context of an autocrine signaling pathway. EpoR-induced cell signaling and Schwann cell proliferation may protect injured peripheral nerves and promote regeneration.

  19. Synthesis and evaluation of cell-permeable biotinylated PU-H71 derivatives as tumor Hsp90 probes

    Directory of Open Access Journals (Sweden)

    Tony Taldone


    Full Text Available The attachment of biotin to a small molecule provides a powerful tool in biology. Here, we present a systematic approach to identify biotinylated analogues of the Hsp90 inhibitor PU-H71 that are capable of permeating cell membranes so as to enable the investigation of Hsp90 complexes in live cells. The identified derivative 2g can isolate Hsp90 through affinity purification and, as we show, represents a unique and useful tool to probe tumor Hsp90 biology in live cells by affinity capture, flow cytometry and confocal microscopy. To our knowledge, 2g is the only reported biotinylated Hsp90 probe to have such combined characteristics.

  20. Synthesis and evaluation of cell-permeable biotinylated PU-H71 derivatives as tumor Hsp90 probes (United States)

    DaGama Gomes, Erica M; Riolo, Matthew; Patel, Hardik J; Alonso-Sabadell, Raul; Zatorska, Danuta; Patel, Maulik R; Kishinevsky, Sarah


    Summary The attachment of biotin to a small molecule provides a powerful tool in biology. Here, we present a systematic approach to identify biotinylated analogues of the Hsp90 inhibitor PU-H71 that are capable of permeating cell membranes so as to enable the investigation of Hsp90 complexes in live cells. The identified derivative 2g can isolate Hsp90 through affinity purification and, as we show, represents a unique and useful tool to probe tumor Hsp90 biology in live cells by affinity capture, flow cytometry and confocal microscopy. To our knowledge, 2g is the only reported biotinylated Hsp90 probe to have such combined characteristics. PMID:23616796

  1. Current understanding of tyrosine kinase BMX in inflammation and its inhibitors

    Directory of Open Access Journals (Sweden)

    Le Qiu


    Full Text Available Tec family kinases, which include tyrosine kinase expressed in hepatocellular carcinoma (TEC, Bruton's tyrosine kinase (BTK, interleukin (IL-2-inducible T-cell kinase (ITK, tyrosine-protein kinase (TXK, and bone marrow tyrosine kinase on chromosome X (BMX, are the second largest group of non-receptor tyrosine kinases and have a highly conserved carboxyl-terminal kinase domain. BMX was identified in human bone marrow cells, and was demonstrated to have been expressed in myeloid hematopoietic lineages cells, endothelial cells, and several types of cancers. Significant progress in this area during the last decade revealed an important role for BMX in inflammation and oncologic disorders. This review focuses on BMX biology, its role in inflammation and possible signaling pathways, and the potential of selective BMX inhibitors.

  2. Current understanding of tyrosine kinase BMX in inflammation and its inhibitors. (United States)

    Qiu, Le; Wang, Fei; Liu, Sheng; Chen, Xu-Lin


    Tec family kinases, which include tyrosine kinase expressed in hepatocellular carcinoma (TEC), Bruton's tyrosine kinase (BTK), interleukin (IL)-2-inducible T-cell kinase (ITK), tyrosine-protein kinase (TXK), and bone marrow tyrosine kinase on chromosome X (BMX), are the second largest group of non-receptor tyrosine kinases and have a highly conserved carboxyl-terminal kinase domain. BMX was identified in human bone marrow cells, and was demonstrated to have been expressed in myeloid hematopoietic lineages cells, endothelial cells, and several types of cancers. Significant progress in this area during the last decade revealed an important role for BMX in inflammation and oncologic disorders. This review focuses on BMX biology, its role in inflammation and possible signaling pathways, and the potential of selective BMX inhibitors.

  3. Expression, isolation, and purification of soluble and insoluble biotinylated proteins for nerve tissue regeneration. (United States)

    McCormick, Aleesha M; Jarmusik, Natalie A; Endrizzi, Elizabeth J; Leipzig, Nic D


    Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing  a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

  4. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection. (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar


    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  5. Carbonic Anhydrase I Is Recognized by an SOD1 Antibody upon Biotinylation of Human Spinal Cord Extracts

    Directory of Open Access Journals (Sweden)

    Robert Bowser


    Full Text Available We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1 antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS. This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to ALS patients. To further characterize this protein, we enriched the protein by column chromatography and determined its protein identity by mass spectrometry. The protein that gave rise to the 32 kDa species upon biotinylation was identified as carbonic anhydrase I (CA I. Biotinylation of CA I from ALS spinal cord resulted in the generation of a novel epitope recognized by the SOD1 antibody. This epitope could also be generated by biotinylation of extracts from cultured cells expressing human CA I. Peptide competition assays identified the amino acid sequence in carbonic anhydrase I responsible for binding the SOD1 antibody. We conclude that chemical modifications used to identify pathogenic protein conformations can lead to the identification of unanticipated proteins that may participate in disease pathogenesis.

  6. 组蛋白的生物素酰化修饰%The biological functions of histone biotinylation

    Institute of Scientific and Technical Information of China (English)

    王燕飞; 鲍宝龙


    Histories are subject to a wide variety of posttranslational modifications. Histone biotinylation, covalent binding of the vitamin biotin to lysine residues in histones, is a novel posttranslational modification. Evidence has been provided that biotinylation of histones plays a role in cell proliferation, gene silencing and cellular response to DNA damage. In this paper, we reviewed biotin metabolism, the mechanism of histone biotinylated by holocarbonxylase synthesis and the physiologic function of histone biotinylation. Finally, several issues relevant to histone biotinylation were discussed.%组蛋白修饰对基因表达的表现遗传学调控起着重要作用.组蛋白生物素酰化修饰是近年来新发现的一种组蛋白修饰,具有重要的生物学功能.有i证据表明组蛋白生物素酰化在细胞增殖、DNA修复、维持基因组稳定等方面发挥作用.组蛋白的生物素酰化修饰是由羧化全酶合成酶与组蛋白直接相互作用的结果.本文主要介绍了组蛋白生物素酰化发现的过程,并对近年来在组蛋白生物素酰化催化机制和组蛋白生物素酰化功能方面的研究进展进行了综述,最后对组蛋白生物素酰化研究领域存在的问题进行了探讨.

  7. Traditional Chinese medicinal herbs combined with epidermal growth factor receptor tyrosine kinase inhibitor for advanced non-small cell lung cancer:a systematic review and meta-analysis

    Institute of Scientific and Technical Information of China (English)

    Zhong-liang Liu; Wei-rong Zhu; Wen-chao Zhou; Hai-feng Ying; Lan Zheng; Yuan-biao Guo; Jing-xian Chen; Xiao-heng Shen


    BACKGROUND: Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) targeted treatment has been a standard therapy for advanced non-small cell lung cancer (NSCLC), but it is not tolerated well by all patients. In China, some studies have reported that traditional Chinese medicinal herbs (TCMHs) may increase efifcacy and reduce toxicity when combined with EGFR-TKI, but outside of China few studies of this kind have been attempted. OBJECTIVE:This study is intended to systematically review the existing clinical evidence on TCMHs combined with EGFR-TKI for treatment of advanced NSCLC. SEARCH STRATEGY:PubMed, the Cochrane Library, the Excerpta Medica Database (EMBASE), the China BioMedical Literature (CBM), and the China National Knowledge Infrastructure (CNKI) and web site of the American Society of Clinical Oncology (ASCO), the European Society for Medical Oncology (ESMO), the World Conference of Lung Cancer (WCLC) were searched; the search included all documents published in English or Chinese before October 2013. INCLUSION CRITERIA:We selected randomized controlled trials based on speciifc criteria, the most important of which was that a TCMH plus EGFR-TKI treatment group was compared with an EGFR-TKI control group in patients with advanced NSCLC. DATA EXTRACTION AND ANALYSIS: The modiifed Jadad scale was used to assess the quality of studies. For each included study, patient characteristics, treatment details, therapeutic approach and clinical outcomes were collected on a standardized form. When disagreements on study inclusion or data extracted from a study emerged, the consensus of all coauthors provided the resolution. The clinical outcome metrics consisted of objective response rate (ORR; complete response + partial response divided by the total number of patients), disease control rate (DCR; complete response + partial response + no change divided by the total number of patients), survival rate, improved or stabilized Karnofsky performance status

  8. N-methyl-N'-nitro- N-nitrosoguanidine suppresses protein tyrosine kinase activity of epidermal growth factor receptor%N-甲基-N'-硝基-N-亚硝胍抑制细胞表皮生长因子受体酪氨酸激酶活性

    Institute of Scientific and Technical Information of China (English)

    王为民; 高永生


    OBJECTIVE: To elucidate the molecular mechanism of low concentration of N-methyl=N'-nitro-N-nitrosoguanidine(MNNG) that interfered with the function of EGFR-mediated cellular signal transduction pathway, by inducing the epidermal growth factor receptor(EGFR)clustering, which was similar to that of epidermal growth factor treatment. METHODS: Eukaryotic expression vector of EGFR cytoplasmic domain gene was constructed and transfected into Lec-1 cells. Then the recombinant EGFR cytoplasmic domain protein was purified, and treated with MNNG for 1 h, at concentrations of 0.25, 0.5, and 1.0 μmol/L. Enzyme linked immunoassay was used to measure the protein tyrosine kinase activity of recombinant EGFR cytoplasmic domain. Untreated recombinant cytoplasmic domain protein tyrosine kinase was used as control. RESULTS: Compared with normal control group, tyrosine kinase activity of EGFR was significantly inhibited by 0.5 μmol/L MNNG (P<0.0l). CONCLUSION: Low concentration of MNNG interfered with the signal transduction pathway of EGFR by supressing its tyrosine kinase activity.%目的:研究低浓度N-甲基-N’-硝基-N-亚硝胍(MNNG)诱发细胞表皮生长因子受体(EGFR)形成的二聚体,干扰了EGFR介导的下游信号转导功能的分子机制.方法:构建EGFR胞内域真核表达的载体,转染Lec-1细胞,纯化EGFR胞内域重组蛋白质后,分别以不同浓度的MNNG(0.25、0.5和1.0μmol/L MNNG)处理EGFR 1 h,应用酶联免疫吸附测定法检测MNNG各浓度对重组EGFR胞内域蛋白质酪氨酸激酶活性的影响.结果:MNNG浓度大于0.5μmol/L即可显著下调EGFR胞内域酪氨酸活性(P<0.01).结论:MNNG通过抑制EGFR胞内域的酪氨酸激酶活性而阻断其对下游信号的传递.

  9. Drug resistance of epidermal growth factor receptor-tyrosine kinase inhibitors in treatment of advanced non-small cell lung cancer——A new dawn in challenge%晚期非小细胞肺癌EGFR-TKIs治疗的耐药机制研究——挑战中蕴含新的曙光

    Institute of Scientific and Technical Information of China (English)

    吴国明; 钱桂生


    Currently, molecularly target therapy has increasingly altered the strategies in advanced non-small cell lung cancer ( NSCLC). Epidermal growth factor receptor tyrosine kinase inhibitors ( EGFR-TKIs) , gefitinib and erlotinib, are regarded as the most successful target drugs. However, EGFR-TKIs resistance has become a major clinical challenge. EGFR-TKIs resistance includes the primary resistance and the acquired resistance. The primary resistance' s mechanisms are associated with other non-sensitive EGFR mutations such as exon 20 insertions and other gene mutations such as KRAS, BRAF and EML4-ALK. The acquired resistance' s mechanisms are often associated with the secondary T790 mutation and MET gene amplification. At present, new strategies in overcoming EGFR-TKIs resistance are mainly focusing on irreversible EGFR inhibitors and MET inhibitors.

  10. 表皮生长因子受体与酪氨酸激酶抑制剂在肿瘤防治中的应用进展%Progress of epidermal growth factor receptor and tyrosine kinase inhibitors in the cancer therapy

    Institute of Scientific and Technical Information of China (English)

    张敏; 张新; 白春学


    表皮生长因子受体(epidermal growth factor receptor,EGFR)信号传递系统可调控细胞周期,调节细胞生长与分化,促进损伤修复.EGFR在包括非小细胞肺癌(non-small-cell lung cancer,NSCLC)在内的多种上皮源性肿瘤中过表达预示存活率低、预后差、转移可能性大,可作为肿瘤基因治疗的靶位.酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)可选择性抑制EGFR酪氨酸激酶活性,抑制肿瘤生长,增加放化疗敏感性.

  11. Tyrosine supplementation in chronic experimental uremia. (United States)

    Abitbol, C L; Mandel, S; Mrozinska, K; Wapnir, R A


    The occurrence of low tyrosine tissue levels in uremic subjects, possibly due to impaired phenylalanine hydroxylation, suggests that tyrosine may be an essential amino acid in uremia. Additional dietary tyrosine may thus re-dress the deficiency. This study examined growth and tyrosine/phenylalanine metabolism in uremic rats during tyrosine supplementation. Rats made uremic (U) by 7/8 nephrectomy were compared to pair-fed (CP) and ad libitum-fed (CA), sham-operated controls. Two sets of each group of rats were studied after 21 days on the respective diets: I = Purina Lab Chow; II = same + 3.5% tyrosine. Plasma tyrosine was below normal in U and CP-fed diet I. With diet II, the tyrosine:phenylalanine ratio in U was lower than both CA and CP. In rats fed diet II, the tyrosine:phenylalanine ratio became indistinguishable among the three groups. Growth parameters in U and CP were similar, regardless of the diet. Body weight gain, tibial length, muscle mass, and tissue protein did not improve in uremic animals supplemented with tyrosine. The specific activity of liver phenylalanine hydroxylase in U was not different from CA or CP. However, loss of cortical renal mass appeared to be the major determinant of decreased kidney phenylalanine hydroxylation in experimental uremia. This alteration is likely to be the greatest contributory factor to the alteration of plasma levels of tyrosine and phenylalanine. The data presented do not support a proposed essentiality of tyrosine in uremia.

  12. Wnt signaling through the Ror receptor in the nervous system

    NARCIS (Netherlands)

    Petrova, Iveta M; Malessy, Martijn J; Verhaagen, J.; Fradkin, Lee G; Noordermeer, Jasprina N


    The receptor tyrosine kinase-like orphan receptor (Ror) proteins are conserved tyrosine kinase receptors that play roles in a variety of cellular processes that pattern tissues and organs during vertebrate and invertebrate development. Ror signaling is required for skeleton and neuronal development

  13. 非小细胞肺癌EGFR-TKI耐药预测生物标志物研究进展%Predictive biomarkers of the resistance of non-small cell lung cancer to epidermal growth factor receptor tyrosine kinase inhibitor

    Institute of Scientific and Technical Information of China (English)



    随着对肿瘤发病机制及其生物学行为的深入研究,分子靶向治疗成为目前治疗非小细胞肺癌(non-small cell lung cancer,NSCLC)最具前景的研究领域.其中表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitor,EGFR-TKI)可延长患者无进展生存期并明显提高患者生活质量,然而耐药已成为影响该类药物临床应用的最大障碍.因此对EGFR-TKI耐药机制的研究已成为关注的热点.现已发现其耐药可能与受体突变、细胞内信号转导相关蛋白、EGFR以外的TK受体介导的通路活化等有关.文中就NSCLC对EGFR-TKI耐药机制的最新研究进展进行综述.%With deeper insights into the pathogenesis and biological behavior of cancer, molecular targeted therapy has become a most promising area in the studies of non-small cell lung cancer ( NSCLC ). The epidermal growth factor receptor tyrosine kinase inhibitor ( EGFR-TKI ) can significantly improve the quality of life and prolong progression-free survival of the patient, but the resis tance to EGFR-TKI has emerged as the biggest obstacle to its application. Accordingly, the mechanism of EGFR-TKI resistance is becoming the focus of attention in this field. Recent studies show that EGFR-TKI resistance may be associated with the mutation of receptors, intracellular signal transduction related proteins, and other than EGFR-TKI receptor-mediated pathway. This article updates the mechanisms of the resistance of NSCLC to EGFR-TKI.

  14. Microglial Immunoreceptor Tyrosine-Based Activation and Inhibition Motif Signaling in Neuroinflammation


    Bettina Linnartz; Yiner Wang; Harald Neumann


    Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis. In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAP12-associated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- (ITAM-) Syk-signaling cascade. Microglial triggering receptor expressed on myeloid cells-2 (TREM2), signal regulatory protein- 1, and complement re...

  15. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  16. Tyrosine kinase blockers: new hope for successful cancer therapy. (United States)

    Pytel, Dariusz; Sliwinski, Tomasz; Poplawski, Tomasz; Ferriola, Deborah; Majsterek, Ireneusz


    Tyrosine kinases (TKs) are attractive targets for cancer therapy, as quite often their abnormal signaling has been linked with tumor development and growth. Constitutive activated TKs stimulate multiple signaling pathways responsible for DNA repair, apoptosis, and cell proliferation. During the last few years, thorough analysis of the mechanism underlying tyrosine kinase's activity led to novel cancer therapy using TKs blockers. These drugs are remarkably effective in the treatment of various human tumors including head and neck, gastric, prostate and breast cancer and leukemias. The most successful example of kinase blockers is Imatinib (Imatinib mesylate, Gleevec, STI571), the inhibitor of Bcr/Abl oncoprotein, which has become a first-line therapy for chronic myelogenous leukemia. The introduction of STI571 for the treatment of leukemia in clinical oncology has had a dramatic impact on how this disease is currently managed. Others kinase inhibitors used recently in cancer therapy include Dasatinib (BMS-354825) specific for ABL non-receptor cytoplasmic kinase, Gefitinib (Iressa), Erlotinib (OSI-774, Tarceva) and Sunitinib (SU 11248, Sutent) specific for VEGF receptor kinase, AMN107 (Nilotinib) and INNO-406 (NS-187) specific for c-KIT kinase. The following TK blockers for treatment of various human tumors are in clinical development: Lapatinib (Lapatinib ditosylate, Tykerb, GW-572016), Canertinib (CI-1033), Zactima (ZD6474), Vatalanib (PTK787/ZK 222584), Sorafenib (Bay 43-9006, Nexavar), and Leflunomide (SU101, Arava). Herein, we discuss the chemistry, biological activity and clinical potential of new drugs with tyrosine kinase blockers for cancer treatment.

  17. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten


    phosphorylation. Protein-tyrosine phosphorylation in bacteria is particular with respect to very low occupancy of phosphorylation sites in vivo; this has represented a major challenge for detection techniques. Only the recent breakthroughs in gel-free high resolution mass spectrometry allowed the systematic...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  18. Application of biotinylated and 32P probes for detection of P-fimbriae in urinary E. coli. (United States)

    Jusková, E; Ciznár, I


    Escherichia coli is the common causative agent of urinary tract infections. Twenty-six strains of Escherichia coli were isolated from children with pyelonephritis, symptomatic urinary tract infections and asymptomatic bacteriuria. Biotinylated and 32P-DNA probes were prepared for detection of P-fimbriae in the isolates. Of the 13 strains isolated from patients with pyelonephritis 11 were positive for the presence of the P gene by both probes. Strains isolated from cases of symptomatic urinary tract infections revealed the presence of P gene only in three samples of the total of nine isolated. None of the isolated E. coli strains from asymptomatic bacteriuria was found positive for the presence of the P gene. The biotinylated probe was simple and easily applicable in standard laboratory conditions and therefore the authors recommend it for use in diagnostic laboratories.

  19. Therapeutic Implications for Striatal-Enriched Protein Tyrosine Phosphatase (STEP) in Neuropsychiatric Disorders


    Goebel-Goody, Susan M.; Baum, Matthew; Paspalas, Constantinos D.; Fernandez, Stephanie M.; Carty, Niki C.; Kurup, Pradeep; LOMBROSO, PAUL J.


    Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase that modulates key signaling molecules involved in synaptic plasticity and neuronal function. Targets include extracellular-regulated kinase 1 and 2 (ERK1/2), stress-activated protein kinase p38 (p38), the Src family tyrosine kinase Fyn, N-methyl-d-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). STEP-mediated dephosphorylation of ERK1/2, p38, and Fyn...

  20. Robust versatile tyrosine kinase assay for HTS in drug discovery (United States)

    Deshpande, Sudhir S.; Mineyev, I.; Owicki, John C.


    A fluorescence polarization assay was developed as an alternative to the radiolabeled SPA assays currently used to monitor the activity of tyrosine kinases in drug discovery. The assay can be used with enzymes having substrate specificity similar to that of the insulin receptor, the EGF receptor and the Src kinase receptor enzymes. The assay is easy to configure in 96, 384 and 1536-well microplates in assay volumes ranging from (mu) L with minimal efforts. The reconstituted reagents are stable for up to 24 hr at ambient temperatures, thereby minimizing the need for replenishing the stock solutions during the course of a high-throughput screen. Because of the stability and equilibrium kinetics, the assay allows the user the luxury of scheduling the reading of plates any time up to 24 hr after the completion of the assay without substantial deterioration in the assay signal. The antibody and the tracer solutions can also be premixed and added as a preformed complex in a single step. The performance of the assay with the insulin receptor kinase is described. In addition, given the diversity of the substrates used in measuring the activity of different tyrosine kinases, LJL's on-going efforts to provide different antibodies of wide ranging specificity and sensitivity are described.

  1. 表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)继发性耐药的机制及对策%The Mechanism and Countermeasures on the Secondary Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (EGFR-TKI)

    Institute of Scientific and Technical Information of China (English)

    吴鹏飞; 朱益平; 杨春晖; 王远飞; 王根和


    Lung cancer, in which about 80% are non-small cell lung cancers (NSCLC), is one of the most common malignant tumors, and also the leading cause of cancer death currently. The epidermal growth factor receptor (EGFR) driver gene plays an important role in the occurrence and development of lung cancer. In recent years, the epidermal growth factor receptor tyrosine ki-nase inhibitors (EGFR-TKI), especially gefitinib and erlotinib, have played a significant role in the molecular and targeted therapy of NSCLC, and brought in good news for NSCLC patients. However, no matter how effective it is in the short term, the patients will inevitably develop into drug resistance and progressive disease in the long run. In this paper, we made a review on the mechanism of EGFR-TKI secondary drug resistance and the countermeasures on drug resistance after treatment, so as to guide the treatment of NSCLC better.%肺癌是最常见的恶性肿瘤之一,也是目前癌症死亡的首要原因,其中约80%为非小细胞肺癌(Non-small Cell Lung Cancers,NSCLC)。表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)驱动基因在肺癌的发生发展过程中起重要作用,近年来,以吉非替尼和厄洛替尼为代表的表皮生长因子受体酪氨酸激酶抑制剂(Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors,EGFR-TKI)在 NSCLC 的分子靶向治疗中发挥了巨大的作用,给NSCLC 患者带来了福音。然而,无论近期效果如何,最终患者都不可避免地产生耐药及病情进展。本文主要对近年来EGFR-TKI 继发性耐药的发生机制及耐药后的对策作一综述,以期更好地指导 NSCLC 的治疗。

  2. Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

    DEFF Research Database (Denmark)

    Confalonieri, S; Salcini, A E; Puri, C;


    for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function...... of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular...... determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis....

  3. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jin-Bao [School of Pharmacy, Weifang Medical University, Weifang 261053 (China); Tang, Ying [Affiliated Hospital of Weifang Medical University, Weifang 261041 (China); Yang, Hong-Ming, E-mail: [School of Pharmacy, Weifang Medical University, Weifang 261053 (China)


    Highlights: • An efficient signal amplification strategy for label-free EIA is proposed. • Divalent biotinylated AP and monovalent biotinylated ZZ were prepared via Avitag–BirA system. • The above site-specific biotinylated fusion proteins form complex via SA–biotin interaction. • The mechanism relies on the ZZ–Avi-B/SA/AP–(Avi-B){sub 2} complex. • The analytical signals are enhanced (32-fold) by the proposed strategy. - Abstract: Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable

  4. Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18. (United States)

    Bao, Baolong; Pestinger, Valerie; Hassan, Yousef I; Borgstahl, Gloria E O; Kolar, Carol; Zempleni, Janos


    Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [(3)H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.

  5. Collagen/chitosan film containing biotinylated glycol chitosan nanoparticles for localized drug delivery. (United States)

    Chen, Ming-Mao; Huang, Yu-Qing; Cao, Huan; Liu, Yan; Guo, Hao; Chen, Lillian S; Wang, Jian-Hua; Zhang, Qi-Qing


    The objective of this study was to design a drug delivery system consisting of biotinylated cholesterol-modified glycol chitosan (Bio-CHGC) nanoparticles and fish collagen/chitosan (Col/Ch) film for localized chemotherapy. Bio-CHGC was synthesized, and then its self-assembled nanoparticles were prepared by probe sonication. Doxorubicin (DOX)-loaded Bio-CHGC (DBC) nanoparticles prepared by dialysis had spherical shape, and their sizes were in the range of 330-397 nm. Col/Ch/DBC nanoparticle films were fabricated by freeze-drying. SEM showed that the DBC nanoparticles were uniformly distributed into the films, and the films retained their structural integrity. A higher degradation and swelling rate of the drug films led to a higher diffusion rate of the nanoparticles from the films, resulting in an increase in the drug release from nanoparticles. The release of DOX from the films or Bio-CHGC nanoparticles was sensitive to the pH value of the release medium. In addition, the DOX release ratio of the drug films was lower than that of the nanoparticles alone, suggesting that the drug films had a double-sustained effect on the drug release. MTT assay implied that the DBC nanoparticle film showed a higher inhibitory ratio than the film containing nanoparticles without biotin, indicating that biotin moieties in the nanoparticles played an important role in exerting a cytotoxic effect. These data demonstrate that Col/Ch/DBC nanoparticle film has the potential to be used as a localized delivery system for hydrophobic antitumor drugs.

  6. Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.

    Directory of Open Access Journals (Sweden)

    Adriana Ariza

    Full Text Available Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B as a tool for the study of protein haptenation by amoxicillin (AX. AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams.

  7. The rotational spectrum of tyrosine. (United States)

    Pérez, Cristóbal; Mata, Santiago; Cabezas, Carlos; López, Juan C; Alonso, José L


    In this work neutral tyrosine has been generated in the gas phase by laser ablation of solid samples, and its most abundant conformers characterized through their rotational spectra. Their identification has been made by comparison between the experimental and ab initio values of the rotational and quadrupole coupling constants. Both conformers are stabilized by an O-H•••N hydrogen bond established within the amino acid skeleton chain and an additional weak N-H•••π hydrogen bond. The observed conformers differ in the orientation of the phenolic -OH group.

  8. Bruton's tyrosine kinase regulates the activation of gene rearrangements at the lambda light chain locus in precursor B cells in the mouse

    NARCIS (Netherlands)

    G.M. Dingjan (Gemma); S. Middendorp; K. Dahlenborg; A. Maas (Alex); R.W. Hendriks (Rudi); F.G. Grosveld (Frank)


    textabstractBruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an approximately 50% reduction in the frequency of immunoglobulin (Ig) lambda light chai

  9. 非小细胞肺癌中微小RNA与表皮生长因子受体-酪氨酸激酶抑制剂敏感性关系的研究进展%MicroRNAs and epidermal growth factor receptor-tyrosine kinase inhibitors sensitivity in non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    裴冬; 陈晓锋; 束永前


    表皮生长因子受体(epidermal growth factor receptor,EGFR) -酪氨酸激酶抑制剂(tyrosine kinase inhibitor,TKI)被广泛应用于肿瘤的治疗,尤其是伴有EGFR基因突变的非小细胞肺癌(non-small cell lung cancer,NSCLC)的治疗,但EGFR- TKI耐药性的产生已成为制约其疗效的主要瓶颈.微小RNA (microRNAs,miRNAs)是一类非编码小分子RNA,在转录后水平调控基因的表达.近年来的研究发现,miRNAs在多个环节参与调节NSCLC对EGFR-TKI的敏感性,提示其可能在TKI耐药中扮演了重要角色,并有可能成为一种新的预测TKI敏感性的生物学标志物.因此,本文就有关NSCLC对EGFR-TKI的敏感性与miRNAs之间关系的最新研究进展进行综述.%Epidermal growth factor receptor-tyrosine kinase inhibitors (ECFR-TKIs) have been wildly used in cancer treatment, especially in non-small cell lung cancer (NSCLC) with EGFR mutation. However, the resistance to EGFR-TKIs has been a major problem which has resulted in a limitation for therapeutic effectiveness of ECFR-TKIs. MiRNAs (microRNAs) are a group of small non-coding RNAs, which are involved in the post-transcriptional regulation of gene expression. More recently, miRNAs have also been found to be involved in the regulation of EGFR-TKIs sensitivity in the treatment of NSCLC through multiple pathways, it suggests that EGFR-TKIs may play an important role in the resistance to TKI and it may be used as a new biomarker in the prediction of TKI sensitivity. This review summarizes the advance in the relationship between miRNAs and EGFR-TKI sensitivity in NSCLC.

  10. 脑缺血时NMDA受体通过Src激酶和Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ调控ERKs激活%N-methyl-D-aspartate receptors mediate diphosphorylation of extracellular signal-regulated kinases through Src family tyrosine kinases and Ca2+/calmodulin-dependent protein kinase Ⅱ in rat hippocampus after cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    吴辉文; 李洪福; 郭军


    目的 ERKs是钙依赖性激活蛋白,本研究旨在探讨钙依赖性蛋白激酶是否参与了脑缺血后ERK级联的调控.方法 采用四动脉结扎诱导大鼠前脑缺血,用免疫印迹的方法观察几个钙依赖性蛋白激酶含量及活性的变化.结果 致死性脑缺血以NMDA受体依赖的方式激活ERKs,并差异性上调Src和Ca2+/钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的活性.Src激酶和CaMKⅡ的抑制剂PP2和KN62能显著的阻止缺血诱导的ERKs激活.然而,缺血诱导的Src过度激活也伴随着ERKs的活性抑制.结论 致死性脑缺血刺激NMDA受体通过Src激酶和CaMKⅡ介导ERKs活性上调,但是脑缺血诱导的Src过度激活可能也参与了ERKs信号通路的负性调控.%Objective: Extracellular signal-regulated kinases (ERKs) can be activated by calcium signals. In this study, we investigated whether calcium-dependent kinases were involved in ERKs cascade activation after global cerebral ischemia.Methods Cerebral ischemia was induced by four-vessel occlusion, and the calcium-dependent proteins were detected by immunoblot. Results Lethal-simulated ischemia significantly resulted in ERKs activation in N-methyl-D-aspartate (NMDA)receptor-dependent manner, accompanying with differential upregulation of Src kinase and Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) activities. With the inhibition of Src family tyrosine kinases or CaMKⅡ by administration of PP2or KN62, the phosphorylation of ERKs was impaired dramatically during post-ischemia recovery. However, ischemic challenge also repressed ERKs activity when Src kinase was excessively activated. Conclusions Src family tyrosine kinases and CaMKⅡ might be involved in the activation of ERKs mediated by NMDA receptor in response to acute ischemic stimuli in vivo, but the intense activation of Src kinase resulted from ischemia may play a reverse role in the ERKs cascade.

  11. Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays

    DEFF Research Database (Denmark)

    Amanchy, Ramars; Zhong, Jun; Molina, Henrik;


    c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human...... embryonic kidney 293T cells. We have identified 10 known substrates and interactors of c-Src and Src family kinases along with 26 novel substrates. We have experimentally validated 4 of the novel proteins (NICE-4, RNA binding motif 10, FUSE-binding protein 1 and TRK-fused gene) as direct substrates of c......-Src using in vitro kinase assays and cotransfection experiments. Significantly, using a c-Src specific inhibitor, we were also able to implicate 3 novel substrates (RNA binding motif 10, EWS1 and Bcl-2 associated transcription factor) in PDGF signaling. Finally, to identify the exact tyrosine residues...

  12. Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

    Directory of Open Access Journals (Sweden)

    Kevin A James

    Full Text Available The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced "superacceptor" activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD motif in the catalytic loop and the Asp-Phe-Gly (DFG motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not

  13. Protein tyrosine phosphatases: structure-function relationships. (United States)

    Tabernero, Lydia; Aricescu, A Radu; Jones, E Yvonne; Szedlacsek, Stefan E


    Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.

  14. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti


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

  15. Metabolite profiling of the multiple tyrosine kinase inhibitor lenvatinib : a cross-species comparison

    NARCIS (Netherlands)

    Dubbelman, Anne Charlotte; Nijenhuis, Cynthia M.; Jansen, Robert S; Rosing, Hilde; Mizuo, Hitoshi; Kawaguchi, Shinki; Critchley, David; Shumaker, Robert; Schellens, Jan H M; Beijnen, Jos H.


    Lenvatinib is an oral, multiple receptor tyrosine kinase inhibitor. Preclinical drug metabolism studies showed unique metabolic pathways for lenvatinib in monkeys and rats. A human mass balance study demonstrated that lenvatinib related material is mainly excreted via feces with a small fraction as

  16. Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation

    DEFF Research Database (Denmark)

    Daum, G; Regenass, S; Sap, J


    Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G...

  17. Biotinylated dextran amine as a neural tracer in the rat corticospinal tract

    Institute of Scientific and Technical Information of China (English)

    Biao Gong; Changqing Li; Xiaofeng Li; Ying Wang


    BACKGROUND: The corticospinal tract is the core structure of cerebral control of extremity movement and plasticity, which are prerequisites for movement rehabilitation after brain injury. The measurement and assessment of plasticity changes within the corticospinal tract has become one of the key goals in this field.OBJECTIVE: To explore the effects of biotinylated dextran amine (BDA) as a neural tracer in the rat corticospinal tract and the possibilities of assessing plasticity within the corticospinal tract.DESIGN: An observational experiment.SETTING: Department of Acupuncture of Chinese Medical College, Chongqing Medical University, Department of Neurology, the Second Affiliated Hospital, Chongqing Medical University.MATERIALS: Eighteen male adult Sprague Dawley (SD) rats of clean grade, weighing 200-250 g, were provided by the experimental animal center of Chongqing Medical University. The animal procedures in this study were in accordance with the animal ethics standards. BDA was provided by Vector Laboratories Company (USA, catalogue Sp-1140; serial number R0721).METHODS: This experiment was performed in the Laboratory of Chongqing Medical University between September and December 2006. Adult SD rats were used in the experiment and 15% BDA was injected slowly with a mini-syringe through two round (3 mm diameter) holes into the left sensory and motor cortex. The center of one hole was located 3 mm anterior from the anterior fontanel and 1.5 mm left of the midline; the second hole was located 1.5 mm posterior from the anterior fontanel and 4 mm left of the midline. Three injections were made at each hole at three different levels: 1.4, 1.2, and 1 mm ventral from the surface of the flat skull. After 14 days, the brains and spinal cords were removed and frozen. Sections were cut on a cryostat and BDA transportation absorbed by axons was observed under a fluorescence microscope.MAIN OUTCOME MEASURES: Axonal absorption and transportation of BDA was observed under

  18. Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch.

    Directory of Open Access Journals (Sweden)

    Sudharsana R Ande

    Full Text Available Prohibitin (PHB or PHB1 is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked beta-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114 and tyrosine 259 (Tyr259 in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121 and threonine 258 (Thr258 respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s or known tyrosine phosphorylation site(s revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.

  19. Convenient synthesis of a propargylated cyclic (3'-5') diguanylic acid and its "click" conjugation to a biotinylated azide. (United States)

    Grajkowski, Andrzej; Cieślak, Jacek; Gapeev, Alexei; Schindler, Christian; Beaucage, Serge L


    The ribonucleoside building block, N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine, was prepared from commercial N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-propargyl guanosine in a yield of 91%. The propargylated guanylyl(3'-5')guanosine phosphotriester was synthesized from the reaction of N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine with N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl] guanosine and isolated in a yield of 88% after P(III) oxidation, 3'-/5'-deprotection, and purification. The propargylated guanylyl(3'-5')guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester, which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups from the propargylated c-di-GMP triester were performed by treatment with aqueous ammonia at ambient temperature. The final 2'-desilylation reaction was effected by exposure to triethylammonium trihydrofluoride affording the desired propargylated c-di-GMP diester, the purity of which exceeded 95%. Biotinylation of the propargylated c-di-GMP diester was easily accomplished through its cycloaddition reaction with a biotinylated azide derivative under click conditions to produce the biotinylated c-di-GMP conjugate of interest in an isolated yield of 62%.

  20. Advances in epidermal growth factor receptor tyrosine kinase inhibitors combined with chemotherapy for lung cancer treatment%表皮生长因子受体酪氨酸激酶抑制剂联合化疗治疗肺癌的研究进展

    Institute of Scientific and Technical Information of China (English)

    张贝贝; 宋正波; 张沂平


    Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show good efficacy for the lung cancer patients with EGFR mutations.Chemotherapy drugs are the first choices for the lung cancer patients with EGFR wild-type.In basic research,EGFR-TKIs combination with chemotherapy drugs show good synergy.But in clinical research,the timing of EGFR-TKIs combination with chemotherapy drugs is related to the efficacy.%表皮生长因子受体(EGFR)酪氨酸激酶抑制剂(EGFR-TKI)对EGFR突变肺癌患者表现出很好的疗效,对于EGFR野生型肺癌患者化疗药物是首选,EGFR-TKI和化疗药物联合在基础研究方面表现出良好的协同作用,而在临床研究方面与其两药联合的时序有关.

  1. 非小细胞肺癌表皮生长因子受体-酪氨酸激酶抑制剂的一线治疗%Epidermal growth factor receptor tyrosine kinase inhibitors as first-line treatment in advanced non-small-cell lung cancer

    Institute of Scientific and Technical Information of China (English)



    Several models about epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKI)as first-line treatment in advanced NSCLC show different results.Combination with chemotherapy and EG-FR-TKIs did not indicate survival advantage.However,sequential chemotherapy in EGFR-TKIs and a single agent of EGFR-TKIs as first-line treatment of advanced NSCLC have made tremendous breakthrough,and give a new path in individual treatment of advanced NSCLC.Also EGFR mulatious in targeted therapy of advanced NSCLc has a sood predictive value of the etticacy.%表皮生长因子受体-酪氨酸激酶抑制剂(EGFR-TKI)参与的晚期非小细胞肺癌(NSCLC)一线治疗的几种模式显示出不同的结果.化疗与EGFR-TKI联合未显示生存优势,化疗序贯EGFR-TKI和EGFR-TKI单药这两种模式取得了巨大的突破,为晚期NSCLC个体化治疗开辟了新的道路.同时发现EGFR突变在晚期NSCLC靶向治疗方面有很好的疗效预测价值.

  2. EGFR-TKIs获得性耐药及耐药后治疗的进展%Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer and the clinical studies of overcoming the acquired resistance

    Institute of Scientific and Technical Information of China (English)

    王慧娟; 李鹏; 王启鸣


    Reversible epidermal growth factor receptor tyrosine kinase inhibitors ( EGFR-TKIs) such as gefitinib and erlotinib are used for the treatment of non-small cell lung cancer following failure of cytotoxic chemotherapy frequently. Somatic activating mutations of the EGFR gene have been associated with dramatic tumor responses and favorable clinical outcomes. All patients, however, ultimately develop resistance to these agents, and there is no standard treatment after acquired resistance yet. This paper mainly discuss the current definition of acquired resistance to EGFR-TKIs and the clinical studies of overcoming the acquired resistance.%表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)吉非替尼和厄洛替尼是用于化疗后失败或复发的晚期非小细胞肺癌的常用药物,随着对它们的深入研究,发现ECFR-TKIs的疗效与EGFR突变相关.然而,几乎所有接受EGFR-TKIs治疗的患者都会出现耐药,而获得性耐药后目前没有标准治疗方案.本文主要探讨EGFR-TKIs获得性耐药的定义,并对目前克服获得性耐药的临床研究作一综述.

  3. Application of epidermal growth factor receptor tyrosine kinase inhibitor in treatment of non-smal cel lung cancer%表皮生长因子受体酪氨酸激酶抑制剂在非小细胞肺癌治疗中的应用

    Institute of Scientific and Technical Information of China (English)

    申燕华; 钱叶长


      肺癌是当今世界上严重威胁人类健康与生命的恶性肿瘤,NSCLC占肺癌总数的80-85﹪.EGFR-TKI在EGFR突变的NSCLC患者的治疗中取得了良好的效果,但NSCLC对EGFR-TKI产生耐药性也逐渐受到重视.本文主要对EGFR基因突变的检测、EGFR-TKI对NSCLC的治疗以及NSCLC对EGFR-TKI耐药的机制及对策进行综述.%Lung cancer is a highly malignant tumor which is the most threatening disease of the human health and life, and 80-85﹪ of the disease is non-smal cel lung cancer (NSCLC). Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has obtained the good effect in the treatment of NSCLC patients with EGFR mutation. But it is also getting increasing attention about NSCLC drug resistance to EGFR-TKI. This paper summarized the detection of the EGFR gene mutation, the effect of EGRF-TKI on the treatment of NSCLC patients, and the pathogenesis and countermeasures about NSCLC drug resistance to EGFR-TKI.

  4. Biotinylation of a propargylated cyclic (3'-5') diguanylic acid and of its mono-6-thioated analog under "click" conditions. (United States)

    Grajkowski, Andrzej; Cieślak, Jacek; Schindler, Christian; Beaucage, Serge L


    Commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(propargyl)guanosine is converted to its 3'-O-levulinyl ester in a yield of 91%. The reaction of commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine with N(2)-isobutyryl-2'-O-propargyl-3'-O-(levulinyl)guanosine provides, after P(III) oxidation, 3'-/5'-deprotection, and purification, the 2'-O-propargylated guanylyl(3'-5')guanosine 2-cyanoethyl phosphate triester in a yield of 88%. Phosphitylation of this dinucleoside phosphate triester with 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole, followed by an in situ intramolecular cyclization, gives the propargylated cyclic dinucleoside phosphate triester, which is isolated in a yield of 40% after P(III) oxidation and purification. Complete removal of the nucleobases, phosphates, and 2'-O-tert-butyldimethylsilyl protecting groups leads to the desired propargylated c-di-GMP diester. Cycloaddition of a biotinylated azide with the propargylated c-di-GMP diester under click conditions provides the biotinylated c-di-GMP conjugate in an isolated yield of 62%. Replacement of the 6-oxo function of N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-levulinyl-2'-O-(propargyl)guanosine with a 2-cyanoethylthio group is effected by treatment with 2,4,6-triisopropybenzenesulfonyl chloride and triethylamine to give a 6-(2,4,6-triisopropylbenzenesulfonic acid) ester intermediate. Reaction of this key intermediate with 3-mercaptoproprionitrile and triethylamine, followed by 5'-dedimethoxytritylation, affords the 6-(2-cyanoethylthio)guanosine derivative in a yield of 70%. The 5'-hydroxy function of this derivative is reacted with commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine. The reaction product is then converted to the mono-6-thioated c-di- GMP biotinylated conjugate under

  5. Phage-free peptide ELISA for ochratoxin A detection based on biotinylated mimotope as a competing antigen. (United States)

    Zou, Xuqiang; Chen, Chaochao; Huang, Xiaolin; Chen, Xuelan; Wang, Lv; Xiong, Yonghua


    To perform the biopanning of a mimotope peptide with reduced affinity to anti-ochratoxin A (OTA) monoclonal antibodies (mAbs), we executed two improved biopanning approaches with a commercial 7-mer peptide library. In the first approach, anti-mouse IgG antibodies were used to erect the anti-OTA mAbs; in the second approach, an ultralow OTA concentration (0.1 ng/mL) was used to perform the competitive elution of phage particles. After the fourth round of biopanning was completed, 30 identified clones were positive phage particles; of these phage particles, 16 exhibited strong competitive inhibition with a low OTA concentration of 0.1 ng/mL. DNA sequencing results revealed that the 16 phage particles represented six different peptide sequences. Among these particles, the phage particle with a peptide sequence of "GMVQTIF" showed the highest sensitivity to OTA detection. The biotinylated 12-mer peptide "GMVQTIF-GGGSK-biotin" was designed as a competing antigen to develop a competitive peptide ELISA. Under the optimal parameters, the proposed peptide ELISA with the biotinylated 12-mer peptide as a competing antigen exhibited good dynamic linear detection for OTA in the range of 0.005 ng/mL-0.2 ng/mL with a detection limit of 0.001 ng/mL. The median inhibition concentration of OTA was 0.024 ng/mL (n=6), which is approximately fivefold more efficient as a competing antigen than the OTA-HRP conjugates. Reaction kinetics revealed that the biotinylated 12-mer peptide exhibited lower affinity to anti-OTA mAbs than the conventional chemical OTA antigen. The practicality of the proposed peptide ELISA was compared with a conventional ELISA method. In summary, this study demonstrated a novel concept of the development of phage-free peptide ELISA for the detection of OTA by using a biotinylated mimotope peptide as a competing antigen. This novel strategy can be applied to sensitively detect other toxic small molecules during food safety monitoring.

  6. RhoA/phosphatidylinositol 3-kinase/protein kinase B/mitogen-activated protein kinase signaling after growth arrest-specific protein 6/mer receptor tyrosine kinase engagement promotes epithelial cell growth and wound repair via upregulation of hepatocyte growth factor in macrophages. (United States)

    Lee, Ye-Ji; Park, Hyun-Jung; Woo, So-Youn; Park, Eun-Mi; Kang, Jihee Lee


    Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.


    Directory of Open Access Journals (Sweden)

    Elisabetta Abruzzese


    Full Text Available The management of patients with chronic myeloid leukemia (CML during pregnancy has became recently a matter of continuous debate.  The introduction of the Tyrosine Kinase Inhibitors (TKIs in clinical practice has dramatically changed the prognosis of CML patients.  Patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy.  This fact has come the necessity to address issues relating to fertility and pregnancy. Physicians are not infrequently being asked for advice regarding the need for, and or the appropriateness of, stopping treatment in order to conceive. In this report we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for all the approved TKIs, as well as suggest how to manage a planned and/or unplanned pregnancy.

  8. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins. (United States)

    Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming


    Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein.

  9. Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid. (United States)

    Polychronakos, C; Piscina, R; Fantus, I G


    Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia.

  10. Signal transduction through the IL-4 and insulin receptor families. (United States)

    Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H


    Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Targeting at the Nanoscale: A Novel S-Layer Fusion Protein Enabling Controlled Immobilization of Biotinylated Molecules. (United States)

    Varga, Melinda


    With the aim of constructing an S-layer fusion protein that combines both excellent self-assembly and specific ligand i.e., biotin binding ability, streptavidin (aa 16-133) was fused to the S-layer protein of Sporosarcina ureae ATCC 13881 (SslA) devoid of its N-terminal 341 and C-terminal 172 amino acids. The genetically engineered chimeric protein could be successfully produced in E. coli, isolated, and purified via Ni affinity chromatography. In vitro recrystallisation experiments performed with the purified chimeric protein in solution and on a silicon wafer have demonstrated that fusion of the streptavidin domain does not interfere with the self-assembling properties of the S-layer part. The chimeric protein self-assembled into multilayers. More importantly, the streptavidin domain retained its full biotin-binding ability, a fact evidenced by experiments in which biotinylated quantum dots were coupled to the fusion protein monomers and adsorbed onto the in vitro recrystallised fusion protein template. In this way, this S-layer fusion protein can serve as a functional template for the controlled immobilization of biotinylated and biologically active molecules.

  12. Non-covalent ligand conjugation to biotinylated DNA nanoparticles using TAT peptide genetically fused to monovalent streptavidin. (United States)

    Sun, Wenchao; Fletcher, David; van Heeckeren, Rolf Christiaan; Davis, Pamela B


    DNA nanoparticles (DNA NPs), which self-assemble from DNA plasmids and poly-L-lysine (pLL)-polyethylene glycol (PEG) block copolymers, transfect several cell types in vitro and in vivo with minimal toxicity and immune response. To further enhance the gene transfer efficiency of DNA NP and control its tropism, we established a strategy to efficiently attach peptide ligands to DNA NPs. The non-covalent biotin-streptavidin (SA) interaction was used for ligand conjugation to overcome problems associated with covalent conjugation methods. A fusion protein of SA with the HIV-1 TAT peptide was cloned, expressed, purified and attached to biotinylated DNA NPs. A modified SA system with tetrameric structure but monovalent biotin binding capacity was adopted and shown to reduce the aggregation of biotinylated DNA NPs compared to neutravidin. Compared to unmodified DNA NPs, TAT modified DNA NPs significantly enhanced in vitro gene transfer, particularly at low DNA concentrations. Studies of cellular uptake and cellular distribution of the DNA NPs indicated that attaching TAT enhanced binding of DNA NPs to cell surface but not internalization at high DNA concentrations. In vivo studies showed that TAT modified DNA NPs mediated equal level of gene transfer to the mouse airways via the luminal route compared to unmodified DNA NPs.

  13. Membrane labeling of coral gastrodermal cells by biotinylation: the proteomic identification of surface proteins involving cnidaria-dinoflagellate endosymbiosis.

    Directory of Open Access Journals (Sweden)

    Hsing-Hui Li

    Full Text Available The cellular and molecular-scale processes underlying the stability of coral-Symbiodinium endosymbioses remain unclear despite decades of investigation. As the coral gastroderm is the only tissue layer characterized by this unique symbiotic association, the membranes of these symbiotic gastrodermal cells (SGCs may play important roles in the initiation and maintenance of the endosymbiosis. In order to elucidate the interactions between the endosymbiotic dinoflagellates and their coral hosts, a thorough characterization of SGC membranes is therefore required. Cell surface proteins of isolated SGCs were biotinylated herein by a cell impermeant agent, biotin-XX sulfosuccinimidyl ester. The in situ distribution of these biotinylated proteins was uncovered by both fluorescence and transmission electron microscopic imaging of proteins bound to Alexa Fluor® 488-conjugated streptavidin. The identity of these proteins was then determined by two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. Nineteen (19 proteins were identified, and they are known to be involved in the molecular chaperone/stress response, cytoskeletal remodeling, and energy metabolism. These results not only reveal the molecular characters of the host SGC membrane, but also provide critical insight into understanding the possible role of host membranes in this ecologically important endosymbiotic association.

  14. A Crystallographic Snapshot of Tyrosine Trans-phosphorylation in Action

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Xu, C; Ma, J; Eliseenkova, A; Li, W; Pollock, P; Pitteloud, N; Miller, W; Neubert, T; Mohammadi, M


    Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 {angstrom} away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

  15. 成年大鼠脊髓全横断损伤后酪氨酸激酶受体C在脊髓和大脑皮层的表达%Expression of tyrosine kinase receptor C in the segments of the spinal cord and the cerebral cortex after cord transection in adult rats

    Institute of Scientific and Technical Information of China (English)

    钱东翔; 张洪钿; 蔡颖谦; 罗鹏; 徐如祥


    目的 研究神经营养因子3(neurotrophin-3,NT-3)的受体-酪氨酸激酶受体C(tyrosine kinase receptor C,TrkC)在脊髓损伤(spinal cord injury,SCI)后神经重塑中的作用.方法 研究脊髓全横断损伤大鼠手术后第1、3、7和14 d时,低位胸髓节段和大脑中央前回TrkC的表达.结果 损伤节段(T10-T11)双侧和临近节段(T9和T12)的TrkC蛋白水平在术后1-7 d显著下调,而在术后14 d快速增强.此外,TrkC mRNA表达水平的暂时性变化与TrkC蛋白的变化模式相似.TrkC蛋白和mRNA在损伤节段(T10-T11)的水平显著高于在临近节段(T9和T12)的水平.此外,TrkC蛋白和mRNA在吻侧节段的水平高于在尾侧节段的水平.与脊髓不同的是,运动皮层中并未检测到TrkC蛋白,并且TrkC mRNA的表达水平也很低.结论 TrkC可能与脊髓损伤后的神经功能重塑有关.%Objective To investigate the role of tyrosine kinase receptor C (TrkC), the receptor of neurotrophin-3 (NT-3), in neuroplasticity following spinal cord injury (SCI). Methods Rats with cord transection were allowed to survive for 1, 3, 7 and 14 d post operation (dpo). TrkC expressions at lower thoracic levels of the spinal cord and in precentral gyrus of cerebral cortex were investigated. Results TrkC protein levels at both the site of injury (T10-T11) and the neighboring segments (T9 and T12) in the spinal cord decreased significantly at 1-7 dpo, followed by a rapid increase at 14 dpo. The temporal changes in TrkC mRNA expression level showed a similar pattern with that of TrkC protein. In addition, the levels of TrkC protein and mRNA at the site of injury (T10-T11) were significantly higher than those at the neighboring spinal segments (T9 and T12). Besides, the levels of TrkC protein and mRNA were higher at the rostral segment than at the caudal segment. However, in the motor cortex, TrkC protein was not detected and TrkC mRNA was expressed at a very low level. Conclusion These results suggest that TrkC may be

  16. Tyrosine Phosphorylation Modulates the Vascular Responses of Mesenteric Arteries from Human Colorectal Tumors

    Directory of Open Access Journals (Sweden)

    Eduardo Ferrero


    Full Text Available The aim of this study was to analyze whether tyrosine phosphorylation in tumoral arteries may modulate their vascular response. To do this, mesenteric arteries supplying blood flow to colorectal tumors or to normal intestine were obtained during surgery and prepared for isometric tension recording in an organ bath. Increasing tyrosine phosphorylation with the phosphatase inhibitor, sodium orthovanadate produced arterial contraction which was lower in tumoral than in control arteries, whereas it reduced the contraction to noradrenaline in tumoral but not in control arteries and reduced the relaxation to bradykinin in control but not in tumoral arteries. Protein expression of VEGF-A and of the VEGF receptor FLT1 was similar in control and tumoral arteries, but expression of the VEGF receptor KDR was increased in tumoral compared with control arteries. This suggests that tyrosine phosphorylation may produce inhibition of the contraction in tumoral mesenteric arteries, which may increase blood flow to the tumor when tyrosine phosphorylation is increased by stimulation of VEGF receptors.

  17. Biotinylated dextran amine is an ideal anterograde tracer for the corticospinal tract in a goat model of ischemic corticospinal tract injury

    Institute of Scientific and Technical Information of China (English)

    Xiaoyu Yang; Yufei Gao; Zhigang Qin; Xia Cao; Xinquan Gu


    Existing visualized tracer studies of the corticospinal tract have been focused on rodents, which have markedly different spinal cord structures compared with humans. In this study, the segmental artery feeding the spinal cord was embolized with digital subtraction angiography to establish a goat model of ischemic spinal cord injury. Biotinylated dextran amine was injected into the motor function areas of the cortex in goats with ischemic spinal cord injury. The corticospinal tract originates from the cerebral cortex motor function area, and travels towards the lateral funiculus at the contralateral spinal dorsal horn after decussation at the pyramid. The number of corticospinal tract positive fibers was found to be gradually reduced. These findings indicate that digital subtraction angiography can be applied to a goat model of ischemic spinal cord injury. Biotinylated dextran amine visualizes the course of the goat corticospinal tract in the spinal cord, which is similar to the human spinal cord. Biotinylated dextran amine is an ideal tracer for the corticospinal tract.

  18. Identification of AcMNPV GP64-binding proteins through a combinational use of a self-biotinylated virus and the cross-linking method. (United States)

    Ke, Xianliang; Zhang, Yuan; Liu, Yan; Wang, Hanzhong


    Baculoviruses are potential vectors of gene therapy for the ability to transfer gene high efficiently into mammalian cells. However, cell membrane proteins which interact with baculoviral glycoproteins have not been identified. In this study, we developed a self-biotinylated AcMNPV bearing biotinylated GP64 glycoproteins. This recombinant virus demonstrated the capability to infect insect cells and to transduct mammalian cells. Using this biotinylated virus, a protein >170Kda which could specifically interact with GP64 proteins was identified from virus transducted BHK-21 cells through cross-linking and streptavidin purification. Our study provides a useful approach for identifying cell membrane proteins that interact with baculovirus surface proteins or proteins involved in virus attachment.

  19. Analysis of tyrosine-O-sulfation

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Sen, J.W.; Johnsen, A.H.


    Tyrosine O-sulfation was first described about 50 years ago as a post-translational modification of fibrinogen. In the following 30 years it was considered to be a rare modification affecting only a few proteins and peptides. However, in the beginning of the 1980s tyrosine (Tyr) sulfation was shown...... the presence of radioactively labeled tyrosine. These techniques have been described in detail previously. The aim of this chapter is to present alternative analytical methods of Tyr sulfation than radioisotope incorporation before analysis Udgivelsesdato: 2008...

  20. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    DEFF Research Database (Denmark)

    Norris, K; Norris, F; Kono, D H;


    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain...... PTPs. Surprisingly, PTP-LAR, previously suggested to be a major regulator of the insulin receptor tyrosine kinase, was expressed in extremely low levels in skeletal muscle, whereas the related receptor-type PTP-sigma and PTP-alpha were expressed in relatively high levels in all four tissues. The low...... a representative picture, all tissues were analyzed by PCR using three different primer sets corresponding to conserved regions of known PTPs. A total of 24 different PTPs were identified. A multiprobe RNase protection assay was developed to obtain a semiquantitative measure of the expression levels of selected...

  1. Neuroblastoma tyrosine kinase signaling networks involve FYN and LYN in endosomes and lipid rafts.

    Directory of Open Access Journals (Sweden)

    Juan Palacios-Moreno


    Full Text Available Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.

  2. Syntheses of L-tyrosine-related amino acids by tyrosine phenol-lyase of Citrobacter intermedius. (United States)

    Nagasawa, T; Utagawa, T; Goto, J; Kim, C J; Tani, Y; Kumagai, H; Yamada, H


    Degradation of tyrosine to phenol, pyruvate and ammonia by tyrosine phenol-lyase from Citrobacter intermedius (formerly named Escherichia intermedia) is readily reversible at high concentrations of pyruvate and ammonia. Spectrophotometric studies indicate that ammonia is the first substrate which interacts with bound pyridoxal 5'-phosphate. Kinetic results show that pyruvate is the second substrate bound, hence phenol must be the third. When an appropriate phenol derivative is substituted for phenol, the corresponding tyrosine analogue can be synthesized. 3-Fluoro-, 2-fluoro-, 3-chloro-, 2-chloro-, 3-bromo-, 2-bromo-, 2-iodo-, 3-methyl-, 2-methyl- and 2-methoxy-L-tyrosines have been synthesized by this reaction. By using various phenol derivatives or tyrosine analogues as substrates, the substrate specificity of tyrosine phenol-lyase is investigated and the situation of its active site is discussed.

  3. Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96: Potential use of extracorporeal immunoadsorption with enhanced tumor radioactivity retention of Iodine, Indium and Rhenium

    Energy Technology Data Exchange (ETDEWEB)

    Chen, JianQing


    In this thesis, methodology of radiolabeling and simultaneous biotinylation for internalizing monoclonal antibody chimeric BR96 have been investigated by using three element groups of potential therapeutic radionuclides iodine, indium and rhenium, and their different labeling methods. The biodistribution and kinetics of biotinylated and radiolabeled chiBR96 have been studied in colon carcinoma isografted rats. The potential use of ECIA, based on the biotin-avidin concept, has been evaluated and compared with the approach of avidin `chase` in the same animal tumor model with respect to an enhancement of tumor-to-normal tissue (T/N) activity ratio. 131 refs.

  4. Effects of coffee on insulin receptor substance and its tyrosine/serine phosphorylation in rats with insulin resistance%咖啡对胰岛素抵抗大鼠胰岛素受体底物及其酪氨酸、丝氨酸磷酸化的影响

    Institute of Scientific and Technical Information of China (English)

    陈琳; 喻明; 夏娟; 高月锦


    目的:观察咖啡对胰岛素抵抗大鼠脂肪组织胰岛素受体底物( IRS)-1、IRS-2及其酪氨酸、丝氨酸磷酸化的影响,评估咖啡对脂肪组织胰岛素受体后信号转导途径影响。方法 Wistar大鼠40只随机分为正常NC、CC、DC、IR组,每组10只。 NC组予正常饮食,CC组、DC组及IR组给予高脂饮食,CC组及DC组每日分别以含咖啡因咖啡和去咖啡因咖啡灌胃。12周后行口服葡萄糖耐量试验( OGTT);根据空腹血清胰岛素和空腹血糖水平及HO-MA-胰岛素抵抗指数( HOMA-IR)反映外周组织胰岛素敏感性;RT-PCR检测大鼠脂肪组织IRS-1、IRS-2 mRNA及蛋白表达;Western blot测定脂肪组织IRS-1酪氨酸磷酸化和丝氨酸磷酸化程度。结果 OGTT显示IR组60和120 min血糖高于NC组( P分别<0.05、0.01)。 IR组、CC 组及DC组空腹血清胰岛素水平均高于NC 组( P均<0.05)。 IR组HOMA-IR高于NC组、CC组和DC组(P均<0.05)。 NC组、CC组脂肪组织IRS-1 mRNA相对表达量均高于IR组(P均<0.01),IRS-2 mRNA表达各组无显著差异(P均>0.05)。各组大鼠脂肪组织IRS-1、IRS-2蛋白含量无明显差异( P均>0.05)。 NC组、CC 组、DC组脂肪组织IRS-1酪氨酸磷酸化水平高于IR组( P均<0.05),丝氨酸磷酸化水平低于IR组(P均<0.05)。结论咖啡灌胃可改善高脂饮食大鼠胰岛素抵抗,其机制可能通过改变脂肪组织IRS-1转录及其酪氨酸/丝氨酸磷酸化水平,影响胰岛素抵抗大鼠胰岛素受体后信号转导。%Objective To observe the effect of coffee on the expression of insulin receptor substance ( IRS)-1 and IRS-2 and their tyrosine/serine phosphorylation in rats with insulin resistance ( IR) and to evaluate its effect on the post-re-ceptor signal transduction in adipose tissues .Methods Totally 40 Wistar rats were randomly divided into the normal con-trol ( NC) group, caffeinated coffee

  5. The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice (United States)

    Yan, Jian; Aboshi, Takako; Teraishi, Masayoshi; Strickler, Susan R.; Spindel, Jennifer E.; Tung, Chih-Wei; Takata, Ryo; Matsumoto, Fuka; Maesaka, Yoshihiro; McCouch, Susan R.; Okumoto, Yutaka; Mori, Naoki; Jander, Georg


    Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-β-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with β-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated β-tyrosine production in Nipponbare. Rice cultivars that do not produce β-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although β-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant β-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As β-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice. PMID:25901084

  6. 脑源性神经生长因子及酪氨酸激酶受体B在子宫内膜癌中的表达及其意义%Expressions and Significances of Brain-Derived Neurotrophic Factor and Tyrosine Kinase Recep-tor B in Endometrial Carcinoma

    Institute of Scientific and Technical Information of China (English)

    陈旭娜; 叶志中


    [Objective]To explore the expressions of brain-derived neurotrophic factor (BDNF)and tyro-sine kinase receptor B (TrkB)in endometrial carcinoma and analyze their clinical significances.[Methods]The expressions of BDNF and TrkB were detected by immunohistochemistry in normal endometrium (n = 22), hyperplasia endometrium (n =32)and endometrial carcinoma (n =62).And the relationship with their clini-copathological characteristics were analyzed.[Results]BDNF and TrkB were negatively or weakly positively expressed in normal endometrium while positively in hyperplasia endometrium and endometrial carcinoma.And statistically significant differences existed among three groups (P < 0.05 ).The expressions of BDNF and TrkB in endometrial carcinoma were significantly correlated with tumor cell differentiation,clinicopathologic staging,myometrial invasion and lymph node metastasis of endometrial carcinoma (P <0.05).[Conclusion]The interaction of BDNF with TrkB may play an important synergistic role in the occurrence and development of endometrial carcinoma.%【目的】探讨脑源性神经生长因子(Brain derived neurotrophic factor,BDNF)及酪氨酸激酶受体 B (tyrosine kinase receptor B,TrkB)在子宫内膜癌(endometrial carcinoma,EC)中的表达及其意义。【方法】采用免疫组织化检测22例正常子宫内膜(正常组)、32例增生子宫内膜(增生组)、62例 EC 组织(EC 组)中的BDNF 及 TrkB 表达水平,并分析 EC 组织中 BDNF、TrkB 的表达与其临床病理特征的关系。【结果】增生组、EC 组 BDNF、TrkB 阳性表达率显著高于正常组,而 EC 组 BDNF、TrkB 阳性表达率显著高于增生组,差异均有统计学意义(P <0.05)。EC 组织中 BDNF、TrkB 的表达与肿瘤细胞分化程度、临床病理分期、肌层浸润深度、淋巴结转移的有无均显著相关(P <0.05),且 EC 组织中 BDNF 与 TrkB 的表达具有显著相关性(r =0.508,P <0.05)。【结论】BDNF 及 TrkB 在 EC 的发生、发展中起

  7. Progress on epidermal growth factor receptor tyrosine kinase inhibitor for treatment of advanced non-small cell lung cancer%表皮生长因子受体酪氨酸激酶抑制剂治疗晚期非小细胞肺癌研究进展

    Institute of Scientific and Technical Information of China (English)

    陈佳艳; 周彩存


    表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)已成为晚期非小细胞肺癌治疗的研究热点.在有EGFR突变的患者中,一线单药EGFR-TKI能显著延长患者无进展生存期;EGFR突变是EGFR-TKI治疗获益的有效预测指标.虽然EGFR-TKI与化疗同步联合未能显示出生存优势,但在序贯联合的研究中能延长无进展生存期.EGFR-TKI维持治疗能显著延长无进展生存期.EGFR-TKI二线治疗较安慰剂有显著生存获益,与标准二线治疗总生存期相似,但安全性更好.%Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been the focus of clinical trials for treatment of advanced non-small cell lung cancer. In patients with EGFR mutation, first-line EGFR-TKI monotherapy can significantly improve progression-free survival. EGFR mutation is an effective predictor of benefit treatment. Although the use of EGFR-TKI in combination with chemotherapy fails to show a survival advantage,progression-free survival can be prolonged in the sequential study. EGFR-TKI maintenance treatment can significantly prolong progression-free survival. Second-line treatment of EGFR-TKI shows significant survival benefit compared with placebo, has similar overall survival with the standard second-line treatment, but better security.

  8. Updates on resistance mechanisms of epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer%非小细胞肺癌表皮生长因子受体酪氨酸激酶抑制剂耐药机制研究进展

    Institute of Scientific and Technical Information of China (English)

    董迪; 周彩存


    Efficacy of epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKI)such as gefitinib and erlotinib is outstanding in the second or third-line treatment of some non-small cell lung cancer patients.However,disease in nearly all patients will aquire resistance to the drugs.Therefore,a better understanding of the mechanisms of resistance to EGFR kinase inhibitor is critical to further improve the efficacy of this class of agents.Genetic mutations of EGFR,cellular phenotypic changes,internalization of EGFR,the constitutive activation of downstream signal molecules or bypass signal pathway activation may provide tumor cells the ability of being independent of EGFR signaling and acquiring resistance tO EGFRTKI.%以吉非替尼和厄罗替尼为代表的表皮生长因子受体酪氨酸激酶抑制剂在部分非小细胞肺癌患者中的治疗效果显著,但几乎所有患者最终均表现为耐药.因此,为提高该类药物的效用,对耐药性的研究便显得至关重要.基因突变、细胞表型改变、受体内化、信号通路成分及其调节因子的基因表达改变等都可以导致肿瘤细胞摆脱对表皮生长因子受体信号通路的专一性依赖而产生耐药.

  9. Research progress of non-receptor tyrosine kinase signal transducer and activator of transcription as a new target in colon cancer%非受体酪氨酸激酶-信号转导和转录激活因子信号通路作为结肠癌治疗新靶点的研究进展

    Institute of Scientific and Technical Information of China (English)

    曲林琳(综述); 续薇(审校)


    癌变的结肠细胞增殖、侵袭和转移时依赖血管生成及有利的肿瘤微环境。非受体酪氨酸激酶-信号转导和转录激活因子(JAK-STAT)信号通路参与慢性炎症相关的肿瘤发生、癌细胞增殖、肿瘤血管生成、免疫逃逸、肿瘤转移多个过程,因此有望成为分子靶向治疗的新靶点。现对JAK-STAT信号通路在结肠癌发生、发展中的研究进展进行综述,并初步探讨JAK-STAT在靶向治疗及预后评估方面的应用前景。%Malignant colon cells require angiogenesis and optimal tumor microenvironment for proliferation, invasion and metastasis. The non-receptor tyrosine kinase signal transducer and activator of transcription (JAK-STAT) pathway are involved in multiple processes, including chronic inflammation-associated tumorigenesis, cancer proliferation, tumor angiogenesis, immune evasion and facilitating tumor metastasis, making JAK-STAT a new target in molecular target therapy. In this review, advances on JAK-STAT pathway as a new target in colon tumorigenesis are updated, and a preliminary estimation of JAK-STAT in prognosis and target therapy will be mentioned to show a potential future of JAK-STAT in colon cancer.

  10. Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

    DEFF Research Database (Denmark)

    Kazi, Julhash U; Sun, Jianmin; Phung, Bengt;


    The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases...... that can regulate receptor tyrosine kinase signal transduction. In this study, we analyzed the role of SOCS6 in Flt3 signal transduction. The results show that ligand stimulation of Flt3 can induce association of SOCS6 and Flt3 and tyrosine phosphorylation of SOCS6. Phosphopeptide fishing indicated...... that SOCS6 binds directly to phosphotyrosines 591 and 919 of Flt3. By using stably transfected Ba/F3 cells with Flt3 and/or SOCS6, we show that the presence of SOCS6 can enhance ubiquitination of Flt3, as well as internalization and degradation of the receptor. The presence of SOCS6 also induces weaker...

  11. Inhibition of tyrosine phosphorylation of sperm flagellar proteins, outer dense fiber protein-2 and tektin-2, is associated with impaired motility during capacitation of hamster spermatozoa. (United States)

    Mariappa, Daniel; Aladakatti, Ravindranath H; Dasari, Santosh K; Sreekumar, Arun; Wolkowicz, Michael; van der Hoorn, Frans; Seshagiri, Polani B


    In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

  12. Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

    Institute of Scientific and Technical Information of China (English)


    Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.

  13. Amplified voltammetric characterization of cleavage of the biotinylated peptide by BACE1 and screening of BACE1 inhibitors. (United States)

    Yi, Xinyao; Han, Hongxing; Zhang, Yu; Wang, Jianxiu; Zhang, Yi; Zhou, Feimeng


    Cleavage of amyloid precursor protein (APP) by the β-site APP cleaving enzyme 1 (BACE1) is a key step in the formation of amyloid beta (Aβ) peptide, the main component of amyloid plaques in Alzheimer's disease (AD). Suppression of BACE1 activity has thus become an efficient way for the treatment of AD. In this study, BACE1 in the absence or presence of BACE1 inhibitors was exposed to the biotinylated peptide substrate-modified electrode. This step was followed by the attachment of ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. Due to the blockage of the BACE1 activity by select inhibitors, well-defined voltammetric peaks of high signal intensity were obtained. However, featureless voltammogram was obtained upon initiating the cleavage reaction. The proposed method is simple, sensitive, and suitable for monitoring of BACE1 activity and screening of BACE1 inhibitors.

  14. Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection. (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar


    Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

  15. In vitro evaluation of avidin antibody pretargeting using 211At-labeled and biotinylated poly-L-lysine as effector molecule

    DEFF Research Database (Denmark)

    Frost, Sofia H L; Jensen, Holger Lau; Lindegren, Sture


    Pretargeting is an approach for enhancing the therapeutic index of radioimmunotherapy by separating the administrations of tumor-targeting substance and radiolabel. In this study, a pretargeting model system of avidin-conjugated monoclonal antibody trastuzumab and biotinylated, (211)At-labeled po...

  16. Quantitative Analysis of Cu(I) Concentration in Click Chemistry : Biotinylation at Side Chain of Propargylglycine Using Click Chemistry under Heating Conditions


    Ogasawara, Yui; Murai, Yuta; Sakihama, Yasuko; Hashidoko, Yasuyuki; Hashimoto, Makoto


    The click reaction is one of the latest techniques for the chemical modification of bioactive compounds. Chemical modifications of α-amino acid side chains are gaining significance as useful and important tools for biochemical research. Biotinylation at side chain of propargylglycine using click reaction was examined. The detail quantitative analysis of Cu(I) concentration are performed to proceed the click reaction effectively.

  17. A Fusion Protein between Streptavidin and the Endogenous TLR4 Ligand EDA Targets Biotinylated Antigens to Dendritic Cells and Induces T Cell Responses In Vivo

    Directory of Open Access Journals (Sweden)

    Laura Arribillaga


    Full Text Available The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA, an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC, are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10−14 mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF-κβ by TLR4-expressing cells, as well as the production of TNF-α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

  18. ACK1/TNK2 tyrosine kinase: molecular signaling and evolving role in cancers. (United States)

    Mahajan, K; Mahajan, N P


    Deregulated tyrosine kinase signaling alters cellular homeostasis to drive cancer progression. The emergence of a non-receptor tyrosine kinase (non-RTK), ACK1 (also known as activated Cdc42-associated kinase 1 or TNK2) as an oncogenic kinase, has uncovered novel mechanisms by which tyrosine kinase signaling promotes cancer progression. Although early studies focused on ACK1 as a cytosolic effector of activated transmembrane RTKs, wherein it shuttles between the cytosol and the nucleus to rapidly transduce extracellular signals from the RTKs to the intracellular effectors, recent data unfold a new aspect of its functionality as an epigenetic regulator. ACK1 interacts with the estrogen receptor (ER)/histone demethylase KDM3A (JHDM2a) complex, which modifies KDM3A by tyrosine phosphorylation to regulate the transcriptional outcome at HOXA1 locus to promote the growth of tamoxifen-resistant breast cancer. It is also well established that ACK1 regulates the activity of androgen receptor (AR) by tyrosine phosphorylation to fuel the growth of hormone-refractory prostate cancers. Further, recent explosion in genomic sequencing has revealed recurrent ACK1 gene amplification and somatic mutations in a variety of human malignancies, providing a molecular basis for its role in neoplastic transformation. In this review, we will discuss the various facets of ACK1 signaling, including its newly uncovered epigenetic regulator function, which enables cells to bypass the blockade to major survival pathways to promote resistance to standard cancer treatments. Not surprisingly, cancer cells appear to acquire an 'addiction' to ACK1-mediated survival, particularly under stress conditions, such as growth factor deprivation or genotoxic insults or hormone deprivation. With the accelerated development of potent and selective ACK1 inhibitors, targeted treatment for cancers harboring aberrant ACK1 activity may soon become a clinical reality.

  19. Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eung-Yoon; Choi, Young-Jin [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of); Park, Chan-Won [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Kang, In-Cheol, E-mail: [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of)


    Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-{gamma}-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

  20. Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action


    Eldar-Finkelman, Hagit; Krebs, Edwin G.


    The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glyc...

  1. HER2-targeted therapy in breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors

    DEFF Research Database (Denmark)

    Nielsen, Dorte Lisbet; Andersson, Michael; Kamby, Claus


    There is strong clinical evidence that trastuzumab, a monoclonal antibody targeting the human epidermal growth factor receptor (HER) two tyrosine kinase receptor, is an important component of first-line treatment of patients with HER2-positive metastatic breast cancer. In particular the combination...... with taxanes and vinorelbine has been established. In the preoperative setting inclusion of trastuzumab has significantly increased the pathological complete response rate. Results from large phase III trials evaluating adjuvant therapy in HER2-positive early breast cancer indicate that the addition...... of trastuzumab to chemotherapy improves disease-free and overall survival. The use of lapatinib, a dual tyrosine kinase inhibitor of both HER1 and HER2, in combination with capecitabine in the second-line treatment of HER2-positive patients with metastatic breast cancer previously treated with trastuzumab has...

  2. Tyrosine Phosphorylation of Pyk2 Is Selectively Regulated by Fyn During TCR Signaling



    The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyros...

  3. Microglial Immunoreceptor Tyrosine-Based Activation and Inhibition Motif Signaling in Neuroinflammation

    Directory of Open Access Journals (Sweden)

    Bettina Linnartz


    Full Text Available Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis. In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAP12-associated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- (ITAM- Syk-signaling cascade. Microglial triggering receptor expressed on myeloid cells-2 (TREM2, signal regulatory protein-1, and complement receptor-3 (CD11b/CD18 signal via the adaptor protein DAP12 and activate phagocytic activity of microglia. Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- (ITIM- signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs. Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec-11 with sialic acids on the neuronal glycocalyx. Thus, ITAM- and ITIM-signaling receptors modulate microglial phagocytosis and cytokine expression during neuroinflammatory processes. Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation.

  4. Haemophilus ducreyi LspA proteins are tyrosine phosphorylated by macrophage-encoded protein tyrosine kinases. (United States)

    Deng, Kaiping; Mock, Jason R; Greenberg, Steven; van Oers, Nicolai S C; Hansen, Eric J


    The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.


    NARCIS (Netherlands)



    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumour promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as

  6. Analysis of Ligand Binding ErbB Receptor Tyrosine Kinases (United States)


    concentration), R, increases in a linear fashion We also used MALLS to analyze the ability of until one molar equivalent of EGF has been added to NRGl -j31 induce formation of s-erbBl "" homo-oligomers (assumed dimers). As with MALLS, 2.5 Ti sedimentation equilibrium studies of s-erbB4: NRGl -131 mixtures...those for the Fig. 3. MALLS studies of EGF-induced homodimenzation of s.erbBl- s-erbB3:NRGI-f31 mixture fit best as free NRGl -131 (A) and NRGl1ll

  7. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39. (United States)

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng


    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

  8. Expression of protein tyrosine phosphatase alpha (RPTPalpha) in human breast cancer correlates with low tumor grade, and inhibits tumor cell growth in vitro and in vivo

    DEFF Research Database (Denmark)

    Ardini, E; Agresti, R; Tagliabue, E;


    Tyrosine phosphorylation is controlled by a balance of tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Whereas the contribution of PTKs to breast tumorigenesis is the subject of intense scrutiny, the potential role of PTPs is poorly known. RPTPalpha is implicated in the activation......% of cases manifesting significant overexpression. High RPTPalpha protein levels correlated significantly with low tumor grade and positive estrogen receptor status. Expression of RPTPalpha in breast carcinoma cells led to growth inhibition, associated with increased accumulation in G0 and G1, and delayed...

  9. 神经生长因子NGF及其受体TrkA在雌性水牛生殖器官的表达定位%Immunohistochemical Localization of Nerve Growth Factor and Its Receptors Tyrosine Kinase A on the Genital in Female Buffalo

    Institute of Scientific and Technical Information of China (English)

    朱晓玲; 李成娇; 侯晓峰; 石德顺; 刘庆友; 周虚


    The distribution and localization of the Nerve Growth Factor(NGF) and its Receptors Tyrosine Kinase A (TrkA) in the genital of female buffalo were studied. The localization of NGF and TrkA in adult buffalo ovaries, uterus and oviduct during the follicular phase and the luteal phase were determined by immunohistochemical ABC techniques. The results showed that NGF/TrkA immunoreactive substance was mainly distributed in the thecal cells and granulose cells in the ovary, the endometrial epithelial cells and glandular cells in uterus, the ciliated colunmar epitheliar cells in the oviduct. The expression of NGF and TrkA depended on the stage of the estrus cycles, with the estrus being different, NGF/TrkA expression was also different, the expression of NGF and TrkA in ovarian had no significant differences; the expression of NGF in uterus was higher during the follicle phase than during the luteal phase, and the expression of TrkA in uterus had no significant differences; the expression of NGF and TrkA in oviduct was higher during the follicle phase than that during the luteal phase.%为研究神经生长因子NGF及其受体TrkA在广西雌性水牛生殖器官中表达定位情况,运用免疫组织化学ABC法对处于发情周期不同阶段(卵泡期、黄体期)成年水牛的卵巢、子宫、输卵管NGF、TrkA分别进行染色定位.结果表明:NGF/TrkA阳性细胞在卵巢主要见于卵泡内膜细胞、颗粒细胞及黄体细胞;在子宫主要见于子宫内膜上皮细胞、腺体细胞;在输卵管主要见于输卵管的柱状上皮纤毛细胞.不同阶段NGF、TrkA的表达量也有所差异,卵巢中NGF、TrkA的表达量在卵泡期与黄体期相比没有显著差异;子宫中卵泡期NGF的表达量高于黄体期,TrkA的表达量在卵泡期与黄体期相比没有明显差异;输卵管中卵泡期NGF、TrkA的表达量均高于相应黄体期.

  10. The effect of receptor tyrosine kinase inhibitor genistein on phenotype expression of collagen and proteoglycan of chondrocyte%三羟基异黄酮对关节软骨细胞合成胶原和蛋白多糖的影响

    Institute of Scientific and Technical Information of China (English)

    张超; 陈飞雁; 夏军; 姜建元; 黄煌渊


    Objective To explore the protective role of the receptor tyrosine kinase inhibitor in chondrocyte in vitro.MethodsReverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry (ICC) and toluidine blue staining were used to understand the changes of phenotype expression of collagenⅠ, Ⅱ and proteoglycan in chondrocyte in vitro. Results Genistein of 20 μg/mL and 25 μg/mL concentrations decreased the expression of collagen Ⅰ and increased the expression of collagen Ⅱ and proteoglycan (P< 0. 05). Conclusions This study showes that genistein can effectively inhibit the expression of collagen Ⅰ above certain concentration, and it can improve the expression of collagen Ⅱ and increase the synthesis of proteoglycan.%目的 通过对体外培养软骨细胞的实验研究,探寻受体酪氨酸激酶抑制剂对软骨细胞在细胞分子水平的影响及其可能机制.方法 对软骨细胞进行原代及传代培养.通过对培养细胞形态学及甲苯胺蓝染色观察,选择适合传代软骨细胞,应用三羟基异黄酮干预后1、2、3、4天RT-PCR、ICC及甲苯胺蓝染色观察法比较药物干预对体外培养软骨细胞Ⅰ、Ⅱ型胶原的转录和翻译水平及蛋白多糖的表达变化.结果 20 μg/mL及25μg/mL 三羟基异黄酮干预可以抑制Ⅰ型胶原表达.同时增加Ⅱ型胶原及蛋白多糖的表达.结论 三羟基异黄酮可以抑制脂多糖刺激诱导的软骨细胞反分化,对体外培养关节软骨细胞的正常表型具有一定的保护作用,其保护作用与三羟基异黄酮浓度呈现一定的相关性.

  11. Identification and Targeting of Upstream Tyrosine Kinases Mediating PI3 Kinase Activation in PTEN Deficient Prostate Cancer (United States)


    pAkt, phospho-Akt; Ab, antibody ; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GPCR , G protein-coupled receptor...tyrosine phosphorylated proteins, but they were not recognized by an anti-pYxxM motif antibody and were not found in PTEN deficient PC3 PCa cells. LC/MS/MS...immunoblotted the p85 immunoprecipitates with a pYxxM motif specific antibody . This antibody weakly detected several discrete p85 associated proteins

  12. Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium. (United States)

    Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi


    Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

  13. Masitinib (AB1010, a potent and selective tyrosine kinase inhibitor targeting KIT.

    Directory of Open Access Journals (Sweden)

    Patrice Dubreuil

    Full Text Available BACKGROUND: The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010, a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. METHODOLOGY/PRINCIPAL FINDINGS: In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC(50 of 200+/-40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC(50 of 150+/-80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. CONCLUSIONS: Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity.

  14. Morphological characteristics of the striatal neural pathway by biotinylated dextran amine tracing in rats

    Institute of Scientific and Technical Information of China (English)

    Bingbing Liu; Shuhua Mu; Lisi Ouyang; Yaxi Zhu; Keyi Li; Mali Zhan; Zongwei Liu; Yu Jia; Wanlong Lei


    Using neural pathway tracing and immunohistochemical technique, the striato-direct pathway (BDA3 kDa injected into the rat lateral globus pallidus) and striato-indirect pathway (BDA3 kDa injected into the substantia nigra pars reticulata) neurons were specifically labeled, and then subjected to double-labeled immunohistochemistry for mu-OPIOID Receptor (specifically-labeled striatal patch compartment), D1, and D2, respectively. The experimental findings showed that there are no statistically significant differences in the soma diameter and the number of primary dendrites between the striato-direct (substantia nigra pars reticularis) and indirect (globus pallidum externum) neurons labeled retrograde by BDA3 kDa. In addition, these two kinds of projection neurons revealed no obvious coexistence. This evidence indicates that as a highly sensitive neural pathway tracer, BDA could yield reliably and exquisitely detailed labeling of target neurons and synaptic structures. The variance of the morphologic structures and the localization of neurons were not statistically significant between the striato-substantia nigra pars reticularis and the globus pallidum externum projection neurons. Mesencephalic and thalamic neurons correlated with striatal neurons in morphology. Especially the latter which make typical excitatory synaptic contacts with striato-direct and -indirect neurons. Thus, this evidence suggests that thalamic neurons may extensively excite striatal neurons.

  15. Sulfated tyrosines 27 and 29 in the N-terminus of human CXCR3 participate in binding native IP-10

    Institute of Scientific and Technical Information of China (English)

    Jinming GAO; Ruolan XIANG; Lei JIANG; Wenhui LI; Qiping FENG; Zijiang GUO; Qi SUN; Zhengpei ZENG; Fude FANG


    Aim:Human CXCR3,a seven-transmembrane segment (7TMS),is predominantly expressed in Th1-mediated responses.Interferon-γ-inducible protein 10 (IP-10) is an important ligand for CXCR3.Their interaction is pivotal for leukocyte migra-tion and activation.Tyrosine sulfation in 7TMS is a posttranslational modification that contributes substantially to ligand binding.We aimed to study the role of tyrosine sulfation of CXCR3 in the protein's binding to IP-10.Methods: Plasmids encoding CXCR3 and its mutants were prepared by PCR and site-directed mutagenesis.HEK 293T cells were transfected with plasmids encoding CXCR3 or its variants using calcium phosphate.Transfected cells were labeled with [35S]-cysteine and methionine or [35S]-Na2SO3 and then analyzed by immunoprecipitation to measure sulfation.Experi-ments with 125I-labeled IP-10 were carried out to evaluate the affinity of CXCR3 for its ligand.Calcium influx assays were used to measure intercellular signal transduction.Results: Our data show that sulfate moieties are added to tyrosines 27 and 29 of CXCR3.Mutation of these two tyrosines to phenylalanines substantially decreases binding of CXCR3 to IP-10 and appears to eliminate the associated signal transduc-tion.Tyrosine sulfation of CXCR3 is enhanced by tyrosyl protein sulfotransferases (TPSTs),and it is weakened by shRNA constructs.The binding ability of CXCR3 to IP-10 is increased by TPSTs and decreased by shRNAs.Conclusion: This study identifies two sulfated tyrosines in the N-terminus of CXCR3 as part of the binding site for IP-10,and it underscores the fact that tyrosine sulfation in the N-termini of 7TMS receptors is functionally important for ligand interactions.Our study suggests a molecular target for inhibiting this ligand-receptor interaction.

  16. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.


    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  17. Phenylketonuria : Tyrosine beyond the phenylalanine-restricted diet

    NARCIS (Netherlands)

    van Spronsen, FJ; Smit, PGA; Koch, R


    Controversies exist on the role of tyrosine in the pathogenesis of phenylketonuria (PKU) and, consequently, on the therapeutic role of tyrosine. This review examines data and theoretical considerations on the role of tyrosine in the pathogenesis and treatment of PKU. It is concluded that treatment w

  18. Development of a candidate method for forensic microbial genotyping using multiplex pyrosequencing combined with a universal biotinylated primer. (United States)

    Gu, Yan; Mao, Xuhu; Zha, Lagabaiyila; Hou, Yiping; Yun, Libing


    Bacterial genotyping can be used for crime scene investigations and contribute to the attribution of biological attacks for microbial forensics. PyroMark ID Pyrosequencer as an accurate detection platform for single nucleotide polymorphisms (SNPs) has been applied to identify and resolve microorganisms involved in closely Escherichia coli O157:H7 (E. coli O157:H7). To explore more applications and improve the efficiency for pyrosequencing in this field, we developed a method integrated multiplex pyrosequencing with a universal primer. Two multiplex pyrosequencing assays with a universal biotinylated primer were designed to analyze five SNPs located in four gene of E. coli O157:H7 strain. The accuracy of the established assays was validated by genotyping reference strain E. coli O157:H7 EDL933 and E. coli K-12. We also demonstrated that two multiplex pyrosequencing assays were specific and sensitive for genotyping closely related E. coli O157 strains. Reproducibility of results and multiplexing capability were evaluated by a comparison of this method with the monoplex pyrosequencing. Furthermore, these two multiplex pyrosequencing assays have been successfully applied to detect 11 E. coli O157 strains isolated from 1504 Chinese livestock samples. This method reduces costs and time consumption in the process of pyrosequencing analysis, and potentially serves as a rapid tool and reliable candidate strategy for the microbial identification and other genotyping application.

  19. Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt) Cry1Ac Toxin (United States)

    Li, Min; Zhu, Min; Zhang, Cunzheng; Liu, Xianjin; Wan, Yakun


    Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system. PMID:25474492

  20. Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt Cry1Ac Toxin

    Directory of Open Access Journals (Sweden)

    Min Li


    Full Text Available Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies sandwich-ELISA (DAS-ELISA assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac were selected as capture antibody (Nb61 and detection antibody (Nb44. The capture antibody (Nb61 was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.

  1. A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine. (United States)

    Ibarrola, Nieves; Molina, Henrik; Iwahori, Akiko; Pandey, Akhilesh


    Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeli