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Sample records for factor receptor single-chain

  1. [Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

    Science.gov (United States)

    Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

    2009-06-01

    Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

  2. [Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

    Science.gov (United States)

    Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

    2010-01-01

    To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

  3. Antiproliferative and apoptotic effects of a specific anti-insulin-like growth factor I receptor single chain antibody on breast cancer cells.

    Science.gov (United States)

    Motallebnezhad, Morteza; Younesi, Vahid; Aghebati-Maleki, Leili; Nickho, Hamid; Safarzadeh, Elham; Ahmadi, Majid; Movassaghpour, Ali Akbar; Hosseini, Ahmad; Yousefi, Mehdi

    2016-11-01

    Insulin-like growth factor I receptor (IGF-IR) is expressed on breast cancer cells and involves in metastasis, survival, and proliferation. Currently, application of IGF-IR-targeting monoclonal antibodies (mAbs), alone or in combination with other drugs, is a promising strategy for breast cancer therapy. Single-chain fragment variable (scFv) antibodies have been introduced as appropriate tools for tumor-targeting purposes because of their advantages over whole antibodies. In the present study, we employed a naïve phage library and isolated scFvs against a specific epitope from extracellular domain of IGF-IR by panning process. The selected scFvs were further characterized using polyclonal and monoclonal phage ELISA, soluble monoclonal ELISA, and colony PCR and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies on breast cancer cell lines were also evaluated by MTT and Annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the IGF-IR peptide, and analyses of PCR product and sequencing confirmed the presence of full length V H and Vκ inserts. Treatment of MCF7 and SKBR3 cells with anti-IGF-IR scFv led to a significant growth inhibition. The results also showed that scFv treatment significantly augmented trastuzumab growth inhibitory effects on SKBR3 cells. The percentage of the apoptotic MCF7 and SKBR3 cells after 24-h treatment with scFv was 39 and 30.70 %, respectively. Twenty-four-hour treatment with scFv in combination with trastuzumab resulted in 44.75 % apoptosis of SKBR3 cells. Taken together, our results demonstrate that the targeting of IGF-IR by scFv can be an effective strategy in the treatment of breast cancer and provide further evidence for effectiveness of dual targeting of HER2 and IGF-IR in breast cancer therapy.

  4. A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors

    NARCIS (Netherlands)

    Annenkov, A.; Rigby, A.; Amor, S.; Zhou, D.M.; Yousaf, N.; Hemmer, B.; Chernajovsky, Y.

    2011-01-01

    In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 (IGF1R)

  5. A chimeric receptor of the insulin-like growth factor receptor type 1 (IGFR1) and a single chain antibody specific to myelin oligodendrocyte glycoprotein activates the IGF1R signalling cascade in CG4 oligodendrocyte progenitors.

    Science.gov (United States)

    Annenkov, Alexander; Rigby, Anne; Amor, Sandra; Zhou, Dun; Yousaf, Nasim; Hemmer, Bernhard; Chernajovsky, Yuti

    2011-08-01

    In order to generate neural stem cells with increased ability to survive after transplantation in brain parenchyma we developed a chimeric receptor (ChR) that binds to myelin oligodendrocyte glycoprotein (MOG) via its ectodomain and activates the insulin-like growth factor receptor type 1 ‎‎(IGF1R) signalling cascade. Activation of this pro-survival pathway in response to ligand broadly available in the brain might increase neuroregenerative potential of transplanted precursors. The ChR was produced by fusing a MOG-specific single ‎chain antibody with the extracellular boundary of the IGF1R transmembrane segment. The ChR is expressed on the cellular surface, predominantly as a monomer, and is not N-glycosylated. To show MOG-dependent functionality of the ChR, neuroblastoma cells B104 expressing this ChR were stimulated with monolayers of cells expressing recombinant MOG. The ChR undergoes MOG-dependent tyrosine phosphorylation and homodimerisation. It promotes insulin and IGF-independent growth of the oligodendrocyte progenitor cell line CG4. The proposed mode of the ChR activation is by MOG-induced dimerisation which promotes kinase domain transphosphorylation, by-passing the requirement of conformation changes known to be important for IGF1R activation. Another ChR, which contains a segment of the β-chain ectodomain, was produced in an attempt to recapitulate some of these conformational changes, but proved non-functional. 2011 Elsevier B.V. All rights reserved.

  6. Population pharmacokinetics of recombinant coagulation factor VIII-SingleChain in patients with severe hemophilia A.

    Science.gov (United States)

    Zhang, Y; Roberts, J; Tortorici, M; Veldman, A; St Ledger, K; Feussner, A; Sidhu, J

    2017-06-01

    Essentials rVIII-SingleChain is a unique recombinant factor VIII (FVIII) molecule. A population pharmacokinetic model was based on FVIII activity of severe hemophilia A patients. The model was used to simulate factor VIII activity-time profiles for various dosing scenarios. The model supports prolonged dosing of rVIII-SingleChain with intervals of up to twice per week. Background Single-chain recombinant coagulation factor VIII (rVIII-SingleChain) is a unique recombinant coagulation factor VIII molecule. Objectives To: (i) characterize the population pharmacokinetics (PK) of rVIII-SingleChain in patients with severe hemophilia A; (ii) identify correlates of variability in rVIII-SingleChain PK; and (iii) simulate various dosing scenarios of rVIII-SingleChain. Patients/Methods A population PK model was developed, based on FVIII activity levels of 130 patients with severe hemophilia A (n = 91 for ≥ 12-65 years; n = 39 for  85% and > 93% of patients were predicted to maintain FVIII activity level above 1 IU dL -1 , at all times with three-times-weekly dosing (given on days 0, 2, and 4.5) at the lowest (20 IU kg -1 ) and highest (50 IU kg -1 ) doses, respectively. For twice weekly dosing (days 0 and 3.5) of 50 IU kg -1 rVIII-SingleChain, 62-80% of patients across all ages were predicted to maintain a FVIII activity level above 1 IU dL -1 at day 7. Conclusions The population PK model adequately characterized rVIII-SingleChain PK, and the model can be utilized to simulate FVIII activity-time profiles for various dosing scenarios. © 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

  7. Single-chain vascular endothelial growth factor variant with antagonist activity

    DEFF Research Database (Denmark)

    Boesen, Thomas P; Soni, Bobby; Schwartz, Thue W

    2002-01-01

    receptor molecules and inducing dimerization. By mixing two vascular endothelial growth factor monomers, each with different substitutions, heterodimers with only one active receptor binding site have previously been prepared. These heterodimers bind the receptor molecule but are unable to induce...... dimerization and activation. However, preparation of heterodimers is cumbersome, involving separate expression of different monomers, refolding the mixture, and separating heterodimers from homodimers. Here we show that a fully functional ligand can efficiently be expressed as a single protein chain containing...

  8. Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

    Directory of Open Access Journals (Sweden)

    Yang J

    2014-05-01

    Full Text Available Jingjing Yang,1,3,* Xiaoping Huang,1,3,* Fanghong Luo,1 Xiaofeng Cheng,3 Lianna Cheng,3 Bin Liu,4 Lihong Chen,2 Ruyi Hu,1,3 Chunyan Shi,1,3 Guohong Zhuang,1,3 Ping Yin2 1Anti-Cancer Research Center, Medical College, Xiamen University, Fujian, People's Republic of China, 2The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, People's Republic of China, 3Organ transplantation institution, Xiamen University, Xiamen, People's Republic of China, 4Jilin Vocational College of Industry and Technology, Jilin, People's Republic of China  *These authors contributed equally to this work Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action. Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors. Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected

  9. Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA

    Directory of Open Access Journals (Sweden)

    Bogen Bjarne

    2010-02-01

    Full Text Available Abstract Background Efficient expression systems exist for antibody (Ab molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs. Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc TCRs. Results The effect of 1 over-expression of the periplasmic chaperon FkpA, 2 culture conditions and 3 molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules. Conclusion The general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1 high yield recovery sufficient for biophysical characterization and 2 high throughput screening of such molecules following molecular engineering.

  10. Advances in single chain technology.

    Science.gov (United States)

    Gonzalez-Burgos, Marina; Latorre-Sanchez, Alejandro; Pomposo, José A

    2015-10-07

    The recent ability to manipulate and visualize single atoms at atomic level has given rise to modern bottom-up nanotechnology. Similar exquisite degree of control at the individual polymeric chain level for producing functional soft nanoentities is expected to become a reality in the next few years through the full development of so-called "single chain technology". Ultra-small unimolecular soft nano-objects endowed with useful, autonomous and smart functions are the expected, long-term valuable output of single chain technology. This review covers the recent advances in single chain technology for the construction of soft nano-objects via chain compaction, with an emphasis in dynamic, letter-shaped and compositionally unsymmetrical single rings, complex multi-ring systems, single chain nanoparticles, tadpoles, dumbbells and hairpins, as well as the potential end-use applications of individual soft nano-objects endowed with useful functions in catalysis, sensing, drug delivery and other uses.

  11. Pressure effects on single chain magnets

    Energy Technology Data Exchange (ETDEWEB)

    Mito, M. E-mail: mitoh@elcs.kyutech.ac.jp; Shindo, N.; Tajiri, T.; Deguchi, H.; Takagi, S.; Miyasaka, H.; Yamashita, M.; Clerac, R.; Coulon, C

    2004-05-01

    Pressure effects on a single chain magnet [Mn{sub 2}(saltmen){sub 2}Ni(pao){sub 2}(py){sub 2}](ClO{sub 4}){sub 2} (saltmen{sup 2-}=N,N'-(1,1,2,2-tetramethylethylene)bis(salicylideneiminate), and pao{sup -}=pyridine-2-aldoximate) have been investigated through AC magnetic measurements under pressure (P). The slow relaxation of the magnetization depends on pressure. Both the blocking temperature (T{sub B}) and energy barrier ({delta}) increase by pressurization, and those enhancements saturate at around P=7 kbar.

  12. Elementary excitations in single-chain magnets

    Science.gov (United States)

    Lutz, Philipp; Aguilà, David; Mondal, Abhishake; Pinkowicz, Dawid; Marx, Raphael; Neugebauer, Petr; Fâk, Björn; Ollivier, Jacques; Clérac, Rodolphe; van Slageren, Joris

    2017-09-01

    Single-chain magnets (SCMs) are one-dimensional coordination polymers or spin chains that display slow relaxation of the magnetization. Typically their static magnetic properties are described by the Heisenberg model, while the description of their dynamic magnetic properties is based on an Ising-like model. The types of excitations predicted by these models (collective vs localized) are quite different. Therefore we probed the nature of the elementary excitations for two SCMs abbreviated Mn2Ni and Mn2Fe , as well as a mononuclear derivative of the Mn2Fe chain, by means of high-frequency electron paramagnetic resonance spectroscopy (HFEPR) and inelastic neutron scattering (INS). We find that the HFEPR spectra of the chains are clearly distinct from those of the monomer. The momentum transfer dependence of the INS intensity did not reveal significant dispersion, indicating an essentially localized nature of the excitations. At the lowest temperatures these are modified by the occurrence of short-range correlations.

  13. Domain walls in single-chain magnets

    Science.gov (United States)

    Pianet, Vivien; Urdampilleta, Matias; Colin, Thierry; Clérac, Rodolphe; Coulon, Claude

    2017-12-01

    The topology and creation energy of domain walls in different magnetic chains (called Single-Chain Magnets or SCMs) are discussed. As these domain walls, that can be seen as "defects", are known to control both static and dynamic properties of these one-dimensional systems, their study and understanding are necessary first steps before a deeper discussion of the SCM properties at finite temperature. The starting point of the paper is the simple regular ferromagnetic chain for which the characteristics of the domain walls are well known. Then two cases will be discussed (i) the "mixed chains" in which isotropic and anisotropic classical spins alternate, and (ii) the so-called "canted chains" where two different easy axis directions are present. In particular, we show that "strictly narrow" domain walls no longer exist in these more complex cases, while a cascade of phase transitions is found for canted chains as the canting angle approaches 45∘. The consequence for thermodynamic properties is briefly discussed in the last part of the paper.

  14. Fibroblast growth factor receptors in breast cancer.

    Science.gov (United States)

    Wang, Shuwei; Ding, Zhongyang

    2017-05-01

    Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

  15. Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.

    Science.gov (United States)

    Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi

    2017-12-01

    The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.

  16. Single-Chain Folding of Synthetic Polymers: A Critical Update.

    Science.gov (United States)

    Altintas, Ozcan; Barner-Kowollik, Christopher

    2015-11-23

    The current contribution serves as a critical update to a previous feature article from us (Macromol. Rapid Commun. 2012, 33, 958-971), and highlights the latest advances in the preparation of single chain polymeric nanoparticles and initial-yet promising-attempts towards mimicking the structure of natural biomacromolecules via single-chain folding of well-defined linear polymers via so-called single chain selective point folding and repeat unit folding. The contribution covers selected examples from the literature published up to ca. September 2015. Our aim is not to provide an exhaustive review but rather highlight a selection of new and exciting examples for single-chain folding based on advanced macromolecular precision chemistry. Initially, the discussion focuses on the synthesis and characterization of single-chain folded structures via selective point folding. The second part of the feature article addresses the folding of well-defined single-chain polymers by means of repeat unit folding. The current state of the art in the field of single-chain folding indicates that repeat unit folding-driven nanoparticle preparation is well-advanced, while initial encouraging steps towards building selective point folding systems have been taken. In addition, a summary of the-in our view-open key questions is provided that may guide future biomimetic design efforts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Abstract. Purpose: To develop specific single chain variable fragments (scFv) against ... libraries. The binding ability of the selected scFv antibody fragments against the IBDV particles was ..... Hermelink H, Koscielniak E. A human recombinant.

  18. Optimization of the crystallizability of a single-chain antibody fragment

    Czech Academy of Sciences Publication Activity Database

    Škerlová, Jana; Král, Vlastimil; Fábry, Milan; Sedláček, Juraj; Veverka, Václav; Řezáčová, Pavlína

    2014-01-01

    Roč. 70, č. 12 (2014), s. 1701-1706 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : single-chain antibody fragment * Thermofluor assay * differential scanning fluorimetry * crystallizability optimization * oligomerization * crystallization Subject RIV: CE - Biochemistry Impact factor: 0.527, year: 2014

  19. The relationship between the connecting peptide of recombined single chain insulin and its biological function

    Institute of Scientific and Technical Information of China (English)

    HUANG; Yiding; (

    2001-01-01

    [1]Straus, D. S., Growth-stimulatory of insulin in vitro and in vivo, Endocr. Rev., 1984, 5(2): 356-369.[2]Svenningsen, A. F., Kanje, M., Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H] thuymidine incorporation through their respective receptors, Glia, 1996, 18(1): 68-72.[3]Ogihara, S., Yamada, M., Saito, T. et al., Insulin potentiates mitogenic effect of epidermal growth factor on cultured guinea pig gastric mucous cells, Am. J. Physiol., 1996, 271(1 Pt 1): G104-121.[4]Steiner, D. F., Oyer, P. E., The biosynthesis of insulin and a probable precursor of insulin by a human islet cell adenoma, Proc. Nalt. Acad. Sci. USA, 1967, 57(2): 473-480.[5]King, G. L., Kahn, C. R., The growth-promoting effects of insulin, in Growth and Maturation Factors(ed. Guroff, G.), New York: John Wiley & Sons, 1984, 223-265.[6]Peavy, D. E., Brunner, M. R., Duckworth, W. C. et al., Receptor binding and biological potency of several split forms (conversion intermediates) of human proinsulin, Studies in cultured IM-9 lymphocytes and in vivo and in vitro in rats, J. Biol. Chem., 1985, 260: 13989-13994.[7]Derewenda, U., Derewenda, Z., Dodson, E. J. et al., X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue, J. Mol. Biol., 1991, 220: 425-433.[8]Hua, Q. X., Shoelson, S. E., Kochoyan, M. et al., Receptor binding redefined by a structural switch in a mutant human insulin, Nature, 1991, 354: 238-241.[9]Hua, Q. X., Gozani, S. N., Chance, R. E. et al., Structure of a protein in a kinetic trap, Nat. Struc. Boil, 1995, 2: 129-138.[10]Kristensen, C., Andersen, A. S., Hach, M., A single-chain insulin-like growth factor I/insulin hybrid binds with high affinity to the insulin receptor, Biochem. J., 1995, 305: 981-986.[11]Humbel, R. E., Insulin-like growth factors I and II, Euro. J. Biochem., 1990, 190: 445-462.[12]Cooke, R. M

  20. Production of a phage-displayed single chain variable fragment ...

    African Journals Online (AJOL)

    Purpose: To develop specific single chain variable fragments (scFv) against infectious bursal disease virus (IBDV) via phage display technology. Methods: Purified viruses were initially applied for iterative panning rounds of scFv phage display libraries. The binding ability of the selected scFv antibody fragments against the ...

  1. Expression, production and renaturation of a functional single-chain ...

    African Journals Online (AJOL)

    The single-chain variable antibody fragment (scFv) against human intercellular adhesion molecule-1 (ICAM-1) was expressed at a high level in Escherichia coli as inclusion bodies. We attempted to refold the scFv by ion-exchange chromatography (IEC), dialysis and dilution. The results show that the column ...

  2. Development of two dimensional electrophoresis method using single chain DNA

    International Nuclear Information System (INIS)

    Ikeda, Junichi; Hidaka, So

    1998-01-01

    By combining a separation method due to molecular weight and a method to distinguish difference of mono-bases, it was aimed to develop a two dimensional single chain DNA labeled with Radioisotope (RI). From electrophoretic pattern difference of parent and variant strands, it was investigated to isolate the root module implantation control gene. At first, a Single Strand Conformation Polymorphism (SSCP) method using concentration gradient gel was investigated. As a result, it was formed that intervals between double chain and single chain DNAs expanded, but intervals of both single chain DNAs did not expand. On next, combination of non-modified acrylic amide electrophoresis method and Denaturing Gradient-Gel Electrophoresis (DGGE) method was examined. As a result, hybrid DNA developed by two dimensional electrophoresis arranged on two lines. But, among them a band of DNA modified by high concentration of urea could not be found. Therefore, in this fiscal year's experiments, no preferable result could be obtained. By the used method, it was thought to be impossible to detect the differences. (G.K.)

  3. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment

    International Nuclear Information System (INIS)

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-01-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies

  4. Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

    Science.gov (United States)

    Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

    2018-01-01

    Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

  5. SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

    Directory of Open Access Journals (Sweden)

    Oliinyk O. S.

    2014-02-01

    Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

  6. Monte Carlo simulated dynamical magnetization of single-chain magnets

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jun; Liu, Bang-Gui, E-mail: bgliu@iphy.ac.cn

    2015-03-15

    Here, a dynamical Monte-Carlo (DMC) method is used to study temperature-dependent dynamical magnetization of famous Mn{sub 2}Ni system as typical example of single-chain magnets with strong magnetic anisotropy. Simulated magnetization curves are in good agreement with experimental results under typical temperatures and sweeping rates, and simulated coercive fields as functions of temperature are also consistent with experimental curves. Further analysis indicates that the magnetization reversal is determined by both thermal-activated effects and quantum spin tunnelings. These can help explore basic properties and applications of such important magnetic systems. - Highlights: • Monte Carlo simulated magnetization curves are in good agreement with experimental results. • Simulated coercive fields as functions of temperature are consistent with experimental results. • The magnetization reversal is understood in terms of the Monte Carlo simulations.

  7. Novel scenario of the folding transition of a single chain

    International Nuclear Information System (INIS)

    Yoshikawa, Kenichi; Yoshinaga, Natsuhiko

    2005-01-01

    Unique characteristics of a single polymer chain with the effects of stiffness and charge are discussed. It has been well established that a flexible polymer chain undergoes a continuous transition from an elongated coil to a compact globule, corresponding to the transition between disordered gas-like and disordered liquid-like states. Here, we will show that a semiflexible chain exhibits a discrete transition from coil to compact states, corresponding to a disorder-order transition to an ordered crystalline state. We will propose a novel strategy to obtain various kinds of nano-ordered structures from single chains connecting a pair of chains of different stiffness. We will also discuss the effect of charge, putting emphasis on intramolecular segregation in a single polyelectrolyte chain

  8. Targeting nanodisks via a single chain variable antibody - Apolipoprotein chimera

    International Nuclear Information System (INIS)

    Iovannisci, David M.; Beckstead, Jennifer A.; Ryan, Robert O.

    2009-01-01

    Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that α-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

  9. Genetically engineered multivalent single chain antibody constructs for cancer therapy

    International Nuclear Information System (INIS)

    Surinder Batra

    2006-01-01

    increase its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

  10. Epidermal Growth Factor Receptor in Pancreatic Cancer

    International Nuclear Information System (INIS)

    Oliveira-Cunha, Melissa; Newman, William G.; Siriwardena, Ajith K.

    2011-01-01

    Pancreatic cancer is the fourth leading cause of cancer related death. The difficulty in detecting pancreatic cancer at an early stage, aggressiveness and the lack of effective therapy all contribute to the high mortality. Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein, which is expressed in normal human tissues. It is a member of the tyrosine kinase family of growth factors receptors and is encoded by proto-oncogenes. Several studies have demonstrated that EGFR is over-expressed in pancreatic cancer. Over-expression correlates with more advanced disease, poor survival and the presence of metastases. Therefore, inhibition of the EGFR signaling pathway is an attractive therapeutic target. Although several combinations of EGFR inhibitors with chemotherapy demonstrate inhibition of tumor-induced angiogenesis, tumor cell apoptosis and regression in xenograft models, these benefits remain to be confirmed. Multimodality treatment incorporating EGFR-inhibition is emerging as a novel strategy in the treatment of pancreatic cancer

  11. Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Yijia [Department; Ford, Nicole R. [Marine; Hecht, Karen A. [Marine; Roesijadi, Guritno [Marine; Department; Squier, Thomas C. [Department

    2017-10-12

    Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39 amino-acid targeting sequence (Sil3T8) to direct a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundances in excess of 200,000 proteins per frustule. The fluorescence of either a derivative of trinitrotoluene (TNT) bound to the scFv or the endogenous fluorescence of EGFP was used to monitor pro-tein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. We find that proteins within isolated frustules undergo isotropic rotational motions with two-fold increases in rotational correlation times, which are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibod-ies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). These results argue that dramatic increases in protein conforma-tional stability within the biosilica frustule matrices arise through molecular crowding, acting to retain native protein folds and associated functionality to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

  12. Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis.

    Science.gov (United States)

    Shigemori, Suguru; Ihara, Masaki; Sato, Takashi; Yamamoto, Yoshinari; Nigar, Shireen; Ogita, Tasuku; Shimosato, Takeshi

    2017-01-01

    Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.

  13. Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

    Science.gov (United States)

    Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

    2017-02-01

    Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Pattern of hormone receptors and human epidermal growth factor ...

    African Journals Online (AJOL)

    Introduction: Breast cancer is the most common cancer among women globally. With immunohistochemistry (IHC), breast cancer is classified into four groups based on IHC profile of estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2/neu) expression, positive (+) and/or ...

  15. Enantiopure heterobimetallic single-chain magnets from the chiral Ru(III) building block.

    Science.gov (United States)

    Ru, Jing; Gao, Feng; Wu, Tao; Yao, Min-Xia; Li, Yi-Zhi; Zuo, Jing-Lin

    2014-01-21

    A pair of one-dimensional enantiomers based on the versatile chiral dicyanoruthenate(III) building block have been synthesized and they are chiral single-chain magnets with the effective spin-reversal barrier of 28.2 K.

  16. Molecular analysis of the nerve growth factor receptor

    International Nuclear Information System (INIS)

    Hempstead, B.; Patil, N.; Olson, K.; Chao, M.

    1988-01-01

    An essential molecule in the translocation of information by nerve growth factor (NGF) to responsive cells is the cell-surface receptor for NGF. This paper presents information on the genomic structure of the NGF receptor gene, NGF receptor models, and transfection of NGF receptors. Equilibrium binding of [ 125 I]NGF to cells reveals two distinct affinity states for the NGF receptor. The human NGF receptor gene is a single-copy gene, consisting of six exons that span 23 kb. The receptor gene is capable of being transferred to fibroblast cells from human genomic DNA and expressed at high levels. The constitutive nature of the receptor promoter sequence is a partial explanation of why this tissue-specific gene is expressed efficiently in a variety of nonneuronal cells after genomic gene transfer. The two kinetic forms of the NGF receptor appear to be encoded by the same protein, which is the product of a single gene

  17. Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering

    NARCIS (Netherlands)

    van Belzen, N.; Spaargaren, M.; Verkleij, A. J.; Boonstra, J.

    1990-01-01

    Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction,

  18. A family of rare-earth-based single chain magnets: playing with anisotropy.

    Science.gov (United States)

    Bernot, Kevin; Bogani, Lapo; Caneschi, Andrea; Gatteschi, Dante; Sessoli, Roberta

    2006-06-21

    The first family of rare-earth-based single chain magnets is presented. Compounds of general formula [M(hfac)3(NITPhOPh)], where M = Eu, Gd, Tb, Dy, Ho, Er, or Yb, and PhOPh is the nitronyl-nitroxide radical (2,4'-benzoxo-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), have been structurally characterized and found to be isostructural. The characterization of both static and dynamic magnetic properties of the whole family is reported. Dy, Tb, and Ho compounds display slow relaxation of the magnetization, and ac susceptibility shows a thermally activated regime with energy barriers of 69, 45, and 34 K for Dy, Tb, and Ho compounds, respectively, while only a frequency-dependent susceptibility is observed for Er below 2.0 K. In Gd and Yb derivatives, antiferromagnetic interactions dominate. The pre-exponential factors differ by about 4 orders of magnitude. Finite size effects, due to naturally occurring defects, affect the static and dynamic properties of the compounds differently.

  19. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A

    Science.gov (United States)

    Mahlangu, Johnny; Kuliczkowski, Kazimierz; Karim, Faraizah Abdul; Stasyshyn, Oleksandra; Kosinova, Marina V.; Lepatan, Lynda Mae; Skotnicki, Aleksander; Boggio, Lisa N.; Klamroth, Robert; Oldenburg, Johannes; Hellmann, Andrzej; Santagostino, Elena; Baker, Ross I.; Fischer, Kathelijn; Gill, Joan C.; P’Ng, Stephanie; Chowdary, Pratima; Escobar, Miguel A.; Khayat, Claudia Djambas; Rusen, Luminita; Bensen-Kennedy, Debra; Blackman, Nicole; Limsakun, Tharin; Veldman, Alex; St. Ledger, Katie

    2016-01-01

    Recombinant VIII (rVIII)-SingleChain is a novel B-domain–truncated recombinant factor VIII (rFVIII), comprised of covalently bonded factor VIII (FVIII) heavy and light chains. It was designed to have a higher binding affinity for von Willebrand factor (VWF). This phase 1/3 study investigated the efficacy and safety of rVIII-SingleChain in the treatment of bleeding episodes, routine prophylaxis, and surgical prophylaxis. Participants were ≥12 years of age, with severe hemophilia A (endogenous FVIII <1%). The participants were allocated by the investigator to receive rVIII-SingleChain in either an on-demand or prophylaxis regimen. Of the 175 patients meeting study eligibility criteria, 173 were treated with rVIII-SingleChain, prophylactically (N = 146) or on-demand (N = 27). The total cumulative exposure was 14 306 exposure days (EDs), with 120 participants reaching ≥50 EDs and 52 participants having ≥100 EDs. Hemostatic efficacy was rated by the investigator as excellent or good in 93.8% of the 835 bleeds treated and assessed. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.0, 2.4) and the median overall annualized bleeding rate (ABR) was 1.14 (Q1, Q3: 0.0, 4.2). Surgical hemostasis was rated as excellent/good in 100% of major surgeries by the investigator. No participant developed FVIII inhibitors. In conclusion, rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy in surgery and in the control of bleeding events, low ABR in patients on prophylaxis, and a favorable safety profile in this large clinical study. This trial was registered at www.clinicaltrials.gov as #NCT01486927. PMID:27330001

  20. Safety, efficacy and pharmacokinetics of rVIII-SingleChain in children with severe hemophilia A: results of a multicenter clinical trial.

    Science.gov (United States)

    Stasyshyn, O; Djambas Khayat, C; Iosava, G; Ong, J; Abdul Karim, F; Fischer, K; Veldman, A; Blackman, N; St Ledger, K; Pabinger, I

    2017-04-01

    Essentials rVIII-SingleChain is a novel recombinant factor VIII with covalently bonded heavy and light chains. Efficacy, safety and pharmacokinetics were studied in pediatric patients with severe hemophilia A. Across all prophylaxis regimens, the median annualized spontaneous bleeding rate was 0.00. rVIII-SingleChain showed excellent hemostatic efficacy and a favorable safety profile. Background rVIII-SingleChain is a novel B-domain truncated recombinant factor VIII (rFVIII) comprised of covalently bonded FVIII heavy and light chains, demonstrating a high binding affinity to von Willebrand factor. Objectives This phase III study investigated the safety, efficacy and pharmacokinetics of rVIII-SingleChain in previously treated pediatric patients hemophilia A. Patients/Methods Patients could be assigned to prophylaxis or on-demand therapy by the investigator. For patients assigned to prophylaxis, the treatment regimen and dose were based on the bleeding phenotype. For patients receiving on-demand therapy, dosing was guided by World Federation of Hemophilia recommendations. The primary endpoint was treatment success, defined as a rating of 'excellent' or 'good' on the investigator's clinical assessment of hemostatic efficacy for all treated bleeding events. Results The study enrolled 84 patients (0 to 50 EDs. In the 347 bleeds treated and evaluated by the investigator, hemostatic efficacy was rated as excellent or good in 96.3%. The median annualized spontaneous bleeding rate was 0.00 (Q1, Q3: 0.00, 2.20), and the median annualized bleeding rate was 3.69 (Q1, Q3: 0.00, 7.20) across all prophylaxis regimens. No participant developed an inhibitor. Conclusions rVIII-SingleChain is a novel rFVIII molecule showing excellent hemostatic efficacy and a favorable safety profile in a clinical study in children hemophilia A. © 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and

  1. Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    Directory of Open Access Journals (Sweden)

    Begoña Barriuso

    2016-06-01

    Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

  2. Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

    Science.gov (United States)

    Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-06-10

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

  3. Urokinase-type plasminogen activator receptor (uPAR), tissue factor (TF) and epidermal growth factor receptor (EGFR)

    DEFF Research Database (Denmark)

    Christensen, Anders; Kiss, Katalin; Lelkaitis, Giedrius

    2017-01-01

    Background: Tumor-specific biomarkers are a prerequisite for the development of targeted imaging and therapy in oral squamous cell carcinoma (OSCC). urokinase-type Plasminogen Activator Receptor (uPAR), Tissue Factor (TF) and Epidermal Growth Factor Receptor (EGFR) are three biomarkers that exhib...... with a reduced survival. uPAR seems to be a prognostic biomarker in oral cancer....

  4. Radiotherapy and receptor of epidermal growth factor

    International Nuclear Information System (INIS)

    Deberne, M.

    2009-01-01

    The expression level of the receptor of the epidermal growth factor is in correlation with the tumor cells radiosensitivity. An overexpression of the E.G.F.R. is often present in the bronchi cancer, epidermoid carcinomas of the O.R.L. sphere, esophagus, uterine cervix, and anal duct but also in the rectum cancers and glioblastomas. At the clinical level, the E.G.F.R. expression is in correlation with an unfavourable prognosis after radiotherapy in numerous tumoral localizations. In the rectum cancers it is an independent prognosis factor found in multifactorial analysis: increase of the rate of nodes and local recurrence when the E.G.F.R. is over expressed. In the uterine cervix cancers, the survival is is negatively affected in multifactorial analysis by the E.G.F.R. membranes expression level. At the therapy level, the development of anti E.G.F.R. targeted therapies (tyrosine kinase inhibitors and monoclonal antibodies) opens a new therapy field at radio-sensitivity potentiality. The irradiation makes an activation of the E.G.F.R. way that would be partially responsible of the post irradiation tumoral repopulation. This activation leads the phosphorylation of the PI3 kinase ways and M.A.P. kinase ones, then the Akt protein one that acts an apoptotic modulator part. It has been shown that blocking the E.G.F.R. way acts on three levels: accumulation of ells in phase G1, reduction of the cell repair and increasing of apoptosis. he inhibition of post irradiation action of the E.G.F.R. signal way is a factor explaining the ionizing radiation - anti E.G.F.R. synergy. The preclinical data suggest that the E.G.F.R. blocking by the monoclonal antibodies is more important than the use of tyrosine kinase inhibitors. A first positive randomized study with the cetuximab, published in 2006 in the epidermoid carcinomas of the O.R.L. sphere lead to its authorization on the market with the radiotherapy for this localization. The use of cetuximab in other indication with or in

  5. Supramolecular Nanoparticles via Single-Chain Folding Driven by Ferrous Ions.

    Science.gov (United States)

    Wang, Fei; Pu, Hongting; Jin, Ming; Wan, Decheng

    2016-02-01

    Single-chain nanoparticles can be obtained via single-chain folding assisted by intramolecular crosslinking reversibly or irreversibly. Single-chain folding is also an efficient route to simulate biomacromolecules. In present study, poly(N-hydroxyethylacrylamide-co-4'-(propoxy urethane ethyl acrylate)-2,2':6',2''-terpyridine) (P(HEAm-co-EMA-Tpy)) is synthesized via reversible addition fragmentation chain transfer polymerization. Single-chain folding and intramolecular crosslinking of P(HEAm-co-EMA-Tpy) are achieved via metal coordination chemistry. The intramolecular interaction is characterized on ultraviolet/visible spectrophotometer (UV-vis spectroscopy), proton nuclear magnetic resonance ((1)H NMR), and differential scanning calorimetry (DSC). The supramolecular crosslinking mediated by Fe(2+) plays an important role in the intramolecular collapsing of the single-chain and the formation of the nanoparticles. The size and morphology of the nanoparticles can be controlled reversibly via metal coordination chemistry, which can be characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), and atomic force microscope (AFM). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Structure of single-chain single crystals of isotactic polystyrene and their radiation resistance

    International Nuclear Information System (INIS)

    Bu Haishan; Cao Jie; Xu Shengyong; Zhang Ze

    1997-01-01

    The structure of the single-chain single crystals of isotactic polystyrene (i-PS) was investigated by electron diffraction (ED) and high resolution electron microscopy (HREM). The nano-scale single-chain single crystals were found to be very stable to electron irradiation. According to the unit cell of i-PS crystals, the reflection rings in ED pattern and the lattice fringes in HREM images could be indexed, but the lower-index diffractions were not found. It is proposed that the single-chain single crystals are very small, thus secondary electrons may be allowed to escape and radiation damage is highly reduced, and that there are less lower-index lattice planes in the single-chain single crystals to provide sufficient diffraction intensity for recording. HREM images can be achieved at room temperature in the case of single-chain single crystals because of its stability to electron irradiation, therefore, this might be a novel experimental approach to the study of crystal structure of macromolecules

  7. Analysis of the relationship between end-to-end distance and activity of single-chain antibody against colorectal carcinoma.

    Science.gov (United States)

    Zhang, Jianhua; Liu, Shanhong; Shang, Zhigang; Shi, Li; Yun, Jun

    2012-08-22

    We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G4S)n (where n = 1-9) as the linker to connect VH and VL, and estimated the 3D structure of single-chain Fv antibody (scFv) by homologous modeling. After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: rn=ABn-ABO2+CDn-CDO2+BCn-BCst2. The relationship between immunoreactivity and r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity.

  8. Exploring single chain amphiphile self-assembly and their possible roles in light transduction

    DEFF Research Database (Denmark)

    Monnard, Pierre-Alain

    2011-01-01

    Self-assembled structures of single-chain amphiphiles have been used as hosts for biochemical, and chemical reactions. Their use as models for protocells (i.e., precursors to the first biological cells) has been extensively researched by various groups because the availability of single chain......: the medium composition in terms of ionic strengths and the medium physical parameters, such as temperature, significantly influence the formation of structures, as well as their subsequent stability. In addition, membranes composed of a single amphiphile type seem to be implausible as no potential amphiphile...... source studied to date can supply one single type of amphiphile at concentrations conducive to self-assembly. Mixtures of single-chain amphiphiles were therefore proposed to better model primitive membranes and potentially enhance their structural integrity1-3. Recently, we have established that complex...

  9. Steroid hormone and epidermal growth factor receptors in meningiomas.

    Science.gov (United States)

    Horsfall, D J; Goldsmith, K G; Ricciardelli, C; Skinner, J M; Tilley, W D; Marshall, V R

    1989-11-01

    A prospective study of steroid hormone and epidermal growth factor receptor expression in 57 meningiomas is presented. Scatchard analysis of radioligand binding identified 20% of meningiomas as expressing classical oestrogen receptors (ER) at levels below that normally accepted for positivity, the remainder being negative. ER could not be visualized in any meningioma using immunocytochemistry. Alternatively, 74% of meningiomas demonstrated the presence of progesterone receptors (PR) by Scatchard analysis, the specificity of which could not be attributed to glucocorticoid or androgen receptors. Confirmation of classical PR presence was determined by immunocytochemical staining. The presence of epidermal growth factor receptor (EGFR) was demonstrated in 100% of meningiomas using immunocytochemical staining. These data are reviewed in the context of previously reported results and are discussed in relation to the potential for medical therapy as an adjunct to surgery.

  10. Andrographolide regulates epidermal growth factor receptor and transferrin receptor trafficking in epidermoid carcinoma (A-431) cells

    Science.gov (United States)

    Tan, Y; Chiow, KH; Huang, D; Wong, SH

    2010-01-01

    Background and purpose: Andrographolide is the active component of Andrographis paniculata, a plant used in both Indian and Chinese traditional medicine, and it has been demonstrated to induce apoptosis in different cancer cell lines. However, not much is known about how it may affect the key receptors implicated in cancer. Knowledge of how andrographolide affects receptor trafficking will allow us to better understand new mechanisms by which andrographolide may cause death in cancer cells. Experimental approach: We utilized the well-characterized epidermal growth factor receptor (EGFR) and transferrin receptor (TfR) expressed in epidermoid carcinoma (A-431) cells as a model to study the effect of andrographolide on receptor trafficking. Receptor distribution, the total number of receptors and surface receptors were analysed by immunofluorescence, Western blot as well as flow-cytometry respectively. Key results: Andrographolide treatment inhibited cell growth, down-regulated EGFRs on the cell surface and affected the degradation of EGFRs and TfRs. The EGFR was internalized into the cell at an increased rate, and accumulated in a compartment that co-localizes with the lysosomal-associated membrane protein in the late endosomes. Conclusion and implications: This study sheds light on how andrographolide may affect receptor trafficking by inhibiting receptor movement from the late endosomes to lysosomes. The down-regulation of EGFR from the cell surface also indicates a new mechanism by which andrographolide may induce cancer cell death. PMID:20233216

  11. Synthesis and functionalization of dextran-based single-chain nanoparticles in aqueous media

    OpenAIRE

    Gracia R.; Marradi M.; Cossío U.; Benito A.; Pérez-San Vicente A.; Gómez-Vallejo V.; Grande H.-J.; Llop J.; and Loinaz I.

    2017-01-01

    Water-dispersible dextran-based single-chain polymer nanoparticles (SCPNs) were prepared in aqueous media and under mild conditions. Radiolabeling of the resulting biocompatible materials allowed the study of lung deposition of aqueous aerosols after intratracheal nebulization by means of single-photon emission computed tomography (SPECT), demonstrating their potential use as imaging contrast agents.

  12. Force Spectroscopy of Hyaluronan by AFM; From H-bonded Networks Towards Single Chain Behavior

    NARCIS (Netherlands)

    Giannotti, M.I.; Rinaudo, Marguerite; Vancso, Gyula J.

    2007-01-01

    The conformational behavior of hyaluronan (HA) polysaccharide chains in aqueous NaCl solution was characterized directly at the single-molecule level. This comunication reports on one of the first single-chain atomic force microscopy (AFM) experiments performed at variable temperatures,

  13. Jet and ultrasonic nebulization of single chain urokinase plasminogen activator (scu-PA)

    DEFF Research Database (Denmark)

    Münster, Anna-Marie; Bendstrup, E; Jensen, J.I.

    2000-01-01

    locally by nebulization in a recombinant zymogen form as single chain urokinase plasminogen activator (scu-PA). We aimed to characterize the particle size distribution, drug output, and enzymatic activity of scu-PA after nebulization with a Ventstream jet nebulizer (Medic-Aid, Bognor Regis, UK) and a Syst...

  14. Surface water retardation around single-chain polymeric nanoparticles: critical for catalytic function?

    Science.gov (United States)

    Stals, Patrick J M; Cheng, Chi-Yuan; van Beek, Lotte; Wauters, Annelies C; Palmans, Anja R A; Han, Songi; Meijer, E W

    2016-03-01

    A library of water-soluble dynamic single-chain polymeric nanoparticles (SCPN) was prepared using a controlled radical polymerisation technique followed by the introduction of functional groups, including probes at targeted positions. The combined tools of electron paramagnetic resonance (EPR) and Overhauser dynamic nuclear polarization (ODNP) reveal that these SCPNs have structural and surface hydration properties resembling that of enzymes.

  15. Well-defined single-chain polymer nanoparticles via thiol-Michael addition

    NARCIS (Netherlands)

    Kröger, A. Pia P.; Boonen, Roy J.E.A.; Paulusse, Jos M.J.

    2017-01-01

    A synthetic strategy has been developed giving facile access to well-defined single-chain polymer nanoparticles (SCNPs) from styrene-, acrylate- and methacrylate-based polymers. Random copolymers (polydispersity indices 1.10–1.15) of methyl (meth)acrylate, benzyl methacrylate or styrene containing

  16. Inactivation of single-chain urokinase-type plasminogen activator by thrombin in human subjects

    NARCIS (Netherlands)

    Braat, E. A.; Levi, M. [=Marcel M.; Bos, R.; Haverkate, F.; Lassen, M. R.; de Maat, M. P.; Rijken, D. C.

    1999-01-01

    Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), a process that may protect a blood clot from early fibrinolysis. It is not known under what circumstances tcu-PA/T can be generated in vivo. We have studied the occurrence

  17. Signal transduction by the platelet-derived growth factor receptor

    International Nuclear Information System (INIS)

    Williams, L.T.; Escobedo, J.A.; Keating, M.T.; Coughlin, S.R.

    1988-01-01

    The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

  18. Novel Drosophila receptor that binds multiple growth factors

    International Nuclear Information System (INIS)

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-01-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10 -6 to 10 -8 M. The 100 kDa protein can be affinity-labeled with these 125 I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by 125 I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors

  19. Assembly and activation of neurotrophic factor receptor complexes.

    Science.gov (United States)

    Simi, Anastasia; Ibáñez, Carlos F

    2010-04-01

    Neurotrophic factors play important roles in the development and function of both neuronal and glial elements of the central and peripheral nervous systems. Their functional diversity is in part based on their ability to interact with alternative complexes of receptor molecules. This review focuses on our current understanding of the mechanisms that govern the assembly and activation of neurotrophic factor receptor complexes. The realization that many, if not the majority, of these complexes exist in a preassembled form at the plasma membrane has forced the revision of classical ligand-mediated oligomerization models, and led to the discovery of novel mechanisms of receptor activation and generation of signaling diversity which are likely to be shared by many different classes of receptors.

  20. Single Chain Fv Constructs of Anti-Ganglioside GD2 Antibodies for Radioimaging and Radioimmunotherapy

    International Nuclear Information System (INIS)

    Cheung, Nai-Kong

    2003-01-01

    because of its broad and usually homogeneous distribution in human solid tumors, and most importantly, their absence on cell membranes of normal human tissues. In separate experiments, we have shown that T-cells transduced with the herpes simplex viral thymidine kinase (HSV-tk) gene can be radiolabeled with 131 I-FIAU to a safe nuclear radiation dose. Using a dicistronic construct we are inserting chimeric immune receptor plus HSV-tk into T-cells to allow such their trafficking to be radioactively monitored. We plan to study the role of cytokines, chemoreceptors and CD4 helper T-cells in recruiting CD8+ transduced T-cells to the tumor site. These studies should provide us with an adoptive cell therapy approach to target cytotoxicity to human tumors, and a lymphocyte tracking tool to study delivery to the tumor sites, to determine if they proliferate locally and/or recirculate. Such pharmacologic information is crucial for optimizing gene-modified T-cells in future clinical trials. Single chain FV constructs of anti-ganglioside GD2 antibodies for radioimaging

  1. Efficient Synthesis of Single-Chain Polymer Nanoparticles via Amide Formation

    Directory of Open Access Journals (Sweden)

    Ana Sanchez-Sanchez

    2015-01-01

    Full Text Available Single-chain technology (SCT allows the transformation of individual polymer chains to folded/collapsed unimolecular soft nanoparticles. In this work we contribute to the enlargement of the SCT toolbox by demonstrating the efficient synthesis of single-chain polymer nanoparticles (SCNPs via intrachain amide formation. In particular, we exploit cross-linking between active methylene groups and isocyanate moieties as powerful “click” chemistry driving force for SCNP construction. By employing poly(methyl methacrylate- (PMMA- based copolymers bearing β-ketoester units distributed randomly along the copolymer chains and bifunctional isocyanate cross-linkers, SCNPs were successfully synthesized at r.t. under appropriate reaction conditions. Characterization of the resulting SCNPs was carried out by means of a combination of techniques including size exclusion chromatography (SEC, infrared (IR spectroscopy, proton nuclear magnetic resonance (1H NMR spectroscopy, dynamic light scattering (DLS, and elemental analysis (EA.

  2. The in vivo characteristics of genetically engineered divalent and tetravalent single-chain antibody constructs

    International Nuclear Information System (INIS)

    Wittel, Uwe A.; Jain, Maneesh; Goel, Apollina; Chauhan, Subhash C.; Colcher, David; Batra, Surinder K.

    2005-01-01

    Engineered multivalent single-chain Fv (scFv) constructs have been demonstrated to exhibit rapid blood clearance and better tumor penetration. To understand the short plasma half-life of multivalent single-chain antibody fragments, the pharmacokinetic properties of covalent dimeric scFv [sc(Fv) 2 ], noncovalent tetrameric scFv {[sc(Fv) 2 ] 2 } and IgG of MAb CC49 were examined. The scFvs displayed an ability to form higher molecular aggregates in vivo. A specific proteolytic cleavage of the linker sequence of the covalent dimeric or a deterioration of the noncovalent association of the dimeric scFv into tetravalent scFv constructs was not observed. In conclusion, sc(Fv) 2 and [sc(Fv) 2 ] 2 are stable in vivo and have significant potential for diagnostic and therapeutic applications

  3. Topical administration of adrenergic receptor pharmaceutics and nerve growth factor

    Directory of Open Access Journals (Sweden)

    Jena J Steinle

    2010-06-01

    Full Text Available Jena J SteinleDepartments of Ophthalmology and Anatomy and Neurobiology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN 38163, USAAbstract: Topical application of nerve growth factor (NGF and adrenergic receptor pharmaceutics are currently in use for corneal ulcers and glaucoma. A recent interest in the neuroprotective abilities of NGF has led to a renewed interest in NGF as a therapeutic for retinal and choroidal diseases. NGF can promote cell proliferation through actions of the TrkA receptor or promote apoptosis through receptor p75NTR. This understanding has led to novel interest in the role of NGF for diseases of the posterior eye. The role of β-adrenergic receptor agonists and antagonists for treatments of glaucoma, diabetic retinopathy, and their potential mechanisms of action, are still under investigation. This review discusses the current knowledge and applications of topical NGF and adrenergic receptor drugs for ocular disease.Keywords: NGF, β-adrenergic receptor agents, α-adrenergic receptor agents, retina, cornea, glaucoma

  4. Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

    Science.gov (United States)

    Bakherad, Hamid; Gargari, Seyed Latif Mousavi; Sepehrizadeh, Zargham; Aghamollaei, Hossein; Taheri, Ramezan Ali; Torshabi, Maryam; Yazdi, Mojtaba Tabatabaei; Ebrahimizadeh, Walead; Setayesh, Neda

    2017-09-01

    It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models. Copyright © 2017. Published by Elsevier Masson SAS.

  5. IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST.

    Science.gov (United States)

    Giudicelli, Véronique; Duroux, Patrice; Kossida, Sofia; Lefranc, Marie-Paule

    2017-06-26

    IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain. The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation. For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino

  6. Single-Chain Conformation for Interacting Poly(N-isopropylacrylamide in Aqueous Solution

    Directory of Open Access Journals (Sweden)

    Boualem Hammouda

    2015-04-01

    Full Text Available The demixing phase behavior of Poly(N-isopropylacrylamide (PNIPAM aqueous solution is investigated using small-angle neutron scattering. This polymer phase separates upon heating and demixes around 32 °C. The pre-transition temperature range is characterized by two scattering modes; a low-Q (large-scale signal and a high-Q dissolved chains signal. In order to get insight into this pre-transition region, especially the origin of the low-Q (large-scale structure, the zero average contrast method is used in order to isolate single-chain conformations even in the demixing polymers transition region. This method consists of measuring deuterated and non-deuterated polymers dissolved in mixtures of deuterated and non-deuterated water for which the polymer scattering length density matches the solvent scattering length density. A fixed 4% polymer mass fraction is used in a contrast variation series where the d-water/h-water fraction is varied in order to determine the match point. The zero average contrast (match point sample displays pure single-chain scattering with no interchain contributions. Our measurements prove that the large scale structure in this polymer solution is due to a transient polymer network formed through hydrophobic segment-segment interactions. Scattering intensity increases when the temperature gets close to the phase boundary. While the apparent radius of gyration increases substantially at the Lower Critical Solution Temperature (LCST transition due to strong interchain correlation, the single-chain true radius of gyration has been found to decrease slightly with temperature when approaching the transition.

  7. Insulin and insulin-like growth factor receptors and responses

    International Nuclear Information System (INIS)

    Roth, R.A.; Steele-Perkins, G.; Hari, J.; Stover, C.; Pierce, S.; Turner, J.; Edman, J.C.; Rutter, W.J.

    1988-01-01

    Insulin is a member of a family of structurally related hormones with diverse physiological functions. In humans, the best-characterized members of this family include insulin, insulin-like growth factor (IGF)-I, and IGF-II. Each of these three polypeptide hormones has its own distinct receptor. The structures of each of these receptors have now been deduced from analyses of isolated cDNA clones. To study further the responses mediated through these three different receptors, the authors have been studying cells expressing the proteins encoded by these three cDNAs. The isolated cDNAs have been transfected into Chinese hamster ovary (CHO) cells, and the resulting transfected cell lines have been characterized as to the ligand-binding activities and signal-transducing activities of the expressed proteins

  8. Early recognition is important when multiple magnets masquerade as a single chain after foreign body ingestion

    Directory of Open Access Journals (Sweden)

    Auriel August

    2016-10-01

    Full Text Available Ingestions of multiple magnets can lead to serious damage to the gastrointestinal tract. Moreover, these foreign bodies can take deceptive shapes such as single chains which may mislead clinicians. We report the case of a ten-year-old boy who swallowed 33 magnets, the most yet reported, which took on the appearance of a single loop in the stomach, while actually being located in the stomach, small bowel, and colon. Early recognition and prompt intervention are necessary to avoid complications of this foreign body misadventure.

  9. Finite-size effects on the static properties of a single-chain magnet

    Science.gov (United States)

    Bogani, L.; Sessoli, R.; Pini, M. G.; Rettori, A.; Novak, M. A.; Rosa, P.; Massi, M.; Fedi, M. E.; Giuntini, L.; Caneschi, A.; Gatteschi, D.

    2005-08-01

    We study the role of defects in the “single-chain magnet” CoPhOMe by inserting a controlled number of diamagnetic impurities. The samples are analyzed with unprecedented accuracy with the particle induced x-ray emission technique, and with ac and dc magnetic measurements. In an external applied field the system shows an unexpected behavior, giving rise to a double peak in the susceptibility. The static thermodynamic properties of the randomly diluted Ising chain with alternating g values are then exactly obtained via a transfer matrix approach. These results are compared to the experimental behavior of CoPhOMe, showing qualitative agreement.

  10. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

    OpenAIRE

    Matsukizono, Miho; Kamegawa, Mariko; Tanaka, Koichi; Kohra, Shinya; Arizono, Koji; Hamazoe, Yuta; Sugimura, Kazuhisa

    2013-01-01

    An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv) clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg) but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competi...

  11. Distribution of corticotropin-releasing factor receptors in primate brain

    International Nuclear Information System (INIS)

    Millan, M.A.; Jacobowitz, D.M.; Hauger, R.L.; Catt, K.J.; Aguilera, G.

    1986-01-01

    The distribution and properties of receptors for corticotropin-releasing factor (CRF) were analyzed in the brain of cynomolgus monkeys. Binding of [ 125 I]tyrosine-labeled ovine CRF to frontal cortex and amygdala membrane-rich fractions was saturable, specific, and time- and temperature-dependent, reaching equilibrium in 30 min at 23 0 C. Scatchard analysis of the binding data indicated one class of high-affinity sites with a K/sub d/ of 1 nM and a concentration of 125 fmol/mg. As in the rat pituitary and brain, CRF receptors in monkey cerebral cortex and amygdala were coupled to adenylate cyclase. Autoradiographic analysis of specific CRF binding in brain sections revealed that the receptors were widely distributed in the cerebral cortex and limbic system. Receptor density was highest in the pars tuberalis of the pituitary and throughout the cerebral cortex, specifically in the prefrontal, frontal, orbital, cingulate, insular, and temporal areas, and in the cerebellar cortex. A low binding density was present in the superior colliculus, locus coeruleus, substantia gelatinosa, preoptic area, septal area, and bed nucleus of the stria terminalis. These data demonstrate that receptors for CRF are present within the primate brain at areas related to the central control of visceral function and behavior, suggesting that brain CRF may serve as a neurotransmitter in the coordination of endocrine and neural mechanisms involved in the response to stress

  12. Platelet-derived growth factor receptors in the human central nervous system : autoradiographic distribution and receptor densities in multiple sclerosis

    NARCIS (Netherlands)

    De Keyser, J; Wilczak, N

    1997-01-01

    Platelet derived growth factor (PDGF) receptors were studied in postmortem adult human brain and cervical spinal cord using autoradiography with human recombinant I-125-PDGF-BB. PDGF-BB binds to the three different dimers of PDGF receptors (alpha alpha, alpha beta and beta beta) PDGF receptors were

  13. Epidermal growth factor receptor expression in urinary bladder cancer

    Directory of Open Access Journals (Sweden)

    Dayalu S.L. Naik

    2011-01-01

    Full Text Available Objective : To evaluate the expression pattern of epidermal growth factor receptor (EGFR in urinary bladder cancer and its association with human epidermal growth factor receptor 2 (HER2, epidermal growth factor (EGF, interleukin-6 (IL-6, and high risk human papilloma virus (HPV types 16 and 18. Materials and Methods : Thirty cases of urothelial carcinoma were analyzed. EGFR, HER2, EGF, and IL-6 expressions in the tissue were evaluated by immunohistochemical staining. For HPV, DNA from tissue samples was extracted and detection of HPV was done by PCR technique. Furthermore, evaluation of different intracellular molecules associated with EGFR signaling pathways was performed by the western blot method using lysates from various cells and tissues. Results : In this study, the frequencies of immunopositivity for EGFR, HER2, EGF, and IL-6 were 23%, 60%, 47%, and 80%, respectively. No cases were positive for HPV-18, whereas HPV-16 was detected in 10% cases. Overall, expression of EGFR did not show any statistically significant association with the studied parameters. However, among male patients, a significant association was found only between EGFR and HER2. Conclusions : Overexpression of EGFR and/or HER2, two important members of the same family of growth factor receptors, was observed in a considerable proportion of cases. Precise knowledge in this subject would be helpful to formulate a rational treatment strategy in patients with urinary bladder cancer.

  14. Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

    International Nuclear Information System (INIS)

    Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture

  15. Selection rules for single-chain-magnet behaviour in non-collinear Ising systems

    Energy Technology Data Exchange (ETDEWEB)

    Vindigni, Alessandro [Laboratorium fuer Festkoerperphysik, ETH Zuerich, CH-8093 Zuerich (Switzerland); Pini, Maria Gloria [Istituto dei Sistemi Complessi, Consiglio Nazionale delle Ricerche, Via Madonna del Piano 10, I-50019 Sesto Fiorentino (Italy)], E-mail: vindigni@phys.ethz.ch

    2009-06-10

    The magnetic behaviour of molecular single-chain magnets is investigated in the framework of a one-dimensional Ising model with single spin-flip Glauber dynamics. Opportune modifications to the original theory are required in order to account for non-collinearity of local anisotropy axes between themselves and with respect to the crystallographic (laboratory) frame. The extension of Glauber's theory to the case of a collinear Ising ferrimagnetic chain is also discussed. Within this formalism, both the dynamics of magnetization reversal in zero field and the response of the system to a weak magnetic field, oscillating in time, are studied. Depending on the experimental geometry, selection rules are found for the occurrence of slow relaxation of the magnetization at low temperatures, as well as for resonant behaviour of the a.c. susceptibility as a function of temperature at low frequencies. The present theory applies successfully to some real systems, namely Mn-, Dy- and Co-based molecular magnetic chains, showing that single-chain-magnet behaviour is not only a feature of collinear ferro- and ferrimagnetic, but also of canted antiferromagnetic chains.

  16. Cell surface expression of single chain antibodies with applications to imaging of gene expression in vivo

    International Nuclear Information System (INIS)

    Northrop, Jeffrey P.; Bednarski, Mark; Li, King C.; Barbieri, Susan O.; Lu, Amy T.; Nguyen, Dee; Varadarajan, John; Osen, Maureen; Star-Lack, Josh

    2003-01-01

    Imaging of gene expression in vivo has many potential uses for biomedical research and drug discovery, ranging from the study of gene regulation and cancer to the non-invasive assessment of gene therapies. To streamline the development of imaging marker gene technologies for nuclear medicine, we propose a new approach to the design of reporter/probe pairs wherein the reporter is a cell surface-expressed single chain antibody variable fragment that has been raised against a low molecular weight imaging probe with optimized pharmacokinetic properties. Proof of concept of the approach was achieved using a single chain antibody variable fragment that binds with high affinity to fluorescein and an imaging probe consisting of fluorescein isothiocyanate coupled to the chelator diethylene triamine penta-acetic acid labeled with the gamma-emitter 111 In. We demonstrate specific high-affinity binding of this probe to the cell surface-expressed reporter in vitro and assess the in vivo biodistribution of the probe both in wild-type mice and in mice harboring tumor xenografts expressing the reporter. Specific uptake of the probe by, and in vivo imaging of, tumors expressing the reporter are shown. Since ScFvs with high affinities can be raised to almost any protein or small molecule, the proposed methodology may offer a new flexibility in the design of imaging tracer/reporter pairs wherein both probe pharmacokinetics and binding affinities can be readily optimized. (orig.)

  17. Selection rules for single-chain-magnet behaviour in non-collinear Ising systems

    International Nuclear Information System (INIS)

    Vindigni, Alessandro; Pini, Maria Gloria

    2009-01-01

    The magnetic behaviour of molecular single-chain magnets is investigated in the framework of a one-dimensional Ising model with single spin-flip Glauber dynamics. Opportune modifications to the original theory are required in order to account for non-collinearity of local anisotropy axes between themselves and with respect to the crystallographic (laboratory) frame. The extension of Glauber's theory to the case of a collinear Ising ferrimagnetic chain is also discussed. Within this formalism, both the dynamics of magnetization reversal in zero field and the response of the system to a weak magnetic field, oscillating in time, are studied. Depending on the experimental geometry, selection rules are found for the occurrence of slow relaxation of the magnetization at low temperatures, as well as for resonant behaviour of the a.c. susceptibility as a function of temperature at low frequencies. The present theory applies successfully to some real systems, namely Mn-, Dy- and Co-based molecular magnetic chains, showing that single-chain-magnet behaviour is not only a feature of collinear ferro- and ferrimagnetic, but also of canted antiferromagnetic chains.

  18. Topical administration of adrenergic receptor pharmaceutics and nerve growth factor

    OpenAIRE

    Steinle, Jena

    2010-01-01

    Jena J SteinleDepartments of Ophthalmology and Anatomy and Neurobiology, Hamilton Eye Institute, University of Tennessee Health Science Center, Memphis, TN 38163, USAAbstract: Topical application of nerve growth factor (NGF) and adrenergic receptor pharmaceutics are currently in use for corneal ulcers and glaucoma. A recent interest in the neuroprotective abilities of NGF has led to a renewed interest in NGF as a therapeutic for retinal and choroidal diseases. NGF can promote cell proliferati...

  19. 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

    1992-01-01

    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 ...

  20. Decreased expression of serum and microvascular vascular endothelial growth factor receptor-2 in meningococcal sepsis*.

    NARCIS (Netherlands)

    Flier, M. van der; Baerveldt, E.M.; Miedema, A.; Hartwig, N.G.; Hazelzet, J.A.; Emonts, M.; Groot, R. de; Prens, E.P.; Vught, A.J. van; Jansen, N.J.

    2013-01-01

    OBJECTIVES: To determine the skin microvessel expression of vascular endothelial growth factor receptor 2 and serum-soluble vascular endothelial growth factor receptor 2 levels in children with meningococcal sepsis. DESIGN: Observational study. SETTING: Two tertiary academic children hospital PICUs.

  1. Epidermal Growth Factor Receptor (EGFR) Crosstalks in Liver Cancer

    International Nuclear Information System (INIS)

    Berasain, Carmen; Latasa, María Ujue; Urtasun, Raquel; Goñi, Saioa; Elizalde, María; Garcia-Irigoyen, Oihane; Azcona, María; Prieto, Jesús; Ávila, Matías A.

    2011-01-01

    Hepatocarcinogenesis is a complex multistep process in which many different molecular pathways have been implicated. Hepatocellular carcinoma (HCC) is refractory to conventional chemotherapeutic agents, and the new targeted therapies are meeting with limited success. Interreceptor crosstalk and the positive feedback between different signaling systems are emerging as mechanisms of targeted therapy resistance. The identification of such interactions is therefore of particular relevance to improve therapeutic efficacy. Among the different signaling pathways activated in hepatocarcinogenesis the epidermal growth factor receptor (EGFR) system plays a prominent role, being recognized as a “signaling hub” where different extracellular growth and survival signals converge. EGFR can be transactivated in response to multiple heterologous ligands through the physical interaction with multiple receptors, the activity of intracellular kinases or the shedding of EGFR-ligands. In this article we review the crosstalk between the EGFR and other signaling pathways that could be relevant to liver cancer development and treatment

  2. Epidermal growth factor receptor in primary human lung cancer

    International Nuclear Information System (INIS)

    Yu Xueyan; Hu Guoqiang; Tian Keli; Wang Mingyun

    1996-01-01

    Cell membranes were prepared from 12 human lung cancers for the study of the expression of epidermal growth factor receptors (EGFR). EGFR concentration was estimated by ligand binding studies using 125 I-radiolabeled EGF. The dissociation constants of the high affinity sites were identical, 1.48 nmol and 1.1 nmol in cancer and normal lung tissues, the EGFR contents were higher in lung cancer tissues (range: 2.25 to 19.39 pmol·g -1 membrane protein) than that in normal tissues from the same patients (range: 0.72 to 7.43 pmol·g -1 membrane protein). These results suggest that EGF and its receptor may play a role in the regulatory mechanisms in the control of lung cellular growth and tumor promotion

  3. Single-ion and single-chain magnetism in triangular spin-chain oxides

    Science.gov (United States)

    Seikh, Md. Motin; Caignaert, Vincent; Perez, Olivier; Raveau, Bernard; Hardy, Vincent

    2017-05-01

    S r4 -xC axM n2Co O9 oxides (x =0 and x =2 ) are found to exhibit magnetic responses typical of single-chain magnets (SCMs) and single-ion magnets (SIMs), two features generally investigated in coordination polymers or complexes. The compound x =0 appears to be a genuine SCM, in that blocking effects associated with slow spin dynamics yield remanence and coercivity in the absence of long-range ordering (LRO). In addition, SIM signatures of nearly identical nature are detected in both compounds, coexisting with SCM in x =0 and with LRO in x =2 . It is also observed that a SCM response can be recovered in x =2 after application of magnetic field. These results suggest that purely inorganic systems could play a valuable role in the topical issue of the interplay among SIM, SCM, and LRO phenomena in low-dimensional magnetism.

  4. Screened ion-ion interaction in mercury-chain compounds: Single chain

    International Nuclear Information System (INIS)

    Mohan, M.M.; Griffin, A.

    1985-01-01

    At room temperature, the mercury chains in Hg/sub 3-delta/AsF 6 exhibit phonons characteristic of a one-dimensional lattice. We calculate the screening of the Hg ion-ion interaction in a single chain by electrons moving in a cylindrical potential of finite radius, within the random-phase approximation. The resulting Bohm-Staver-type expression for the phonon velocity is (Z 2 mN/sub I//MN/sub e/)/sup 1/2/v/sub F/, where Z is the Hg ionic charge and N/sub I/ (N/sub e/) is the number of ions (electrons) per unit length. Use of the Tomonaga-Luttinger solution for the electronic response function (keeping only the small-momentum scattering processes) just renormalizes the Fermi velocity in this expression

  5. Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

    International Nuclear Information System (INIS)

    Ceridono, Mara; Belleudi, Francesca; Ceccarelli, Simona; Torrisi, Maria Rosaria

    2005-01-01

    Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR

  6. Epidermal growth factor and its receptors in human pancreatic carcinoma

    International Nuclear Information System (INIS)

    Chen, Y.F.; Pan, G.Z.; Hou, X.; Liu, T.H.; Chen, J.; Yanaihara, C.; Yanaihara, N.

    1990-01-01

    The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells

  7. Expression of epidermal growth factor receptors in human endometrial carcinoma

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Ottesen, B

    1993-01-01

    Little data exist on the expression of epidermal growth factor receptors (EGF-Rs) in human endometrial cancer. EGF-R status was studied in 65 patients with endometrial carcinomas and in 26 women with nonmalignant postmenopausal endometria, either inactive/atrophic endometrium or adenomatous...... hyperplasia. EGF-R was identified on frozen tissue sections by means of an indirect immunoperoxidase technique with a monoclonal antibody against the external domain of the EGF-R. Seventy-one percent of the carcinomas expressed positive EGF-R immunoreactivity. In general, staining was most prominent...

  8. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials

    DEFF Research Database (Denmark)

    Voldborg, B R; Damstrup, L; Spang-Thomsen, M

    1997-01-01

    The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation upon activation through the binding of one of its ligands. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intr...... aspects of therapeutic targeting of EGFR....

  9. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    International Nuclear Information System (INIS)

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H.

    1990-01-01

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

  10. Single-chain magnet features in 1D [MnR4TPP][TCNE] compounds

    International Nuclear Information System (INIS)

    Balanda, Maria; Tomkowicz, Zbigniew; Rams, Michal; Haase, Wolfgang

    2011-01-01

    Molecular chains of antiferrimagnetically coupled Mn III -ion (S = 2) and TCNE (tetracyanoethylene) radical moments (s = 1/2 ) show different behaviour depending on group R substituted to TPP (tetraphenylporphyrin) and on the substitution site. The compound with R = F in Ortho position is a Single-Chain Magnet (SCM) with blocking temperature T b = 6.6K, while that with R = F in Meta position shows both blocking (T b = 5.4 K) and magnetic ordering transition (T c = 10 K). For bulky groups R = OC n H 2n+1 , the magnetically ordered phase is observed (T c ∼ 22 K), which does not however prevent slow relaxation at T c of 2 T at 2.3 K is like that of SCM. The frequency dependent AC susceptibility in the superimposed DC field reveals common features of all systems. The energy of intrachain ferromagnetic coupling between effective spin units 3/2, relevant at low temperatures, is determined for all compounds and the interchain dipolar coupling is estimated. It is concluded that slow relaxation is inherent for all quasi one-dimensional compounds and for the magnetically ordered ones shows up in the high enough magnetic field.

  11. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis

    Directory of Open Access Journals (Sweden)

    Chi-Hsin Lee

    2017-10-01

    Full Text Available Russell’s vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs containing 3.4 × 107 and 5.5 × 107 transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

  12. Reaction pathway towards formation of cobalt single chain magnets and nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Balaji, G.; Desilva, Rohini M.; Palshin, V. [Center for Advanced Microstructures and Devices, Louisiana State University, 6980 Jefferson Highway, Baton Rouge, LA 70806 (United States); Desilva, N. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Palmer, G. [Department of Biochemistry and Cell Biology, Rice University, MS 140, 6100 Main street, Houston, TX 77251 (United States); Kumar, Challa S.S.R., E-mail: ckumar1@lsu.ed [Center for Advanced Microstructures and Devices, Louisiana State University, 6980 Jefferson Highway, Baton Rouge, LA 70806 (United States)

    2010-03-15

    With the advent of molecular magnets the quest for suitable high density magnetic storage materials has fuelled further research in this area. Here in this report, we present a detailed mechanistic investigation of thermal decomposition of cyclopentadienyl cobalt [CoCp(CO){sub 2}] precursor where Cp is the cyclopentadienyl moiety. The reaction revealed the formation of cobalt nanoparticles (Co-NPs) through an isolable reaction intermediate characterized as a Single Chain Magnet (SCM), [Co(Cp){sub 2}]{sub 2}CoCl{sub 4} (1). The SQUID magnetic measurements showed the presence of very strong antiferromagnetic interactions between Co{sup 2+} ions. The zero-field cooled (ZFC) and field cooled (FC) magnetization curves branch out below 5 K and there is evidence for frequency dependent complex susceptibility along with a maximum observed around 2.5 K. The optical studies indicated that the Co{sup 2+} d-d transition is influenced by the polarity of the solvents. The cobalt nanoparticles (Co-NPs) were obtained, either directly from 1 or from its precursor. They are spherical in shape with a mean size 15 nm, have fcc crystal structure and were found to be ferromagnetic at room temperature.

  13. Single Chain Antibody Fragment against Venom from the Snake Daboia russelii formosensis.

    Science.gov (United States)

    Lee, Chi-Hsin; Lee, Yu-Ching; Lee, Yueh-Lun; Leu, Sy-Jye; Lin, Liang-Tzung; Chen, Chi-Ching; Chiang, Jen-Ron; Mwale, Pharaoh Fellow; Tsai, Bor-Yu; Hung, Ching-Sheng; Yang, Yi-Yuan

    2017-10-27

    Russell's vipers containing hemotoxic and neurotoxic venom commonly cause snake envenomation. Horse-derived antivenom is a specific antidote, but its production is expensive and has side effects. Developing a cost-effective and more tolerable therapeutic strategy is favorable. In this study, using glutaraldehyde-attenuated Daboia russelii formosensis (DRF) venom proteins to immunize chickens, polyclonal yolk-immunoglobulin (IgY) antibodies were generated and showed a specific binding affinity. Phage display technology was used to generate two antibody libraries of single-chain variable fragments (scFvs) containing 3.4 × 10⁷ and 5.5 × 10⁷ transformants, respectively. Phage-based ELISA indicated that specific clones were enriched after bio-panning. The nucleotide sequences of scFv-expressing clones were analyzed and classified into six groups in the short linker and four groups in the long linker. These scFv antibodies specifically bound to DRF proteins, but not other venom proteins. Mass spectrometric data suggested that these scFv antibodies may recognize phospholipase A2 RV-4 or RV-7. In vivo studies showed that anti-DRF IgY exhibited complete protective effects and mixed scFv antibodies increased the survival rate and time of mice challenged with a lethal dose of DRF proteins. These antibodies can be potentially applied in a rapid diagnostic method or for treatment in the future.

  14. Single-chain statistics and the upper wave-vector cutoff in polymer blends

    International Nuclear Information System (INIS)

    Holyst, R.; Vilgis, T.A.

    1994-01-01

    We derive the equation for the single-chain correlation function in polymer blends. The chains in the incompressible blend have a radius of gyration smaller than the radius of gyration for ideal chains. The chains shrink progressively as we approach the critical temperature T c . The correction responsible for shrinking is proportional to 1/ √N , where N is the polymerization index. At T=T c and for N=1000, the size of the chain has been estimated to be 10% smaller than the size of the ideal coil. The estimate relies on the appropriate cutoff. In the limit of N→∞ the chains approach the random walk limit. Additionally, we propose in this paper a self-consistent determination of the radius of gyration and the upper wave-vector cutoff. Our model is free from any divergences such as were encountered in the previous mean-field studies; we make an estimate of the chain size at the true critical temperature and not the mean-field one

  15. Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

    Science.gov (United States)

    Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Shi, Linqing; Zhang, Renxia; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2017-04-28

    The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

  16. Characterization of a Single Chain Fv Antibody that Reacts with Free Morphine

    Directory of Open Access Journals (Sweden)

    Kazuhisa Sugimura

    2013-02-01

    Full Text Available An immune phage library derived from mice, hyperimmunized with morphine-conjugated BSA, was used to isolate a single-chain Fv (scFv clone, M86, with binding activity to morphine-conjugated thyroglobulin (morphine-C-Tg but not to codeine-, cocaine-, or ketamine-conjugated Tg. Surface plasmon resonance analysis using a morphine-C-Tg-coupled CM5 sensor chip showed that the Kd value was 1.26 × 10−8 M. To analyze its binding activity to free morphine and related compounds, we performed a competitive ELISA with M86 and morphine-C-Tg in the absence or presence of varying doses of free morphine and related compounds. IC50 values for opium, morphine, codeine, and heroin were 257 ng/mL, 36.4, 7.3, and 7.4 nM, respectively. Ketamine and cocaine exhibited no competitive binding activity to M86. Thus, we established a phage library-derived scFv, M86, which recognized not only free morphine and codeine as opium components but also heroin. This characteristic of M86 may be useful for developing therapeutic reagents for opiate addiction and as a free morphine-specific antibody probe.

  17. Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

    NARCIS (Netherlands)

    Arafat, WO; Gomez-Navarro, J; Buchsbaum, DJ; Xiang, J; Casado, E; Barker, SD; Mahasreshti, PJ; Haisma, HJ; Barnes, MN; Siegal, GP; Alvarez, RD; Hemminki, A; Nettelbeck, DM; Curiel, DT

    Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of

  18. Structural and Functional Characterization of a Single-chain Peptide-MHC Molecule that Modulates both Naive and Activated CD8plus T Cells

    Energy Technology Data Exchange (ETDEWEB)

    D Samanta; G Mukherjee; U Ramagopal; R Chaparro; S Nathenson; T DiLorenzo; S Almo

    2011-12-31

    Peptide-MHC (pMHC) multimers, in addition to being tools for tracking and quantifying antigen-specific T cells, can mediate downstream signaling after T-cell receptor engagement. In the absence of costimulation, this can lead to anergy or apoptosis of cognate T cells, a property that could be exploited in the setting of autoimmune disease. Most studies with class I pMHC multimers used noncovalently linked peptides, which can allow unwanted CD8{sup +} T-cell activation as a result of peptide transfer to cellular MHC molecules. To circumvent this problem, and given the role of self-reactive CD8{sup +} T cells in the development of type 1 diabetes, we designed a single-chain pMHC complex (scK{sup d}.IGRP) by using the class I MHC molecule H-2K{sup d} and a covalently linked peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP{sub 206-214}), a well established autoantigen in NOD mice. X-ray diffraction studies revealed that the peptide is presented in the groove of the MHC molecule in canonical fashion, and it was also demonstrated that scK{sup d}.IGRP tetramers bound specifically to cognate CD8{sup +} T cells. Tetramer binding induced death of naive T cells and in vitro- and in vivo-differentiated cytotoxic T lymphocytes, and tetramer-treated cytotoxic T lymphocytes showed a diminished IFN-{gamma} response to antigen stimulation. Tetramer accessibility to disease-relevant T cells in vivo was also demonstrated. Our study suggests the potential of single-chain pMHC tetramers as possible therapeutic agents in autoimmune disease. Their ability to affect the fate of naive and activated CD8{sup +} T cells makes them a potential intervention strategy in early and late stages of disease.

  19. Primitive-path statistics of entangled polymers: mapping multi-chain simulations onto single-chain mean-field models

    International Nuclear Information System (INIS)

    Steenbakkers, Rudi J A; Schieber, Jay D; Tzoumanekas, Christos; Li, Ying; Liu, Wing Kam; Kröger, Martin

    2014-01-01

    We present a method to map the full equilibrium distribution of the primitive-path (PP) length, obtained from multi-chain simulations of polymer melts, onto a single-chain mean-field ‘target’ model. Most previous works used the Doi–Edwards tube model as a target. However, the average number of monomers per PP segment, obtained from multi-chain PP networks, has consistently shown a discrepancy of a factor of two with respect to tube-model estimates. Part of the problem is that the tube model neglects fluctuations in the lengths of PP segments, the number of entanglements per chain and the distribution of monomers among PP segments, while all these fluctuations are observed in multi-chain simulations. Here we use a recently proposed slip-link model, which includes fluctuations in all these variables as well as in the spatial positions of the entanglements. This turns out to be essential to obtain qualitative and quantitative agreement with the equilibrium PP-length distribution obtained from multi-chain simulations. By fitting this distribution, we are able to determine two of the three parameters of the model, which govern its equilibrium properties. This mapping is executed for four different linear polymers and for different molecular weights. The two parameters are found to depend on chemistry, but not on molecular weight. The model predicts a constant plateau modulus minus a correction inversely proportional to molecular weight. The value for well-entangled chains, with the parameters determined ab initio, lies in the range of experimental data for the materials investigated. (paper)

  20. Expression of nerve growth factor and its receptor, tyrosine kinase receptor A, in rooster testes.

    Science.gov (United States)

    Ma, Wei; Wang, Chunqiang; Su, Yuhong; Tian, Yumin; Zhu, Hongyan

    2015-10-01

    Nerve growth factor (NGF), which is required for the survival and differentiation of the nervous system, is also thought to play an important role in the development of mammalian reproductive tissues. To explore the function of NGF in the male reproductive system of non-mammalian animals, we determined the presence of NGF and its receptor, tyrosine kinase receptor A (TrkA), in rooster testes and investigated the regulation of NGF and TrkA expression by follicle-stimulating hormone (FSH). The mRNA and protein levels of NGF and TrkA in 6-week-old rooster testes were lower than those in 12-, 16- or 20-week age groups; levels were highest in the 16-week group. Immunohistochemistry showed that NGF and TrkA were both detected in spermatogonia, spermatocytes and spermatids. NGF immunoreactivity was observed in Leydig cells and strong TrkA signals were present in Sertoli cells. Meanwhile, FSH increased TrkA transcript levels in rooster testes in a dose-dependent manner. We present novel evidence for the developmental and FSH-regulated expression of the NGF/TrkA system, and our findings suggest that the NGF/TrkA system may play a prominent role in chicken spermatogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Personalized Radiation Oncology: Epidermal Growth Factor Receptor and Other Receptor Tyrosine Kinase Inhibitors.

    Science.gov (United States)

    Higgins, Geoff S; Krause, Mechthild; McKenna, W Gillies; Baumann, Michael

    Molecular biomarkers are currently evaluated in preclinical and clinical studies in order to establish predictors for treatment decisions in radiation oncology. The receptor tyrosine kinases (RTK) are described in the following text. Among them, the most data are available for the epidermal growth factor receptor (EGFR) that plays a major role for prognosis of patients after radiotherapy, but seems also to be involved in mechanisms of radioresistance, specifically in repopulation of tumour cells between radiotherapy fractions. Monoclonal antibodies against the EGFR improve locoregional tumour control and survival when applied during radiotherapy, however, the effects are heterogeneous and biomarkers for patient selection are warranted. Also other RTK´s such as c-Met and IGF-1R seem to play important roles in tumour radioresistance. Beside the potential to select patients for molecular targeting approaches combined with radiotherapy, studies are also needed to evluate radiotherapy adaptation approaches for selected patients, i.e. adaptation of radiation dose, or, more sophisticated, of target volumes.

  2. Nerve growth factor receptor immunostaining suggests an extrinsic origin for hypertrophic nerves in Hirschsprung's disease.

    OpenAIRE

    Kobayashi, H; O'Briain, D S; Puri, P

    1994-01-01

    The expression of nerve growth factor receptor in colon from 20 patients with Hirshsprung's disease and 10 controls was studied immunohistochemically. The myenteric and submucous plexuses in the ganglionic bowel and hypertrophic nerve trunks in the aganglionic bowel displayed strong expression of nerve growth factor receptor. The most important finding was the identical localisation of nerve growth factor receptor immunoreactivity on the perineurium of both hypertrophic nerve trunks in Hirshs...

  3. Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; Boonstra, J.; de Laat, S. W.

    1991-01-01

    The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized

  4. Single-chain-in-mean-field simulations of weak polyelectrolyte brushes

    Science.gov (United States)

    Léonforte, F.; Welling, U.; Müller, M.

    2016-12-01

    Structural properties of brushes which are composed of weak acidic and basic polyelectrolytes are studied in the framework of a particle-based approach that implicitly accounts for the solvent quality. Using a semi-grandcanonical partition function in the framework of the Single-Chain-in-Mean-Field (SCMF) algorithm, the weak polyelectrolyte is conceived as a supramolecular mixture of polymers in different dissociation states, which are explicitly treated in the partition function and sampled by the SCMF procedure. One obtains a local expression for the equilibrium acid-base reaction responsible for the regulation of the charged groups that is also incorporated to the SCMF sampling. Coupled to a simultaneous treatment of the electrostatics, the approach is shown to capture the main features of weak polyelectrolyte brushes as a function of the bulk pH in the solution, the salt concentration, and the grafting density. Results are compared to experimental and theoretical works from the literature using coarse-grained representations of poly(acrylic acid) (PAA) and poly(2-vinyl pyridine) (P2VP) polymer-based brushes. As the Born self-energy of ions can be straightforwardly included in the numerical approach, we also study its effect on the local charge regulation mechanism of the brush. We find that its effect becomes significant when the brush is dense and exposed to high salt concentrations. The numerical methodology is then applied (1) to the study of the kinetics of collapse/swelling of a P2VP brush and (2) to the ability of an applied voltage to induce collapse/swelling of a PAA brush in a pH range close to the pKa value of the polymer.

  5. General model of phospholipid bilayers in fluid phase within the single chain mean field theory

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Yachong; Baulin, Vladimir A. [Departament d’Enginyeria Química, Universitat Rovira i Virgili, Av. dels Paisos Catalans 26, 43007 Tarragona (Spain); Pogodin, Sergey [Institute of Chemical Research of Catalonia, ICIQ, Av. Paisos Catalans 16, 43007 Tarragona (Spain)

    2014-05-07

    Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120°, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins.

  6. Effect of chain stiffness on the structure of single-chain polymer nanoparticles

    Science.gov (United States)

    Moreno, Angel J.; Bacova, Petra; Lo Verso, Federica; Arbe, Arantxa; Colmenero, Juan; Pomposo, José A.

    2018-01-01

    Polymeric single-chain nanoparticles (SCNPs) are soft nano-objects synthesized by purely intramolecular cross-linking of single polymer chains. By means of computer simulations, we investigate the conformational properties of SCNPs as a function of the bending stiffness of their linear polymer precursors. We investigate a broad range of characteristic ratios from the fully flexible case to those typical of bulky synthetic polymers. Increasing stiffness hinders bonding of groups separated by short contour distances and increases looping over longer distances, leading to more compact nanoparticles with a structure of highly interconnected loops. This feature is reflected in a crossover in the scaling behaviour of several structural observables. The scaling exponents change from those characteristic for Gaussian chains or rings in θ-solvents in the fully flexible limit, to values resembling fractal or ‘crumpled’ globular behaviour for very stiff SCNPs. We characterize domains in the SCNPs. These are weakly deformable regions that can be seen as disordered analogues of domains in disordered proteins. Increasing stiffness leads to bigger and less deformable domains. Surprisingly, the scaling behaviour of the domains is in all cases similar to that of Gaussian chains or rings, irrespective of the stiffness and degree of cross-linking. It is the spatial arrangement of the domains which determines the global structure of the SCNP (sparse Gaussian-like object or crumpled globule). Since intramolecular stiffness can be varied through the specific chemistry of the precursor or by introducing bulky side groups in its backbone, our results propose a new strategy to tune the global structure of SCNPs.

  7. End-anchored polymers in good solvents from the single chain limit to high anchoring densities.

    Science.gov (United States)

    Whitmore, Mark D; Grest, Gary S; Douglas, Jack F; Kent, Michael S; Suo, Tongchuan

    2016-11-07

    An increasing number of applications utilize grafted polymer layers to alter the interfacial properties of solid substrates, motivating refinement in our theoretical understanding of such layers. To assess existing theoretical models of them, we have investigated end-anchored polymer layers over a wide range of grafting densities, σ, ranging from a single chain to high anchoring density limits, chain lengths ranging over two orders of magnitude, for very good and marginally good solvent conditions. We compare Monte Carlo and molecular dynamics simulations, numerical self-consistent field calculations, and experimental measurements of the average layer thickness, h, with renormalization group theory, the Alexander-de Gennes mushroom theory, and the classical brush theory. Our simulations clearly indicate that appreciable inter-chain interactions exist at all simulated areal anchoring densities so that there is no mushroom regime in which the layer thickness is independent of σ. Moreover, we find that there is no high coverage regime in which h follows the predicted scaling, h ∼ Nσ 1/3 , for classical polymer brushes either. Given that no completely adequate analytic theory seems to exist that spans wide ranges of N and σ, we applied scaling arguments for h as a function of a suitably defined reduced anchoring density, defined in terms of the solution radius of gyration of the polymer chains and N. We find that such a scaling approach enables a smooth, unified description of h in very good solvents over the full range of anchoring density and chain lengths, although this type of data reduction does not apply to marginal solvent quality conditions.

  8. Arctigenin induced gallbladder cancer senescence through modulating epidermal growth factor receptor pathway.

    Science.gov (United States)

    Zhang, Mingdi; Cai, Shizhong; Zuo, Bin; Gong, Wei; Tang, Zhaohui; Zhou, Di; Weng, Mingzhe; Qin, Yiyu; Wang, Shouhua; Liu, Jun; Ma, Fei; Quan, Zhiwei

    2017-05-01

    Gallbladder cancer has poor prognosis and limited therapeutic options. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms involved in the antitumor effect of arctigenin on gallbladder cancer have not been fully elucidated. The expression levels of epidermal growth factor receptor were examined in 100 matched pairs of gallbladder cancer tissues. A positive correlation between high epidermal growth factor receptor expression levels and poor prognosis was observed in gallbladder cancer tissues. Pharmacological inhibition or inhibition via RNA interference of epidermal growth factor receptor induced cellular senescence in gallbladder cancer cells. The antitumor effect of arctigenin on gallbladder cancer cells was primarily achieved by inducing cellular senescence. In gallbladder cancer cells treated with arctigenin, the expression level of epidermal growth factor receptor significantly decreased. The analysis of the activity of the kinases downstream of epidermal growth factor receptor revealed that the RAF-MEK-ERK signaling pathway was significantly inhibited. Furthermore, the cellular senescence induced by arctigenin could be reverted by pcDNA-epidermal growth factor receptor. Arctigenin also potently inhibited the growth of tumor xenografts, which was accompanied by the downregulation of epidermal growth factor receptor and induction of senescence. This study demonstrates arctigenin could induce cellular senescence in gallbladder cancer through the modulation of epidermal growth factor receptor pathway. These data identify epidermal growth factor receptor as a key regulator in arctigenin-induced gallbladder cancer senescence.

  9. ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

    Science.gov (United States)

    Spencer, Brian; Emadi, Sharareh; Desplats, Paula; Eleuteri, Simona; Michael, Sarah; Kosberg, Kori; Shen, Jay; Rockenstein, Edward; Patrick, Christina; Adame, Anthony; Gonzalez, Tania; Sierks, Michael; Masliah, Eliezer

    2014-10-01

    Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.

  10. Developmental regulation of human truncated nerve growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    DiStefano, P.S.; Clagett-Dame, M.; Chelsea, D.M.; Loy, R. (Abbott Laboratories, Abbott Park, IL (USA))

    1991-01-01

    Monoclonal antibodies (designated XIF1 and IIIG5) recognizing distinct epitopes of the human truncated nerve growth factor receptor (NGF-Rt) were used in a two-site radiometric immunosorbent assay to monitor levels of NGF-Rt in human urine as a function of age. Urine samples were collected from 70 neurologically normal subjects ranging in age from 1 month to 68 years. By using this sensitive two-site radiometric immunosorbent assay, NGF-Rt levels were found to be highest in urine from 1-month old subjects. By 2.5 months, NGF-Rt values were half of those seen at 1 month and decreased more gradually between 0.5 and 15 years. Between 15 and 68 years, urine NGF-Rt levels were relatively constant at 5% of 1-month values. No evidence for diurnal variation of adult NGF-Rt was apparent. Pregnant women in their third trimester showed significantly elevated urine NGF-Rt values compared with age-matched normals. Affinity labeling of NGF-Rt with 125I-NGF followed by immunoprecipitation with ME20.4-IgG and gel autoradiography indicated that neonatal urine contained high amounts of truncated receptor (Mr = 50 kd); decreasingly lower amounts of NGF-Rt were observed on gel autoradiograms with development, indicating that the two-site radiometric immunosorbent assay correlated well with the affinity labeling technique for measuring NGF-Rt. NGF-Rt in urines from 1-month-old and 36-year-old subjects showed no differences in affinities for NGF or for the monoclonal antibody IIIG5. These data show that NGF-Rt is developmentally regulated in human urine, and are discussed in relation to the development and maturation of the peripheral nervous system.

  11. Developmental regulation of human truncated nerve growth factor receptor

    International Nuclear Information System (INIS)

    DiStefano, P.S.; Clagett-Dame, M.; Chelsea, D.M.; Loy, R.

    1991-01-01

    Monoclonal antibodies (designated XIF1 and IIIG5) recognizing distinct epitopes of the human truncated nerve growth factor receptor (NGF-Rt) were used in a two-site radiometric immunosorbent assay to monitor levels of NGF-Rt in human urine as a function of age. Urine samples were collected from 70 neurologically normal subjects ranging in age from 1 month to 68 years. By using this sensitive two-site radiometric immunosorbent assay, NGF-Rt levels were found to be highest in urine from 1-month old subjects. By 2.5 months, NGF-Rt values were half of those seen at 1 month and decreased more gradually between 0.5 and 15 years. Between 15 and 68 years, urine NGF-Rt levels were relatively constant at 5% of 1-month values. No evidence for diurnal variation of adult NGF-Rt was apparent. Pregnant women in their third trimester showed significantly elevated urine NGF-Rt values compared with age-matched normals. Affinity labeling of NGF-Rt with 125I-NGF followed by immunoprecipitation with ME20.4-IgG and gel autoradiography indicated that neonatal urine contained high amounts of truncated receptor (Mr = 50 kd); decreasingly lower amounts of NGF-Rt were observed on gel autoradiograms with development, indicating that the two-site radiometric immunosorbent assay correlated well with the affinity labeling technique for measuring NGF-Rt. NGF-Rt in urines from 1-month-old and 36-year-old subjects showed no differences in affinities for NGF or for the monoclonal antibody IIIG5. These data show that NGF-Rt is developmentally regulated in human urine, and are discussed in relation to the development and maturation of the peripheral nervous system

  12. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

    DEFF Research Database (Denmark)

    Pandey, A; Podtelejnikov, A V; Blagoev, B

    2000-01-01

    Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2...

  13. Development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor receptor and insulin receptor expression in equine tissue

    Directory of Open Access Journals (Sweden)

    Stephen B. Hughes

    2013-08-01

    Full Text Available The insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins and insulin are essential to muscle metabolism and most aspects of male and female reproduction. Insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. In order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation, real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. The assays were sensitive: 192 copies/µLand 891 copies/µL for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95%limit of detection, and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. The assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor. This allowed for analysis of very small amounts of messenger ribonucleic acid. Low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. The assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1

  14. Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

    International Nuclear Information System (INIS)

    Meramveliotaki, Chrysi; Kotsifaki, Dina; Androulaki, Maria; Hountas, Athanasios; Eliopoulos, Elias; Kokkinidis, Michael

    2007-01-01

    PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4 2 , with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement

  15. Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling

    Directory of Open Access Journals (Sweden)

    Inês Gomes Ferreira

    2018-02-01

    Full Text Available Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1 by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2 through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3 by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and β1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

  16. Epidermal growth factor receptor mutation in gastric cancer.

    Science.gov (United States)

    Liu, Zhimin; Liu, Lina; Li, Mei; Wang, Zhaohui; Feng, Lu; Zhang, Qiuping; Cheng, Shihua; Lu, Shen

    2011-04-01

    Epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) mutations have been identified as predictors of response to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer. We aimed to screen the mutations of both genes in gastric carcinoma to detect the suitability of EGFR TKIs for patients with gastric carcinoma. We screened EGFR mutation in exons 19-21 and KRAS mutation in exon 2 in 58 gastric adenocarcinomas from China using high resolution melting analysis (HRMA). Positive samples were confirmed by DNA sequencing. Three EGFR missense mutations (5.2%) and 22 single nucleotide polymorphisms (SNP, Q787Q, 37.9%) were identified. To our knowledge, we report for the first time three mutation patterns of EGFR, Y801C, L858R and G863D, in gastric carcinoma. Two samples with EGFR mutation were mucinous adenocarcinoma. These three samples were collected from male patients aged over 75 years old. The frequency of KRAS mutation was 10.3% (6/58). The exclusiveness of EGFR and KRAS mutations was proven for the first time in gastric cancer. Gastric carcinoma of the mucinous adenocarcinoma type collected from older male patients may harbour EGFR mutations. The small subset of gastric adenocarcinoma patients may respond to EGFR TKIs.

  17. Construction and Self-Assembly of Single-Chain Polymer Nanoparticles via Coordination Association and Electrostatic Repulsion in Water.

    Science.gov (United States)

    Zhu, Zhengguang; Xu, Na; Yu, Qiuping; Guo, Lei; Cao, Hui; Lu, Xinhua; Cai, Yuanli

    2015-08-01

    Simultaneous coordination-association and electrostatic-repulsion interactions play critical roles in the construction and stabilization of enzymatic function metal centers in water media. These interactions are promising for construction and self-assembly of artificial aqueous polymer single-chain nanoparticles (SCNPs). Herein, the construction and self-assembly of dative-bonded aqueous SCNPs are reported via simultaneous coordination-association and electrostatic-repulsion interactions within single chains of histamine-based hydrophilic block copolymer. The electrostatic-repulsion interactions are tunable through adjusting the imidazolium/imidazole ratio in response to pH, and in situ Cu(II)-coordination leads to the intramolecular association and single-chain collapse in acidic water. SCNPs are stabilized by the electrostatic repulsion of dative-bonded block and steric shielding of nonionic water-soluble block, and have a huge specific surface area of function metal centers accessible to substrates in acidic water. Moreover, SCNPs can assemble into micelles, networks, and large particles programmably in response to the solution pH. These unique media-sensitive phase-transformation behaviors provide a general, facile, and versatile platform for the fabrication of enzyme-inspired smart aqueous catalysts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance.

    Science.gov (United States)

    Patel, Rekha; Andrien, Bruce A

    2010-01-01

    Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field.

  19. Association of coatomer proteins with the beta-receptor for platelet-derived growth factor

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Rorsman, C

    1997-01-01

    The nonreceptor tyrosine kinase Src binds to and is activated by the beta-receptor for platelet-derived growth factor (PDGF). The interaction leads to Src phosphorylation of Tyr934 in the kinase domain of the receptor. In the course of the functional characterization of this phosphorylation, we...... of intracellular vesicle transport. In order to explore the functional significance of the interaction between alpha- and beta'-COP and the PDGF receptor, a receptor mutant was made in which the conserved histidine residue 928 was mutated to an alanine residue. The mutant receptor, which was unable to bind alpha...

  20. The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

    Science.gov (United States)

    Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2012-08-03

    N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

  1. A transcription factor active on the epidermal growth factor receptor gene

    International Nuclear Information System (INIS)

    Kageyama, R.; Merlino, G.T.; Pastan, I.

    1988-01-01

    The authors have developed an in vitro transcription system for the epidermal growth factor receptor (EGFR) oncogene by using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce EGFR. They found that a nuclear factor, termed EGFR-specific transcription factor (ETF), specifically stimulated EGFR transcription by 5- to 10-fold. In this report, ETF, purified by using sequence-specific oligonucleotide affinity chromatography, is shown by renaturing material eluted from a NaDodSO 4 /polyacrylamide gel to be a protein with a molecular mass of 120 kDa. ETF binds to the promoter region, as measured by DNase I footprinting and gel-mobility-shift assays, and specifically stimulates the transcription of the EGFR gene in a reconstituted in vitro transcription system. These results suggest that ETF could play a role in the overexpression of the cellular oncogene EGFR

  2. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    Science.gov (United States)

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  3. Host factors that modify Plasmodium falciparum adhesion to endothelial receptors.

    Science.gov (United States)

    Mahamar, Almahamoudou; Attaher, Oumar; Swihart, Bruce; Barry, Amadou; Diarra, Bacary S; Kanoute, Moussa B; Cisse, Kadidia B; Dembele, Adama B; Keita, Sekouba; Gamain, Benoît; Gaoussou, Santara; Issiaka, Djibrilla; Dicko, Alassane; Duffy, Patrick E; Fried, Michal

    2017-10-24

    P. falciparum virulence is related to adhesion and sequestration of infected erythrocytes (IE) in deep vascular beds, but the endothelial receptors involved in severe malaria remain unclear. In the largest ever study of clinical isolates, we surveyed adhesion of freshly collected IE from children under 5 years of age in Mali to identify novel vascular receptors, and examined the effects of host age, hemoglobin type, blood group and severe malaria on levels of IE adhesion to a panel of endothelial receptors. Several novel molecules, including integrin α3β1, VE-cadherin, ICAM-2, junctional adhesion molecule-B (JAM-B), laminin, and cellular fibronectin, supported binding of IE from children. Severe malaria was not significantly associated with levels of IE adhesion to any of the 19 receptors. Hemoglobin AC, which reduces severe malaria risk, reduced IE binding to the receptors CD36 and integrin α5β1, while hemoglobin AS did not modify IE adhesion to any receptors. Blood groups A, AB and B significantly reduced IE binding to ICAM-1. Severe malaria risk varies with age, but age significantly impacted the level of IE binding to only a few receptors: IE binding to JAM-B decreased with age, while binding to CD36 and integrin α5β1 significantly increased with age.

  4. Baculovirus display of single chain antibody (scFv using a novel signal peptide

    Directory of Open Access Journals (Sweden)

    Gonzalez Gaëlle

    2010-11-01

    Full Text Available Abstract Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17, was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2 to another scFv recognizing CD147 (scFv-M6-1B9 conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV

  5. Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression.

    Science.gov (United States)

    Feng, Shu; Shao, Longjiang; Yu, Wendong; Gavine, Paul; Ittmann, Michael

    2012-07-15

    Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1-3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer.

  6. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

    International Nuclear Information System (INIS)

    Ayyagari, R.R.; Khan-Dawood, F.S.

    1987-01-01

    Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol 125 I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function

  7. Napsin A and Thyroid Transcription Factor-1-Positive Cerebellar Tumor with Epidermal Growth Factor Receptor Mutation

    Directory of Open Access Journals (Sweden)

    Taiji Kuwata

    2011-12-01

    Full Text Available We present a very rare case of cerebellar metastasis of unknown origin, in which a primary lung adenocarcinoma was diagnosed by pathological examination of a cerebellar metastatic tumor, using immunohistochemical markers and epidermal growth factor receptor (EGFR mutation of primary lung cancer. A 69-year-old woman was admitted to our hospital because of a hemorrhagic cerebellar tumor and multiple small brain tumors. She underwent cerebellar tumor resection. On pathological examination, the tumor was diagnosed as adenocarcinoma. However, the primary tumor site was unidentifiable even with several imaging inspections. On immunohistochemical analysis, the resected tumor was positive for napsin A and thyroid transcription factor-1. In addition, an EGFR mutation was detected in the tumor. Therefore, primary lung cancer was diagnosed and the patient was started on gefitinib (250 mg/day therapy.

  8. Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

    International Nuclear Information System (INIS)

    Hirose, Tetsuro; Terajima, Hiroaki; Yamauchi, Akira

    1997-01-01

    Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125 I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3 H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125 I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125 I-insulin specific binding was not affected. The decrease in 125 I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125 I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125 I-epidermal growth factor binding

  9. Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

    Directory of Open Access Journals (Sweden)

    Marchal Joëlle

    2005-10-01

    Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

  10. Receptores do factor estimulante de colónias de macrófagos do robalo

    OpenAIRE

    Oliveira, Márcio Adriano Guiomar de

    2010-01-01

    O receptor do factor estimulante de colónias de macrófagos, também conhecido como receptor do factor estimulante de colónias-1 (CSF1R), é um receptor de um factor de crescimento hematopoiético que é especificamente expresso em células do sistema fagocítico-mononuclear e desempenha um papel essencial no desenvolvimento e regulação destas células. O CSF1R já foi descrito em vários mamíferos e a sua biologia tem sido exaustivamente caracterizada nestes vertebrados mas o conheci...

  11. Multistep change in epidermal growth factor receptors during spontaneous neoplastic progression in Chinese hamster embryo fibroblasts

    International Nuclear Information System (INIS)

    Wakshull, E.; Kraemer, P.M.; Wharton, W.

    1985-01-01

    Whole Chinese hamster embryo lineages have been shown to undergo multistep spontaneous neoplastic progression during serial passage in culture. The authors have studied the binding, internalization, and degradation of 125 I-labeled epidermal growth factor at four different stages of transformation. The whole Chinese hamster embryo cells lost cell surface epidermal growth factor receptors gradually during the course of neoplastic progression until only 10% of the receptor number present in the early-passage cells (precrisis) were retained in the late-passage cells (tumorigenic). No differences in internalization rates, chloroquine sensitivity, or ability to degrade hormone between the various passage levels were seen. No evidence for the presence in conditioned medium of transforming growth factors which might mask or down-regulate epidermal growth factor receptor was obtained. These results suggest that a reduction in cell surface epidermal growth factor receptor might be an early event during spontaneous transformation in whole Chinese hamster embryo cells

  12. Hierarchical classification strategy for Phenotype extraction from epidermal growth factor receptor endocytosis screening

    NARCIS (Netherlands)

    L. Cao (Lu); M. Graauw (Marjo de); K. Yan (Kuan); L.C.J. Winkel (Leah C.J.); F.J. Verbeek (Fons)

    2016-01-01

    textabstractBackground: Endocytosis is regarded as a mechanism of attenuating the epidermal growth factor receptor (EGFR) signaling and of receptor degradation. There is increasing evidence becoming available showing that breast cancer progression is associated with a defect in EGFR endocytosis. In

  13. Targeting of the tumor necrosis factor receptor superfamily for cancer immunotherapy

    NARCIS (Netherlands)

    Bremer, Edwin

    2013-01-01

    The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamilies constitute an important regulatory axis that is pivotal for immune homeostasis and correct execution of immune responses. TNF ligands and receptors are involved in diverse biological processes ranging from the selective

  14. Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

    International Nuclear Information System (INIS)

    Xu, Ling; Hausmann, Martin; Dietmaier, Wolfgang; Kellermeier, Silvia; Pesch, Theresa; Stieber-Gunckel, Manuela; Lippert, Elisabeth; Klebl, Frank; Rogler, Gerhard

    2010-01-01

    Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab

  15. Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Kellermeier Silvia

    2010-06-01

    Full Text Available Abstract Background Cholangiocarcinoma (CC is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Methods Expression of EGFR (epithelial growth factor receptor, HGFR (hepatocyte growth factor receptor IGF1R (insulin-like growth factor 1 receptor, IGF2R (insulin-like growth factor 2 receptor and VEGFR1-3 (vascular endothelial growth factor receptor 1-3 were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1. The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. Results EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml, with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D. HuH28, OZ and TFK-1 lacked KRAS mutation. Conclusion CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

  16. Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    Directory of Open Access Journals (Sweden)

    Ahnen Dennis

    2005-01-01

    Full Text Available Abstract Background Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs is associated with a decreased mortality from colorectal cancer (CRC. NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2 signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF receptor (EGFR. Methods HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068, total EGFR, phosphorylated ERK1/2 (pERK1/2, total ERK1/2, activated caspase-3, and α-tubulin. Results EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. Conclusion These results suggest that

  17. Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

    Science.gov (United States)

    Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

  18. Regulation of insulin-like growth factor I receptors on vascular smooth muscle cells by growth factors and phorbol esters.

    Science.gov (United States)

    Ververis, J J; Ku, L; Delafontaine, P

    1993-06-01

    Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells. To characterize regulation of vascular IGF I receptors, we performed radioligand displacement experiments using rat aortic smooth muscle cells (RASMs). Serum deprivation for 48 hours caused a 40% decrease in IGF I receptor number. Exposure of quiescent RASMs to platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), or angiotensin II (Ang II) caused a 1.5-2.0-fold increase in IGF I receptors per cell. After FGF exposure, there was a marked increase in the mitogenic response to IGF I. IGF I downregulated its receptors in the presence of platelet-poor plasma. Stimulation of protein kinase C (PKC) by exposure of quiescent RASMs to phorbol 12-myristate 13-acetate caused a biphasic response in IGF I binding; there was a 42% decrease in receptor number at 45 minutes and a 238% increase at 24 hours. To determine the role of PKC in growth factor-induced regulation of IGF I receptors, we downregulated PKC by exposing RASMs to phorbol 12,13-dibutyrate (PDBu) for 48 hours. PDGF- and FGF- but not Ang II-mediated upregulation of IGF I receptors was completely inhibited in PDBu-treated cells. Thus, acute PKC activation by phorbol esters inhibits IGF I binding, whereas chronic PKC activation increases IGF I binding. PDGF and FGF but not Ang II regulate vascular IGF I receptors through a PKC-dependent pathway. These data provide new insights into the regulation of vascular smooth muscle cell IGF I receptors in vitro and are of potential importance in characterizing vascular proliferative responses in vivo.

  19. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Directory of Open Access Journals (Sweden)

    Shibaguchi H

    2017-08-01

    Full Text Available Hirotomo Shibaguchi,1,* Naixiang Luo,1,* Naoto Shirasu,1,* Motomu Kuroki,2 Masahide Kuroki1 1Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan; 2School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan *These authors equally contributed to this work Abstract: Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. Keywords: chimeric antigen receptor, fusion protein, human scFv, CEA, combination therapy

  20. EphA2 is a functional receptor for the growth factor progranulin.

    Science.gov (United States)

    Neill, Thomas; Buraschi, Simone; Goyal, Atul; Sharpe, Catherine; Natkanski, Elizabeth; Schaefer, Liliana; Morrione, Andrea; Iozzo, Renato V

    2016-12-05

    Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. © 2016 Neill et al.

  1. Signal transduction by growth factor receptors: signaling in an instant

    DEFF Research Database (Denmark)

    Dengjel, Joern; Akimov, Vyacheslav; Blagoev, Blagoy

    2007-01-01

    Phosphorylation-based signaling events happening within the first minute of receptor stimulation have so far only been analyzed by classical cell biological approaches like live-cell microscopy. The development of a quench flow system with a time resolution of one second coupled to a read...

  2. Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system.

    Science.gov (United States)

    Walsh, Matthew C; Lee, JangEun; Choi, Yongwon

    2015-07-01

    Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factorreceptors (TGFβR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Tumor necrosis factor receptor associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system

    Science.gov (United States)

    Walsh, Matthew C.; Lee, JangEun; Choi, Yongwon

    2016-01-01

    Summary Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adaptor protein that mediates a wide array of protein-protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of IL-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the toll-like receptor (TLR) family, tumor growth factorreceptors (TGFβR), and T cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor (IRF) pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system, but also for maintaining immune tolerance, and more recent works have begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs. PMID:26085208

  4. A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

    Science.gov (United States)

    Zhou, Xin X; Zou, Xinzhi; Chung, Hokyung K; Gao, Yuchen; Liu, Yanxia; Qi, Lei S; Lin, Michael Z

    2018-02-16

    Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

  5. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

    Science.gov (United States)

    McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

    1992-01-01

    The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

  6. Increase of tumor necrosis factor receptor 1 expression in women with unexplained early spontaneous abortion

    Institute of Scientific and Technical Information of China (English)

    YAN Chun-fang; YU Xue-wen; JIN Hui; LI Xu

    2004-01-01

    To investigate membrane tumor necrosis factor receptor 1 protein expression level in decidua andconcentration of soluble tumor necrosis factor receptor 1 in serum in women with unexplained early spontaneous abortion,threatened abortion, and compare the levels with healthy pregnant women. Methods: Thirty-seven women with unexplainedearly spontaneous abortion, 27 women with threatened abortion, and 34 healthy pregnant women undergoing artificial abortionof pregnancy at 6 - 10 weeks of gestation were selected. Decidual samples were collected when women were undergoing arti-ficial abortion, and blood samples were collected at the same time. The level of membrane tumor necrosis factor receptor 1 indecidua was detected by flow cytometer, and the concentration of soluble tumor necrosis factor receptor 1 in sera was mea-sured with an enzyme-linked immunosorbent assay. Results: The ercentages of membrane tumor necrosis factor receptor 1positive decidual cells were 16.42 ± 7.10 Mean ± SD for women with unexplained early spontaneous abortion and 13.14 ±6.30 for healthy pregnant women ( P < 0.05). Serum oncentration of soluble tumor necrosis factor receptor 1 was signifi-cantly higher in women with unexplained early spontaneous abortion than in healthy pregnant women and in women withthreatened abortion, and no difference was found between healthy pregnant women and women with threatened abortion.Conclusion: Women with unexplained early spontaneous abortion present significantly higher expression of tumor necrosisfactor receptor 1 than healthy pregnant women, suggesting that over-expression of tumor necrosis factor receptor 1 may cont-ribute to the development of early spontaneous abortion.

  7. Immunoautoradiographic analysis of epidermal growth factor receptors: a sensitive method for the in situ identification of receptor proteins and for studying receptor specificity

    International Nuclear Information System (INIS)

    Fernandez-Pol, J.A.

    1982-01-01

    The use of an immunoautoradiographic system for the detection and analysis of epidermal growth factor (EGF) receptors in human epidermoid carcinoma A-431 cells is reported. By utilizing this technique, the interaction between EGF and its membrane receptor in A-431 cells can be rapidly visualized. The procedure is simple, rapid, and very sensitive, and it provides conclusive evidence that the 150K dalton protein is the receptor fo EGF in A-431 cells. In summary, the immunoautoradiographic procedure brings to the analysis of hormone rceptor proteins the power that the radioimmunoassay technique has brought to the analysis of hormones. Thus, this assay system is potentially applicable in a wide spectrum in many fields of nuclear medicine and biology

  8. Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis

    International Nuclear Information System (INIS)

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-01-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [ 125 I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers

  9. Hormonal receptors and vascular endothelial growth factor in juvenile nasopharyngeal angiofibroma: immunohistochemical and tissue microarray analysis.

    Science.gov (United States)

    Liu, Zhuofu; Wang, Jingjing; Wang, Huan; Wang, Dehui; Hu, Li; Liu, Quan; Sun, Xicai

    2015-01-01

    This work demonstrated that juvenile nasopharyngeal angiofibromas (JNAs) express high levels of hormone receptors and vascular endothelial growth factor (VEGF) compared with normal nasal mucosa. The interaction between hormone receptors and VEGF may be involved in the initiation and growth of JNA. JNA is a rare benign tumor that occurs almost exclusively in male adolescents. Although generally regarded as a hormone-dependent tumor, this has not been proven in previous studies. The aim of this study was to investigate the role of hormone receptors in JNA and the relationship with clinical characteristics. Standard immunohistochemical microarray analysis was performed on 70 JNA samples and 10 turbinate tissue samples. Specific antibodies for androgen receptor (AR), estrogen receptor-α (ER-α), estrogen receptor-β (ER-β), progesterone receptor (PR), and VEGF were examined, and the relationships of receptor expression with age, tumor stage, and bleeding were evaluated. RESULTS showed that JNA expressed ER-α (92.9%), ER-β (91.4%), AR (65.7%), PR (12.8%), and VEGF (95.7%) at different levels. High level of VEGF was linked to elevated ER-α and ER-β. There was no significant relationship between hormonal receptors and age at diagnosis, tumor stage or bleeding. However, overexpression of ER-α was found to be an indicator of poor prognosis (p = 0.031).

  10. Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

    International Nuclear Information System (INIS)

    Scott, C.D.; Baxter, R.C.

    1987-01-01

    Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [ 125 I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density

  11. Molecular and functional characterization of pigeon (Columba livia) tumor necrosis factor receptor-associated factor 3.

    Science.gov (United States)

    Zhou, Yingying; Kang, Xilong; Xiong, Dan; Zhu, Shanshan; Zheng, Huijuan; Xu, Ying; Guo, Yaxin; Pan, Zhiming; Jiao, Xinan

    2017-04-01

    Tumor necrosis factor receptor-associated factor 3 (TRAF3) plays a key antiviral role by promoting type I interferon production. We cloned the pigeon TRAF3 gene (PiTRAF3) according to its predicted mRNA sequence to investigate its function. The 1704-bp full-length open reading frame encodes a 567-amino acid protein. One Ring finger, two TRAF-type Zinc fingers, one Coiled coil, and one MATH domain were inferred. RT-PCR showed that PiTRAF3 was expressed in all tissues, with relatively weak expression in the heart and liver. In HEK293T cells, over-expression of wild-type, △Ring, △Zinc finger, and △Coiled coil PiTRAF3, but not a △MATH form, significantly increased IFN-β promoter activity. Zinc finger and Coiled coil domains were essential for NF-κB activation. In chicken HD11 cells, PiTRAF3 increased IFN-β promoter activity and four domains were all contributing. R848 stimulation of pigeon peripheral blood mononuclear cells and splenocytes significantly increased expression of PiTRAF3 and the inflammatory cytokine genes CCL5, IL-8, and IL-10. These data demonstrate TRAF3's innate immune function and improve understanding of its involvement in poultry antiviral defense. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.

    Science.gov (United States)

    Fantl, W J; Escobedo, J A; Martin, G A; Turck, C W; del Rosario, M; McCormick, F; Williams, L T

    1992-05-01

    The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.

  13. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Directory of Open Access Journals (Sweden)

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  14. Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

    Science.gov (United States)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

    2013-01-01

    The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown. PMID:23472148

  15. Growth factor receptors as molecular targets for cancer diagnosis and therapy

    International Nuclear Information System (INIS)

    Zalutsky, M. R.

    1997-01-01

    Growth factor receptors are of great interest as molecular targets for the diagnosis and treatment of cancer. Growth factor receptors are frequently over expressed on malignant cell populations since many cellular oncogenes encode either growth factors of their receptors. The wild-type epidermal growth factor receptor has a molecular weight of 170 kD and is over expressed on gliomas, bladder tumors, squamous cells carcinomas and breast carcinomas. Another growth factor oncogene, c-erb B-2, encodes a 185-kD glycoprotein found on the surface of gliomas, breast and ovarian cancers as well as other carcinomas of epithelial origin. In addition to causing over expression, oncogenic transformation also can result in genomic re-arrangements. An important example from the perspective of targeting is EGFRvIII, a deletion mutant which lacks amino acids 6-273 in the extracellular domain of the epiderma growth factor receptor. The EGFRvIII molecule (145 kD) may be of great value for targeting because it appears to be tumor-specific. Antibodies have been developed with specific reactivity with these growth factor receptors. Since these antibodies are internalized into the cell after receptor binding, it is necessary to use radiolabeling methods which residualize the radioactivity in the tumor cell after intracellular catabolism. To investigate this problem they have evaluated the effect of radioiodination method on the in vitro an in vivo properties of an anti-EGFRvIII antibody. Methods studied were Iodogen, tyramine-cellobiose, and N-succinimidyl 5-iodo-3-pyridine-carboxylate with the last offering optimal localization in a human xenograft model

  16. Epidermal growth factor receptor: an independent predictor of survival in astrocytic tumors given definitive irradiation

    International Nuclear Information System (INIS)

    An Zhu; Shaeffer, James; Leslie, Susan; Kolm, Paul; El-Mahdi, Anas M.

    1996-01-01

    Purpose: To determine whether the expression of epidermal growth factor receptor (EGFR) protein was predictive of patient survival independently of other prognostic factors in astrocytic tumors. Methods and Materials: Epidermal growth factor receptor protein expression was investigated immunohistochemically in formalin-fixed, paraffin-embedded surgical specimens of 55 glioblastoma multiforme, 14 anaplastic astrocytoma, and 2 astrocytomas given definitive irradiation. We evaluated the relationship of EGFR protein expression and tumor grade, histologic features, age at diagnosis, sex, patient survival, and recurrence-free survival. Results: The percentage of tumor cells which were EGFR positive related to reduced survival by Cox regression analysis in both univariate (p = 0.0424) and multivariate analysis (p = 0.0016). Epidermal growth factor receptor positivity was the only 1 of 11 clinical and histological variables associated with decreased recurrence-free survival by either univariate (p = 0.0353) or multivariate (p = 0.0182) analysis. Epidermal growth factor receptor protein expression was not related to patient age, sex, or histologic features. Conclusion: Epidermal growth factor receptor positivity was a significant and independent prognostic indicator for overall survival and recurrence-free survival for irradiated patients with astrocytic gliomas

  17. Inhibition of the release of soluble tumor necrosis factor receptors in experimental endotoxemia by an anti-tumor necrosis factor-alpha antibody

    NARCIS (Netherlands)

    Jansen, J.; van der Poll, T.; Levi, M. [=Marcel M.; ten Cate, H.; Gallati, H.; ten Cate, J. W.; van Deventer, S. J.

    1995-01-01

    The role of tumor necrosis factor-alpha in the shedding of soluble tumor necrosis factor receptors in endotoxemia was investigated. The appearance of the soluble tumor necrosis factor receptors was assessed in four healthy volunteers following an intravenous injection of tumor necrosis factor-alpha

  18. Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

    Science.gov (United States)

    Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

    2014-03-01

    Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

  19. ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.

    Science.gov (United States)

    Hsu, H; Solovyev, I; Colombero, A; Elliott, R; Kelley, M; Boyle, W J

    1997-05-23

    Members of tumor necrosis factor receptor (TNFR) family signal largely through interactions with death domain proteins and TRAF proteins. Here we report the identification of a novel TNFR family member ATAR. Human and mouse ATAR contain 283 and 276 amino acids, respectively, making them the shortest known members of the TNFR superfamily. The receptor is expressed mainly in spleen, thymus, bone marrow, lung, and small intestine. The intracellular domains of human and mouse ATAR share only 25% identity, yet both interact with TRAF5 and TRAF2. This TRAF interaction domain resides at the C-terminal 20 amino acids. Like most other TRAF-interacting receptors, overexpression of ATAR activates the transcription factor NF-kappaB. Co-expression of ATAR with TRAF5, but not TRAF2, results in synergistic activation of NF-kappaB, suggesting potentially different roles for TRAF2 and TRAF5 in post-receptor signaling.

  20. The development of epidermal growth factor receptor molecular imaging in cancer

    International Nuclear Information System (INIS)

    Zhou Xiaoliang; Wang Hao; Shi Peiji; Liu Jianfeng; Meng Aimin

    2013-01-01

    In vivo epidermal growth factor receptor (EGFR) targeted therapy has great potential for cancer diagnosis and the evaluation of curative effects. Enhancement of EGFR-targeted therapy needs a reliable quantitative molecular imaging method which could enable monitoring of receptor drug binding and receptor occupancy in vivo, and identification of the mutation in EGFR. PET or SPECT is the most advanced molecular imaging technology of non-invasively selecting responders, predicting therapeutic outcome and monitoring EGFR-targeted treatment. This review analyzed the present situation and research progress of molecular imaging agents. (authors)

  1. Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

    DEFF Research Database (Denmark)

    Mi, Li-Zhi; Grey, Michael J; Nishida, Noritaka

    2008-01-01

    Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase...... differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane...

  2. Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

    OpenAIRE

    Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

    1992-01-01

    Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protei...

  3. A receptor model for urban aerosols based on oblique factor analysis

    DEFF Research Database (Denmark)

    Keiding, Kristian; Sørensen, Morten S.; Pind, Niels

    1987-01-01

    A procedure is outlined for the construction of receptor models of urban aerosols, based on factor analysis. The advantage of the procedure is that the covariation of source impacts is included in the construction of the models. The results are compared with results obtained by other receptor......-modelling procedures. It was found that procedures based on correlating sources were physically sound as well as in mutual agreement. Procedures based on non-correlating sources were found to generate physically obscure models....

  4. Targeting the epidermal growth factor receptor in solid tumor malignancies

    DEFF Research Database (Denmark)

    Nedergaard, Mette K; Hedegaard, Chris J; Poulsen, Hans S

    2012-01-01

    been proposed as valid targets in many cancer therapy settings. Different strategies have been developed in order to either inhibit EGFR/EGFRvIII activity or to ablate EGFR/EGFRvIII-positive tumor cells. Drugs that inhibit these receptors include monoclonal antibodies (mAbs) that bind...... to the extracellular part of EGFR, blocking the binding sites for the EGFR ligands, and intracellular tyrosine kinase inhibitors (TKIs) that block the ATP binding site of the tyrosine kinase domain. Besides an EGFRvIII-targeted vaccine, conjugated anti-EGFR mAbs have been used in different settings to deliver lethal...... agents to the EGFR/EGFRvIII-positive cells; among these are radio-labelled mAbs and immunotoxins. This article reviews the current status and efficacy of EGFR/EGFRvIII-targeted therapies....

  5. Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

    Science.gov (United States)

    Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

    2007-09-14

    The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

  6. 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

    2012-04-01

    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.

  7. The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization.

    Science.gov (United States)

    Lindsey, Stephan; Papoutsakis, Eleftherios T

    2011-02-01

    We propose that the aryl hydrocarbon receptor (AHR) is a novel transcriptional regulator of megakaryopoietic polyploidization. Functional evidence was obtained that AHR impacts in vivo megakaryocytic differentiation and maturation; compared to wild-type mice, AHR-null mice had lower platelet counts, fewer numbers of newly synthesized platelets, increased bleeding times and lower-ploidy megakaryocytes (Mks). AHR mRNA increased 3·6-fold during ex vivo megakaryocytic differentiation, but reduced or remained constant during parallel isogenic granulocytic or erythroid differentiation. We interrogated the role of AHR in megakaryopoiesis using a validated Mk model of megakaryopoiesis, the human megakaryoblastic leukaemia CHRF cell line. Upon CHRF Mk differentiation, AHR mRNA and protein levels increased, AHR protein shifted from the cytoplasm to the nucleus and AHR binding to its consensus DNA binding sequence increased. Protein and mRNA levels of the AHR transcriptional target HES1 also increased. Mk differentiation of CHRF cells where AHR or HES1 was knocked-down using RNAi resulted in lower ploidy distributions and cells that were incapable of reaching ploidy classes ≥16n. AHR knockdown also resulted in increased DNA synthesis of lower ploidy cells, without impacting apoptosis. Together, these data support a role for AHR in Mk polyploidization and in vivo platelet function, and warrant further detailed investigations. © 2011 Blackwell Publishing Ltd.

  8. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands

    DEFF Research Database (Denmark)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe

    2013-01-01

    after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist....... Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown...... fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand...

  9. Soluble tumor necrosis factor receptor-1 in preterm infants with chronic lung disease.

    Science.gov (United States)

    Sato, Miho; Mori, Masaaki; Nishimaki, Shigeru; An, Hiromi; Naruto, Takuya; Sugai, Toshiyuki; Shima, Yoshio; Seki, Kazuo; Yokota, Shumpei

    2010-04-01

    It is clear that inflammation plays an important role in developing chronic lung disease in preterm infants. The purpose of the present study is to investigate changes of serum soluble tumor necrosis factor receptor-1 levels over time in infants with chronic lung disease. The serum levels of soluble tumor necrosis factor receptor-1 were measured after delivery, and at 7, 14, 21 and 28 days of age in 10 infants with chronic lung disease and in 18 infants without chronic lung disease. The serum level of soluble tumor necrosis factor receptor-1 was significantly higher in infants with chronic lung disease than in infants without chronic lung disease after delivery. The differences between these two groups remained up to 28 days of age. Prenatal inflammation with persistence into postnatal inflammation may be involved in the onset of chronic lung disease.

  10. Identification of a second putative receptor of platelet activating factor on human polymorphonuclear leukocytes

    International Nuclear Information System (INIS)

    Hwang, S.B.

    1987-01-01

    Due to multiple molecular species of platelet activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors on human leukocytes and platelets. Human PMN leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (Kd) of 4.7 (+/- 1.4) x 10 -10 M. The maximal number (B/sub max/) of receptor sites was estimated to be 3.13 (+/- 1.4) x 10 -13 mol/mg protein. They compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One antagonist (Ono-6240) was found to be 8 times less potent at inhibiting the [ 3 H]PAF specific receptor binding to human leukocytes than to human platelets. Mg 2+ , Ca 2+ and K + ions potentiated the [ 3 H]PAF specific binding in both systems. Na + ions inhibited the [ 3 H]PAF specific binding to human platelets but showed no effects in human leukocytes. K + ions decreased the Mg 2+ -potentiated [ 3 H]PAF binding in human leukocytes but showed no effects in human platelets. These results suggest that the PAF specific receptors in human leukocytes are different structurally and possibly functionally from the receptors identified in human platelets

  11. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M

    1992-01-01

    of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell......Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...

  12. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  13. ErbB2 resembles an autoinhibited invertebrate epidermal growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A.; (UPENN-MED)

    2009-09-25

    The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.

  14. Peptides derived from specific interaction sites of the fibroblast growth factor 2 - FGF receptor complexes induce receptor activation and signaling

    DEFF Research Database (Denmark)

    Manfè, Valentina; Kochoyan, Artur; Bock, Elisabeth

    2010-01-01

    J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06718.x Abstract Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic...... biologic actions by binding, dimerizing, and activating FGF receptors (FGFRs). The present study reports the physiologic impact of various FGF2-FGFR1 contact sites employing three different synthetic peptides, termed canofins, designed based on structural analysis of the interactions between FGF2 and FGFR1...

  15. Thyroid hormone and retinoic acid nuclear receptors: specific ligand-activated transcription factors

    International Nuclear Information System (INIS)

    Brtko, J.

    1998-01-01

    Transcriptional regulation by both the thyroid hormone and the vitamin A-derived 'retinoid hormones' is a critical component in controlling many aspects of higher vertebrate development and metabolism. Their functions are mediated by nuclear receptors, which comprise a large super-family of ligand-inducible transcription factors. Both the thyroid hormone and the retinoids are involved in a complex arrangement of physiological and development responses in many tissues of higher vertebrates. The functions of 3,5,3'-triiodothyronine (T 3 ), the thyromimetically active metabolite of thyroxine as well as all-trans retinoic acid, the biologically active vitamin A metabolite are mediated by nuclear receptor proteins that are members of the steroid/thyroid/retinoid hormone receptor family. The functions of all members of the receptor super family are discussed. (authors)

  16. Inactivation of the transforming growth factor beta type II receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Hougaard, S; Nørgaard, P; Abrahamsen, N

    1999-01-01

    Transforming growth factor beta (TGF-beta) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-beta due to lack of type II receptor (RII) has been described in some cancer types including small cell lung...

  17. Type-I Insulin-Like Growth Factor Receptor (IGF1R)-Estrogen Receptor (ER) Crosstalk Contributes to Antiestrogen Therapy Resistance in Breast Cancer Cells

    Science.gov (United States)

    2013-02-01

    vitro have downregulated J GF1R making antibodies directed agai nst th is receptor ineffective. Inhlbition of IH may be necessary to manage ...monoclonal antibody to insulin-like growth factor receptor 1. J Clin Oncol 2009;27:580Q-7. 31. Drury s. Detre s. Leary A, Salter J, Reis-Filho J

  18. Up-regulation of proproliferative genes and the ligand/receptor pair placental growth factor and vascular endothelial growth factor receptor 1 in hepatitis C cirrhosis.

    Science.gov (United States)

    Huang, Xiao X; McCaughan, Geoffrey W; Shackel, Nicholas A; Gorrell, Mark D

    2007-09-01

    Cirrhosis can lead to hepatocellular carcinoma (HCC). Non-diseased liver and hepatitis C virus (HCV)-associated cirrhosis with or without HCC were compared. Proliferation pathway genes, immune response genes and oncogenes were analysed by a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining. Real-time RT-PCR showed up-regulation of genes in HCV cirrhosis including the proliferation-associated genes bone morphogenetic protein 3 (BMP3), placental growth factor 3 (PGF3), vascular endothelial growth factor receptor 1 (VEGFR1) and soluble VEGFR1, the oncogene FYN, and the immune response-associated genes toll-like receptor 9 (TLR9) and natural killer cell transcript 4 (NK4). Expressions of TLR2 and the oncogenes B-cell CLL/lymphoma 9 (BCL9) and PIM2 were decreased in HCV cirrhosis. In addition, PIM2 and TLR2 were increased in HCV cirrhosis with HCC compared with HCV cirrhosis. The ligand/receptor pair PGF and VEGFR1 was intensely expressed by the portal tract vascular endothelium. VEGFR1 was expressed in reactive biliary epithelial structures in fibrotic septum and in some stellate cells and macrophages. PGF and VEGFR1 may have an important role in the pathogenesis of the neovascular response in cirrhosis.

  19. Characterization of receptors for recombinant human tumor necrosis factor-alpha from human placental membranes

    International Nuclear Information System (INIS)

    Aiyer, R.A.; Aggarwal, B.B.

    1990-01-01

    High affinity receptors for recombinant human tumor necrosis factor-alpha (rhTNF-alpha) were identified on membranes prepared from full term human placenta. Highly purified rhTNF-alpha iodinated by the iodogen method was found to bind placental membranes in a displaceable manner with an approximate dissociation constant (KD) of 1.9 nM. The membrane bound TNF-alpha receptor could be solubilized by several detergents with optimum extraction being obtained with 1% Triton X-100. The binding of 125I-rhTNF-alpha to the solubilized receptor was found to be time and temperature dependent, yielding maximum binding within 1 h, 24 h and 48 h at 37 degrees C, 24 degrees C and 4 degrees C, respectively. However, the maximum binding obtainable at 4 degrees C was only 40% of that at 37 degrees C. The binding 125I-rhTNF-alpha to solubilized placental membrane extracts was displaceable by unlabeled rhTNF-alpha, but not by a related protein recombinant human tumor necrosis factor-beta (rhTNF-beta; previously called lymphotoxin). This is similar to the behavior of TNF-alpha receptors derived from detergent-solubilized cell extracts, although on intact cells, both rhTNF-alpha and rhTNF-beta bind with equal affinity to TNF receptors. The Scatchard analysis of the binding data of the solubilized receptor revealed high affinity binding sites with a KD of approximately 0.5 nM and a receptor concentration of about 1 pmole/mg protein. Gel filtration of the solubilized receptor-ligand complexes on Sephacryl S-300 revealed two different peaks of radioactivity at approximate molecular masses of 50,000 Da and 400,000 Da. The 400,000 dalton peak corresponded to the receptor-ligand complex. Overall, our results suggest that high affinity receptors for TNF-alpha are present on human placental membranes and provide evidence that these receptors may be different from that of rhTNF-beta

  20. Activated platelets in carotid artery thrombosis in mice can be selectively targeted with a radiolabeled single-chain antibody.

    Directory of Open Access Journals (Sweden)

    Timo Heidt

    Full Text Available BACKGROUND: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. METHODOLOGY/PRINCIPAL FINDINGS: LIBS as well as an unspecific control single-chain antibody were labeled with (111Indium ((111In via bifunctional DTPA ( = (111In-LIBS/(111In-control. Autoradiography after incubation with (111In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2, 4010 ± 630 DLU/mm(2 and 4520 ± 293 DLU/mm(2 produced a significantly higher ligand uptake compared to (111In-control (2101 ± 76 DLU/mm(2, 1181 ± 96 DLU/mm(2 and 1866 ± 246 DLU/mm(2 indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2 vs. 17390 ± 7470 DLU/mm(2; P<0.05. These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01. CONCLUSIONS/SIGNIFICANCE: Nuclear imaging with (111In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of

  1. A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

    Science.gov (United States)

    Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

    2016-08-01

    Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

  2. Insulin-like growth factor-II (IGF II) receptor from rat brain is of lower apparent molecular weight than the IGF II receptor from rat liver

    International Nuclear Information System (INIS)

    McElduff, A.; Poronnik, P.; Baxter, R.C.

    1987-01-01

    The binding subunits of the insulin and insulin-like growth factor-I (IGF I) receptors from rat brain are of lower molecular weight than the corresponding receptor in rat liver, possibly due to variations in sialic acid content. We have compared the IGF II receptor from rat brain and rat liver. The brain receptor is of smaller apparent mol wt (about 10 K) on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is independent of ligand binding as it persists in iodinated and specifically immunoprecipitated receptors. From studies of wheat germ agglutinin binding and the effect of neuraminidase on receptor mobility, we conclude that this difference is not simply due to variations in sialic acid content. Treatment with endoglycosidase F results in reduction in the molecular size of both liver and brain receptors and after this treatment the aglycoreceptors are of similar size. We conclude that in rat brain tissue the IGF II receptor like the binding subunits of the insulin and IGF I receptors is of lower molecular size than the corresponding receptors in rat liver. This difference is due to differences in N-linked glycosylation

  3. Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation

    International Nuclear Information System (INIS)

    Yarden, Y.; Schlessinger, J.

    1987-01-01

    The membrane receptor for epidermal growth factor (EGF) is a 170,000 dalton glycoprotein composed of an extracellular EGF-binding domain and a cytoplasmic kinase domain connected by a stretch of 23 amino acids traversing the plasma membrane. The binding of EGF to the extracellular domain activates the cytoplasmic kinase function even in highly purified preparations of EGF receptor, suggesting that the activation occurs exclusively within the EGF receptor moiety. Conceivably, kinase activation may require the transfer of a conformational change through the single transmembrane region from the ligand binding domain to the cytoplasmic kinase region. Alternatively, ligand-induced receptor-receptor interactions may activate the kinase and thus bypass this requirement. Both mechanisms were contrasted by employing independent experimental approaches. On the basis of these results, an allosteric aggregation model is formulated for the activation of the cytoplasmic kinase function of the receptor by EGF. This model may be relevant to the mechanism by which the mitogenic signal of EGF is transferred across the membrane

  4. Sperm Impairment by Sperm Agglutinating Factor Isolated from Escherichia coli: Receptor Specific Interactions

    Directory of Open Access Journals (Sweden)

    Kiranjeet Kaur

    2013-01-01

    Full Text Available In an earlier work done in our laboratory, we have been able to isolate a sperm agglutinating strain of Escherichia coli from the semen sample of a male attending infertility clinic. Further, factor responsible for sperm agglutination (SAF was isolated and purified, and, using SAF as a tool, corresponding SAF binding receptor from human spermatozoa has been purified. Characterization of SAF and SAF binding receptor using MALDI-TOF showed homology to glutamate decarboxylase and MHC class I molecule, respectively. Coincubation of SAF with spermatozoa not only resulted in spermagglutination but could also compromise other sperm parameters, namely, Mg2+ dependent ATPase activity and apoptosis. Intravaginal administration of SAF could lead to infertility in Balb/c mice. SAF induced impairment of sperm parameters, and infertility was observed to be due to interaction of SAF with sperm surface receptor component as, when purified receptor was introduced, receptor completely inhibited all the detrimental effects induced by SAF. From these results, it could be concluded that interaction of SAF with spermatozoa is receptor mediated.

  5. The under-appreciated promiscuity of the epidermal growth factor receptor family.

    Directory of Open Access Journals (Sweden)

    Sean P Kennedy

    2016-08-01

    Full Text Available Each member of the epidermal growth factor receptor (EGFR family plays a key role in normal development, homeostasis and a variety of pathophysiological conditions, most notably in cancer. According to the prevailing dogma, these four receptor tyrosine kinases (RTKs; EGFR, ERBB2, ERBB3 and ERBB4 function exclusively through the formation of homodimers and heterodimers within the EGFR family. These combinatorial receptor interactions are known to generate increased interactome diversity and therefore influence signalling output, subcellular localization and function of the heterodimer. This molecular plasticity is also thought to play a role in the development of resistance towards targeted cancer therapies aimed at these known oncogenes. Interestingly, many studies now challenge this dogma and suggest that the potential for EGFR family receptors to interact with more distantly related RTKs is much greater than currently appreciated. Here we discuss how the promiscuity of these oncogenic receptors may lead to the formation of many unexpected receptor pairings and the significant implications for the efficiency of many targeted cancer therapies.

  6. Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells

    International Nuclear Information System (INIS)

    Vale, R.D.; Ignatius, M.J.; Shooter, E.M.

    1985-01-01

    Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. 125 I-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin

  7. Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

    Science.gov (United States)

    Mochizuki, Michiyo; Kori, Masakuni; Kobayashi, Katsumi; Yano, Takahiko; Sako, Yuu; Tanaka, Maiko; Kanzaki, Naoyuki; Gyorkos, Albert C; Corrette, Christopher P; Cho, Suk Young; Pratt, Scott A; Aso, Kazuyoshi

    2016-03-24

    Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

  8. Characterization of cell-surface receptors for monoclonal-nonspecific suppressor factor (MNSF)

    International Nuclear Information System (INIS)

    Nakamura, M.; Ogawa, H.; Tsunematsu, T.

    1990-01-01

    Monoclonal-nonspecific suppressor factor (MNSF) is a lymphokine derived from murine T cell hybridoma. The target tissues are both LPS-stimulated B cells and Con A-stimulated T cells. Since the action of MNSF may be mediated by its binding to specific cell surface receptors, we characterized the mode of this binding. The purified MNSF was labeled with 125 I, using the Bolton-Hunter reagent. The labeled MNSF bound specifically to a single class of receptor (300 receptors per cell) on mitogen-stimulated murine B cells or T cells with an affinity of 16 pM at 24 degrees C, in the presence of sodium azide. Competitive experiments showed that MNSF bound to the specific receptor and that the binding was not shared with IL2, IFN-gamma, and TNF. Various cell types were surveyed for the capacity to specifically bind 125 I-MNSF. 125 I-MNSF bound to MOPC-31C (a murine plasmacytoma line) and to EL4 (a murine T lymphoma line). The presence of specific binding correlates with the capacity of the cells to respond to MNSF. These data support the view that like other polypeptide hormones, the action of MNSF is mediated by specific cell surface membrane receptor protein. Identification of these receptors will provide insight into the apparently diverse activities of MNSF

  9. Soluble receptors for tumor necrosis factor as markers of disease activity in visceral leishmaniasis

    NARCIS (Netherlands)

    Zijlstra, E. E.; van der Poll, T.; Mevissen, M.

    1995-01-01

    Serum concentrations of soluble receptors for tumor necrosis factor (sTNFRs) were measured before and after antimony therapy in 25 Sudanese patients with active visceral leishmaniasis (VL). Both sTNFR types I and II were significantly elevated in patients with VL compared with healthy controls from

  10. Brain-derived neurotrophic factor in human subjects with function-altering melanocortin-4 receptor variants

    Science.gov (United States)

    In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown. The objective of this study is to compare BDNF concentrations of subjects wi...

  11. Changes in epidermal growth factor receptor expression during chemotherapy in non-small cell lung cancer

    DEFF Research Database (Denmark)

    Jakobsen, Jan Nyrop; Santoni-Rugiu, Eric; Sørensen, Jens Benn

    2014-01-01

    BACKGROUND: Antibodies targeting epidermal growth factor receptor (EGFR), such as cetuximab, may potentially improve outcome in non-small cell lung cancer (NSCLC) patients with high EGFR expression. The EGFR expression may be heterogeneously distributed within tumors, and small biopsies may thus...

  12. Amplification of epidermal growth factor receptor gene in renal cell carcinoma

    DEFF Research Database (Denmark)

    El-Hariry, Iman; Powles, Thomas; Lau, Mike R

    2010-01-01

    Expression of epidermal growth factor receptor (EGFR) may be of prognostic value in renal cell cancer (RCC). Gene amplification of EGFR was investigated in a cohort of 315 patients with advanced RCC from a previously reported randomised study. Using fluorescent in situ hybridisation, only 2...

  13. Localization and functional roles of corticotropin-releasing factor receptor type 2 in the cerebellum

    NARCIS (Netherlands)

    Gounko, Natalia V.; Gramsbergen, Albert; van der Want, Johannes J. L.

    The corticotropin-releasing factor (CRF) type 2 receptor has three splice variants alpha, beta, and gamma. In the rodent brain only CRF-R2 alpha is present. In the cerebellum, CRF-R2 alpha has two different isoforms: a full-length form (fl) and truncated (tr). Both forms CRF-R2 have a unique

  14. Orphan nuclear receptor TR4 and fibroblast growth factor 1 in metabolism

    NARCIS (Netherlands)

    Liu, Weilin

    2016-01-01

    Metabolic homeostasis is achieved, in part, through the coordinated activities of members of the Nuclear Receptor (NR) family, a superfamily of ligand-modulated transcription factors (TFs) that mediate responses to a wide range of lipophilic signaling molecules including lipids, steroids, retinoids,

  15. Cell-Cell Adhesion and Insulin-Like Growth Factor I Receptor in Breast Cancer

    National Research Council Canada - National Science Library

    Bartucci, Monica

    2001-01-01

    .... Our goal was to study the role of the insulin-like growth factor I receptor (IGF-IR) in breast cancer. The IGF-IR is a multifunctional tyrosine kinase that has been recently implicated in breast tumor development and progression...

  16. Palliation of bone cancer pain by antagonists of platelet-activating factor receptors.

    Directory of Open Access Journals (Sweden)

    Katsuya Morita

    Full Text Available Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2 protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients.

  17. Single-chain magnet features in 1D [MnR{sub 4}TPP][TCNE] compounds

    Energy Technology Data Exchange (ETDEWEB)

    Balanda, Maria [Institute of Nuclear Physics PAN, Radzikowskiego 152, 31-342 Krakow (Poland); Tomkowicz, Zbigniew; Rams, Michal [Institute of Physics, Jagiellonian University, Reymonta 4, 30-059 Krakow (Poland); Haase, Wolfgang, E-mail: Maria.Balanda@ifj.edu.pl [Institute of Physical Chemistry, Darmstadt University of Technology, 64287 Darmstadt (Germany)

    2011-07-06

    Molecular chains of antiferrimagnetically coupled Mn{sup III}-ion (S = 2) and TCNE (tetracyanoethylene) radical moments (s = 1/2 ) show different behaviour depending on group R substituted to TPP (tetraphenylporphyrin) and on the substitution site. The compound with R = F in Ortho position is a Single-Chain Magnet (SCM) with blocking temperature T{sub b} = 6.6K, while that with R = F in Meta position shows both blocking (T{sub b} = 5.4 K) and magnetic ordering transition (T{sub c} = 10 K). For bulky groups R = OC{sub n}H{sub 2n+1}, the magnetically ordered phase is observed (T{sub c} {approx} 22 K), which does not however prevent slow relaxation at T <8 K. Magnetic hysteresis with coercive field H{sub c} of 2 T at 2.3 K is like that of SCM. The frequency dependent AC susceptibility in the superimposed DC field reveals common features of all systems. The energy of intrachain ferromagnetic coupling between effective spin units 3/2, relevant at low temperatures, is determined for all compounds and the interchain dipolar coupling is estimated. It is concluded that slow relaxation is inherent for all quasi one-dimensional compounds and for the magnetically ordered ones shows up in the high enough magnetic field.

  18. Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells

    Science.gov (United States)

    Nie, Yong-Zhan; He, Feng-Tian; Li, Zhi-Kui; Wu, Kai-Chun; Cao, Yun-Xin; Chen, Bao-Jun; Fan, Dai-Ming

    2002-01-01

    AIM: To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens. METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed. Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens. Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed. The phage of positive clones was identified with competitive ELISA, and infected by E. coli HB2151 to express soluble ScFv. RESULTS: The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60%, 24% and 30%. MC3-ScFv had Mr 32000 confirmed by Western blot. The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab. CONCLUSION: The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies. PMID:12174367

  19. A uranium-based UO_2"+-Mn"2"+ single-chain magnet assembled trough cation-cation interactions

    International Nuclear Information System (INIS)

    Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Pecaut, Jacques; Mazzanti, Marinella; Caciuffo, Roberto; Colineau, Eric; Tuna, Floriana; Magnani, Nicola; Geyer, Arnaud de

    2014-01-01

    Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO_2(salen)(py)][M(py)_4](NO_3)}]_n, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO_2(salen)py][Cp"*_2Co] (Cp"*=pentamethylcyclopentadienyl) with Cd(NO_3)_2 or Mn(NO_3)_2 in pyridine. The infinite UMn chain displays a high relaxation barrier of 134±0.8 K (93±0.5 cm"-"1), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T<6 K, with an impressive coercive field of 3.4 T at 2 K.

  20. A uranium-based UO_2"+-Mn"2"+ single-chain magnet assembled trough cation-cation interactions

    International Nuclear Information System (INIS)

    Mougel, Victor; Chatelain, Lucile; Hermle, Johannes; Pecaut, Jacques; Mazzanti, Marinella; Caciuffo, Roberto; Colineau, Eric; Tuna, Floriana; Magnani, Nicola; Geyer, Arnaud de

    2014-01-01

    Single-chain magnets (SCMs) are materials composed of magnetically isolated one-dimensional (1D) units exhibiting slow relaxation of magnetization. The occurrence of SCM behavior requires the fulfillment of stringent conditions for exchange and anisotropy interactions. Herein, we report the synthesis, the structure, and the magnetic characterization of the first actinide-containing SCM. The 5f-3d heterometallic 1D chains [{[UO_2(salen)(py)][M(py)_4](NO_3)}]_n, (M=Cd (1) and M=Mn (2); py=pyridine) are assembled trough cation-cation interaction from the reaction of the uranyl(V) complex [UO_2(salen)py][Cp*_2Co] (Cp*=pentamethylcyclopentadienyl) with Cd(NO_3)_2 or Mn(NO_3)_2 in pyridine. The infinite UMn chain displays a high relaxation barrier of 134 ±0.8 K (93 ±0.5 cm"-"1), probably as a result of strong intra-chain magnetic interactions combined with the high Ising anisotropy of the uranyl(V) dioxo group. It also exhibits an open magnetic hysteresis loop at T <6 K, with an impressive coercive field of 3.4 T at 2 K. (Copyright copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  1. Arg9 facilitates the translocation and downstream signal inhibition of an anti-HER2 single chain antibody

    Directory of Open Access Journals (Sweden)

    Hu Yi

    2012-07-01

    Full Text Available Abstract Background HER2 plays a critical role in the pathogenesis of many cancers and is linked to poor prognosis or cancer metastases. Monoclonal antibodies, such as Herceptin against HER2-overexpressing cancers, have showed satisfactory clinical therapeutic effect. However, they have difficulty to surmount obstacles to enter cells or blood–brain barrier. Results In this study, a cell-penetrating peptide Arg9 was linked to the C-terminus of anti-HER2 single chain antibody (MIL5scFv. Flow cytometry, confocal microscopy and electron microscopy analysis all revealed that Arg9 peptide facilitated the penetration of MIL5scFv into HER2-negative cell line NIH3T3 and orientate in mitochondria. More interestingly, Western blot assay showed the potential enhanced bioactivity of MIL5scFv-Arg9 in HER2+ cell line SKOV3, indicating that Arg9 could help large molecules (e.g. antibody to penetrate into cells and therefore enhance its anti-neoplastic function. Conclusions Our work represented an attractive by preliminary strategy to enhance the therapeutic effect of existing antibodies by entering cells easier, or more desirable, surmounting the physical barriers, especially in hard-to-reach cancers such as brain metastases cases.

  2. Concentrated Solutions of Single-Chain Nanoparticles: A Simple Model for Intrinsically Disordered Proteins under Crowding Conditions.

    Science.gov (United States)

    Moreno, Angel J; Lo Verso, Federica; Arbe, Arantxa; Pomposo, José A; Colmenero, Juan

    2016-03-03

    By means of large-scale computer simulations and small-angle neutron scattering (SANS), we investigate solutions of single-chain nanoparticles (SCNPs), covering the whole concentration range from infinite dilution to melt density. The analysis of the conformational properties of the SCNPs reveals that these synthetic nano-objects share basic ingredients with intrinsically disordered proteins (IDPs), as topological polydispersity, generally sparse conformations, and locally compact domains. We investigate the role of the architecture of the SCNPs in their collapse behavior under macromolecular crowding. Unlike in the case of linear macromolecules, which experience the usual transition from self-avoiding to Gaussian random-walk conformations, crowding leads to collapsed conformations of SCNPs resembling those of crumpled globules. This behavior is already found at volume fractions (about 30%) that are characteristic of crowding in cellular environments. The simulation results are confirmed by the SANS experiments. Our results for SCNPs--a model system free of specific interactions--propose a general scenario for the effect of steric crowding on IDPs: collapse from sparse conformations at high dilution to crumpled globular conformations in cell environments.

  3. Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

    Science.gov (United States)

    Ghadge, Ghanashyam D; Pavlovic, John D; Koduvayur, Sujatha P; Kay, Brian K; Roos, Raymond P

    2013-08-01

    Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

    Directory of Open Access Journals (Sweden)

    Yanan Sun

    Full Text Available Single chain variable fragments (scFvs against diethylstilbestrol (DES were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3 for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR analysis was used to determine binding kinetics of one clone (30-1. The measured K(D was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

  5. Creation of Single Chain of Nanoscale Skyrmion Bubbles with Record-high Temperature Stability in a Geometrically Confined Nanostripe

    KAUST Repository

    Hou, Zhipeng; Zhang, Qiang; Xu, Guizhou; Gong, Chen; Ding, Bei; Wang, Yue; Li, Hang; Liu, Enke; Xu, Feng; Zhang, Hongwei; Yao, Yuan; Wu, Guangheng; Zhang, Xixiang; Wang, Wenhong

    2018-01-01

    Nanoscale topologically nontrivial spin textures, such as magnetic skyrmions, have been identified as promising candidates for the transport and storage of information for spintronic applications, notably magnetic racetrack memory devices. The design and realization of a single skyrmion chain at room temperature (RT) and above in the low-dimensional nanostructures are of great importance for future practical applications. Here, we report the creation of a single skyrmion bubble chain in a geometrically confined Fe3Sn2 nanostripe with a width comparable to the featured size of a skyrmion bubble. Systematic investigations on the thermal stability have revealed that the single chain of skyrmion bubbles can keep stable at temperatures varying from RT up to a record-high temperature of 630 K. This extreme stability can be ascribed to the weak temperature-dependent magnetic anisotropy and the formation of edge states at the boundaries of the nanostripes. The realization of the highly stable skyrmion bubble chain in a geometrically confined nanostructure is a very important step toward the application of skyrmion-based spintronic devices.

  6. Creation of Single Chain of Nanoscale Skyrmion Bubbles with Record-high Temperature Stability in a Geometrically Confined Nanostripe

    KAUST Repository

    Hou, Zhipeng

    2018-01-04

    Nanoscale topologically nontrivial spin textures, such as magnetic skyrmions, have been identified as promising candidates for the transport and storage of information for spintronic applications, notably magnetic racetrack memory devices. The design and realization of a single skyrmion chain at room temperature (RT) and above in the low-dimensional nanostructures are of great importance for future practical applications. Here, we report the creation of a single skyrmion bubble chain in a geometrically confined Fe3Sn2 nanostripe with a width comparable to the featured size of a skyrmion bubble. Systematic investigations on the thermal stability have revealed that the single chain of skyrmion bubbles can keep stable at temperatures varying from RT up to a record-high temperature of 630 K. This extreme stability can be ascribed to the weak temperature-dependent magnetic anisotropy and the formation of edge states at the boundaries of the nanostripes. The realization of the highly stable skyrmion bubble chain in a geometrically confined nanostructure is a very important step toward the application of skyrmion-based spintronic devices.

  7. A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

    Science.gov (United States)

    Cox, Dianne; Hodgson, Louis

    2014-01-01

    Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized. PMID:24798463

  8. Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

    DEFF Research Database (Denmark)

    Zhao, Heng; Stet, René J M; Skjødt, Karsten

    2008-01-01

    Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable ...... MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association...... antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently......, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC...

  9. Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

    Science.gov (United States)

    Vassbotn, F S; Ostman, A; Siegbahn, A; Holmsen, H; Heldin, C H

    1992-08-05

    The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

  10. Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer.

    Science.gov (United States)

    Wu, Weijuan; Yang, Qing; Fung, Kar-Ming; Humphreys, Mitchell R; Brame, Lacy S; Cao, Amy; Fang, Yu-Ting; Shih, Pin-Tsen; Kropp, Bradley P; Lin, Hsueh-Kung

    2014-03-05

    Neuroendocrine (NE) differentiation has been attributed to the progression of castration-resistant prostate cancer (CRPC). Growth factor pathways including the epidermal growth factor receptor (EGFR) signaling have been implicated in the development of NE features and progression to a castration-resistant phenotype. However, upstream molecules that regulate the growth factor pathway remain largely unknown. Using androgen-insensitive bone metastasis PC-3 cells and androgen-sensitive lymph node metastasis LNCaP cells derived from human prostate cancer (PCa) patients, we demonstrated that γ-aminobutyric acid A receptor (GABA(A)R) ligand (GABA) and agonist (isoguvacine) stimulate cell proliferation, enhance EGF family members expression, and activate EGFR and a downstream signaling molecule, Src, in both PC-3 and LNCaP cells. Inclusion of a GABA(A)R antagonist, picrotoxin, or an EGFR tyrosine kinase inhibitor, Gefitinib (ZD1839 or Iressa), blocked isoguvacine and GABA-stimulated cell growth, trans-phospohorylation of EGFR, and tyrosyl phosphorylation of Src in both PCa cell lines. Spatial distributions of GABAAR α₁ and phosphorylated Src (Tyr416) were studied in human prostate tissues by immunohistochemistry. In contrast to extremely low or absence of GABA(A)R α₁-positive immunoreactivity in normal prostate epithelium, elevated GABA(A)R α₁ immunoreactivity was detected in prostate carcinomatous glands. Similarly, immunoreactivity of phospho-Src (Tyr416) was specifically localized and limited to the nucleoli of all invasive prostate carcinoma cells, but negative in normal tissues. Strong GABAAR α₁ immunoreactivity was spatially adjacent to the neoplastic glands where strong phospho-Src (Tyr416)-positive immunoreactivity was demonstrated, but not in adjacent to normal glands. These results suggest that the GABA signaling is linked to the EGFR pathway and may work through autocrine or paracine mechanism to promote CRPC progression. Copyright © 2013 Elsevier

  11. Wnt signalling via the epidermal growth factor receptor: a role in breast cancer?

    International Nuclear Information System (INIS)

    Musgrove, Elizabeth A

    2004-01-01

    Recent data have suggested the epidermal-growth-factor receptor (EGFR) as a point of convergence for several different classes of receptor. Civenni and colleagues have now demonstrated crosstalk between Wnt signalling and the EGFR, showing that in breast epithelial cells Wnts activate downstream targets of the EGFR, including cyclin D1. Given the role of members of these pathways in the aetiology of breast cancer and as markers of outcome and potential therapeutic targets in breast cancer, this observation has a number of potential implications important for both the basic biology of breast cancer and the clinical management of the disease

  12. The diminished expression of proangiogenic growth factors and their receptors in gastric ulcers of cirrhotic patients.

    Science.gov (United States)

    Luo, Jiing-Chyuan; Peng, Yen-Ling; Hou, Ming-Chih; Huang, Kuang-Wei; Huang, Hui-Chun; Wang, Ying-Wen; Lin, Han-Chieh; Lee, Fa-Yauh; Lu, Ching-Liang

    2013-01-01

    The pathogenesis of the higher occurrence of peptic ulcer disease in cirrhotic patients is complex. Platelets can stimulate angiogenesis and promote gastric ulcer healing. We compared the expressions of proangiogenic growth factors and their receptors in the gastric ulcer margin between cirrhotic patients with thrombocytopenia and those of non-cirrhotic patients to elucidate possible mechanisms. Eligible cirrhotic patients (n = 55) and non-cirrhotic patients (n = 55) who had gastric ulcers were enrolled. Mucosa from the gastric ulcer margin and non-ulcer areas were sampled and the mRNA expressions of the proangiogenic growth factors (vascular endothelial growth factor [VEGF], platelet derived growth factor [PDGF], basic fibroblast growth factor [bFGF]) and their receptors (VEGFR1, VEGFR2, PDGFRA, PDGFRB, FGFR1, FGFR2) were measured and compared. Platelet count and the expressions of these growth factors and their receptors were correlated with each other. The two groups were comparable in terms of gender, ulcer size and infection rate of Helicobacter pylori. However, the cirrhotic group were younger in age, had a lower platelet count than those in the non-cirrhotic group (pexpressions of PDGFB, VEGFR2, FGFR1, and FGFR2 in gastric ulcer margin when compared with those of the non-cirrhotic patients (pexpressions of PDGFB and VEGFR2, FGFR1, and FGFR2 were well correlated with the degree of thrombocytopenia in these cirrhotic patients (ρ>0.5, pimplied that diminished activity of proangiogenic factors and their receptors may contribute to the pathogenesis of gastric ulcers in cirrhotic patients.

  13. An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

    Science.gov (United States)

    Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

    2016-01-01

    Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

  14. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

    Science.gov (United States)

    Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

    1993-11-01

    Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

  15. Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

    International Nuclear Information System (INIS)

    Grob, P.M.; David, E.; Warren, T.C.; DeLeon, R.P.; Farina, P.R.; Homon, C.A.

    1990-01-01

    Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons

  16. Structural basis for receptor recognition of vitamin-B(12)-intrinsic factor complexes

    DEFF Research Database (Denmark)

    Andersen, Christian Brix Folsted; Madsen, Mette; Storm, Tina

    2010-01-01

    Cobalamin (Cbl, vitamin B(12)) is a bacterial organic compound and an essential coenzyme in mammals, which take it up from the diet. This occurs by the combined action of the gastric intrinsic factor (IF) and the ileal endocytic cubam receptor formed by the 460-kilodalton (kDa) protein cubilin...... and the 45-kDa transmembrane protein amnionless. Loss of function of any of these proteins ultimately leads to Cbl deficiency in man. Here we present the crystal structure of the complex between IF-Cbl and the cubilin IF-Cbl-binding-region (CUB(5-8)) determined at 3.3 A resolution. The structure provides...... of how Cbl indirectly induces ligand-receptor coupling. Finally, the comparison of Ca(2+)-binding CUB domains and the low-density lipoprotein (LDL) receptor-type A modules suggests that the electrostatic pairing of a basic ligand arginine/lysine residue with Ca(2+)-coordinating acidic aspartates...

  17. FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs

    Science.gov (United States)

    Schwartz, Carol J.E.; Sampson, Heidi M.; Hlousek, Daniela; Percival-Smith, Anthony; Copeland, John W.R.; Simmonds, Andrew J.; Krause, Henry M.

    2001-01-01

    To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We also show that FTZ contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins. PMID:11157757

  18. Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells

    International Nuclear Information System (INIS)

    Roy, L.M.; Gittinger, C.K.; Landreth, G.E.

    1989-01-01

    Epidermal growth factor receptors (EGF-R) have been shown to be associated with the detergent-insoluble cytoskeleton of A431 cells, where they retained both a functional ligand-binding domain and tyrosine kinase activity. In the present study we have characterized the tyrosine kinase and ligand binding activities of this cytoskeletally associated EGF-R. The tyrosine kinase activity of the cytoskeletally associated EGF-R was stimulated by EGF treatment of intact cells as evidenced by increased autophosphorylation and phosphorylation of the exogenous substrate angiotensin II (AII). The kinetic behavior of the EGF-R associated with cytoskeletons of EGF-treated cells was similar to that of purified receptors. The stimulation of the receptor kinase activity required EGF treatment of intact cells prior to Triton extraction. If cytoskeletons were prepared from untreated cells and then incubated with EGF, there was no stimulation of the detergent-insoluble receptor kinase activity, indicating that the immobilized receptor was unable to undergo EGF-stimulated activation. Comparison of peptide maps from soluble and cytoskeletally associated EGF-R revealed qualitatively similar patterns; however, they are distinguished by a prominent 46 kD band in digests of the cytoskeletal EGF-R. Saturable binding of 125I-EGF to A431 cytoskeletons prepared from adherent and suspended cells demonstrated the presence of specific receptors on the cytoskeleton. High-affinity EGF-R were preferentially retained upon detergent extraction of adherent cells, whereas both low- and high-affinity receptors were solubilized from the cytoskeletons of suspended cells. Suspension of cells resulted in the solubilization of an additional 15% of the EGF-R to that solubilized in adherent cells, indicating that EGF-R can reversibly associate with the structural elements of the cell

  19. Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

    Science.gov (United States)

    Smits, A; Funa, K; Vassbotn, F S; Beausang-Linder, M; af Ekenstam, F; Heldin, C H; Westermark, B; Nistér, M

    1992-03-01

    Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

  20. A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

    Science.gov (United States)

    Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

    2015-05-21

    Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses

    Science.gov (United States)

    Liu, Han-Lin; Lin, Wei-Fang; Hu, Wen-Chi; Lee, Yung-An

    2015-01-01

    Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli. PMID:26209665

  2. Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease.

    Science.gov (United States)

    Ta, H T; Prabhu, S; Leitner, E; Jia, F; von Elverfeldt, D; Jackson, Katherine E; Heidt, T; Nair, A K N; Pearce, H; von Zur Muhlen, C; Wang, X; Peter, K; Hagemeyer, C E

    2011-08-05

    Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

  3. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    Science.gov (United States)

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin. © FASEB.

  4. Efficient sampling of reversible cross-linking polymers: Self-assembly of single-chain polymeric nanoparticles

    Science.gov (United States)

    Oyarzún, Bernardo; Mognetti, Bortolo Matteo

    2018-03-01

    We present a new simulation technique to study systems of polymers functionalized by reactive sites that bind/unbind forming reversible linkages. Functionalized polymers feature self-assembly and responsive properties that are unmatched by the systems lacking selective interactions. The scales at which the functional properties of these materials emerge are difficult to model, especially in the reversible regime where such properties result from many binding/unbinding events. This difficulty is related to large entropic barriers associated with the formation of intra-molecular loops. In this work, we present a simulation scheme that sidesteps configurational costs by dedicated Monte Carlo moves capable of binding/unbinding reactive sites in a single step. Cross-linking reactions are implemented by trial moves that reconstruct chain sections attempting, at the same time, a dimerization reaction between pairs of reactive sites. The model is parametrized by the reaction equilibrium constant of the reactive species free in solution. This quantity can be obtained by means of experiments or atomistic/quantum simulations. We use the proposed methodology to study the self-assembly of single-chain polymeric nanoparticles, starting from flexible precursors carrying regularly or randomly distributed reactive sites. We focus on understanding differences in the morphology of chain nanoparticles when linkages are reversible as compared to the well-studied case of irreversible reactions. Intriguingly, we find that the size of regularly functionalized chains, in good solvent conditions, is non-monotonous as a function of the degree of functionalization. We clarify how this result follows from excluded volume interactions and is peculiar of reversible linkages and regular functionalizations.

  5. Spin canting in a Dy-based single-chain magnet with dominant next-nearest-neighbor antiferromagnetic interactions

    Science.gov (United States)

    Bernot, K.; Luzon, J.; Caneschi, A.; Gatteschi, D.; Sessoli, R.; Bogani, L.; Vindigni, A.; Rettori, A.; Pini, M. G.

    2009-04-01

    We investigate theoretically and experimentally the static magnetic properties of single crystals of the molecular-based single-chain magnet of formula [Dy(hfac)3NIT(C6H4OPh)]∞ comprising alternating Dy3+ and organic radicals. The magnetic molar susceptibility χM displays a strong angular variation for sample rotations around two directions perpendicular to the chain axis. A peculiar inversion between maxima and minima in the angular dependence of χM occurs on increasing temperature. Using information regarding the monomeric building block as well as an ab initio estimation of the magnetic anisotropy of the Dy3+ ion, this “anisotropy-inversion” phenomenon can be assigned to weak one-dimensional ferromagnetism along the chain axis. This indicates that antiferromagnetic next-nearest-neighbor interactions between Dy3+ ions dominate, despite the large Dy-Dy separation, over the nearest-neighbor interactions between the radicals and the Dy3+ ions. Measurements of the field dependence of the magnetization, both along and perpendicularly to the chain, and of the angular dependence of χM in a strong magnetic field confirm such an interpretation. Transfer-matrix simulations of the experimental measurements are performed using a classical one-dimensional spin model with antiferromagnetic Heisenberg exchange interaction and noncollinear uniaxial single-ion anisotropies favoring a canted antiferromagnetic spin arrangement, with a net magnetic moment along the chain axis. The fine agreement obtained with experimental data provides estimates of the Hamiltonian parameters, essential for further study of the dynamics of rare-earth-based molecular chains.

  6. Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein

    International Nuclear Information System (INIS)

    Yazaki, Paul J.; Kassa, Thewodros; Cheung, Chia-wei; Crow, Desiree M.; Sherman, Mark A.; Bading, James R.; Anderson, Anne-Line J.; Colcher, David; Raubitschek, Andrew

    2008-01-01

    Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [ 125 I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [ 111 In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [ 64 Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents

  7. A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Shuli [Immunology and Reproductive Biology Laboratory, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Nanjing Affiliated First Hospital, Nanjing Medical University, Nanjing (China); Zhao, Guangfeng; Xie, Hao; Huang, Yahong [Immunology and Reproductive Biology Laboratory, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Hou, Yayi [Immunology and Reproductive Biology Laboratory, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing (China)

    2012-01-27

    Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex){sub 1.3}(DOX){sub 20}. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8%) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers.

  8. Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.

    Science.gov (United States)

    Khantasup, Kannika; Chantima, Warangkana; Sangma, Chak; Poomputsa, Kanokwan; Dharakul, Tararaj

    2015-12-01

    Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

  9. A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

    International Nuclear Information System (INIS)

    Zhao, Shuli; Zhao, Guangfeng; Xie, Hao; Huang, Yahong; Hou, Yayi

    2012-01-01

    Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex) 1.3 (DOX) 20 . In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8%) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers

  10. Prognostic significance of highly sulfated chondroitin sulfates in ovarian cancer defined by the single chain antibody GD3A11.

    Science.gov (United States)

    van der Steen, Sophieke C H A; van Tilborg, Angela A G; Vallen, Myrtille J E; Bulten, Johan; van Kuppevelt, Toin H; Massuger, Leon F A G

    2016-03-01

    The extracellular matrix (ECM) of ovarian cancer may provide a number of potential biomarkers. Chondroitin sulfate (CS), a class of sulfated polysaccharides, is abundantly present in the ECM of ovarian cancer. Structural alterations of CS chains (i.e. sulfation pattern) have been demonstrated to play a role in cancer development and progression. In this study we investigate the potential of highly sulfated CS as a biomarker in ovarian cancer using the single chain antibody GD3A11 selected by the phage display technology. The specificity of the antibody was determined by an indirect ELISA. GD3A11 epitope expression was assessed by immunohistochemistry in healthy organs, benign and malignant ovarian tumors (N=359) and correlated to clinical parameters. The CHST15 gene, responsible for the biosynthesis of highly sulfated CS was evaluated for mutation and methylation status. The GD3A11 epitope was minimally expressed in normal organs. Intense expression was observed in the ECM of different ovarian cancer subtypes, in contrast to benign ovarian tumors. Expression was independent of tumor grade, FIGO stage, and the use chemotherapy. For the aggressive ovarian cancer phenotype, intense expression was identified as an independent predictor for poor prognosis. CHST15 gene analysis showed no mutations nor an altered methylation status. Specific highly sulfated CS motifs expressed in the tumoral ECM hold biomarker potential in ovarian cancer patients. These matrix motifs constitute a novel class of biomarkers with prognostic significance and may be instrumental for innovative diagnostic and therapeutic applications (e.g. targeted therapy) in management of ovarian cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Insulin-like growth factor II: complexity of biosynthesis and receptor binding

    DEFF Research Database (Denmark)

    Gammeltoft, S; Christiansen, Jan; Nielsen, F C

    1991-01-01

    Insulin-like growth factor II (IGF-II) belongs to the insulin family of peptides and acts as a growth factor in many fetal tissues and tumors. The gene expression of IGF-II is initiated at three different promoters which gives rise to multiple transcripts. In a human rhabdomyosarcoma cell line......, Man-6-P induces cellular responses. We have studied rat brain neuronal precursor cells where Man-6-P acted as a mitogen suggesting that phosphomannosylated proteins may act as growth factors via the Man-6-P/IGF-II receptor. In conclusion, the gene expression and mechanism of action of IGF-II is very...

  12. Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia.

    Science.gov (United States)

    Pawlinski, Rafal; Pedersen, Brian; Schabbauer, Gernot; Tencati, Michael; Holscher, Todd; Boisvert, William; Andrade-Gordon, Patricia; Frank, Rolf Dario; Mackman, Nigel

    2004-02-15

    Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.

  13. Emerging growth factor receptor antagonists for the treatment of renal cell carcinoma.

    Science.gov (United States)

    Zahoor, Haris; Rini, Brian I

    2016-12-01

    The landscape of systemic treatment for metastatic renal cell carcinoma (RCC) has dramatically changed with the introduction of targeted agents including vascular endothelial growth factor (VEGF) inhibitors. Recently, multiple new agents including growth factor receptor antagonists and a checkpoint inhibitor were approved for the treatment of refractory metastatic RCC based on encouraging benefit shown in clinical trials. Areas covered: The background and biological rationale of existing treatment options including a brief discussion of clinical trials which led to their approval, is presented. This is followed by reviewing the limitations of these therapeutic options, medical need to develop new treatments and major goals of ongoing research. We then discuss two recently approved growth factor receptor antagonists i.e. cabozantinib and lenvatinib, and a recently approved checkpoint inhibitor, nivolumab, and issues pertaining to drug development, and future directions in treatment of metastatic RCC. Expert opinion: Recently approved growth factor receptor antagonists have shown encouraging survival benefit but associated drug toxicity is a major issue. Nivolumab, a programmed death 1 (PD-1) checkpoint inhibitor, has similarly shown survival benefit and is well tolerated. With multiple options now available in this patient population, the right sequence of these agents remains to be determined.

  14. Cognitive disorder and changes in cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury

    Institute of Scientific and Technical Information of China (English)

    Weiliang Zhao; Dezhi Kang; Yuanxiang Lin

    2008-01-01

    BACKGROUND: Learning and memory damage is one of the most permanent and the severest symptoms of traumatic brain injury; it can seriously influence the normal life and work of patients. Some research has demonstrated that cognitive disorder is closely related to nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor. OBJECTIVE: To summarize the cognitive disorder and changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury. RETRIEVAL STRATEGY: A computer-based online search was conducted in PUBMED for English language publications containing the key words "brain injured, cognitive handicap, acetylcholine, N-methyl-D aspartate receptors, neural cell adhesion molecule, brain-derived neurotrophic factor" from January 2000 to December 2007. There were 44 papers in total. Inclusion criteria: ① articles about changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury; ② articles in the same researching circle published in authoritative journals or recently published. Exclusion criteria: duplicated articles.LITERATURE EVALUATION: References were mainly derived from research on changes in these four factors following brain injury. The 20 included papers were clinical or basic experimental studies. DATA SYNTHESIS: After craniocerebral injury, changes in these four factors in brain were similar to those during recovery from cognitive disorder, to a certain degree. Some data have indicated that activation of nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor could greatly improve cognitive disorder following brain injury. However, there are still a lot of questions remaining; for example, how do these

  15. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: Evidence for more than one receptor class

    International Nuclear Information System (INIS)

    Gronwald, R.G.K.; Grant, F.J.; Haldeman, B.A.; Hart, C.E.; O'Hara, P.J.; Hagen, F.S.; Ross, R.; Bowen-Pope, D.F.; Murray, M.J.

    1988-01-01

    The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A) + RNA from human dermal fibroblasts detects a major and a minor transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an ∼ 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF. Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class

  16. Metabotropic glutamate receptor 2 and corticotrophin-releasing factor receptor-1 gene expression is differently regulated by BDNF in rat primary cortical neurons

    DEFF Research Database (Denmark)

    Jørgensen, Christinna V; Klein, Anders B; El-Sayed, Mona

    2013-01-01

    Brain-derived neurotrophic factor (BDNF) is important for neuronal survival and plasticity. Incorporation of matured receptor proteins is an integral part of synapse formation. However, whether BDNF increases synthesis and integration of receptors in functional synapses directly is unclear. We...... are particularly interested in the regulation of the 5-hydroxytryptamine receptor 2A (5-HT2A R). This receptor form a functional complex with the metabotropic glutamate receptor 2 (mGluR2) and is recruited to the cell membrane by the corticotrophin-releasing factor receptor 1 (CRF-R1). The effect of BDNF on gene...... expression for all these receptors, as well as a number of immediate-early genes, was pharmacologically characterized in primary neurons from rat frontal cortex. BDNF increased CRF-R1 mRNA levels up to fivefold, whereas mGluR2 mRNA levels were proportionally downregulated. No effect on 5-HT2A R mRNA was seen...

  17. Fibroblast growth factor 10-fibroblast growth factor receptor 2b mediated signaling is not required for adult glandular stomach homeostasis.

    Directory of Open Access Journals (Sweden)

    Allison L Speer

    Full Text Available The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10 and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b, in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22 except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10's main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.

  18. Optimal experimental design in an epidermal growth factor receptor signalling and down-regulation model.

    Science.gov (United States)

    Casey, F P; Baird, D; Feng, Q; Gutenkunst, R N; Waterfall, J J; Myers, C R; Brown, K S; Cerione, R A; Sethna, J P

    2007-05-01

    We apply the methods of optimal experimental design to a differential equation model for epidermal growth factor receptor signalling, trafficking and down-regulation. The model incorporates the role of a recently discovered protein complex made up of the E3 ubiquitin ligase, Cbl, the guanine exchange factor (GEF), Cool-1 (beta -Pix) and the Rho family G protein Cdc42. The complex has been suggested to be important in disrupting receptor down-regulation. We demonstrate that the model interactions can accurately reproduce the experimental observations, that they can be used to make predictions with accompanying uncertainties, and that we can apply ideas of optimal experimental design to suggest new experiments that reduce the uncertainty on unmeasurable components of the system.

  19. Anti-epidermal growth factor receptor skin toxicity: a matter of topical hydration.

    Science.gov (United States)

    Ferrari, Daris; Codecà, Carla; Bocci, Barbara; Crepaldi, Francesca; Violati, Martina; Viale, Giulia; Careri, Carmela; Caldiera, Sarah; Bordin, Veronica; Luciani, Andrea; Zonato, Sabrina; Cassinelli, Gabriela; Foa, Paolo

    2016-02-01

    Skin toxicity is a frequent complication of anti-epidermal growth factor receptor therapy, which can be an obstacle in maintaining the dose intensity and may negatively impact on the clinical outcome of cancer patients. Skin lesions depend on the disruption of the keratinocyte development pathways and no treatment is clearly effective in resolving the cutaneous alterations frequently found during anti-epidermal growth factor receptor therapy. Among systemic treatments, oral tetracycline proved to be useful in preventing skin manifestations. We describe the case of a patient affected by metastatic colorectal cancer, for whom a combination of chemotherapy and cetuximab was used as second-line treatment. The patient developed a symptomatic papulopustular skin rash that disappeared completely after a twice-daily application of a hydrating and moisturizing cream, mainly consisting of a mixture of paraffin, silicone compounds, and macrogol. The marked cutaneous amelioration allowed the patient to continue cetuximab without any further symptoms and was associated with a partial radiological response.

  20. Insulin-Insulin-like Growth Factors Hybrids as Molecular Probes of Hormone:Receptor Binding Specificity

    Czech Academy of Sciences Publication Activity Database

    Křížková, Květoslava; Chrudinová, Martina; Povalová, Anna; Selicharová, Irena; Collinsová, Michaela; Vaněk, Václav; Brzozowski, A. M.; Jiráček, Jiří; Žáková, Lenka

    2016-01-01

    Roč. 55, č. 21 (2016), s. 2903-2913 ISSN 0006-2960 R&D Projects: GA ČR GA15-19018S Institutional support: RVO:61388963 Keywords : alanine scanning mutagenesis * high-affinity binding * type 1 IGF receptor Subject RIV: CE - Biochemistry Impact factor: 2.938, year: 2016 http://pubs.acs.org/doi/pdf/10.1021/acs.biochem.6b00140

  1. Insulin-like Growth Factor Receptor Inhibitors: Baby or the Bathwater?

    OpenAIRE

    Yee, Douglas

    2012-01-01

    The success of targeted therapies for cancer is undisputed; strong preclinical evidence has resulted in the approval of several new agents for cancer treatment. The type I insulin-like growth factor receptor (IGF1R) appeared to be one of these promising new targets. Substantial population and preclinical data have all pointed toward this pathway as an important regulator of tumor cell biology. Although early results from clinical trials that targeted the IGF1R showed some evidence of response...

  2. Optimal Therapeutic Strategy for Non-small Cell Lung Cancer with Mutated Epidermal Growth Factor Receptor

    Directory of Open Access Journals (Sweden)

    Zhong SHI

    2015-02-01

    Full Text Available Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs have been widely used in non-small cell lung cancer (NSCLC patients, it is still controversial about how to combine EGFR-TKI with chemotherapy and other targeted drugs. We have made a summary on the current therapeutic models of EGFR-TKI combined with chemotherapy/bevacizumab in this review and aimed to find the optimal therapeutic strategy for NSCLC patients with EGFR mutation.

  3. Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

    Science.gov (United States)

    Roskoski, Robert

    2005-11-11

    Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

  4. A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yanjuan; Zhang, Shaolian; Wen, Qingqing; Huang, Hongxing; Yang, Peihui, E-mail: typh@jnu.edu.cn

    2015-06-30

    Highlights: • EGF-cytosensor was used for evaluating EGFR expression level on cell surfaces. • CdSQDs and EGF were coated on magnetic beads (MBs) for ECL-probe. • Good sensitivity was achieved due to the signal amplification of ECL-probe. - Abstract: A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4 × 10{sup 6} cells mL{sup −1} with a detection limit of 40 cells mL{sup −1} was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35 × 10{sup 5} with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening.

  5. Hepatocyte growth factor enhances death receptor-induced apoptosis by up-regulating DR5

    International Nuclear Information System (INIS)

    Li, Yang; Fan, Xing; Goodwin, C Rory; Laterra, John; Xia, Shuli

    2008-01-01

    Hepatocyte growth factor (HGF) and its receptor c-MET are commonly expressed in malignant gliomas and embryonic neuroectodermal tumors including medulloblastoma and appear to play an important role in the growth and dissemination of these malignancies. Dependent on cell context and the involvement of specific downstream effectors, both pro- and anti-apoptotic effects of HGF have been reported. Human medulloblastoma cells were treated with HGF for 24–72 hours followed by death receptor ligand TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) for 24 hours. Cell death was measured by MTT and Annexin-V/PI flow cytometric analysis. Changes in expression levels of targets of interest were measured by Northern blot analysis, quantitative reverse transcription-PCR, Western blot analysis as well as immunoprecipitation. In this study, we show that HGF promotes medulloblastoma cell death induced by TRAIL. TRAIL alone triggered apoptosis in DAOY cells and death was enhanced by pre-treating the cells with HGF for 24–72 h prior to the addition of TRAIL. HGF (100 ng/ml) enhanced TRAIL (10 ng/ml) induced cell death by 36% (P < 0.001). No cell death was associated with HGF alone. Treating cells with PHA-665752, a specific c-Met receptor tyrosine kinase inhibitor, significantly abrogated the enhancement of TRAIL-induced cell death by HGF, indicating that its death promoting effect requires activation of its canonical receptor tyrosine kinase. Cell death induced by TRAIL+HGF was predominately apoptotic involving both extrinsic and intrinsic pathways as evidenced by the increased activation of caspase-3, 8, 9. Promotion of apoptosis by HGF occurred via the increased expression of the death receptor DR5 and enhanced formation of death-inducing signal complexes (DISC). Taken together, these and previous findings indicate that HGF:c-Met pathway either promotes or inhibits medulloblastoma cell death via pathway and context specific mechanisms

  6. Identification of fibroblast growth factor receptor 3 (FGFR3 as a protein receptor for botulinum neurotoxin serotype A (BoNT/A.

    Directory of Open Access Journals (Sweden)

    Birgitte P S Jacky

    Full Text Available Botulinum neurotoxin serotype A (BoNT/A causes transient muscle paralysis by entering motor nerve terminals (MNTs where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206 to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs, making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs. Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3 as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

  7. Granulocyte-Colony Stimulating Factor Receptor, Tissue Factor, and VEGF-R Bound VEGF in Human Breast Cancer In Loco.

    Science.gov (United States)

    Wojtukiewicz, Marek Z; Sierko, Ewa; Skalij, Piotr; Kamińska, Magda; Zimnoch, Lech; Brekken, Ralf A; Thorpe, Philip E

    2016-01-01

    Doxorubicin and docetaxel-based chemotherapy regimens used in breast cancer patients are associated with high risk of febrile neutropenia (FN). Granulocyte colony-stimulating factors (G-CSF) are recommended for both treating and preventing chemotherapy-induced neutropenia. Increased thrombosis incidence in G-CSF treated patients was reported; however, the underlying mechanisms remain unclear. The principal activator of blood coagulation in cancer is tissue factor (TF). It additionally contributes to cancer progression and stimulates angiogenesis. The main proangiogenic factor is vascular endothelial growth factor (VEGF). The aim of the study was to evaluate granulocyte-colony stimulating factor receptor (G-CSFR), tissue factor (TF) expression and vascular endothelial growth factor receptor (VEGF-R) bound VEGF in human breast cancer in loco. G-CSFR, TF and VEGFR bound VEGF (VEGF: VEGFR) were assessed in 28 breast cancer tissue samples. Immunohistochemical (IHC) methodologies according to ABC technique and double staining IHC procedure were employed utilizing antibodies against G-CSFR, TF and VEGF associated with VEGFR (VEGF: VEGFR). Expression of G-CSFR was demonstrated in 20 breast cancer tissue specimens (71%). In 6 cases (21%) the expression was strong (IRS 9-12). Strong expression of TF was observed in all investigated cases (100%). Moreover, expression of VEGF: VEGFR was visualized in cancer cells (IRS 5-8). No presence of G-CSFR, TF or VEGF: VEGFR was detected on healthy breast cells. Double staining IHC studies revealed co-localization of G-CSFR and TF, G-CSFR and VEGF: VEGFR, as well as TF and VEGF: VEGFR on breast cancer cells and ECs. The results of the study indicate that GCSFR, TF and VEGF: VEGFR expression as well as their co-expression might influence breast cancer biology, and may increase thromboembolic adverse events incidence.

  8. Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

    Science.gov (United States)

    Doyle, Michael L; Tian, Shin-Shay; Miller, Stephen G; Kessler, Linda; Baker, Audrey E; Brigham-Burke, Michael R; Dillon, Susan B; Duffy, Kevin J; Keenan, Richard M; Lehr, Ruth; Rosen, Jon; Schneeweis, Lumelle A; Trill, John; Young, Peter R; Luengo, Juan I; Lamb, Peter

    2003-03-14

    Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

  9. Thyroid hormone regulation of epidermal growth factor receptor levels in mouse mammary glands

    International Nuclear Information System (INIS)

    Vonderhaar, B.K.; Tang, E.; Lyster, R.R.; Nascimento, M.C.

    1986-01-01

    The specific binding of iodinated epidermal growth factor ([ 125 I]iodo-EGF) to membranes prepared from the mammary glands and spontaneous breast tumors of euthyroid and hypothyroid mice was measured in order to determine whether thyroid hormones regulate the EGF receptor levels in vivo. Membranes from hypothyroid mammary glands of mice at various developmental ages bound 50-65% less EGF than those of age-matched euthyroid controls. Treatment of hypothyroid mice with L-T4 before killing restored binding to the euthyroid control level. Spontaneous breast tumors arising in hypothyroid mice also bound 30-40% less EGF than tumors from euthyroid animals even after in vitro desaturation of the membranes of endogenous growth factors with 3 M MgCl2 treatment. The decrease in binding in hypothyroid membranes was due to a decrease in the number of binding sites, not to a change in affinity of the growth factor for its receptor, as determined by Scatchard analysis of the binding data. Both euthyroid and hypothyroid membranes bound EGF primarily to a single class of high affinity sites [dissociation constant (Kd) = 0.7-1.8 nM]. Euthyroid membranes bound 28.4 +/- (SE) 0.6 fmol/mg protein, whereas hypothyroid membranes bound 15.5 +/- 1.0 fmol/mg protein. These data indicate that EGF receptor levels in normal mammary glands and spontaneous breast tumors in mice are subject to regulation by thyroid status

  10. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    International Nuclear Information System (INIS)

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. [ 3 H]PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 μM. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRPγS and GDPβS, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA)

  11. Structural analysis of the receptors for granulocyte colony-stimulating factor on neutrophils

    International Nuclear Information System (INIS)

    Hanazono, Y.; Hosoi, T.; Kuwaki, T.; Matsuki, S.; Miyazono, K.; Miyagawa, K.; Takaku, F.

    1990-01-01

    We investigated granulocyte colony-stimulating factor (G-CSF) receptors on neutrophils from three patients with chronic myelogenous leukemia (CML) in the chronic phase, in comparison with four normal volunteers. Because we experienced some difficulties in radioiodinating intact recombinant human G-CSF, we developed a new derivative of human G-CSF termed YPY-G-CSF. It was easy to iodinate this protein using the lactoperoxidase method because of two additional tyrosine residues, and its radioactivity was higher than that previously reported. The biological activity of YPY-G-CSF as G-CSF was fully retained. Scatchard analysis demonstrated that CML neutrophils had a single class of binding sites (1400 +/- 685/cell) with a dissociation constant (Kd) of 245 +/- 66 pM. The number of sites and Kd value of CML neutrophils were not significantly different from those of normal neutrophils (p greater than 0.9). Cross-linking studies revealed two specifically labeled bands of [125I]YPY-G-CSF-receptor complexes with apparent molecular masses of 160 and 110 kd on both normal and CML neutrophils. This is the first report describing two receptor proteins on neutrophils. According to the analyses of the proteolytic process of these cross-linked complexes and proteolytic mapping, we assume that alternative splicing or processing from a single gene may generate two distinct receptor proteins that bind specifically to G-CSF but have different fates in intracellular metabolism

  12. Targeting of liposomes to cells bearing nerve growth factor receptors mediated by biotinylated NGF

    International Nuclear Information System (INIS)

    Rosenberg, M.B.

    1986-01-01

    Previous studies of liposome targeting have concentrated on immunological systems, the use of ligand-receptor interactions has received little attention. The protein hormone beta-nerve growth factor (NGF) was modified by biotinylation via carboxyl group substitution (C-bio-NGF) under reaction conditions that yielded an average of 3 biotin additions per NGF subunit. NGF was also biotinylated through amino group substitution to produce derivatives with ratios of 1, 2 and 4 biotin moieties per NGF subunit (N-bio-NGF). These derivatives were compared with native NGF for their ability to compete with 125 I-NGF for binding to NGF receptors on rat pheochromocytoma (PC 12) cells at 4 0 C. C-bio-NGF was as effective as native NGF in binding to NGF receptors, while N-bio-NGF containing 1 biotin per NGF subunit was only 28% as active in binding as native NGF. C-bio-NGF, but not N-bio-NGF, mediated the specific binding of 125 I-streptavidin to PC12 cells. Biocytin-NGF, a derivative of C-bio-NGF with an extended spacer chain, was also synthesized and retained full biological and receptor binding activities. C-bio-NGF and biocytin-NGF were as effective as native NGF in a bioassay involving induction of neurite outgrowth from PC12 cells

  13. Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1

    Energy Technology Data Exchange (ETDEWEB)

    Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly; Xu, H. Eric (Van Andel)

    2009-01-15

    The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

  14. Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

    International Nuclear Information System (INIS)

    Konnai, Satoru; Usui, Tatsufumi; Ikeda, Manabu; Kohara, Junko; Hirata, Toh-ichi; Okada, Kosuke; Ohashi, Kazuhiko; Onuma, Misao

    2005-01-01

    Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-α and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-α-induced responses, in this study we examined the TNF-α-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-α (rTNF-α) was significantly higher than those from AL cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5 + or sIgM + cells and these cells showed resistance to TNF-α-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-α-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection

  15. Expression of transforming growth factor alpha and epidermal growth factor receptor in rat lung neoplasms induced by plutonium-239

    International Nuclear Information System (INIS)

    Stegelmeier, B.L.; Gillett, N.A.; Hahn, F.F.; Kelly, G.; Rebar, A.H.

    1994-01-01

    Ninety-two rat lung proliferative lesions and neoplasms induced by inhaled 239 PuO 2 were evaluated for aberrant expression of transforming growth factor alpha (TGF-α) and epidermal growth factor receptor (EGFR). Expression of TGF-α protein, measured by immunohistochemistry, was higher in 94% of the squamous cell carcinomas and 87% of the foci of alveolar epithelial squamous metaplasia than that exhibited by the normal-appearing, adjacent lung parenchyma. In contrast, only 20% of adenocarcinomas and foci of epithelial hyperplasia expressed elevated levels of TGF-α. Many neoplasms expressing TGF-α also expressed excessive levels of EGFR mRNA. Southern and DNA slot blot analyses showed that the elevated EGFR expression was not due to amplification of the EGFR gene. These data suggest that increased amounts of TGF-α were early alterations in the progression of plutonium-induced squamous cell carcinoma, and these increases may occur in parallel with overexpression of the receptor for this growth factor. Together, these alterations create a potential autocrine loop for sustaining clonal expansion of cells initiated by high-LET radiation. 44 refs., 4 figs., 1 tab

  16. Tissue factor is an angiogenic-specific receptor for factor VII-targeted immunotherapy and photodynamic therapy.

    Science.gov (United States)

    Hu, Zhiwei; Cheng, Jijun; Xu, Jie; Ruf, Wolfram; Lockwood, Charles J

    2017-02-01

    Identification of target molecules specific for angiogenic vascular endothelial cells (VEC), the inner layer of pathological neovasculature, is critical for discovery and development of neovascular-targeting therapy for angiogenesis-dependent human diseases, notably cancer, macular degeneration and endometriosis, in which vascular endothelial growth factor (VEGF) plays a central pathophysiological role. Using VEGF-stimulated vascular endothelial cells (VECs) isolated from microvessels, venous and arterial blood vessels as in vitro angiogenic models and unstimulated VECs as a quiescent VEC model, we examined the expression of tissue factor (TF), a membrane-bound receptor on the angiogenic VEC models compared with quiescent VEC controls. We found that TF is specifically expressed on angiogenic VECs in a time-dependent manner in microvessels, venous and arterial vessels. TF-targeted therapeutic agents, including factor VII (fVII)-IgG1 Fc and fVII-conjugated photosensitizer, can selectively bind angiogenic VECs, but not the quiescent VECs. Moreover, fVII-targeted photodynamic therapy can selectively and completely eradicate angiogenic VECs. We conclude that TF is an angiogenic-specific receptor and the target molecule for fVII-targeted therapeutics. This study supports clinical trials of TF-targeted therapeutics for the treatment of angiogenesis-dependent diseases such as cancer, macular degeneration and endometriosis.

  17. Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Szlachcic A

    2016-08-01

    Full Text Available Anna Szlachcic, Malgorzata Zakrzewska, Michal Lobocki, Piotr Jakimowicz, Jacek Otlewski Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract: Fibroblast growth factor receptors (FGFRs are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V, was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE, and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. Keywords: fibroblast growth factor 1, FGF receptor, targeted cancer therapy, cytotoxic conjugates, FGFR-dependent cancer, MMAE, auristatin

  18. In-vivo fluorescence detection of breast cancer growth factor receptors by fiber-optic probe

    Science.gov (United States)

    Bustamante, Gilbert; Wang, Bingzhi; DeLuna, Frank; Sun, LuZhe; Ye, Jing Yong

    2018-02-01

    Breast cancer treatment options often include medications that target the overexpression of growth factor receptors, such as the proto-oncogene human epidermal growth factor receptor 2 (HER2/neu) and epidermal growth factor receptor (EGFR) to suppress the abnormal growth of cancerous cells and induce cancer regression. Although effective, certain treatments are toxic to vital organs, and demand assurance that the pursued receptor is present at the tumor before administration of the drug. This requires diagnostic tools to provide tumor molecular signatures, as well as locational information. In this study, we utilized a fiber-optic probe to characterize in vivo HER2 and EGFR overexpressed tumors through the fluorescence of targeted dyes. HER2 and EGFR antibodies were conjugated with ICG-Sulfo-OSu and Alexa Fluor 680, respectively, to tag BT474 (HER2+) and MDA-MB-468 (EGFR+) tumors. The fiber was inserted into the samples via a 30-gauge needle. Different wavelengths of a supercontinuum laser were selected to couple into the fiber and excite the corresponding fluorophores in the samples. The fluorescence from the dyes was collected through the same fiber and quantified by a time-correlated single photon counter. Fluorescence at different antibody-dye concentrations was measured for calibration. Mice with subcutaneous HER2+ and/or EGFR+ tumors received intravenous injections of the conjugates and were later probed at the tumor sites. The measured fluorescence was used to distinguish between tumor types and to calculate the concentration of the antibody-dye conjugates, which were detectable at levels as low as 40 nM. The fiber-optic probe presents a minimally invasive instrument to characterize the molecular signatures of breast cancer in vivo.

  19. Macrophage colony-stimulating factor, CSF-1, and its proto-oncogene-encoded receptor

    International Nuclear Information System (INIS)

    Sherr, C.J.; Rettenmier, C.W.; Roussel, M.F.

    1988-01-01

    The macrophage colony-stimulating factor, CSF-1, or M-CSF, is one of a family of hematopoietic growth factors that stimulates the proliferation of monocytes, macrophages, and their committed bone marrow progenitors. Unlike pluripotent hemopoietins such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3 or multi-CSF), which affect the growth of myeloid cells of several different hematopoietic lineages, CSF-1 acts only on cells of the mononuclear phagocyte series to stimulate their growth and enhance their survival. Retroviral transduction of the feline c-fms gene in the Susan McDonough and Hardy Zuckerman-5 (HZ-5) strains of feline sarcoma virus (FeSV) led to genetic alterations that endowed the recombined viral oncogene (v-fms) with the ability to transform cells in culture morphologically and to induce firbrosarcomas and hematopoietic neoplasms in susceptible animals. The v-fms oncogene product differs from the normal CSF-1 receptor in certain of its cardinal biochemical properties, most notably in exhibiting constitutively high basal levels of tyrosine kinase activity in the absence of its ligand. Comparative studies of the c-fms and v-fms genes coupled with analyses of engineered mutants and receptor chimeras have begun to pinpoint pertinent genetic alterations in the normal receptor gene that unmask its latent oncogenic potential. In addition, the availability of biologically active c-fms, v-fms, and CSF-1 cDNAs has allowed these genes to be mobilized and expressed in naive cells, thereby facilitating assays for receptor coupling with downstream components of the mitogenic pathway in diverse cell types

  20. Antibody-induced activation of the epidermal growth factor receptor tyrosine kinase requires the presence of detergent

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; de Laat, S. W.; Boonstra, J.

    1990-01-01

    Activation of the epidermal growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild

  1. Blockade of epidermal growth factor receptors chemosensitizes breast cancer cells through up-regulation of Bnip3L

    NARCIS (Netherlands)

    Real, PJ; Benito, A; Cuevas, J; Berciano, MT; de Juan, A; Coffer, P; Gomez-Roman, J; Lafarga, M; Lopez-Vega, JM; Fernandez-Luna, JL

    2005-01-01

    Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling

  2. Prognostic factors in advanced breast cancer: Race and receptor status are significant after development of metastasis.

    Science.gov (United States)

    Ren, Zhiyong; Li, Yufeng; Shen, Tiansheng; Hameed, Omar; Siegal, Gene P; Wei, Shi

    2016-01-01

    Prognostic factors are well established in early-stage breast cancer (BC), but less well-defined in advanced disease. We analyzed 323 BC patients who had distant relapse during follow-up from 1997 to 2010 to determine the significant clinicopathologic factors predicting survival outcomes. By univariate analysis, race, tumor grade, estrogen and progesterone receptors (ER/PR) and HER2 status were significantly associated with overall survival (OS) and post-metastasis survival (PMS). Applying a Cox regression model revealed that all these factors remained significant for PMS, while race, tumor grade and HER2 were independent factors for OS. Tumor grade was the only significant factor for metastasis-free survival by univariate and multivariate analyses. Our findings demonstrated that being Caucasian, hormonal receptor positive (HR+) and HER2 positive (HER2+) were all associated with a decreased hazard of death and that patients with HR+/HER2+ tumors had superior outcomes to those with HR+/HER2- disease. Further, PR status held a prognostic value over ER, thus reflecting the biologic mechanism of the importance of the functional ER pathway and the heterogeneity in the response to endocrine therapy. These observations indicate that the patients' genetic makeup and the intrinsic nature of the tumor principally govern BC progression and prognosticate the long-term outcomes in advanced disease. Copyright © 2015 Elsevier GmbH. All rights reserved.

  3. Differential role of tumor necrosis factor receptors in mouse brain inflammatory responses in cryolesion brain injury

    DEFF Research Database (Denmark)

    Quintana, Albert; Giralt, Mercedes; Rojas, Santiago

    2005-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF-alpha can induce both apoptosis and necrosis via intracell......Tumor necrosis factor-alpha (TNF-alpha) is one of the mediators dramatically increased after traumatic brain injury that leads to the activation, proliferation, and hypertrophy of mononuclear, phagocytic cells and gliosis. Eventually, TNF-alpha can induce both apoptosis and necrosis via...... intracellular signaling. This cytokine exerts its functions via interaction with two receptors: type-1 receptor (TNFR1) and type-2 receptor (TNFR2). In this work, the inflammatory response after a freeze injury (cryolesion) in the cortex was studied in wild-type (WT) animals and in mice lacking TNFR1 (TNFR1 KO...... signaling also affected the expression of apoptosis/cell death-related genes (Fas, Rip, p53), matrix metalloproteinases (MMP3, MMP9, MMP12), and their inhibitors (TIMP1), suggesting a role of TNFR1 in extracellular matrix remodeling after injury. However, GDNF, NGF, and BDNF expression were not affected...

  4. Expression, purification, and characterization of a diabody against the most important angiogenesis cell receptor: Vascular endothelial growth factor receptor 2

    Directory of Open Access Journals (Sweden)

    Mahdi Behdani

    2012-01-01

    Full Text Available Antibodies and their derivative fragments have long been used as tools in a variety of applications, in fundamental research work, biotechnology, diagnosis, and therapy. Camels produce single heavy-chain antibodies (VHH in addition to usual antibodies. These minimal-sized binders are very robust and bind the antigen with high affinity in a monomeric state. Vascular endothelial growth factor recepror-2 (VEGFR2 is an important tumor-associated receptor that blockade of its signaling can lead to the inhibition of neovascularization and tumor metastasis. Here, we describe the construction, expression, and purification VEGFR2-specific Diabody. Two variable fragments of a same camel anti-VEGFR2 antibody were linked together by the upper hinge segment of antibody to make a diabody. We showed the ability of diabody to recognition of VEGFR2 on the cell surface by FACS. Diabodies can be produced in the low-cost prokaryotic expression system, so they are suitable molecules for diagnostic and therapeutic issues.

  5. Purification of rat intestinal receptor for intrinsic factor-vitamin B12 complex

    International Nuclear Information System (INIS)

    Yamada, Shoji; Itaya, Harutaka; Nakazawa, Osamu; Fukuda, Morimichi.

    1977-01-01

    The intrinsic factor (IF) in a rat gastric mucosal extract was bound efficiently to vitamin B 12 -sepharose without significant change in its nature to produce IF-vitamin B 12 -sepharose. The purification of the intestinal receptor for the IF-vitamin B 12 complex was performed by the affinity chromatography using the IF-vitamin B 12 -sepharose as the affinity adsorbent. As a result of admixing the gastric mucosal extract sample with B 12 -sepharose while stirring for 4 hours, the adsorption was performed without any break through. Further, it was recognized that the B 12 -bound protein purified by the affinity chromatography using B 12 -sepharose was not much changed as compared with that before purification. Furthermore, it was recognized that IF-B 12 -sepharose was able to be made by binding IF with B 12 -sepharose which was made by coupling B 12 with the market-available AH-sepharose. The IF-B 12 -sepharose was washed with buffer solution, and then was loaded with the small intestine mucosal extract. Thereafter, the receptor was eluted by making di-valent cation inert with the buffer solution. After the removal of EDTA in the eluted solution by dialysis, the activity of the receptor was measured. 48.5% of the receptor activity loaded was recovered by the elution with EDTA. The specific activity of the receptor represented by the final amount of B 12 (pg)/the amount of protein (mg) in the purified substance was 335 folds of the original activity. (Iwakiri, K.)

  6. Platelet activating factor receptor binding plays a critical role in jet fuel-induced immune suppression

    International Nuclear Information System (INIS)

    Ramos, Gerardo; Kazimi, Nasser; Nghiem, Dat X.; Walterscheid, Jeffrey P.; Ullrich, Stephen E.

    2004-01-01

    Applying military jet fuel (JP-8) or commercial jet fuel (Jet-A) to the skin of mice suppresses the immune response in a dose-dependant manner. The release of biological response modifiers, particularly prostaglandin E 2 (PGE 2 ), is a critical step in activating immune suppression. Previous studies have shown that injecting selective cyclooxygenase-2 inhibitors into jet fuel-treated mice blocks immune suppression. Because the inflammatory phospholipid mediator, platelet-activating factor (PAF), up-regulates cyclooxygenase-2 production and PGE 2 synthesis by keratinocytes, we tested the hypothesis that PAF-receptor binding plays a role in jet fuel-induced immune suppression. Treating keratinocyte cultures with PAF and/or jet fuel (JP-8 and Jet-A) stimulates PGE 2 secretion. Jet fuel-induced PGE 2 production was suppressed by treating the keratinocytes with specific PAF-receptor antagonists. Injecting mice with PAF, or treating the skin of the mice with JP-8, or Jet-A, induced immune suppression. Jet fuel-induced immune suppression was blocked when the jet fuel-treated mice were injected with PAF-receptor antagonists before treatment. Jet fuel treatment has been reported to activate oxidative stress and treating the mice with anti-oxidants (Vitamins C, or E or beta-hydroxy toluene), before jet fuel application, interfered with immune suppression. These findings confirm previous studies showing that PAF-receptor binding can modulate immune function. Furthermore, they suggest that PAF-receptor binding may be an early event in the induction of immune suppression by immunotoxic environmental agents that target the skin

  7. Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery

    DEFF Research Database (Denmark)

    Schiødt, I; Jensen, Charlotte Harken; Kjaersgaard, E

    2000-01-01

    In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell......-CSF receptor positive, CD34+ progenitor cells were measured by flow cytometry in the leukapheresis product used for transplantation in a subgroup of 15 patients (NHL, n = 8, MM, n = 7). Three factors were identified as having a significant impact on platelet recovery. First, the level of Tpo in blood...... at the time of the nadir (day +7). Second, the percentage of re-infused thrombopoietin receptor positive progenitors and finally, the percentage of Flt3 receptor positive progenitors. On the other hand, none of the analyzed factors significantly predicted myeloid or erythroid recovery. These findings need...

  8. A Premature Termination of Human Epidermal Growth Factor Receptor Transcription in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jihene Elloumi-Mseddi

    2014-01-01

    Full Text Available Our success in producing an active epidermal growth factor receptor (EGFR tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

  9. Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

    Science.gov (United States)

    El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

    2018-01-17

    Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

  10. Mactosylceramide Prevents Glial Cell Overgrowth by Inhibiting Insulin and Fibroblast Growth Factor Receptor Signaling

    DEFF Research Database (Denmark)

    Gerdøe-Kristensen, Stine; Lund, Viktor K; Wandall, Hans H

    2017-01-01

    , in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what...... of the Drosophila Insulin Receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK...... hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of Insulin and Fibroblast Growth Factor Receptors in Drosophila glia. This article is protected by copyright. All rights reserved....

  11. Detection and Quantification of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases in Primary Human Endothelial Cells.

    Science.gov (United States)

    Fearnley, Gareth W; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2015-01-01

    Proteins differ widely in their pattern of expression depending on organism, tissue, and regulation in response to changing conditions. In the mammalian vasculature, the endothelium responds to vascular endothelial growth factors (VEGFs) via membrane-bound receptor tyrosine kinases (VEGFRs) to modulate many aspects of vascular physiology including vasculogenesis, angiogenesis, and blood pressure. Studies on VEGFR biology are thus dependent on detecting expression levels in different cell types and evaluating how changes in protein levels correlate with changing conditions including circulating VEGF levels. Here, we present a robust immunoblot-based protocol for detecting and quantifying VEGFRs in human endothelial cells. Using internal and external standards, we can rapidly evaluate receptor copy number and assess how this is altered in response to the cellular environment.

  12. Receptors for insulin-like growth factors I and II: autoradiographic localization in rat brain and comparison to receptors for insulin

    International Nuclear Information System (INIS)

    Lesniak, M.A.; Hill, J.M.; Kiess, W.; Rojeski, M.; Pert, C.B.; Roth, J.

    1988-01-01

    Receptors for insulin-like growth factor I (IGF-I) in rat brain were visualized using autoradiography with [125I]IGF-I. The binding of the labeled peptide was competed for fully by high concentrations of unlabeled IGF-I. At intermediate concentrations of unlabeled peptide the binding of [125I]IGF-I was competed for by unlabeled IGF-I more effectively than by IGF-II or insulin, which is typical of receptors for IGF-I. Essentially every brain section shows specific binding of IGF-I, and the pattern of binding of IGF-I to its receptors correlated well with the cytoarchitectonic structures. In parallel studies we showed that [125I]IGF-II was bound to tissue sections of rat brain and that the binding was competed for by an excess of unlabeled IGF-II. However, intermediate concentrations of unlabeled peptides gave inconclusive results. To confirm that the binding of [125I]IGF-II was to IGF-II receptors, we showed that antibodies specific for the IGF-II receptor inhibited the binding of labeled IGF-II. Furthermore, the binding of the antibody to regions of the brain section, visualized by the application of [125I]protein-A, gave patterns indistinguishable from those obtained with [125I]IGF-II alone. Again, the binding was very widely distributed throughout the central nervous system, and the patterns of distribution corresponded well to the underlying neural structures. Densitometric analysis of the receptors enabled us to compare the distribution of IGF-I receptors with that of IGF-II receptors as well as retrospectively with that of insulin receptors

  13. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

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

  14. Advanced Research of Fibroblast Growth Factor Receptor 
in Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Dan PU

    2013-11-01

    Full Text Available Lung cancer is severely threatening human health. In recent years, the treatment for lung adenocarcinoma has made a great progress, targeted therapy has been widely applied in clinic, and benefits amount of patients. However, in squamous cell lung cancer, the incidence of epidermal growth factor receptor (EGFR gene mutant and ALK fusion gene are low,and targeted therapy like Tarceva and crizotinib, can hardly work. Since the fibroblast growth factors (fibroblast growth factor, FGF pathway is considered to be related to tumor cell proliferation, metastasis and angiogenesis, more and more researches proved the amplification of fibroblast growth factor receptor (FGFR in squamous cell lung cancer. Experiments in vivo and in vitro found that blocking FGF pathway could reduce the proliferation of tumor cells and inhibit metastasis. The FGF pathway might be a new target for treatment of squamous cell lung cancer. This article reviews the effect of FGFR in tumorigenesis,as well as the prospect as a therapeutic target in non-small cell lung cancer.

  15. Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

    International Nuclear Information System (INIS)

    Ellsworth, J.L.; Brown, C.; Cooper, A.D.

    1988-01-01

    The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the apparent Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column

  16. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  17. Novel targeted approaches to treating biliary tract cancer: the dual epidermal growth factor receptor and ErbB-2 tyrosine kinase inhibitor NVP-AEE788 is more efficient than the epidermal growth factor receptor inhibitors gefitinib and erlotinib.

    Science.gov (United States)

    Wiedmann, Marcus; Feisthammel, Jürgen; Blüthner, Thilo; Tannapfel, Andrea; Kamenz, Thomas; Kluge, Annett; Mössner, Joachim; Caca, Karel

    2006-08-01

    Aberrant activation of the epidermal growth factor receptor is frequently observed in neoplasia, notably in tumors of epithelial origin. Attempts to treat such tumors with epidermal growth factor receptor antagonists resulted in remarkable success in recent studies. Little is known, however, about the efficacy of this therapy in biliary tract cancer. Protein expression of epidermal growth factor receptor, ErbB-2, and vascular endothelial growth factor receptor-2 was assessed in seven human biliary tract cancer cell lines by immunoblotting. In addition, histological sections from 19 patients with extrahepatic cholangiocarcinoma were analyzed for epidermal growth factor receptor, ErbB-2 and vascular endothelial growth factor receptor-2 expression by immunohistochemistry. Moreover, we sequenced the cDNA products representing the entire epidermal growth factor receptor coding region of the seven cell lines, and searched for genomic epidermal growth factor receptor amplifications and polysomy by fluorescence in-situ hybridization. Cell growth inhibition by gefitinib erlotinib and NVP-AEE788 was studied in vitro by automated cell counting. In addition, the anti-tumoral effect of erlotinib and NVP-AEE788 was studied in a chimeric mouse model. The anti-tumoral drug mechanism in this model was assessed by MIB-1 antibody staining, terminal deoxynucleotidyl transfer-mediated dUTP nick end-labelling assay, von Willebrand factor staining, and immunoblotting for p-p42/44 (p-Erk1/2, p-MAPK) and p-AKT. Immunoblotting revealed expression of epidermal growth factor receptor, ErbB-2, and vascular endothelial growth factor receptor-2 in all biliary tract cancer cell lines. EGFR was detectable in six of 19 (32%) extrahepatic human cholangiocarcinoma tissue samples, ErbB-2 in 16 of 19 (84%), and vascular endothelial growth factor receptor-2 in nine of 19 (47%). Neither epidermal growth factor receptor mutations nor amplifications or polysomy were found in the seven biliary tract cancer

  18. Adenovirus E4-ORF1 Dysregulates Epidermal Growth Factor and Insulin/Insulin-Like Growth Factor Receptors To Mediate Constitutive Myc Expression

    OpenAIRE

    Kong, Kathleen; Kumar, Manish; Taruishi, Midori; Javier, Ronald T.

    2015-01-01

    The E4-ORF1 protein encoded by human adenovirus stimulates viral replication in human epithelial cells by binding and activating cellular phosphatidylinositol 3-kinase (PI3K) at the plasma membrane and cellular Myc in the nucleus. In this study, we showed that E4-ORF1 hijacks the tyrosine kinase activities of cellular epidermal growth factor receptor (EGFR) and insulin receptor (InsR)/insulin-like growth factor receptor 1 (IGF1R), as well as the lipid kinase activity of PI3K, to mediate const...

  19. Mammary tumors that become independent of the type I insulin-like growth factor receptor express elevated levels of platelet-derived growth factor receptors

    Directory of Open Access Journals (Sweden)

    Campbell Craig I

    2011-11-01

    Full Text Available Abstract Background Targeted therapies are becoming an essential part of breast cancer treatment and agents targeting the type I insulin-like growth factor receptor (IGF-IR are currently being investigated in clinical trials. One of the limitations of targeted therapies is the development of resistant variants and these variants typically present with unique gene expression patterns and characteristics compared to the original tumor. Results MTB-IGFIR transgenic mice, with inducible overexpression of the IGF-IR were used to model mammary tumors that develop resistance to IGF-IR targeting agents. IGF-IR independent mammary tumors, previously shown to possess characteristics associated with EMT, were found to express elevated levels of PDGFRα and PDGFRβ. Furthermore, these receptors were shown to be inversely expressed with the IGF-IR in this model. Using cell lines derived from IGF-IR-independent mammary tumors (from MTB-IGFIR mice, it was demonstrated that PDGFRα and to a lesser extent PDGFRβ was important for cell migration and invasion as RNAi knockdown of PDGFRα alone or PDGFRα and PDGFRβ in combination, significantly decreased tumor cell migration in Boyden chamber assays and suppressed cell migration in scratch wound assays. Somewhat surprisingly, concomitant knockdown of PDGFRα and PDGFRβ resulted in a modest increase in cell proliferation and a decrease in apoptosis. Conclusion During IGF-IR independence, PDGFRs are upregulated and function to enhance tumor cell motility. These results demonstrate a novel interaction between the IGF-IR and PDGFRs and highlight an important, therapeutically relevant pathway, for tumor cell migration and invasion.

  20. Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

    Science.gov (United States)

    Wang, Linlin; Schulz, Thomas C.; Sherrer, Eric S.; Dauphin, Derek S.; Shin, Soojung; Nelson, Angelique M.; Ware, Carol B.; Zhan, Mei; Song, Chao-Zhong; Chen, Xiaoji; Brimble, Sandii N.; McLean, Amanda; Galeano, Maria J.; Uhl, Elizabeth W.; D'Amour, Kevin A.; Chesnut, Jonathan D.; Rao, Mahendra S.

    2007-01-01

    Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs. PMID:17761519

  1. Involvement of macrophage migration inhibitory factor and its receptor (CD74) in human breast cancer.

    Science.gov (United States)

    Richard, Vincent; Kindt, Nadège; Decaestecker, Christine; Gabius, Hans-Joachim; Laurent, Guy; Noël, Jean-Christophe; Saussez, Sven

    2014-08-01

    Macrophage migration inhibitory factor (MIF) and its receptor CD74 appear to be involved in tumorigenesis. We evaluated, by immunohistochemical staining, the tissue expression and distribution of MIF and CD74 in serial sections of human invasive breast cancer tumor specimens. The serum MIF level was also determined in breast cancer patients. We showed a significant increase in serum MIF average levels in breast cancer patients compared to healthy individuals. MIF tissue expression, quantified by a modified Allred score, was strongly increased in carcinoma compared to tumor-free specimens, in the cancer cells and in the peritumoral stroma, with fibroblasts the most intensely stained. We did not find any significant correlation with histoprognostic factors, except for a significant inverse correlation between tumor size and MIF stromal positivity. CD74 staining was heterogeneous and significantly decreased in cancer cells but increased in the surrounding stroma, namely in lymphocytes, macrophages and vessel endothelium. There was no significant variation according to classical histoprognostic factors, except that CD74 stromal expression was significantly correlated with triple-negative receptor (TRN) status and the absence of estrogen receptors. In conclusion, our data support the concept of a functional role of MIF in human breast cancer. In addition to auto- and paracrine effects on cancer cells, MIF could contribute to shape the tumor microenvironment leading to immunomodulation and angiogenesis. Interfering with MIF effects in breast tumors in a therapeutic perspective remains an attractive but complex challenge. Level of co-expression of MIF and CD74 could be a surrogate marker for efficacy of anti-angiogenic drugs, particularly in TRN breast cancer tumor.

  2. Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

    Science.gov (United States)

    Sun, Wan-Wei; Zhang, Xin-Xu; Wan, Wei-Song; Wang, Shu-Qi; Wen, Xiao-Bo; Zheng, Huai-Ping; Zhang, Yue-Ling; Li, Sheng-Kang

    2017-02-01

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain. Copyright © 2016. Published by Elsevier Ltd.

  3. A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus.

    Science.gov (United States)

    Alejo, Alí; Ruiz-Argüello, M Begoña; Ho, Yin; Smith, Vincent P; Saraiva, Margarida; Alcami, Antonio

    2006-04-11

    Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.

  4. Orexin–Corticotropin-Releasing Factor Receptor Heteromers in the Ventral Tegmental Area as Targets for Cocaine

    Science.gov (United States)

    Navarro, Gemma; Quiroz, César; Moreno-Delgado, David; Sierakowiak, Adam; McDowell, Kimberly; Moreno, Estefanía; Rea, William; Cai, Ning-Sheng; Aguinaga, David; Howell, Lesley A.; Hausch, Felix; Cortés, Antonio; Mallol, Josefa; Casadó, Vicent; Lluís, Carme; Canela, Enric I.

    2015-01-01

    Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R–OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R–OX1R heteromer. Cocaine binding to the σ1R–CRF1R–OX1R complex promotes a long-term disruption of the orexin-A–CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking. PMID:25926444

  5. Orexin-corticotropin-releasing factor receptor heteromers in the ventral tegmental area as targets for cocaine.

    Science.gov (United States)

    Navarro, Gemma; Quiroz, César; Moreno-Delgado, David; Sierakowiak, Adam; McDowell, Kimberly; Moreno, Estefanía; Rea, William; Cai, Ning-Sheng; Aguinaga, David; Howell, Lesley A; Hausch, Felix; Cortés, Antonio; Mallol, Josefa; Casadó, Vicent; Lluís, Carme; Canela, Enric I; Ferré, Sergi; McCormick, Peter J

    2015-04-29

    Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R-OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R-OX1R heteromer. Cocaine binding to the σ1R-CRF1R-OX1R complex promotes a long-term disruption of the orexin-A-CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking. Copyright © 2015 the authors 0270-6474/15/356639-15$15.00/0.

  6. Receptors for insulin-like growth factor II (IGF-II) in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1986-01-01

    Renal glomeruli were isolated by a technique involving renal perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving and isolation over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor-II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabelled IGF-II, with 50% inhibition of binding observed at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked to its receptor with disuccinimidyl suberate in two tissues known to contain IGF-II receptors, the rat chondrosarcoma chondrocyte and the rat kidney tubule, as well as in rat renal glomeruli. In all three cases, a specific high-molecular weight (Mr = 255,000) band could be identified on autoradiograms of dodecyl sulfate polyacrylamide gels. These results indicate that the rat glomerulus contains a high-affinity receptor for IGF-II. This finding is consistent with the hypothesis that IGF-II plays a role in glomerular growth and differentiation

  7. Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family

    Science.gov (United States)

    2012-01-01

    Accumulating evidence suggests that various diseases, including many types of cancer, result from alteration of subcellular protein localization and compartmentalization. Therefore, it is worthwhile to expand our knowledge in subcellular trafficking of proteins, such as epidermal growth factor receptor (EGFR) and ErbB-2 of the receptor tyrosine kinases, which are highly expressed and activated in human malignancies and frequently correlated with poor prognosis. The well-characterized trafficking of cell surface EGFR is routed, via endocytosis and endosomal sorting, to either the lysosomes for degradation or back to the plasma membrane for recycling. A novel nuclear mode of EGFR signaling pathway has been gradually deciphered in which EGFR is shuttled from the cell surface to the nucleus after endocytosis, and there, it acts as a transcriptional regulator, transmits signals, and is involved in multiple biological functions, including cell proliferation, tumor progression, DNA repair and replication, and chemo- and radio-resistance. Internalized EGFR can also be transported from the cell surface to several intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and the mitochondria, in addition to the nucleus. In this review, we will summarize the functions of nuclear EGFR family and the potential pathways by which EGFR is trafficked from the cell surface to a variety of cellular organelles. A better understanding of the molecular mechanism of EGFR trafficking will shed light on both the receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical application. PMID:22520625

  8. Specific, high affinity receptors for insulin-like growth factor II in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1988-01-01

    Rat renal glomeruli were isolated by a technique involving kidney perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving, and concentration over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components, while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabeled IGF-II, with 50% inhibition at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked with disuccinimidyl suberate to its receptor in rat renal glomeruli and a specific high mol wt (255,000) band could be identified on autoradiograms of dodecyl sulfate-polyacrylamide gels. [ 125 I]IGF-II binding and cross-linking to this band was inhibited by a polyclonal antibody against the type II IGF receptor. These results demonstrate for the first time that the isolated rat renal glomerulus contains a high affinity receptor for IGF-II

  9. A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus

    Science.gov (United States)

    Alejo, Alí; Ruiz-Argüello, M. Begoña; Ho, Yin; Smith, Vincent P.; Saraiva, Margarida; Alcami, Antonio

    2006-01-01

    Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis. PMID:16581912

  10. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid

    Science.gov (United States)

    Sarlati, Fatemeh; Sattari, Mandana; Razzaghi, Shilan; Nasiri, Malihe

    2012-01-01

    Background: Osteoclastogenesis is coordinated by the interaction of three members of the tumor necrosis factor (TNF) superfamily: Osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK). The aim of this study was to investigate RANKL and OPG levels, and their relative ratio in gingival crevicular fluid (GCF) of patients with chronic and aggressive periodontitis, as well as healthy controls. Materials and Methods: In this analytical study, GCF was obtained from healthy (n = 10), mild chronic periodontitis (n = 18), moderate chronic periodontitis (n = 18), severe chronic periodontitis (n = 20), and generalized aggressive periodontitis (n = 20) subjects. RANKL and OPG concentrations were measured by enzyme-linked immunosorbent assay. Statistical tests used were Kruskal–Wallis test, Mann–Whitney U rank sum test, and Spearman's rank correlation analysis. The level of statistical significance was set at P chronic periodontitis (mild, moderate, severe), and aggressive periodontitis (P = 0.41). There was statistically significant correlation between the concentration of sRANKL and Clinical Attachment Level (CAL) in moderate chronic periodontitis patients (R = 0.48, P = 0.04). There was also negative correlation between OPG concentration and CAL in moderate chronic periodontitis patients, although not significant (R = −0.13). Conclusion: RANKL was prominent in periodontitis sites, especially in moderate periodontitis patients, whereas OPG was not detectable in some diseased sites with bleeding on probing, supporting the role of these two molecules in the bone loss developed in this disease. PMID:23559954

  11. Involvement of growth factors and their receptors in radon-induced rat lung tumors

    International Nuclear Information System (INIS)

    Leung, F.C.; Dagle, G.E.; Cross, F.T.

    1992-01-01

    In this paper we examine the role of growth factors (GF) and their receptors (GFR) in radon-induced rat lung tumors. Inhalation exposure of radon and its daughters induced lung tumors in rats, but the molecule/cellular mechanisms are not known. Recent evidence suggests that GF/GFR play a critical role in the growth and development of lung cancer in humans and animals. We have developed immunocytochemical methods for identifying sites of production and action of GF/GFR at the cellular level; for example, the avidin-biotin horseradish peroxidase technique. In radon-induced rat epidermoid carcinomas, epidermal growth factor (EGF), EGF-receptors (EGF-R), transforming growth factor alpha (TGF-α), and bombesin were found to be abnormally expressed. These abnormal expressions, mainly associated with epidermoid carcinomas of the lung, were not found in any other lung tumor types. Our data suggest that EGF, EGF-R, TGF-α, and bombesin are involved in radon oncogenesis in rat lungs, especially in epidermoid carcinomas, possibly through the autocrine/paracrine pathway

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential...

  13. Divergent effects of insulin-like growth factor-1 receptor expression on prognosis of estrogen receptor positive versus triple negative invasive ductal breast carcinoma

    NARCIS (Netherlands)

    Hartog, Hermien; Horlings, Hugo M; van der Vegt, Bert; Kreike, Bas; Ajouaou, Abderrahim; van de Vijver, Marc J; Boezen, Hendrika; de Bock, Geertruida H; van der Graaf, Wilhelmina; Wesseling, Jelle

    2011-01-01

    The insulin-like growth factor type 1 receptor (IGF1R) is involved in progression of breast cancer and resistance to systemic treatment. Targeting IGF1R signaling may, therefore, be beneficial in systemic treatment. We report the effect of IGF1R expression on prognosis in invasive ductal breast

  14. P55 tumour necrosis factor receptor in bone marrow-derived cells promotes atherosclerosis development in low-density lipoprotein receptor knock-out mice

    NARCIS (Netherlands)

    Xanthoulea, Sofia; Gijbels, Marion J. J.; van der Made, Ingeborg; Mujcic, Hilda; Thelen, Melanie; Vergouwe, Monique N.; Ambagts, Matheus H. C.; Hofker, Marten H.; de Winther, Menno P. J.

    2008-01-01

    Tumour necrosis factor (TNF) is a pivotal pro-inflammatory cytokine with a clear pathogenic role in many chronic inflammatory diseases, and p55 TNF receptor (TNFR) mediates the majority of TNF responses. The aim of the current study was to investigate the role of p55 TNFR expression in bone

  15. The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Raymond; Matthews, Jason, E-mail: jason.matthews@utoronto.ca

    2013-07-15

    Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important role in mediating cellular protection against reactive oxygen species. NRF2 signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but inhibited by estrogen receptor alpha (ERα). In this study we investigated the crosstalk among NRF2, AHR and ERα in MCF-7 breast cancer cells treated with the NRF2 activator sulforaphane (SFN), a dual AHR and ERα activator, 3,3′-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 17β-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1 (NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after co-treatment with E2. E2-dependent repression of NQO1 and HMOX1 was associated with increased ERα but reduced p300 recruitment and reduced histone H3 acetylation at both genes. In contrast, DIM + SFN or TCDD + SFN induced NQO1 and HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by RNAi-mediated knockdown of AHR. DIM + SFN but not TCDD + SFN also induced recruitment of ERα to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored p300 recruitment and histone H3 acetylation, thereby reversing the ERα-dependent repression of NRF2. Taken together, our study provides further evidence of functional interplay among NRF2, AHR and ERα signaling pathways through altered p300 recruitment to NRF2-regulated target genes. - Highlights: • We examined crosstalk among ERα, AHR, and NRF2 in MCF-7 breast cancer cells. • AHR enhanced the mRNA expression levels of two NRF2 target genes – HMOX1 and NQO1. • ERα repressed HMOX1 and NQO1 expression via decreased histone acetylation. • AHR prevented ERα-dependent repression of HMOX1 and NQO1.

  16. Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies

    Directory of Open Access Journals (Sweden)

    Isabell Lang

    2018-04-01

    Full Text Available Progranulin (PGRN is a secreted anti-inflammatory protein which can be processed by neutrophil proteases to various granulins. It has been reported that at least a significant portion of the anti-inflammatory effects of PGRN is due to direct high affinity binding to tumor necrosis factor receptor-1 (TNFR1 and TNFR2 and inhibition of tumor necrosis factor (TNF-induced TNFR1/2 signaling. Two studies failed to reproduce the interaction of TNFR1 and TNFR2 with PGRN, but follow up reports speculated that this was due to varying experimental circumstances and/or the use of PGRN from different sources. However, even under consideration of these speculations, there is still a striking discrepancy in the literature between the concentrations of PGRN needed to inhibit TNF signaling and the concentrations required to block TNF binding to TNFR1 and TNFR2. While signaling events induced by 0.2–2 nM of TNF have been efficiently inhibited by low, near to equimolar concentrations (0.5–2.5 nM of PGRN in various studies, the reported inhibitory effects of PGRN on TNF-binding to TNFR1/2 required a huge excess of PGRN (100–1,000-fold. Therefore, we investigated the effect of PGRN on TNF binding to TNFR1 and TNFR2 in highly sensitive cellular binding studies. Unlabeled TNF inhibited >95% of the specific binding of a Gaussia princeps luciferase (GpL fusion protein of TNF to TNFR1 and TNFR2 and blocked binding of soluble GpL fusion proteins of TNFR1 and TNFR2 to membrane TNF expressing cells to >95%, too. Purified PGRN, however, showed in both assays no effect on TNF–TNFR1/2 interaction even when applied in huge excess. To rule out that tags and purification- or storage-related effects compromise the potential ability of PGRN to bind TNF receptors, we directly co-expressed PGRN, and as control TNF, in TNFR1- and TNFR2-expressing cells and looked for binding of GpL-TNF. While expression of TNF strongly inhibited binding of GpL-TNF to TNFR1/2, co

  17. The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast cancer cells

    International Nuclear Information System (INIS)

    Lo, Raymond; Matthews, Jason

    2013-01-01

    Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important role in mediating cellular protection against reactive oxygen species. NRF2 signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but inhibited by estrogen receptor alpha (ERα). In this study we investigated the crosstalk among NRF2, AHR and ERα in MCF-7 breast cancer cells treated with the NRF2 activator sulforaphane (SFN), a dual AHR and ERα activator, 3,3′-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 17β-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1 (NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after co-treatment with E2. E2-dependent repression of NQO1 and HMOX1 was associated with increased ERα but reduced p300 recruitment and reduced histone H3 acetylation at both genes. In contrast, DIM + SFN or TCDD + SFN induced NQO1 and HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by RNAi-mediated knockdown of AHR. DIM + SFN but not TCDD + SFN also induced recruitment of ERα to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored p300 recruitment and histone H3 acetylation, thereby reversing the ERα-dependent repression of NRF2. Taken together, our study provides further evidence of functional interplay among NRF2, AHR and ERα signaling pathways through altered p300 recruitment to NRF2-regulated target genes. - Highlights: • We examined crosstalk among ERα, AHR, and NRF2 in MCF-7 breast cancer cells. • AHR enhanced the mRNA expression levels of two NRF2 target genes – HMOX1 and NQO1. • ERα repressed HMOX1 and NQO1 expression via decreased histone acetylation. • AHR prevented ERα-dependent repression of HMOX1 and NQO1.

  18. Co-inhibition of epidermal growth factor receptor and insulin-like growth factor receptor 1 enhances radiosensitivity in human breast cancer cells

    International Nuclear Information System (INIS)

    Li, Ping; Veldwijk, Marlon R; Zhang, Qing; Li, Zhao-bin; Xu, Wen-cai; Fu, Shen

    2013-01-01

    Over-expression of epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor (IGF-1R) have been shown to closely correlate with radioresistance of breast cancer cells. This study aimed to investigate the impact of co-inhibition of EGFR and IGF-1R on the radiosensitivity of two breast cancer cells with different profiles of EGFR and IGF-1R expression. The MCF-7 (EGFR +/−, IGF-1R +++) and MDA-MB-468 (EGFR +++, IGF-1R +++) breast cancer cell lines were used. Radiosensitizing effects were determined by colony formation assay. Apoptosis and cell cycle distribution were measured by flow cytometry. Phospho-Akt and phospho-Erk1/2 were quantified by western blot. In vivo studies were conducted using MDA-MB-468 cells xenografted in nu/nu mice. In MDA-MB-468 cells, the inhibition of IGF-1R upregulated the p-EGFR expression. Either EGFR (AG1478) or IGF-1R inhibitor (AG1024) radiosensitized MDA-MB-468 cells. In MCF-7 cells, radiosensitivity was enhanced by AG1024, but not by AG1478. Synergistical radiosensitizing effect was observed by co-inhibition of EGFR and IGF-1R only in MDA-MB-468 cells with a DMF 10% of 1.90. The co-inhibition plus irradiation significantly induced more apoptosis and arrested the cells at G0/G1 phase in MDA-MB-468 cells. Only co-inhibition of EGFR and IGF-1R synergistically diminished the expression of p-Akt and p-Erk1/2 in MDA-MB-468 cells. In vivo studies further verified the radiosensitizing effects by co-inhibition of both pathways in a MDA-MB-468 xenograft model. Our data suggested that co-inhibition of EGFR and IGF-1R synergistically radiosensitized breast cancer cells with both EGFR and IGF-1R high expression. The approach may have an important therapeutic implication in the treatment of breast cancer patients with high expression of EGFR and IGF-1R

  19. Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

    DEFF Research Database (Denmark)

    Li, Hongbing; Panina, Svetlana; Kaur, Amandeep

    2012-01-01

    Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca2+/CaM complexes, which interact with and activate target proteins....... In the present study the role of Ca2+/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca2+ chelator inhibited...

  20. Strong association of epidermal growth factor receptor status with breast cancer FDG uptake

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Joohee; Moon, Seung Hwan; Hyun, Seung Hyup; Cho, Young Seok; Choi, Joon Young; Kim, Byung-Tae; Lee, Kyung-Han [Sungkyunkwan University School of Medicine, Department of Nuclear Medicine, Samsung Medical Center, Seoul (Korea, Republic of); Lee, Eun Jeong [Seoul Medical Center, Department of Nuclear Medicine, Seoul (Korea, Republic of); Kim, Seokhwi [Sungkyunkwan University School of Medicine, Department of Pathology, Samsung Medical Center, Seoul (Korea, Republic of)

    2017-08-15

    Imaging tumor FDG uptake could complement breast cancer biomarkers of risk and treatment response. Although breast cancer FDG uptake is reputedly influenced by major biomarker states, the role of epidermal growth factor receptor (EGFR) expression remains largely unexplored. This is a retrospective study that included 499 patients with primary breast cancer at initial presentation. Tumor FDG uptake was measured on pretreatment PET/CT as maximum standardized uptake value (SUVmax), and biomarkers were assessed by immunohistochemistry of tumor tissue. Regression analysis was performed for predictors of high tumor FDG uptake (SUVmax ≥ 8.6). SUVmax was higher in ER- (36.5%; 11.2 ± 6.0 vs. 8.3 ± 5.3), PR- (42.3%; 10.9 ± 6.0 vs. 8.2 ± 5.2), and triple-negative tumors (19.8%; 12.0 ± 6.9 vs. 8.7 ± 5.2; all p < 0.0001). EGFR expression (28.5%) was more frequent in ER-, PR-, triple-negative, cytokeratin 5/6 (CK5/6) + and mutant P53 (mP53) + tumors (all p < 0.0001). EGFR+ was associated with higher SUVmax among all tumors (11.9 ± 6.0 vs. 8.3 ± 5.3), ER- tumors (p < 0.0001), PR- and + tumors (p < 0.0001 and 0.027), hormone receptor- and + tumors (p < 0.0001 and 0.004), human epidermal growth factor receptor 2 (HER2)- and + tumors (p < 0.0001 and 0.006), non-triple negative tumors (p < 0.0001), CK5/6- and + tumors (p = 0.021 and <0.0001), and mP53- and + tumors (p < 0.0001 and 0.008). Tumors had high FDG uptake in 73.2% of EGFR+ and 40.6% of EGFR- tumors. On regression analysis, significant multivariate predictors of high tumor FDG uptake were large size, EGFR+ and CK5/6+ for the entire subjects, and EGFR+ and CK5/6+ for ER- and hormone receptor negative subgroups. High FDG uptake was able to sub-stratify EGFR+ tumors that were more likely to be ER- and CK5/6+, and EGFR- tumors more likely to be mP53 +. Primary breast tumor FDG uptake is strongly influenced by EGFR status beyond that by other major biomarkers including hormone receptor and HER2 status, and EGFR

  1. Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

    Science.gov (United States)

    Nayak, Ramesh C.; Keshava, Shiva; Esmon, Charles T.; Pendurthi, Usha R.; Rao, L. Vijaya Mohan

    2013-01-01

    Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa. PMID:23555015

  2. Expression of Vascular Endothelial Growth Factor Receptors in Benign Vascular Lesions of the Orbit: A Case Series.

    Science.gov (United States)

    Atchison, Elizabeth A; Garrity, James A; Castillo, Francisco; Engman, Steven J; Couch, Steven M; Salomão, Diva R

    2016-01-01

    Vascular lesions of the orbit, although not malignant, can cause morbidity because of their location near critical structures in the orbit. For the same reason, they can be challenging to remove surgically. Anti-vascular endothelial growth factor (VEGF) drugs are increasingly being used to treat diseases with prominent angiogenesis. Our study aimed to determine to what extent VEGF receptors and their subtypes are expressed on selected vascular lesions of the orbit. Retrospective case series of all orbital vascular lesions removed by one of the authors (JAG) at the Mayo Clinic. A total of 52 patients who underwent removal of vascular orbital lesions. The pathology specimens from the patients were retrieved, their pathologic diagnosis was confirmed, demographic and clinical information were gathered, and sections from vascular tumors were stained with vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor type 1 (VEGFR1), vascular endothelial growth factor receptor type 2 (VEGFR2), and vascular endothelial growth factor receptor type 3 (VEGFR3). The existence and pattern of staining with VEGF and its subtypes on these lesions. There were 28 specimens of venous malformations, 4 capillary hemangiomas, 7 lymphatic malformations, and 6 lymphaticovenous malformations. All samples stained with VEGF, 55% stained with VEGFR1, 98% stained with VEGFR2, and 96% stained with VEGFR3. Most (94%) of the VEGFR2 staining was diffuse. Most orbital vascular lesions express VEGF receptors, which may suggest a future target for nonsurgical treatment. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  3. Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation.

    LENUS (Irish Health Repository)

    Wakai, A

    2012-02-03

    The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng\\/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5\\'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.

  4. Fibroblast growth factor 21, fibroblast growth factor receptor 1, and β-Klotho expression in bovine growth hormone transgenic and growth hormone receptor knockout mice.

    Science.gov (United States)

    Brooks, Nicole E; Hjortebjerg, Rikke; Henry, Brooke E; List, Edward O; Kopchick, John J; Berryman, Darlene E

    Although growth hormone (GH) and fibroblast growth factor 21 (FGF21) have a reported relationship, FGF21 and its receptor, fibroblast growth factor receptor 1 (FGFR1) and cofactor β-Klotho (KLB), have not been analyzed in chronic states of altered GH action. The objective of this study was to quantify circulating FGF21 and tissue specific expression of Fgf21, Fgfr1, and Klb in mice with modified GH action. Based on previous studies, we hypothesized that bovine GH transgenic (bGH) mice will be FGF21 resistant and GH receptor knockout (GHR-/-) mice will have normal FGF21 action. Seven-month-old male bGH mice (n=9) and wild type (WT) controls (n=10), and GHR-/- mice (n=8) and WT controls (n=8) were used for all measurements. Body composition was determined before dissection, and tissue weights were measured at the time of dissection. Serum FGF21 levels were evaluated by ELISA. Expression of Fgf21, Fgfr1, and Klb mRNA in white adipose tissue (AT), brown AT, and liver were evaluated by reverse transcription quantitative PCR. As expected, bGH mice had increased body weight (p=3.70E -8 ) but decreased percent fat mass (p=4.87E -4 ). Likewise, GHR-/- mice had decreased body weight (p=1.78E -10 ) but increased percent fat mass (p=1.52E -9 ), due to increased size of the subcutaneous AT depot when normalized to body weight (p=1.60E -10 ). Serum FGF21 levels were significantly elevated in bGH mice (p=0.041) and unchanged in GHR-/- mice (p=0.88). Expression of Fgf21, Fgfr1, and Klb mRNA in white AT and liver were downregulated or unchanged in both bGH and GHR-/- mice. The only exception was Fgf21 expression in brown AT of GHR-/-, which trended toward increased expression (p=0.075). In accordance with our hypothesis, we provide evidence that circulating FGF21 is increased in bGH animals, but remains unchanged in GHR-/- mice. Downregulation or no change in Fgf21, Fgfr1, and Klb expression are seen in white AT, brown AT, and liver of bGH and GHR-/- mice when compared to their

  5. Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

    Science.gov (United States)

    Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

    2002-02-01

    We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

  6. Fibroblast growth factor receptor 4 regulates tumor invasion by coupling fibroblast growth factor signaling to extracellular matrix degradation

    DEFF Research Database (Denmark)

    Sugiyama, Nami; Varjosalo, Markku; Meller, Pipsa

    2010-01-01

    /stroma border and tumor invasion front. The strongest overall coexpression was found in prostate carcinoma. Studies with cultured prostate carcinoma cell lines showed that the FGFR4-R388 variant, which has previously been associated with poor cancer prognosis, increased MT1-MMP-dependent collagen invasion......Aberrant expression and polymorphism of fibroblast growth factor receptor 4 (FGFR4) has been linked to tumor progression and anticancer drug resistance. We describe here a novel mechanism of tumor progression by matrix degradation involving epithelial-to-mesenchymal transition in response...... to membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) induction at the edge of tumors expressing the FGFR4-R388 risk variant. Both FGFR4 and MT1-MMP were upregulated in tissue biopsies from several human cancer types including breast adenocarcinomas, where they were partially coexpressed at the tumor...

  7. Development of epidermal growth factor receptor targeted therapy in pancreatic cancer.

    Science.gov (United States)

    Qing, Liu; Qing, Wang

    2018-02-01

    The epidermal growth factor receptor (EGFR) family are a series of important cancer therapeutic targets involved in cancer biology. These genes play an important role in tumor biological characteristics including angiogenesis, cell survival, invasion and glucose metabolism. In recent years, progresses have been achieved upon the cellular and molecular biological characteristics of EGFR and its role in cancer development based on the study of tumor specimens and experimental animal model. EGFR(HER1/ErbB) is overexpressed in over sixty percent of triple-negative breast cancers and occurs in pancreatic, bladder, lung and head-and-neck cancers. Up to now, EGFR inhibitors have been applied in various of cancer, such as lung, breast, bladder and head and neck cancers etc., in which the combination of EGFR inhibitors plus chemotherapeutic agents is now seen as the standard of care for advanced/metastatic pancreatic cancer. For these reasons, EGFR inhibitors and their therapeutic effect for pancreatic cancer is becoming the focus in Laboratory and clinical research. In this paper, research progress of the development of epidermal growth factor receptor targeted therapy in pancreatic cancer is introduced.

  8. Therapies based on inhibitors of the epidermal growth factor receptor: enclosing the future

    International Nuclear Information System (INIS)

    Diaz, Arlhee; Lage, Agustin

    2007-01-01

    The Epidermal Growth Factor Receptor (EGFR) is considered an important target for rational drug design due to its key role in numerous tumors. Potential contribution of EGFR-related signaling pathways to promote tumorigenic processes, including cell proliferation, angiogenesis, and resistance to apoptosis has been well established. Two classes of anti-EGFR agents in late-stage clinical testing include monoclonal antibodies against extracellular EGFR domain (Cetuximab, Nimotuzumab) and small molecules tyrosine kinase inhibitors, which inhibit the receptor enzyme activity (Gefitinib, Erlotinib). A considerable body of evidence has emerged since its introduction in the treatment of cancer patients. However, important questions such as reliable surrogate markers to predict response to the treatment, or optimal sequence and combination of these agents with conventional therapies remain to be addressed. Identify and validate predictive factors to select patients likely to respond to EGFR inhibitors, such as mutations that confer resistance versus those associated with sensitivity is required. A better understanding of molecular mechanisms associated with antitumor activity will useful to predict the interaction of these agents with other therapies in order to avoid antagonisms or overlapping effects resulting in no adding effects. Finally, the benefits derived from EGFR inhibitors as first-line therapy in selected populations, and the optimal doses and ways to delivery to the tumor site resulting in optimal target modulation should be established by the ongoing investigation. (Author)

  9. Early clinical development of epidermal growth factor receptor targeted therapy in breast cancer

    Science.gov (United States)

    Matsuda, Naoko; Lim, Bora; Wang, Xiaoping; Ueno, Naoto T.

    2018-01-01

    Introduction Epidermal growth factor receptor (EGFR) targeted treatment has been evaluated but has not shown a clear clinical benefit for breast cancer. This review article aims to consider the knowledge of the biological background of EGFR pathways in dissecting clinical studies of EGFR targeted treatment in breast cancer. Areas covered This review focuses on the role of the EGFR pathway and the investigational drugs that target EGFR for breast cancer. Expert opinion Recent studies have indicated that EGFR targeted therapy for breast cancer has some promising effects for patients with triple-negative breast cancer, basal-like breast cancer, and inflammatory breast cancer. However, predictive and prognostic biomarkers for EGFR targeted therapy have not been identified. The overexpression or amplification of EGFR itself may not be the true factor of induction of the canonical pathway as an oncogenic driver of breast cancer. Instead, downstream, non-canonical pathways related to EGFR may contribute to some aspects of the biological behavior of breast cancer; therefore, the blockade of the receptor could result in sufficient suppression of downstream pathways to inhibit the aggressive behavior of breast cancer. Mechanistic studies to investigate the dynamic interaction between the EGFR pathway and non-canonical pathways are warranted. PMID:28271910

  10. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

    International Nuclear Information System (INIS)

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena; Martinez-Irujo, Juan Jose; Rouzaut, Ana

    2008-01-01

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 μM triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure

  11. Stepwise Progress in Epidermal Growth Factor Receptor/Radiation Studies for Head and Neck Cancer

    International Nuclear Information System (INIS)

    Harari, Paul M.

    2007-01-01

    The U.S. Food and Drug Administration approval of four new epidermal growth factor receptor (EGFR) inhibitors for cancer therapy (cetuximab, panitumumab, gefitinib, and erlotinib) over the last 3 years is a remarkable milestone in oncology. Indeed, molecular inhibition of EGFR signaling represents one of the most promising current arenas for the development of molecular-targeted cancer therapies. Epidermal growth factor receptor inhibitors from both the monoclonal antibody and tyrosine kinase inhibitor class have demonstrated clinical activity in the treatment of a broad spectrum of common human malignancies. For the discipline of radiation oncology, the 2006 report of a phase III trial demonstrating a survival advantage for advanced head and neck cancer patients with the addition of weekly cetuximab during a 7-week course of radiation is particularly gratifying. Indeed, this is the first phase III trial to confirm a survival advantage with the addition of a molecular-targeted agent to radiation. Furthermore, this result seems to have been achieved with only a modest increment in overall treatment toxicity and with very high compliance to the prescribed treatment regimen. Nevertheless, much remains to be learned regarding the rational integration of EGFR inhibitors into cancer treatment regimens, as well as methods to optimize the selection of patients most likely to benefit from EGFR inhibitor strategies

  12. Early clinical development of epidermal growth factor receptor targeted therapy in breast cancer.

    Science.gov (United States)

    Matsuda, Naoko; Lim, Bora; Wang, Xiaoping; Ueno, Naoto T

    2017-04-01

    Epidermal growth factor receptor (EGFR) targeted treatment has been evaluated but has not shown a clear clinical benefit for breast cancer. This review article aims to consider the knowledge of the biological background of EGFR pathways in dissecting clinical studies of EGFR targeted treatment in breast cancer. Areas covered: This review focuses on the role of the EGFR pathway and the investigational drugs that target EGFR for breast cancer. Expert opinion: Recent studies have indicated that EGFR targeted therapy for breast cancer has some promising effects for patients with triple-negative breast cancer, basal-like breast cancer, and inflammatory breast cancer. However, predictive and prognostic biomarkers for EGFR targeted therapy have not been identified. The overexpression or amplification of EGFR itself may not be the true factor of induction of the canonical pathway as an oncogenic driver of breast cancer. Instead, downstream, non-canonical pathways related to EGFR may contribute to some aspects of the biological behavior of breast cancer; therefore, the blockade of the receptor could result in sufficient suppression of downstream pathways to inhibit the aggressive behavior of breast cancer. Mechanistic studies to investigate the dynamic interaction between the EGFR pathway and non-canonical pathways are warranted.

  13. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Directory of Open Access Journals (Sweden)

    Béatrice Marquèze-Pouey

    Full Text Available Signaling mediated by the epidermal growth factor (EGF is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer. In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  14. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Science.gov (United States)

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  15. Insulin-like growth factor receptor inhibitors: baby or the bathwater?

    Science.gov (United States)

    Yee, Douglas

    2012-07-03

    The success of targeted therapies for cancer is undisputed; strong preclinical evidence has resulted in the approval of several new agents for cancer treatment. The type I insulin-like growth factor receptor (IGF1R) appeared to be one of these promising new targets. Substantial population and preclinical data have all pointed toward this pathway as an important regulator of tumor cell biology. Although early results from clinical trials that targeted the IGF1R showed some evidence of response, larger randomized phase III trials have not shown clear clinical benefit of targeting this pathway in combination with conventional strategies. These disappointing results have resulted in the discontinuation of several anti-IGF1R programs. However, the conduct of these trials has brought to the forefront several important factors that need to be considered in the conduct of future clinical trials. The need to develop biomarkers, a clearer understanding of insulin receptor function, and defining rational combination regimens all require further consideration. In this commentary, the current state of IGF1R inhibitors in cancer therapy is reviewed.

  16. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Yongjun Yin

    2016-05-01

    Full Text Available Activating mutations in fibroblast growth factor receptor 3 (FGFR3 have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9, a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11 with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.

  17. Immunohistochemical detection of epidermal growth factor receptor in radiation-induced lung tumors in Beagle dogs

    Energy Technology Data Exchange (ETDEWEB)

    Gillett, N A; Haley, P J; Hahn, F F

    1988-12-01

    Increased levels of epidermal growth factor receptor have been reported in a variety of tumors, including pulmonary squamous cell carcinomas in man. The purpose of this study was to determine if increased levels of epidermal growth factor (EGFR) were present in lung tumors from Beagle dogs that had been exposed to {sup 239}PuO{sub 2}- Using immunohistochemical techniques, sections from 17 lung tumors were examined for the presence of EGFR. Seven of the tumors were strongly positive for EGFR; the remainder of the tumors and the normal lung sections were negative. The positive immunostaining could not be correlated with the histologic phenotype of the tumors. Work is in progress to determine the level of EGFR in preneoplastic, proliferative epithelial foci in the Iung. (author)

  18. Sex, Receptors and Attachment: A Review of Individual Factors Influencing Response to Oxytocin

    Directory of Open Access Journals (Sweden)

    Kai S Macdonald

    2013-01-01

    Full Text Available As discussed in the larger review in this special issue (MacDonald and Feifel, intranasal oxytocin (IN OT is demonstrating a growing potential as a therapeutic agent in psychiatry. Importantly, research suggests that a variety of individual factors may influence a person’s response to OT. In this mini-review, I provides a review of three: (1 sex and hormonal status; (2 genetic variation in aspects of the OT system (i.e. OT receptors; and (3 attachment history. Each of these factors will be important to monitor as we strive to develop a richer understanding of OT's role in human development, brain-based disease, and the potential for individualized, OT-targeted treatments.

  19. Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Gang [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Department of Anesthesiology, First Affiliated Hospital of China Medical University, Shenyang (China); Hitomi, Hirofumi, E-mail: hitomi@kms.ac.jp [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Hosomi, Naohisa [Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, Kagawa (Japan); Lei, Bai; Nakano, Daisuke [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Deguchi, Kazushi; Mori, Hirohito; Masaki, Tsutomu [Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Ma, Hong [Department of Anesthesiology, First Affiliated Hospital of China Medical University, Shenyang (China); Griendling, Kathy K. [Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA (United States); Nishiyama, Akira [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan)

    2011-10-15

    Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia. -- Highlights: {yields} Mechanical stretch augments insulin-induced VSMC proliferation via IGF-1 receptor. {yields} Src/EGFR-mediated ERK and Akt phosphorylation are augmented in stretched VSMCs. {yields} Similar to in vitro experiment, IGF-1 receptor is increased in hypertensive rats. {yields} Results provide possible mechanisms of vascular remodeling in hypertension with DM.

  20. Potent and long-acting corticotropin releasing factor (CRF) receptor 2 selective peptide competitive antagonists.

    Science.gov (United States)

    Rivier, J; Gulyas, J; Kirby, D; Low, W; Perrin, M H; Kunitake, K; DiGruccio, M; Vaughan, J; Reubi, J C; Waser, B; Koerber, S C; Martinez, V; Wang, L; Taché, Y; Vale, W

    2002-10-10

    We present evidence that members of the corticotropin releasing factor (CRF) family assume distinct structures when interacting with the CRF(1) and CRF(2) receptors. Predictive methods, physicochemical measurements, and structure-activity relationship studies have suggested that CRF, its family members, and competitive antagonists such as astressin [cyclo(30-33)[DPhe(12),Nle(21),Glu(30),Lys(33),Nle(38)]hCRF((12-41))] assume an alpha-helical conformation when interacting with their receptors. We had shown that alpha-helical CRF((9-41)) and sauvagine showed some selectivity for CRF receptors other than that responsible for ACTH secretion(1) and later for CRF2.(2) More recently, we suggested the possibility of a helix-turn-helix motif around a turn encompassing residues 30-33(3) that would confer high affinity for both CRF(1) and CRF(2)(2,4) in agonists and antagonists of all members of the CRF family.(3) On the other hand, the substitutions that conferred ca. 100-fold CRF(2) selectivity to the antagonist antisauvagine-30 [[DPhe(11),His(12)]sauvagine((11-40))] did not confer such property to the corresponding N-terminally extended agonists. We find here that a Glu(32)-Lys(35) side chain to side chain covalent lactam constraint in hCRF and the corresponding Glu(31)-Lys(34) side chain to side chain covalent lactam constraint in sauvagine yield potent ligands that are selective for CRF(2). Additionally, we introduced deletions and substitutions known to increase duration of action to yield antagonists such as cyclo(31-34)[DPhe(11),His(12),C(alpha)MeLeu(13,39),Nle(17),Glu(31),Lys(34)]Ac-sauvagine((8-40)) (astressin(2)-B) with CRF(2) selectivities greater than 100-fold. CRF receptor autoradiography was performed in rat tissue known to express CRF(2) and CRF(1) in order to confirm that astressin(2)-B could indeed bind to established CRF(2) but not CRF(1) receptor-expressing tissues. Extended duration of action of astressin(2)-B vs that of antisauvagine-30 is demonstrated in

  1. Coagulation factor VII is regulated by androgen receptor in breast cancer.

    Science.gov (United States)

    Naderi, Ali

    2015-02-01

    Androgen receptor (AR) is widely expressed in breast cancer; however, there is limited information on the key molecular functions and gene targets of AR in this disease. In this study, gene expression data from a cohort of 52 breast cancer cell lines was analyzed to identify a network of AR co-expressed genes. A total of 300 genes, which were significantly enriched for cell cycle and metabolic functions, showed absolute correlation coefficients (|CC|) of more than 0.5 with AR expression across the dataset. In this network, a subset of 35 "AR-signature" genes were highly co-expressed with AR (|CC|>0.6) that included transcriptional regulators PATZ1, NFATC4, and SPDEF. Furthermore, gene encoding coagulation factor VII (F7) demonstrated the closest expression pattern with AR (CC=0.716) in the dataset and factor VII protein expression was significantly associated to that of AR in a cohort of 209 breast tumors. Moreover, functional studies demonstrated that AR activation results in the induction of factor VII expression at both transcript and protein levels and AR directly binds to a proximal region of F7 promoter in breast cancer cells. Importantly, AR activation in breast cancer cells induced endogenous factor VII activity to convert factor X to Xa in conjunction with tissue factor. In summary, F7 is a novel AR target gene and AR activation regulates the ectopic expression and activity of factor VII in breast cancer cells. These findings have functional implications in the pathobiology of thromboembolic events and regulation of factor VII/tissue factor signaling in breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. MiR-125a TNF receptor-associated factor 6 to inhibit osteoclastogenesis

    International Nuclear Information System (INIS)

    Guo, Li-Juan; Liao, Lan; Yang, Li; Li, Yu; Jiang, Tie-Jian

    2014-01-01

    MicroRNAs (miRNAs) play important roles in osteoclastogenesis and bone resorption. In the present study, we found that miR-125a was dramatically down-regulated during macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) induced osteoclastogenesis of circulating CD14+ peripheral blood mononuclear cells (PBMCs). Overexpression of miR-125a in CD14+ PBMCs inhibited osteoclastogenesis, while inhibition of miR-125a promoted osteoclastogenesis. TNF receptor-associated factor 6 (TRAF6), a transduction factor for RANKL/RANK/NFATc1 signal, was confirmed to be a target of miR-125a. EMSA and ChIP assays confirmed that NFATc1 bound to the promoter of the miR-125a. Overexpression of NFATc1 inhibited miR-125a transcription, and block of NFATc1 expression attenuated RANKL-regulated miR-125a transcription. Here, we reported that miR-125a played a biological function in osteoclastogenesis through a novel TRAF6/ NFATc1/miR-125a regulatory feedback loop. It suggests that regulation of miR-125a expression may be a potential strategy for ameliorating metabolic disease. - Highlights: • MiR-125a was significantly down-regulated in osteoclastogenesis of CD14+ PBMCs. • MiR-125a inhibited osteoclast differentiation by targeting TRAF6. • NFATc1 inhibited miR-125a transciption by binding to the promoter of miR-125a. • TRAF6/NFATc1 and miR-125a form a regulatory feedback loop in osteoclastogenesis

  3. The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis.

    Science.gov (United States)

    Barbieri, M Alejandro; Kong, Chen; Chen, Pin-I; Horazdovsky, Bruce F; Stahl, Philip D

    2003-08-22

    Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.

  4. Nature and regulation of the receptors for insulin-like growth factors

    International Nuclear Information System (INIS)

    Rechler, M.M.; Nissley, S.P.

    1985-01-01

    Two subtypes of IGF receptors have been identified. Type I IGF receptors have a Mr greater than 300,000 and are composed of disulfide-linked 130,000-dalton (alpha) and approximately 90,000-dalton (beta) subunits. Type I receptors preferentially bind IGF-I but also bind IGF-II and, more weakly, insulin. Type II IGF receptors consist of a 250,000-dalton protein that contains internal disulfide bonds but is not linked to other membrane components. Type II receptors bind IGF-II with higher affinity than IGF-I. They do not interact with even very high concentrations of insulin. Type I IGF receptors and insulin receptors are homologous structures. Type II IGF receptors do not appear to be homologous to type I receptors. Type II receptors do not appear to be downregulated. Insulin acutely upregulates type II IGF receptors in intact rat adipose cells by effecting a redistribution of receptors cycling between a large intracellular pool and the plasma membrane. Insulin and the IGFs elicit the same biological responses, either by cross-reacting with one of the receptors for the heterologous ligand or by concurrent activation of convergent effector pathways by binding to the homologous receptor. Which mechanism is utilized appears to depend more on the tissue than on the biological response. Insulin desensitizes rat hepatoma cells to the actions of insulin and IGFs, mediated by both insulin and IGF receptors, by mechanisms distal to hormone binding and possibly common to IGF and insulin effector pathways

  5. Direct demonstration of rapid insulin-like growth factor II receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process

    International Nuclear Information System (INIS)

    Oka, Y.; Rozek, L.M.; Czech, M.P.

    1985-01-01

    The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl- 125 I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl- 125 I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time; and 3) increases the rate of cellular 125 I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody

  6. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

    OpenAIRE

    Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

    1993-01-01

    Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth fac...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    SUMMARY OBJECTIVES: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain-of-function variations in the V2R gene (AVPR2) would lead to higher plasma levels of V...

  8. Expression and Regulation of Corticotropin-Releasing Factor Receptor Type 2 beta in Developing and Mature Mouse Skeletal Muscle

    NARCIS (Netherlands)

    Kuperman, Yael; Issler, Orna; Vaughan, Joan; Bilezikjian, Louise; Vale, Wylie; Chen, Alon

    Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM) tissue where it is suggested to inhibit interactions between insulin signaling pathway components affecting whole-body glucose homeostasis. However, little is known about factors regulating SM CRFR2

  9. Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc.

    Science.gov (United States)

    Le Maitre, Christine L; Richardson, Stephen M A; Baird, Pauline; Freemont, Anthony J; Hoyland, Judith A

    2005-12-01

    Low back pain (LBP) is a common, debilitating and economically important disorder. Current evidence implicates loss of intervertebral disc (IVD) matrix consequent upon 'degeneration' as a major cause of LBP. Degeneration of the IVD involves increases in degradative enzymes and decreases in the extracellular matrix (ECM) component in a process that is controlled by a range of cytokines and growth factors. Studies have suggested using anabolic growth factors to regenerate the normal matrix of the IVD, hence restoring disc height and reversing degenerative disc disease. However, for such therapies to be successful it is vital that the target cells (i.e. the disc cells) express the appropriate receptors. This immunohistochemical study has for the first time investigated the expression and localization of four potentially beneficial growth factor receptors (i.e. TGFbetaRII, BMPRII, FGFR3 and IGFRI) in non-degenerate and degenerate human IVDs. Receptor expression was quantified across regions of the normal and degenerate disc and showed that cells of the nucleus pulposus (NP) and inner annulus fibrosus (IAF) expressed significantly higher levels of the four growth factor receptors investigated. There were no significant differences between the four growth factor expression in non-degenerate and degenerate biopsies. However, expression of TGFbetaRII, FGFR3 and IGFRI, but not BMP RII, were observed in the ingrowing blood vessels that characterize part of the disease aetiology. In conclusion, this study has demonstrated the expression of the four growth factor receptors at similar levels in the chondrocyte-like cells of the NP and IAF in both non-degenerate and degenerate discs, implicating a role in normal disc homeostasis and suggesting that the application of these growth factors to the degenerate human IVD would stimulate matrix production. However, the expression of some of the growth factor receptors on ingrowing blood vessels might be problematic in a therapeutic

  10. NF-κB Protects NKT Cells from Tumor Necrosis Factor Receptor 1-induced Death.

    Science.gov (United States)

    Kumar, Amrendra; Gordy, Laura E; Bezbradica, Jelena S; Stanic, Aleksandar K; Hill, Timothy M; Boothby, Mark R; Van Kaer, Luc; Joyce, Sebastian

    2017-11-15

    Semi-invariant natural killer T (NKT) cells are innate-like lymphocytes with immunoregulatory properties. NKT cell survival during development requires signal processing by activated RelA/NF-κB. Nonetheless, the upstream signal(s) integrated by NF-κB in developing NKT cells remains incompletely defined. We show that the introgression of Bcl-x L -coding Bcl2l1 transgene into NF-κB signalling-deficient IκBΔN transgenic mouse rescues NKT cell development and differentiation in this mouse model. We reasoned that NF-κB activation was protecting developing NKT cells from death signals emanating either from high affinity agonist recognition by the T cell receptor (TCR) or from a death receptor, such as tumor necrosis factor receptor 1 (TNFR1) or Fas. Surprisingly, the single and combined deficiency in PKC-θ or CARMA-1-the two signal transducers at the NKT TCR proximal signalling node-only partially recapitulated the NKT cell deficiency observed in IκBΔN tg mouse. Accordingly, introgression of the Bcl2l1 transgene into PKC-θ null mouse failed to rescue NKT cell development. Instead, TNFR1-deficiency, but not the Fas-deficiency, rescued NKT cell development in IκBΔN tg mice. Consistent with this finding, treatment of thymocytes with an antagonist of the inhibitor of κB kinase -which blocks downstream NF-κB activation- sensitized NKT cells to TNF-α-induced cell death in vitro. Hence, we conclude that signal integration by NF-κB protects developing NKT cells from death signals emanating from TNFR1, but not from the NKT TCR or Fas.

  11. Effect of corticotropin-releasing factor receptor antagonist on psychologically suppressed masculine sexual behavior in rats.

    Science.gov (United States)

    Miwa, Yoshiji; Nagase, Keiko; Oyama, Nobuyuki; Akino, Hironobu; Yokoyama, Osamu

    2011-03-01

    Corticotropin-releasing factor (CRF) coordinates various responses of the body to stress, and CRF receptors are important targets of treatment for stress-related disorders. To investigate the effect of a nonselective CRF receptor antagonist, astressin, on suppression of masculine sexual behavior by psychological stress in rats. First, we investigated the influence of psychological stress, induced 2 hours per day for three consecutive days, on sexual behavior. Then, rats were divided into 4 groups: a control group, an astressin administration group (A), a psychological stress loading group (PS), and a psychological stress loading and astressin administration group (PS + A). The rats were exposed to sham or psychological stress for three consecutive days. After the last stress loading, the rats were injected with vehicle or astressin, and their sexual behavior was observed. We also measured serum levels of adrenocorticotropic hormone (ACTH). The effects of astressin on sexual behavior and serum levels of ACTH in rats affected by psychological stress were determined. Sexual behavior was reduced after psychological stress loading. The PS rats had significantly longer mount, intromission, and ejaculation latencies and lower ejaculation frequency than did the control, A, and PS + A rats. The intromission latency and ejaculation frequency in the PS + A rats did not achieve the level observed in the controls. There was no significant difference in these parameters between the control and A rats. Serum ACTH levels were significantly lower in PS + A rats than in PS rats. Psychologically suppressed masculine sexual behavior could be partially recovered with astressin administration in rats. These data provide a rationale for the further study of CRF receptor antagonists as novel agents for treating psychological sexual disorders. © 2010 International Society for Sexual Medicine.

  12. Characterization of germ cell-specific expression of the orphan nuclear receptor, germ cell nuclear factor.

    Science.gov (United States)

    Katz, D; Niederberger, C; Slaughter, G R; Cooney, A J

    1997-10-01

    Nuclear receptors, such as those for androgens, estrogens, and progesterones, control many reproductive processes. Proteins with structures similar to these receptors, but for which ligands have not yet been identified, have been termed orphan nuclear receptors. One of these orphans, germ cell nuclear factor (GCNF), has been shown to be germ cell specific in the adult and, therefore, may also participate in the regulation of reproductive functions. In this paper, we examine more closely the expression patterns of GCNF in germ cells to begin to define spatio-temporal domains of its activity. In situ hybridization showed that GCNF messenger RNA (mRNA) is lacking in the testis of hypogonadal mutant mice, which lack developed spermatids, but is present in the wild-type testis. Thus, GCNF is, indeed, germ cell specific in the adult male. Quantitation of the specific in situ hybridization signal in wild-type testis reveals that GCNF mRNA is most abundant in stage VII round spermatids. Similarly, Northern analysis and specific in situ hybridization show that GCNF expression first occurs in testis of 20-day-old mice, when round spermatids first emerge. Therefore, in the male, GCNF expression occurs postmeiotically and may participate in the morphological changes of the maturing spermatids. In contrast, female expression of GCNF is shown in growing oocytes that have not completed the first meiotic division. Thus, GCNF in the female is expressed before the completion of meiosis. Finally, the nature of the two different mRNAs that hybridize to the GCNF complementary DNA was studied. Although both messages contain the DNA binding domain, only the larger message is recognized by a probe from the extreme 3' untranslated region. In situ hybridization with these differential probes demonstrates that both messages are present in growing oocytes. In addition, the coding region and portions of the 3' untranslated region of the GCNF complementary DNA are conserved in the rat.

  13. Oncogenic role of fibroblast growth factor receptor 3 in tumorigenesis of urinary bladder cancer.

    Science.gov (United States)

    Pandith, Arshad A; Shah, Zafar A; Siddiqi, Mushtaq A

    2013-05-01

    Bladder cancer is the second most common genitourinary tumor and constitutes a very heterogeneous disease. Molecular and pathologic studies suggest that low-grade noninvasive and high-grade invasive urothelial cell carcinoma (UCC) arise via distinct pathways. Low-grade noninvasive UCC represent the majority of tumors at presentation. A high proportion of patients with low-grade UCC develop recurrences but usually with no progression to invasive disease. At presentation, a majority of the bladder tumors (70%-80%) are low-grade noninvasive (pTa). Several genetic changes may occur in bladder cancer, but activating mutations in the fibroblast growth factor receptor 3 (FGFR3) genes are the most common and most specific genetic abnormality in bladder cancer. Interestingly, these mutations are associated with bladder tumors of low stage and grade, which makes the FGFR3 mutation the first marker that can be used for diagnosis of noninvasive bladder tumors. Since the first report of FGFR3 involvement in bladder tumors, numerous studies have been conducted to understand its function and thereby confirm the oncogenic role of this receptor particularly in noninvasive groups. Efforts are on to exploit this receptor as a therapeutic target, which holds much promise in the treatment of bladder cancer, particularly low-grade noninvasive tumors. Further studies need to explore the potential use of FGFR3 mutations in bladder cancer diagnosis, prognosis, and in surveillance of patients with bladder cancer. This review focuses on the role of FGFR3 in bladder tumors in the backdrop of various studies published. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. The epidermal growth factor receptor as a target for gastrointestinal cancer therapy.

    Science.gov (United States)

    Tedesco, Karen L; Lockhart, A Craig; Berlin, Jordan D

    2004-10-01

    The epidermal growth factor receptor (EGFR) is a member of the family of transmembrane protein kinase receptors known as the erbB or HER receptor family. When activated, EGFR phosphorylates and activates other intracellular proteins that affect cell signaling pathways, cellular proliferation, control of apoptosis and angiogenesis. EGFR signaling is best thought of as a network of activating and inactivating proteins with EGFR as the entry point into the network. EGFR overexpression occurs in most GI malignancies and while data are not entirely consistent, EGFR overexpression often confers a poor prognosis in those GI malignancies that have been studied. It often correlates with poorly differentiated histology, more advanced stage and other known poor prognostic markers. The EGFR is a tempting target because of its presence and overexpression on so many tumor types. However, downstream of the EGFR are several proteins that may be activated without EGFR thus allowing blockade to be overcome. Therefore, while blocking the activity of the EGFR protein appears to be a promising anticancer strategy, a simplistic strategy of blocking only EGFR is likely to only impact a minority of patients. It is time for the laboratory and clinical researchers to work closely together to develop this treatment strategy, moving back and forth from clinical to laboratory to best understand how to block this network effectively enough to produce a broader antitumor effect. While multiple methods of targeting the EGFR pathway are under development, including the inhibition of downstream proteins, only two modalities have entered clinical trials in GI malignancies: small molecule inhibitors of the intracellular kinase domain of EGFR and antibodies designed to block the extracellular ligand-binding domain of EGFR. EGFR inhibitors are still experimental in every GI malignancy with the notable exception of cetuximab that is approved for second or third-line therapy of metastatic colorectal

  15. Targeting the epidermal growth factor receptor in radiotherapy: radiobiological mechanisms, preclinical and clinical results

    International Nuclear Information System (INIS)

    Baumann, Michael; Krause, Mechthild

    2004-01-01

    Background and purpose: Inhibition of the epidermal growth factor receptor (EGFR) is a fastly developing field in preclinical and clinical cancer research. This review presents the current status of knowledge and discusses radiobiological mechanisms which may underly the efficacy of EGFR inhibitors combined with irradiation. Materials and methods: Preclinical and clinical results on combined targeting of the EGFR and irradiation from the literature and from this laboratory are reviewed. Focus is given to the radiobiological rationale of this approach and to endpoints of experimental radiotherapy. Results: Overexpression of the EGFR is associated with decreased local tumour control after radiotherapy, especially when the overall treatment time is long. Inhibition of the EGFR either alone or in combination with irradiation decreases the growth rate of tumours expressing this receptor. Preclinical data provide proof-of-principle that local tumour control may be improved by combining irradiation with C225 mAb. In a randomised phase III clinical trial, simultaneous irradiation and treatment with the EGFR antibody Cetuximab (Erbitux[reg]; C225) in head and neck cancer patients resulted in significantly improved locoregional tumour control and survival compared to curative irradiation alone. Acute skin reactions increased in the experimental arm. The underlying mechanisms of enhanced radiation effects of combined EGFR inhibition with irradiation and of the partly conflicting results in different studies are poorly understood. There is increasing evidence, that important intertumoral heterogeneity in the response to EGFR inhibition alone and combined with irradiation exists, which appears to be at least partly dependent on specific mutations of the receptor as well as of molecules that are involved in the intracellular signal transduction pathway. Conclusions and outlook: Further investigations at all levels of the translational research chain exploring the mechanisms of

  16. Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

    Science.gov (United States)

    Taniguchi, Hirokazu; Takeuchi, Shinji; Fukuda, Koji; Nakagawa, Takayuki; Arai, Sachiko; Nanjo, Shigeki; Yamada, Tadaaki; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

    2017-01-01

    Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  17. Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

    Science.gov (United States)

    Tiong, Kai Hung; Tan, Boon Shing; Choo, Heng Lungh; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Tan, Si Hoey; Khor, Nelson Tze Woei; Wong, Shew Fung; See, Sze-Jia; Tan, Yuen-Fen; Rosli, Rozita; Cheong, Soon-Keng; Leong, Chee-Onn

    2016-09-06

    Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

  18. Association of Polymorphisms in Connective Tissue Growth Factor and Epidermal Growth Factor Receptor Genes With Human Longevity.

    Science.gov (United States)

    Donlon, Timothy A; Morris, Brian J; He, Qimei; Chen, Randi; Masaki, Kamal H; Allsopp, Richard C; Willcox, D Craig; Tranah, Gregory J; Parimi, Neeta; Evans, Daniel S; Flachsbart, Friederike; Nebel, Almut; Kim, Duk-Hwan; Park, Joobae; Willcox, Bradley J

    2017-08-01

    Growth pathways play key roles in longevity. The present study tested single-nucleotide polymorphisms (SNPs) in the connective tissue growth factor gene (CTGF) and the epidermal growth factor receptor gene (EGFR) for association with longevity. Comparison of allele and genotype frequencies of 12 CTGF SNPs and 41 EGFR SNPs between 440 American men of Japanese ancestry aged ≥95 years and 374 men of average life span revealed association with longevity at the p cases, consistent with heterozygote advantage in living to extreme old age. No associations of the most significant SNPs were observed in whites or Koreans. In conclusion, the present findings indicate that genetic variation in CTGF and EGFR may contribute to the attainment of extreme old age in Japanese. More research is needed to confirm that genetic variation in CTGF and EGFR contributes to the attainment of extreme old age across human populations. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Tebar, A.; Barde, Y.A.

    1988-09-01

    Brain-derived neurotrophic factor (BDNF), a protein known to support the survival of embryonic sensory neurons and retinal ganglion cells, was derivatized with 125I-Bolton-Hunter reagent and obtained in a biologically active, radioactive form (125I-BDNF). Using dorsal root ganglion neurons from chick embryos at 9 d of development, the basic physicochemical parameters of the binding of 125I-BDNF with its receptors were established. Two different classes of receptors were found, with dissociation constants of 1.7 x 10(-11) M (high-affinity receptors) and 1.3 x 10(-9) M (low-affinity receptors). Unlabeled BDNF competed with 125I-BDNF for binding to the high-affinity receptors with an inhibition constant essentially identical to the dissociation constant of the labeled protein: 1.2 x 10(-11) M. The association and dissociation rates from both types of receptors were also determined, and the dissociation constants calculated from these kinetic experiments were found to correspond to the results obtained from steady-state binding. The number of high-affinity receptors (a few hundred per cell soma) was 15 times lower than that of low-affinity receptors. No high-affinity receptors were found on sympathetic neurons, known not to respond to BDNF, although specific binding of 125I-BDNF to these cells was detected at a high concentration of the radioligand. These results are discussed and compared with those obtained with nerve growth factor on the same neuronal populations.

  20. Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo

    International Nuclear Information System (INIS)

    Rodriguez-Tebar, A.; Barde, Y.A.

    1988-01-01

    Brain-derived neurotrophic factor (BDNF), a protein known to support the survival of embryonic sensory neurons and retinal ganglion cells, was derivatized with 125I-Bolton-Hunter reagent and obtained in a biologically active, radioactive form (125I-BDNF). Using dorsal root ganglion neurons from chick embryos at 9 d of development, the basic physicochemical parameters of the binding of 125I-BDNF with its receptors were established. Two different classes of receptors were found, with dissociation constants of 1.7 x 10(-11) M (high-affinity receptors) and 1.3 x 10(-9) M (low-affinity receptors). Unlabeled BDNF competed with 125I-BDNF for binding to the high-affinity receptors with an inhibition constant essentially identical to the dissociation constant of the labeled protein: 1.2 x 10(-11) M. The association and dissociation rates from both types of receptors were also determined, and the dissociation constants calculated from these kinetic experiments were found to correspond to the results obtained from steady-state binding. The number of high-affinity receptors (a few hundred per cell soma) was 15 times lower than that of low-affinity receptors. No high-affinity receptors were found on sympathetic neurons, known not to respond to BDNF, although specific binding of 125I-BDNF to these cells was detected at a high concentration of the radioligand. These results are discussed and compared with those obtained with nerve growth factor on the same neuronal populations

  1. Increased expression of protease-activated receptor 4 and Trefoil factor 2 in human colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Guoyu Yu

    Full Text Available Protease-activated receptor 4 (PAR4, a member of G-protein coupled receptors family, was recently reported to exhibit decreased expression in gastric cancer and esophageal squamous cancer, yet increased expression during the progression of prostate cancer. Trefoil factor 2 (TFF2, a small peptide constitutively expressed in the gastric mucosa, plays a protective role in restitution of gastric mucosa. Altered TFF2 expression was also related to the development of gastrointestinal cancer. TFF2 has been verified to promote cell migration via PAR4, but the roles of PAR4 and TFF2 in the progress of colorectal cancer are still unknown. In this study, the expression level of PAR4 and TFF2 in colorectal cancer tissues was measured using real-time PCR (n = 38, western blotting (n=38 and tissue microarrays (n = 66. The mRNA and protein expression levels of PAR4 and TFF2 were remarkably increased in colorectal cancer compared with matched noncancerous tissues, especially in positive lymph node and poorly differentiated cancers. The colorectal carcinoma cell LoVo showed an increased response to TFF2 as assessed by cell invasion upon PAR4 expression. However, after intervention of PAR4 expression, PAR4 positive colorectal carcinoma cell HT-29 was less responsive to TFF2 in cell invasion. Genomic bisulfite sequencing showed the hypomethylation of PAR4 promoter in colorectal cancer tissues and the hypermethylation in the normal mucosa that suggested the low methylation of promoter was correlated to the increased PAR4 expression. Taken together, the results demonstrated that the up-regulated expression of PAR4 and TFF2 frequently occurs in colorectal cancer tissues, and that overexpression of PAR4 may be resulted from promoter hypomethylation. While TFF2 promotes invasion activity of LoVo cells overexpressing PAR4, and this effect was significantly decreased when PAR4 was knockdowned in HT-29 cells. Our findings will be helpful in further investigations into the

  2. Emerging role of epidermal growth factor receptor inhibition in therapy for advanced malignancy: focus on NSCLC

    International Nuclear Information System (INIS)

    Langer, Corey J.

    2004-01-01

    Combination chemotherapy regimens have emerged as the standard approach in advanced non-small-cell lung cancer. Meta-analyses have demonstrated a 2-month increase in median survival after platinum-based therapy vs. best supportive care, and an absolute 10% improvement in the 1-year survival rate. Just as importantly, cytotoxic therapy has produced benefits in symptom control and quality of life. Newer agents, including the taxanes, vinorelbine, gemcitabine, and irinotecan, have expanded our therapeutic options in the treatment of advanced non-small-cell lung cancer. Despite their contributions, we have reached a therapeutic plateau, with response rates seldom exceeding 30-40% in cooperative group studies and 1-year survival rates stable between 30% and 40%. It is doubtful that substituting one agent for another in various combinations will lead to any further improvement in these rates. The thrust of current research has focused on targeted therapy, and epidermal growth factor receptor inhibition is one of the most promising clinical strategies. Epidermal growth factor receptor inhibitors currently under investigation include the small molecules gefitinib (Iressa, ZD1839) and erlotinib (Tarceva, OSI-774), as well as monoclonal antibodies such as cetuximab (IMC-225, Erbitux). Agents that have only begun to undergo clinical evaluation include CI-1033, an irreversible pan-erbB tyrosine kinase inhibitor, and PKI166 and GW572016, both examples of dual kinase inhibitors (inhibiting epidermal growth factor receptor and Her2). Preclinical models have demonstrated synergy for all these agents in combination with either chemotherapy or radiotherapy, leading to great enthusiasm regarding their ultimate contribution to lung cancer therapy. However, serious clinical challenges persist. These include the identification of the optimal dose(s); the proper integration of these agents into popular, established cytotoxic regimens; and the selection of the optimal setting(s) in which

  3. Pluripotency factors and Polycomb Group proteins repress aryl hydrocarbon receptor expression in murine embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Chia-I Ko

    2014-01-01

    Full Text Available The aryl hydrocarbon receptor (AHR is a transcription factor and environmental sensor that regulates expression of genes involved in drug-metabolism and cell cycle regulation. Chromatin immunoprecipitation analyses, Ahr ablation in mice and studies with orthologous genes in invertebrates suggest that AHR may also play a significant role in embryonic development. To address this hypothesis, we studied the regulation of Ahr expression in mouse embryonic stem cells and their differentiated progeny. In ES cells, interactions between OCT3/4, NANOG, SOX2 and Polycomb Group proteins at the Ahr promoter repress AHR expression, which can also be repressed by ectopic expression of reprogramming factors in hepatoma cells. In ES cells, unproductive RNA polymerase II binds at the Ahr transcription start site and drives the synthesis of short abortive transcripts. Activation of Ahr expression during differentiation follows from reversal of repressive marks in Ahr promoter chromatin, release of pluripotency factors and PcG proteins, binding of Sp factors, establishment of histone marks of open chromatin, and engagement of active RNAPII to drive full-length RNA transcript elongation. Our results suggest that reversible Ahr repression in ES cells holds the gene poised for expression and allows for a quick switch to activation during embryonic development.

  4. Insulin and insulin-like growth factor receptors in the brain: physiological and pathological aspects.

    Science.gov (United States)

    Werner, Haim; LeRoith, Derek

    2014-12-01

    The involvement of insulin, the insulin-like growth factors (IGF1, IGF2) and their receptors in central nervous system development and function has been the focus of scientific interest for more than 30 years. The insulin-like peptides, both locally-produced proteins as well as those transported from the circulation into the brain via the blood-brain barrier, are involved in a myriad of biological activities. These actions include, among others, neuronal survival, neurogenes, angiogenesis, excitatory and inhibitory neurotransmission, regulation of food intake, and cognition. In recent years, a linkage between brain insulin/IGF1 and certain neuropathologies has been identified. Epidemiological studies have demonstrated a correlation between diabetes (mainly type 2) and Alzheimer׳s disease. In addition, an aberrant decline in IGF1 values was suggested to play a role in the development of Alzheimer׳s disease. The present review focuses on the expression and function of insulin, IGFs and their receptors in the brain in physiological and pathological conditions. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

  5. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Yuxin Feng

    Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

  6. The transcription factor ERG increases expression of neurotransmitter receptors on prostate cancer cells

    International Nuclear Information System (INIS)

    Kissick, Haydn T.; On, Seung T.; Dunn, Laura K.; Sanda, Martin G.; Asara, John M.; Pellegrini, Kathryn L.; Noel, Jonathan K.; Arredouani, Mohamed S.

    2015-01-01

    The TMPRSS2-ERG gene fusion occurs in about half of prostate cancer (PCa) cases and results in overexpression of the transcription factor ERG. Overexpression of ERG has many effects on cellular function. However, how these changes enhance cell growth and promote tumor development is unclear. To investigate the role of ERG, LNCaP and PC3 cells were transfected with ERG and gene expression and metabolic profile were analyzed. Our data show that expression of ERG induces overexpression of many nicotinicacetylcholine receptors (nAChRs). In addition, metabolic profiling by LC-MS/MS revealed elevated production of several neurotransmitters in cells expressing ERG. Consistently, treatment of ERG-expressing cells with nicotine induced elevated calcium influx, GSK3β (Ser9) phosphorylation and cell proliferation. Finally, we show that PCa patientswho are smokers have larger tumors if their tumors are TMPRSS2-ERG gene fusion positive. Collectively, our data suggest that ERG sensitizes prostate tumor cells to neurotransmitter receptor agonists like nicotine. The online version of this article (doi:10.1186/s12885-015-1612-3) contains supplementary material, which is available to authorized users

  7. Characterization of the receptors for mycobacterial cord factor in Guinea pig.

    Directory of Open Access Journals (Sweden)

    Kenji Toyonaga

    Full Text Available Guinea pig is a widely used animal for research and development of tuberculosis vaccines, since its pathological disease process is similar to that present in humans. We have previously reported that two C-type lectin receptors, Mincle (macrophage inducible C-type lectin, also called Clec4e and MCL (macrophage C-type lectin, also called Clec4d, recognize the mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM. Here, we characterized the function of the guinea pig homologue of Mincle (gpMincle and MCL (gpMCL. gpMincle directly bound to TDM and transduced an activating signal through ITAM-bearing adaptor molecule, FcRγ. Whereas, gpMCL lacked C-terminus and failed to bind to TDM. mRNA expression of gpMincle was detected in the spleen, lymph nodes and peritoneal macrophages and it was strongly up-regulated upon stimulation of zymosan and TDM. The surface expression of gpMincle was detected on activated macrophages by a newly established monoclonal antibody that also possesses a blocking activity. This antibody potently suppressed TNF production in BCG-infected macrophages. Collectively, gpMincle is the TDM receptor in the guinea pig and TDM-Mincle axis is involved in host immune responses against mycobacteria.

  8. Characterization of the receptors for mycobacterial cord factor in Guinea pig.

    Science.gov (United States)

    Toyonaga, Kenji; Miyake, Yasunobu; Yamasaki, Sho

    2014-01-01

    Guinea pig is a widely used animal for research and development of tuberculosis vaccines, since its pathological disease process is similar to that present in humans. We have previously reported that two C-type lectin receptors, Mincle (macrophage inducible C-type lectin, also called Clec4e) and MCL (macrophage C-type lectin, also called Clec4d), recognize the mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM). Here, we characterized the function of the guinea pig homologue of Mincle (gpMincle) and MCL (gpMCL). gpMincle directly bound to TDM and transduced an activating signal through ITAM-bearing adaptor molecule, FcRγ. Whereas, gpMCL lacked C-terminus and failed to bind to TDM. mRNA expression of gpMincle was detected in the spleen, lymph nodes and peritoneal macrophages and it was strongly up-regulated upon stimulation of zymosan and TDM. The surface expression of gpMincle was detected on activated macrophages by a newly established monoclonal antibody that also possesses a blocking activity. This antibody potently suppressed TNF production in BCG-infected macrophages. Collectively, gpMincle is the TDM receptor in the guinea pig and TDM-Mincle axis is involved in host immune responses against mycobacteria.

  9. Regulation of granulocyte colony-stimulating factor receptor-mediated granulocytic differentiation by C-mannosylation.

    Science.gov (United States)

    Otani, Kei; Niwa, Yuki; Suzuki, Takehiro; Sato, Natsumi; Sasazawa, Yukiko; Dohmae, Naoshi; Simizu, Siro

    2018-04-06

    Granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is a type I cytokine receptor which is involved in hematopoietic cell maturation. G-CSFR has three putative C-mannosylation sites at W253, W318, and W446; however, it is not elucidated whether G-CSFR is C-mannosylated or not. In this study, we first demonstrated that G-CSFR was C-mannosylated at only W318. We also revealed that C-mannosylation of G-CSFR affects G-CSF-dependent downstream signaling through changing ligand binding capability but not cell surface localization. Moreover, C-mannosylation of G-CSFR was functional and regulated granulocytic differentiation in myeloid 32D cells. In conclusion, we found that G-CSFR is C-mannosylated at W318 and that this C-mannosylation has role(s) for myeloid cell differentiation through regulating downstream signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Targeting Epidermal Growth Factor Receptor-Related Signaling Pathways in Pancreatic Cancer.

    Science.gov (United States)

    Philip, Philip A; Lutz, Manfred P

    2015-10-01

    Pancreatic cancer is aggressive, chemoresistant, and characterized by complex and poorly understood molecular biology. The epidermal growth factor receptor (EGFR) pathway is frequently activated in pancreatic cancer; therefore, it is a rational target for new treatments. However, the EGFR tyrosine kinase inhibitor erlotinib is currently the only targeted therapy to demonstrate a very modest survival benefit when added to gemcitabine in the treatment of patients with advanced pancreatic cancer. There is no molecular biomarker to predict the outcome of erlotinib treatment, although rash may be predictive of improved survival; EGFR expression does not predict the biologic activity of anti-EGFR drugs in pancreatic cancer, and no EGFR mutations are identified as enabling the selection of patients likely to benefit from treatment. Here, we review clinical studies of EGFR-targeted therapies in combination with conventional cytotoxic regimens or multitargeted strategies in advanced pancreatic cancer, as well as research directed at molecules downstream of EGFR as alternatives or adjuncts to receptor targeting. Limitations of preclinical models, patient selection, and trial design, as well as the complex mechanisms underlying resistance to EGFR-targeted agents, are discussed. Future clinical trials must incorporate translational research end points to aid patient selection and circumvent resistance to EGFR inhibitors.

  11. Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

    International Nuclear Information System (INIS)

    Diermeier, Simone; Horvath, Gabor; Knuechel-Clarke, Ruth; Hofstaedter, Ferdinand; Szoellosi, Janos; Brockhoff, Gero

    2005-01-01

    Background: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. Methods: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. Results: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. Conclusion: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and

  12. Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

    LENUS (Irish Health Repository)

    Gleeson, Eimear M

    2013-07-19

    Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

  13. Increased Eps15 homology domain 1 and RAB11FIP3 expression regulate breast cancer progression via promoting epithelial growth factor receptor recycling.

    Science.gov (United States)

    Tong, Dandan; Liang, Ya-Nan; Stepanova, A A; Liu, Yu; Li, Xiaobo; Wang, Letian; Zhang, Fengmin; Vasilyeva, N V

    2017-02-01

    Recent research indicates that the C-terminal Eps15 homology domain 1 is associated with epithelial growth factor receptor-mediated endocytosis recycling in non-small-cell lung cancer. The aim of this study was to determine the clinical significance of Eps15 homology domain 1 gene expression in relation to phosphorylation of epithelial growth factor receptor expression in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Eps15 homology domain 1, RAB11FIP3, and phosphorylation of epithelial growth factor receptor expression via immunohistochemistry. The clinical significance was assessed via a multivariate Cox regression analysis, Kaplan-Meier curves, and the log-rank test. Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor were upregulated in 60.46% (185/306) and 53.92% (165/306) of tumor tissues, respectively, as assessed by immunohistochemistry. The statistical correlation analysis indicated that Eps15 homology domain 1 overexpression was positively correlated with the increases in phosphorylation of epithelial growth factor receptor ( r = 0.242, p breast cancer for the overall survival in the total, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. However, the use of combined expression of Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor markers is more effective for the disease-free survival in the overall population, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. Moreover, the combined markers are also significant prognostic markers of breast cancer in the human epidermal growth factor receptor 2 (+), estrogen receptor (+), and estrogen receptor (-) groups. Eps15 homology domain 1 has a tumor suppressor function, and the combined marker of Eps15 homology domain 1/phosphorylation of epithelial growth factor receptor expression was identified as a better prognostic marker in breast cancer diagnosis

  14. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    Energy Technology Data Exchange (ETDEWEB)

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Marco, Ario de, E-mail: ario.demarco@ung.si [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Dept. Environmental Sciences, University of Nova Gorica (UNG), Vipavska 13, P.O. Box 301-SI-5000, Rozna Dolina, Nova Gorica (Slovenia)

    2011-05-20

    Highlights: {yields} Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. {yields} These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. {yields} The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  15. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    International Nuclear Information System (INIS)

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo; Marco, Ario de

    2011-01-01

    Highlights: → Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. → These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. → The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  16. Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform*

    OpenAIRE

    Cha, Jiyoung Y.; Maddileti, Savitri; Mitin, Natalia; Harden, T. Kendall; Der, Channing J.

    2009-01-01

    Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine r...

  17. 99m Tc-anti-epidermal growth factor receptor nanobody for tumor imaging.

    Science.gov (United States)

    Piramoon, Majid; Hosseinimehr, Seyed Jalal; Omidfar, Kobra; Noaparast, Zohreh; Abedi, Seyed Mohammad

    2017-04-01

    Nanobodies are important biomolecules for tumor targeting. In this study, we synthesized and labeled anti-epidermal growth factor receptor (EGFR) nanobody OA-cb6 with 99m Tc(CO) 3 + and evaluated its characteristics for targeting the EGFR in the A431 human epidermal carcinoma cell line. Nanobody radiolabeling was achieved with high yield and radiochemical purity, and the radioconjugate was stable. Biodistribution results in nude mice exhibited a favorable tumor-to-muscle ratio at 4-hr postinjection, and tumor location was visualized at 4 hr after injection of radiolabeled nanobody. Our result showed that the OA-cb6- 99m Tc-tricarbonyl radiolabeled nanobody is a promising radiolabeled biomolecule for tumor imaging in cancers with high EGFR overexpression. © 2016 John Wiley & Sons A/S.

  18. No evidence for a role of the serotonin 4 receptor in five-factor personality traits

    DEFF Research Database (Denmark)

    Stenbæk, Dea Siggaard; Dam, Vibeke Høyrup; Fisher, Patrick MacDonald

    2017-01-01

    Serotonin (5-HT) brain architecture appears to be implicated in normal personality traits as supported by genetic associations and studies using molecular brain imaging. However, so far, no studies have addressed potential contributions to variation in normal personality traits from in vivo...... serotonin 4 receptor (5-HT4R) brain availability, which has recently become possible to image with Positron Emission Tomography (PET). This is particularly relevant since availability of 5-HT4R has been shown to adapt to synaptic levels of 5-HT and thus offers information about serotonergic tone...... in the healthy brain. In 69 healthy participants (18 females), the associations between personality traits assessed with the five-factor NEO Personality Inventory-Revised (NEO PI-R) and regional cerebral 5-HT4R binding in neocortex, amygdala, hippocampus, and anterior cingulate cortex (ACC) were investigated...

  19. Urokinase plasminogen activator receptor on invasive cancer cells: A prognostic factor in distal gastric adenocarcinoma

    DEFF Research Database (Denmark)

    Alpizar, Warner Enrique Alpizar; Christensen, Ib Jarle; Santoni-Rugiu, Eric

    2012-01-01

    Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation...... by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. u...... association between the expression of uPAR on tumor cells in the peripheral invasion zone and overall survival of gastric cancer patients (HR = 2.16; 95% CI: 1.13-4.14; p = 0.02). Multivariate analysis showed that uPAR immunoreactivity in cancer cells at the invasive front is an independent prognostic factor...

  20. Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans

    Directory of Open Access Journals (Sweden)

    Jinah Han

    2015-02-01

    Full Text Available Neural stem cells (NSCs continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs, VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

  1. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins.

    Science.gov (United States)

    Mizumoto, Shuji; Yamada, Shuhei; Sugahara, Kazuyuki

    2015-10-01

    Recent functional studies on chondroitin sulfate-dermatan sulfate (CS-DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS-DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS-DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS-DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS-DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. Copyright © 2015. Published by Elsevier Ltd.

  2. Upregulation of vascular endothelial growth factor receptor-1 contributes to sevoflurane preconditioning–mediated cardioprotection

    Directory of Open Access Journals (Sweden)

    Qian B

    2018-04-01

    Full Text Available Bin Qian,1 Yang Yang,2 Yusheng Yao,3 Yanling Liao,3 Ying Lin3 1Department of Anesthesiology, People’s Hospital Affiliated to Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China; 2Department of Anesthesiology, West China Hospital, Sichuan University, Chengdu, Sichuan, China; 3Department of Anesthesiology, The Shengli Clinical Medical College, Fujian Medical University, Fuzhou, Fujian, China Purpose: Sevoflurane preconditioning (SPC can provide myocardial protective effects similar to ischemic preconditioning. However, the exact mechanism of SPC remains unclear. Previous studies indicate that vascular endothelial growth factor receptor 1 (VEGFR-1 is involved in ischemic preconditioning-mediated cardioprotection. This study was designed to determine the significance of VEGFR-1 signaling in SPC-mediated cardioprotection.Materials and methods: Myocardial ischemia–reperfusion (I/R rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, after 15 min of baseline equilibration, the isolated hearts were pretreated with 2.5% sevoflurane, 2.5% sevoflurane+MF1 10 µmol/L, or 2.5% sevoflurane+placental growth factor 10 µmol/L, and then subjected to 30 min of global ischemia and 120 min of reperfusion. The changes in hemodynamic parameters, myocardial infarct size, and the levels of creatine kinase-MB, lactate dehydrogenase, cardiac troponin-I, tumor necrosis factor-α, and interleukin 6 in the myocardium were evaluated.Results: Compared to the I/R group, pretreatment with 2.5% sevoflurane significantly improved the cardiac function, limited myocardial infarct size, reduced cardiac enzyme release, upregulated VEGFR-1 expression, and decreased inflammation. In addition, the selective VEGFR-1 agonist, placental growth factor, did not enhance the cardioprotection and anti-inflammation effects of sevoflurane, while the specific VEGFR-1 inhibitor, MF1, completely reversed these effects

  3. Facile labelling of an anti-epidermal growth factor receptor Nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET.

    Science.gov (United States)

    Vosjan, Maria J W D; Perk, Lars R; Roovers, Rob C; Visser, Gerard W M; Stigter-van Walsum, Marijke; van Bergen En Henegouwen, Paul M P; van Dongen, Guus A M S

    2011-04-01

    The ∼15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies®) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with (68)Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for (89)Zr immuno-positron emission tomography (PET). Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified (68)Ga was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml(-1) gentisic acid, pH 5.5) at 4°C or in human serum at 37°C, a mixture of (67)Ga and (68)Ga was used. Biodistribution and immuno-PET studies of (68)Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using (89)Zr-Df-Bz-NCS-7D12 as the reference conjugate. The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall (68)Ga radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. (68/67)Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 ± 1.3 to 7.2 ± 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate (89)Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study. Via a rapid

  4. Facile labelling of an anti-epidermal growth factor receptor nanobody with {sup 68}Ga via a novel bifunctional desferal chelate for immuno-PET

    Energy Technology Data Exchange (ETDEWEB)

    Vosjan, Maria J.W.D.; Perk, Lars R.; Stigter van Walsum, Marijke [VU University Medical Center, Department of Otolaryngology/Head and Neck Surgery, De Boelelaan 1117, P.O. Box 7057, Amsterdam (Netherlands); Roovers, Rob C.; Bergen en Henegouwen, Paul M.P. van [Utrecht University, Cellular Dynamics, Science Faculty, Utrecht (Netherlands); Visser, Gerard W.M. [VU University Medical Center, Department of Nuclear Medicine and PET Research, Amsterdam (Netherlands); Dongen, Guus A.M.S. van [VU University Medical Center, Department of Otolaryngology/Head and Neck Surgery, De Boelelaan 1117, P.O. Box 7057, Amsterdam (Netherlands); VU University Medical Center, Department of Nuclear Medicine and PET Research, Amsterdam (Netherlands)

    2011-04-15

    The {proportional_to}15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies {sup registered}) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with {sup 68}Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for {sup 89}Zr immuno-positron emission tomography (PET). Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified {sup 68}Ga was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml{sup -1} gentisic acid, pH 5.5) at 4 C or in human serum at 37 C, a mixture of {sup 67}Ga and {sup 68}Ga was used. Biodistribution and immuno-PET studies of {sup 68}Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using {sup 89}Zr-Df-Bz-NCS-7D12 as the reference conjugate. The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall {sup 68}Ga radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. {sup 68/67}Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (<2% radioactivity loss). In biodistribution studies the {sup 68}Ga-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 {+-} 1.3 to 7.2 {+-} 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was

  5. Facile labelling of an anti-epidermal growth factor receptor nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET

    International Nuclear Information System (INIS)

    Vosjan, Maria J.W.D.; Perk, Lars R.; Stigter van Walsum, Marijke; Roovers, Rob C.; Bergen en Henegouwen, Paul M.P. van; Visser, Gerard W.M.; Dongen, Guus A.M.S. van

    2011-01-01

    The ∝15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies registered ) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with 68 Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for 89 Zr immuno-positron emission tomography (PET). Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified 68 Ga was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml -1 gentisic acid, pH 5.5) at 4 C or in human serum at 37 C, a mixture of 67 Ga and 68 Ga was used. Biodistribution and immuno-PET studies of 68 Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using 89 Zr-Df-Bz-NCS-7D12 as the reference conjugate. The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall 68 Ga radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. 68/67 Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period ( 68 Ga-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 ± 1.3 to 7.2 ± 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate 89 Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study. Via a rapid

  6. Mechanisms of Inhibition of the Epidermal Growth Factor Receptor: Implications for Novel Anti-Cancer Therapies

    National Research Council Canada - National Science Library

    Klein, Daryl E

    2005-01-01

    .... No secreted or extracellular ErbB receptor inhibitors have been reported in mammals. However, two natural inhibitors of the highly homologous Drosophila EGF receptor are found in Drosophila melanogaster...

  7. Epidermal Growth Factor Receptor Activating Mutations in Squamous Histology of Lung Cancer Patients of Southern Bulgaria

    Directory of Open Access Journals (Sweden)

    Genova Silvia N.

    2015-12-01

    Full Text Available There is only limited data on the prevalence of epidermal growth factor receptor (EGFR activating mutations in squamous cell carcinomas and adenosquamous carcinomas of the lung in patients of the Southern Bulgarian region and the efficacy of EGFR tyrosine kinase inhibitors. AIM: Previous reports for Bulgarian population showed high incidence of EGFR mutations in the squamous cell carcinomas, so we set the goal to investigate their frequency in Southern Bulgaria, after precise immunohistochemical verification of lung cancers. MATERIALS AND METHODS: Two hundred and thirty-six lung carcinomas were included in this prospective study. All biopsies were initially analysed with p63, TTF1, Napsin A, CK7, CK34βE12, synaptophysin, CK20 and CDX2. Two hundred and twenty-five non-small cell lung carcinomas were studied with real-time PCR technology to assess the status of the EGFR gene. RESULTS: We detected 132 adenocarcinomas (58.7%, 89 squamous cell carcinomas (39.2%, 4 adenosquamous carcinomas (1.8%, 9 large cell neuroendocrine carcinomas (3.8% and 2 metastatic colorectal adenocarcinomas (0.8%. Activating mutations in the EGF receptor had 3 out of 89 squamous cell carcinomas (3.37%. We have established mutations in L858R, deletion in exon 19 and rare mutation in S7681. One out of four adenosquamous carcinomas had a point mutation in the L858R (25%. CONCLUSIONS: The frequency of EGFR mutations we found in lung squamous cell carcinomas in a Southern Bulgarian region is lower than that in European countries. Ethnic diversity in the region does not play role of an independent predictive factor in terms of mutation frequency.

  8. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Do, Ji Yeon; Choi, Young Keun [Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University School of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Kook, Hyun [Department of Pharmacology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Suk, Kyoungho [Department of Pharmacology, Brain Science & Engineering Institute, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Lee, In-Kyu [Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University School of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Division of Endocrinology and Metabolism, Department of Internal Medicine, Research Institute of Aging and Metabolism, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Park, Dong Ho, E-mail: sarasate2222@gmail.com [Department of Ophthalmology, Kyungpook National University School of Medicine, Daegu (Korea, Republic of)

    2015-05-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O{sub 2}). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice.

  9. Molecular Cloning and Characterization of Growth Factor Receptor Bound-Protein in Clonorchis sinensis

    Science.gov (United States)

    Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong

    2014-01-01

    Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892

  10. Molecular cloning and characterization of growth factor receptor bound-protein in Clonorchis sinensis.

    Directory of Open Access Journals (Sweden)

    Xuelian Bai

    Full Text Available BACKGROUND: Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2 is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2 from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. CONCLUSION: Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths.

  11. Inhibition of fibroblast growth factor receptor with AZD4547 mitigates juvenile nasopharyngeal angiofibroma.

    Science.gov (United States)

    Le, Tran; New, Jacob; Jones, Joel W; Usman, Shireen; Yalamanchali, Sreeya; Tawfik, Ossama; Hoover, Larry; Bruegger, Dan E; Thomas, Sufi Mary

    2017-10-01

    Juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor that presents in adolescent males. Although surgical excision is the mainstay of treatment, recurrences complicate treatment. There is a need to develop less invasive approaches for management. JNA tumors are composed of fibroblasts and vascular endothelial cells. We identified fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor (VEGF) expression in JNA-derived fibroblasts. FGFR influences fibroblast proliferation and VEGF is necessary for angiogenesis. We hypothesized that targeting FGFR would mitigate JNA fibroblast proliferation, invasion, and migration, and that targeting the VEGF receptor would attenuate endothelial tubule formation. After informed consent, fibroblasts from JNA explants of 3 patients were isolated. Fibroblasts were treated with FGFR inhibitor AZD4547, 0 to 25 μg/mL for 72 hours and proliferation was quantified using CyQuant assay. Migration and invasion of JNA were assessed using 24-hour transwell assays with subsequent fixation and quantification. Mitigation of FGFR and downstream signaling was evaluated by immunoblotting. Tubule formation was assessed in human umbilical vein endothelial cells (HUVECs) treated with vehicle control (dimethylsulfoxide [DMSO]) or semaxanib (SU5416) as well as in serum-free media (SFM) or JNA conditioned media (CM). Tubule length was compared between treatment groups. Compared to control, AZD4547 inhibited JNA fibroblast proliferation, migration, and invasion through inhibition of FGFR and downstream signaling, specifically phosphorylation of - p44/42 mitogen activated protein kinase (p44/42 MAPK). JNA fibroblast CM significantly increased HUVEC tubule formation (p = 0.0039). AZD4547 effectively mitigates FGFR signaling and decreases JNA fibroblast proliferation, migration, and invasion. SU5416 attenuated JNA fibroblast-induced tubule formation. AZD4547 may have therapeutic potential in the treatment of JNA. © 2017 ARS

  12. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

    International Nuclear Information System (INIS)

    Do, Ji Yeon; Choi, Young Keun; Kook, Hyun; Suk, Kyoungho; Lee, In-Kyu; Park, Dong Ho

    2015-01-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O 2 ). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice

  13. Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

    Directory of Open Access Journals (Sweden)

    Chen Qian

    Full Text Available Platelet-derived growth factor receptor alpha (PDGFRα is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures.To address the temporal requirement of Pdgfra in embryonic development.We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies.Current study showed that (i conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5 resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives.Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b if mutations / sequence variations of these regulatory elements cause these anomalies.

  14. Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor

    International Nuclear Information System (INIS)

    Roy, L.M.; Gittinger, C.K.; Landreth, G.E.

    1991-01-01

    Epidermal growth factor receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization

  15. Structure and function of the Juxta membrane domain of the human epidermal growth factor receptor by NMR spectroscopy

    International Nuclear Information System (INIS)

    Choowongkomon, Kiattawee; Carlin, Cathleen; Sonnichsen, Frank D.

    2005-10-01

    The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family involved in the regulation of cellular proliferation and differentiation. Its juxta membrane domain (JX), the region located between the transmembrane and kinase domains, plays important roles in receptor trafficking since both basolateral sorting in polarized epithelial cells and lysosomal sorting signals are identified in this region. In order to understand the regulation of these signals, we characterized the structural properties of recombinant JX domain in dodecyl phosphocholine detergent (DPC) by nuclear magnetic resonance (NMR) spectroscopy. In DPC micelles, structures derived from NMR data showed three amphipathic, helical segments. Two equivalent average structural models on the surface of micelles were obtained that differ only in the relative orientation between the first and second helices. Our data suggests that the activity of sorting signals may be regulated by their membrane association and restricted accessibility in the intact receptor

  16. Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues

    DEFF Research Database (Denmark)

    Magni, M; Pandiella, A; Helin, K

    1991-01-01

    a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather...... in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed......, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion...

  17. Frequency of Epidermal Growth Factor Receptor Mutation in Smokers with Lung Cancer Without Pulmonary Emphysema.

    Science.gov (United States)

    Takeda, Kenichi; Yamasaki, Akira; Igishi, Tadashi; Kawasaki, Yuji; Ito-Nishii, Shizuka; Izumi, Hiroki; Sakamoto, Tomohiro; Touge, Hirokazu; Kodani, Masahiro; Makino, Haruhiko; Yanai, Masaaki; Tanaka, Natsumi; Matsumoto, Shingo; Araki, Kunio; Nakamura, Hiroshige; Shimizu, Eiji

    2017-02-01

    Chronic obstructive pulmonary disease is a smoking-related disease, and is categorized into the emphysema and airway dominant phenotypes. We examined the relationship between emphysematous changes and epidermal growth factor receptor (EGFR) mutation status in patients with lung adenocarcinoma. The medical records for 250 patients with lung adenocarcinoma were retrospectively reviewed. All patients were categorized into the emphysema or non-emphysema group. Wild-type EGFR was detected in 136 (54%) and mutant EGFR in 48 (19%). Emphysematous changes were observed in 87 (36%) patients. EGFR mutation was highly frequent in the non-emphysema group (p=0.0014). Multivariate logistic regression analysis showed that emphysema was an independent risk factor for reduced frequency of EGFR mutation (Odds Ratio=3.47, p=0.005). Our data showed a relationship between emphysematous changes and EGFR mutation status. There might be mutually exclusive genetic risk factors for carcinogenesis and development of emphysematous changes. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  18. Tumor necrosis factor-alpha induces activation of coagulation and fibrinolysis in baboons through an exclusive effect on the p55 receptor

    NARCIS (Netherlands)

    van der Poll, T.; Jansen, P. M.; van Zee, K. J.; Welborn, M. B.; de Jong, I.; Hack, C. E.; Loetscher, H.; Lesslauer, W.; Lowry, S. F.; Moldawer, L. L.

    1996-01-01

    Tumor necrosis factor-alpha (TNF-alpha) can bind to two distinct transmembrane receptors, the p55 and p75 TNF receptors. We compared the capability of two mutant TNF proteins with exclusive affinity for the p55 or p75 TNF receptor with that of wild type TNF, to activate the hemostatic mechanism in

  19. From bench to bedside: What do we know about hormone receptor-positive and human epidermal growth factor receptor 2-positive breast cancer?

    Science.gov (United States)

    Wu, Victoria Shang; Kanaya, Noriko; Lo, Chiao; Mortimer, Joanne; Chen, Shiuan

    2015-09-01

    Breast cancer is a heterogeneous disease. Thanks to extensive efforts from research scientists and clinicians, treatment for breast cancer has advanced into the era of targeted medicine. With the use of several well-established biomarkers, such as hormone receptors (HRs) (i.e., estrogen receptor [ER] and progesterone receptor [PgR]) and human epidermal growth factor receptor-2 (HER2), breast cancer patients can be categorized into multiple subgroups with specific targeted treatment strategies. Although therapeutic strategies for HR-positive (HR+) HER2-negative (HER2-) breast cancer and HR-negative (HR-) HER2-positive (HER2+) breast cancer are well-defined, HR+ HER2+ breast cancer is still an overlooked subgroup without tailored therapeutic options. In this review, we have summarized the molecular characteristics, etiology, preclinical tools and therapeutic options for HR+ HER2+ breast cancer. We hope to raise the attention of both the research and the medical community on HR+ HER2+ breast cancer, and to advance patient care for this subtype of disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Diagnostic values of vascular endothelial growth factor and epidermal growth factor receptor for benign and malignant hydrothorax.

    Science.gov (United States)

    Gu, Yan; Zhang, Min; Li, Guo-Hua; Gao, Jun-Zhen; Guo, Liping; Qiao, Xiao-Juan; Wang, Li-Hong; He, Lan; Wang, Mei-Ling; Yan, Li; Fu, Xiu-Hua

    2015-02-05

    Hydrothorax, as one of the common complications of malignant tumors, still cannot be sensitively detected in clinical practice, thus requiring a sensitive, specific method for diagnosis. The aim of this study was to analyze the correlation between levels of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) in patients with benign and malignant hydrothorax. The contents of VEGF in the pleural effusion and serum of the patients with malignant pleural effusion (n = 35) and benign pleural effusion (n = 30) were detected by double antibody sandwich enzyme linked immunosorbent assay. The gene copy number level of EGFR in pleural effusion was detected by fluorescence in situ hybridization (FISH). The points with the highest sensitivity and specificity were selected as the critical values to calculate the diagnostic value of the VEGF in pleural effusion and serum, and EGFR gene copy number in pleural effusion. The contents of VEGF in pleural effusion and serum of patients with malignant hydrothorax were (384.91 ± 120.18), and (129.62 ± 46.35) ng/L, respectively, which were significantly higher than those of the patients with benign hydrothorax (207.97 ± 64.04), (63.49 ± 24.58) ng/L (P benign and malignant hydrothorax. The sensitivity and specificity of serum were 74.3% and 96.7%, respectively (the boundary value was 99.21 ng/L) for diagnosing benign and malignant hydrothorax. The diagnostic efficiencies of EGFR and VEGF in hydrothorax were similar. There was a significant correlation between EGFR and VEGF in hydrothorax (P benign and malignant pleural effusions, which contributed to differential diagnosis results of benign and malignant pleural effusions. It is feasible to detect the gene copy number of the pleural effusion cell mass EGFR by FISH technique. Joint detection can improve the diagnostic sensitivity.

  1. Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

    DEFF Research Database (Denmark)

    Pedersen, Mikkel W; Pedersen, Nina; Damstrup, Lars

    2005-01-01

    moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes...... by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1...

  2. Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

    Science.gov (United States)

    Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

    2015-05-30

    Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

  3. Identification and Functional Characterisation of Nod Factor Receptor (NFR) Paralogs in Lotus japonicus

    DEFF Research Database (Denmark)

    Vestergaard, Gitte; Radutoiu, Elena Simona; Stougaard, Jens

    an important missing link in plant-bacterial communication. This picture changed with the cloning of LysM-domain containing receptor-like kinases (LysM-RLKs) in different legume species. In Lotus japonicus, two LysM-RLKs, Nod Factor Receptor 1 (NFR1) and Nod Factor Receptor 5 (NFR5), are believed to bind Nod...... using the sequences of NFR1 and NFR5. Microsattelite markers were developed from each TAC clone containing the LysM-RLK, permitting us to locate the genes on a genetic map of Lotus japonicus. In order to get more insight into the function of these genes an inverse genetic approach using RNAi has been...

  4. Systemic factors related to soluble (prorenin receptor in plasma of patients with proliferative diabetic retinopathy.

    Directory of Open Access Journals (Sweden)

    Keitaro Hase

    Full Text Available (Prorenin receptor [(PRR], a new component of the tissue renin-angiotensin system (RAS, plays a crucial role in inflammation and angiogenesis in the eye, thus contributing to the development of proliferative diabetic retinopathy (PDR. In this study, we investigated systemic factors related to plasma levels of soluble form of (PRR [s(PRR] in patients with PDR. Twenty type II diabetic patients with PDR and 20 age-matched, non-diabetic patients with idiopathic macular diseases were enrolled, and plasma levels of various molecules were measured by enzyme-linked immunosorbent assays. Human retinal microvascular endothelial cells were stimulated with several diabetes-related conditions to evaluate changes in gene expression using real-time quantitative PCR. Of various systemic parameters examined, the PDR patients had significantly higher blood sugar and serum creatinine levels than non-diabetic controls. Protein levels of s(PRR, prorenin, tumor necrosis factor (TNF-α, complement factor D (CFD, and leucine-rich α-2-glycoprotein 1 (LRG1 significantly increased in the plasma of PDR subjects as compared to non-diabetes, with positive correlations detected between s(PRR and these inflammatory molecules but not prorenin. Estimated glomerular filtration rate and serum creatinine were also correlated with plasma s(PRR, but not prorenin, levels. Among the inflammatory molecules correlated with s(PRR in the plasma, TNF-α, but not CFD or LRG1, application to retinal endothelial cells upregulated the mRNA expression of (PRR but not prorenin, while stimulation with high glucose enhanced both (PRR and prorenin expression. These findings suggested close relationships between plasma s(PRR and diabetes-induced factors including chronic inflammation, renal dysfunction, and hyperglycemia in patients with PDR.

  5. Matrix metalloproteinase-3, vitamin D receptor gene polymorphisms, and occupational risk factors in lumbar disc degeneration.

    Science.gov (United States)

    Zawilla, N H; Darweesh, H; Mansour, N; Helal, S; Taha, F M; Awadallah, M; El Shazly, R

    2014-06-01

    Lumbar disc degeneration (LDD) is a process that begins early in life, contributing to the development of low back pain. LDD is a consequence of a variety of factors, and its etiology remains poorly understood. Objectives to investigate occupational and genetic risk factors inducing lumbar disc degeneration, and to evaluate the possible association of genetic polymorphisms of matrix metalloproteinase 3 (MMP-3) and vitamin D receptor (VDR) with the severity of LDD in an Egyptian population. A case control study involving 84 LDD and 60 controls was carried out. Five types of work related factors were investigated by questionnaire, complete neurological examination for all subjects and MRI for the cases. Polymerase chain reaction and restriction fragment length polymorphism methods were applied to detect polymorphisms in MMP-3 Promoter (-1,171 6A/5A) (rs 731236) and VDR-Apa (rs 35068180). We found that family history, back injury, smoking, high level of sitting, bending/twisting, physical workload, lifting, whole body vibration, mutant allele 5A of MMP-3 and mutant allele T of VDR were significantly associated with LDD (OR = 2.9, 3.1, 2.1, 11.1, 15.9, 11.7, 8.2, 12.6, 2.5 and 3.1 respectively, p < 0.05). Cases that carry allele 5A and/or allele T were associated with LDD severity. LDD is closely associated in occurrence and severity with occupational, environmental risk factors and susceptibility genes namely MMP-3, and VDR (ApaI). This study throws light on the importance of screening for early detection of susceptible individuals and disease prevention.

  6. Expression of tumor necrosis factor-alpha and receptor I(P55in pterygium

    Directory of Open Access Journals (Sweden)

    Bing Wu

    2014-06-01

    Full Text Available AIM:To observe the expression of tumor necrosis factor- alpha(TNF-αand its receptor I(P55in different pterygium and discuss the role of TNF-α and receptor I(P55in pterygium.METHODS: Immunohistochemistical staining method(PVwas adopted to detect the expression of TNF-α and receptor I in pterygium(72 eyesand para-pterygium conjunctival tissue(30 eyes. The relationship between the expression and clinical-pathological parameters was also analyzed. RESULTS: The positive rates of TNF-α were 65.3%(47/72, 26.7%(8/30in pterygium and para-pterygium conjunctival tissue. The positive expression of TNF-α had statistic difference between the two groups(χ2=12.706, Pχ2=13.875, Pχ2=6.547, P=0.011. There had no statistically significance of the expression intensity between the two groups(F=1.288, P=0.393; the positive rate in advanced pterygium group was higher than quiescent pterygium group(χ2=4.082, P=0.043. The expression intensity had no statistically significance between the two groups(F=0.489, P=0.708. The positive rate of P55 in recurrent pterygium group was higher than primary pterygium group(χ2=9.907, P=0.002. There had no statistically significance of the two group's expression intensity(F=1.175, P=0.424; the positive rate in advanced pterygium group was higher than in quiescent pterygium group(χ2=11.140, P=0.001. The expression intensity had no statistically significance between the two groups(F=0.665, P=0.621. CONCLUSION:The expression of TNF-α and P55 are changing according to the development of clinical staging and onset. The expression of TNF-α and P55 may be related to clinical classification, staging and patient's working conditions of pterygium. There has no significant difference expression intensity of TNF-α and P55 in clinical staging and onset of pterygium.

  7. Vascular endothelial growth factor receptor-3 directly interacts with phosphatidylinositol 3-kinase to regulate lymphangiogenesis.

    Directory of Open Access Journals (Sweden)

    Sanja Coso

    Full Text Available BACKGROUND: Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF family is a major regulator of lymphatic endothelial cell (LEC function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. METHODS AND RESULTS: Here we delineate the VEGF-C/VEGF receptor (VEGFR-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCγ1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. CONCLUSIONS: Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis.

  8. Targeting Insulin-Like Growth Factor 1 Receptor Inhibits Pancreatic Cancer Growth and Metastasis

    Science.gov (United States)

    Subramani, Ramadevi; Lopez-Valdez, Rebecca; Arumugam, Arunkumar; Nandy, Sushmita; Boopalan, Thiyagarajan; Lakshmanaswamy, Rajkumar

    2014-01-01

    Pancreatic cancer is one of the most lethal cancers. Increasing incidence and mortality indicates that there is still much lacking in detection and management of the disease. This is partly due to a lack of specific symptoms during early stages of the disease. Several growth factor receptors have been associated with pancreatic cancer. Here, we have investigated if an RNA interference approach targeted to IGF-IR could be effective and efficient against pancreatic cancer growth and metastasis. For that, we evaluated the effects of IGF-1R inhibition using small interfering RNA (siRNAs) on tumor growth and metastasis in HPAC and PANC-1 pancreatic cancer cell lines. We found that silencing IGF-1R inhibits pancreatic cancer growth and metastasis by blocking key signaling pathways such AKT/PI3K, MAPK, JAK/STAT and EMT. Silencing IGF-1R resulted in an anti-proliferative effect in PANC-1 and HPAC pancreatic cancer cell lines. Matrigel invasion, transwell migration and wound healing assays also revealed a role for IGF-1R in metastatic properties of pancreatic cancer. These results were further confirmed using Western blotting analysis of key intermediates involved in proliferation, epithelial mesenchymal transition, migration, and invasion. In addition, soft agar assays showed that silencing IGF-1R also blocks the colony forming capabilities of pancreatic cancer cells in vitro. Western blots, as well as, flow cytometric analysis revealed the induction of apoptosis in IGF-1R silenced cells. Interestingly, silencing IGF-1R also suppressed the expression of insulin receptor β. All these effects together significantly control pancreatic cancer cell growth and metastasis. To conclude, our results demonstrate the significance of IGF-1R in pancreatic cancer. PMID:24809702

  9. Osteoblast-specific transcription factor Osterix increases vitamin D receptor gene expression in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Chi Zhang

    Full Text Available Osterix (Osx is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

  10. The pluripotency factor Nanog is directly upregulated by the androgen receptor in prostate cancer cells.

    Science.gov (United States)

    Kregel, Steven; Szmulewitz, Russell Z; Vander Griend, Donald J

    2014-11-01

    The Androgen Receptor (AR) is a nuclear hormone receptor that functions as a critical oncogene in all stages of prostate cancer progression, including progression to castration-resistance following androgen-deprivation therapy. Thus, identifying and targeting critical AR-regulated genes is one potential method to block castration-resistant cancer proliferation. Of particular importance are transcription factors that regulate stem cell pluripotency; many of these genes are emerging as critical oncogenes in numerous tumor cell types. Of these, Nanog has been previously shown to increase the self-renewal and stem-like properties of prostate cancer cells. Thus, we hypothesized that Nanog is a candidate AR target gene that may impart castration-resistance. We modulated AR signaling in LNCaP prostate cancer cells and assayed for Nanog expression. Direct AR binding to the NANOG promoter was tested using AR Chromatin Immunoprecipation (ChIP) and analyses of publically available AR ChIP-sequencing data-sets. Nanog over-expressing cells were analyzed for cell growth and cytotoxicity in response to the AR antagonist enzalutamide and the microtubule stabilizing agent docetaxel. AR signaling upregulates Nanog mRNA and protein. AR binds directly to the NANOG promoter, and was not identified within 75 kb of the NANOGP8 pseudogene, suggesting the NANOG gene locus was preferentially activated. Nanog overexpression in LNCaP cells increases overall growth, but does not increase resistance to enzalutamide or docetaxel. Nanog is a novel oncogenic AR target gene in prostate cancer cells, and stable expression of Nanog increases proliferation and growth of prostate cancer cells, but not resistance to enzalutamide or docetaxel. © 2014 Wiley Periodicals, Inc.

  11. Insulin like growth factor 2 regulation of aryl hydrocarbon receptor in MCF-7 breast cancer cells

    International Nuclear Information System (INIS)

    Tomblin, Justin K.; Salisbury, Travis B.

    2014-01-01

    Highlights: •IGF-2 stimulates concurrent increases in AHR and CCND1 expression. •IGF-2 promotes the binding of AHR to the endogenous cyclin D1 promoter. •AHR knockdown inhibits IGF-2 stimulated increases in CCND1 mRNA and protein. •AHR knockdown inhibits IGF-2 stimulated increases in MCF-7 proliferation. -- Abstract: Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancer proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (P < .001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (P < .001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (P < .001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (P < .001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR

  12. Insulin like growth factor 2 regulation of aryl hydrocarbon receptor in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tomblin, Justin K.; Salisbury, Travis B., E-mail: salisburyt@marshall.edu

    2014-01-17

    Highlights: •IGF-2 stimulates concurrent increases in AHR and CCND1 expression. •IGF-2 promotes the binding of AHR to the endogenous cyclin D1 promoter. •AHR knockdown inhibits IGF-2 stimulated increases in CCND1 mRNA and protein. •AHR knockdown inhibits IGF-2 stimulated increases in MCF-7 proliferation. -- Abstract: Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancer proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (P < .001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (P < .001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (P < .001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (P < .001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR.

  13. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    Energy Technology Data Exchange (ETDEWEB)

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Rodrigues, Michele A. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Department of General Pathology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Gomes, Dawidson A., E-mail: dawidson@ufmg.br [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil)

    2016-09-09

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  14. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    International Nuclear Information System (INIS)

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.; Rodrigues, Michele A.; Gomes, Dawidson A.

    2016-01-01

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  15. Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors*

    Science.gov (United States)

    Pontejo, Sergio M.; Alejo, Ali; Alcami, Antonio

    2015-01-01

    The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs. PMID:25940088

  16. Comparative Biochemical and Functional Analysis of Viral and Human Secreted Tumor Necrosis Factor (TNF) Decoy Receptors.

    Science.gov (United States)

    Pontejo, Sergio M; Alejo, Ali; Alcami, Antonio

    2015-06-26

    The blockade of tumor necrosis factor (TNF) by etanercept, a soluble version of the human TNF receptor 2 (hTNFR2), is a well established strategy to inhibit adverse TNF-mediated inflammatory responses in the clinic. A similar strategy is employed by poxviruses, encoding four viral TNF decoy receptor homologues (vTNFRs) named cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE. These vTNFRs are differentially expressed by poxviral species, suggesting distinct immunomodulatory properties. Whereas the human variola virus and mouse ectromelia virus encode one vTNFR, the broad host range cowpox virus encodes all vTNFRs. We report the first comprehensive study of the functional and binding properties of these four vTNFRs, providing an explanation for their expression profile among different poxviruses. In addition, the vTNFRs activities were compared with the hTNFR2 used in the clinic. Interestingly, CrmB from variola virus, the causative agent of smallpox, is the most potent TNFR of those tested here including hTNFR2. Furthermore, we demonstrate a new immunomodulatory activity of vTNFRs, showing that CrmB and CrmD also inhibit the activity of lymphotoxin β. Similarly, we report for the first time that the hTNFR2 blocks the biological activity of lymphotoxin β. The characterization of vTNFRs optimized during virus-host evolution to modulate the host immune response provides relevant information about their potential role in pathogenesis and may be used to improve anti-inflammatory therapies based on soluble decoy TNFRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Normal epidermal growth factor receptor signaling is dispensable for bone anabolic effects of parathyroid hormone.

    Science.gov (United States)

    Schneider, Marlon R; Dahlhoff, Maik; Andrukhova, Olena; Grill, Jessica; Glösmann, Martin; Schüler, Christiane; Weber, Karin; Wolf, Eckhard; Erben, Reinhold G

    2012-01-01

    Although the bone anabolic properties of intermittent parathyroid hormone (PTH) have long been employed in the treatment of osteoporosis, the molecular mechanisms behind this action remain largely unknown. Previous studies showed that PTH increases the expression and the activity of epidermal growth factor receptor (EGFR) in osteoblasts, and activation of ERK1/2 by PTH in osteoblasts was demonstrated to induce the proteolytical release of EGFR ligands and EGFR transactivation. However, conclusive evidence for an important role of the EGFR system in mediating the anabolic actions of intermittent PTH on bone in vivo is lacking. Here, we evaluated the effects of intermittent PTH on bone in Waved-5 (Wa5) mice which carry an antimorphic Egfr allele whose product acts as a dominant negative receptor. Heterozygous Wa5 females and control littermates received a subcutaneous injection of PTH (80 μg/kg) or buffer on 5 days per week for 4 weeks. Wa5 mice had slightly lower total bone mineral density (BMD), but normal cancellous bone volume and turnover in the distal femoral metaphysis. The presence of the antimorphic Egfr allele neither influenced the PTH-induced increase in serum osteocalcin nor the increases in distal femoral BMD, cortical thickness, cancellous bone volume, and cancellous bone formation rate. Similarly, the PTH-induced rise in lumbar vertebral BMD was unchanged in Wa5 relative to wild-type mice. Wa5-derived osteoblasts showed considerably lower basal extracellular signal-regulated kinase 1/2 (ERK1/2) activation as compared to control osteoblasts. Whereas activation of ERK1/2 by the EGFR ligand amphiregulin was largely blocked in Wa5 osteoblasts, treatment with PTH induced ERK1/2 activation comparable to that observed in control osteoblasts, relative to baseline levels. Our data indicate that impairment of EGFR signaling does not affect the anabolic action of intermittent PTH on cancellous and cortical bone. Copyright © 2011. Published by Elsevier Inc.

  18. Human epidermal growth factor receptor2 expression in unresectable gastric cancers: Relationship with CT characteristics

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Sub [Dept. of Radiology, Jeju National University Hospital, Jeju (Korea, Republic of); Kim, Se Hyung; Im, Seock Ah; Kim, Min A; Han, Joon Koo [Seoul National University Hospital, Seoul (Korea, Republic of)

    2017-09-15

    To retrospectively analyze the qualitative CT features that correlate with human epidermal growth factor receptor 2 (HER2)-expression in pathologically-proven gastric cancers. A total of 181 patients with pathologically-proven unresectable gastric cancers with HER2-expression (HER2-positive [n = 32] and negative [n = 149]) were included. CT features of primary gastric and metastatic tumors were reviewed. The prevalence of each CT finding was compared in both groups. Thereafter, binary logistic regression determined the most significant differential CT features. Clinical outcomes were compared using Kaplan-Meier method. HER2-postive cancers showed lower clinical T stage (21.9% vs. 8.1%; p = 0.015), hyperattenuation on portal phase (62.5% vs. 30.9%; p = 0.003), and was more frequently metastasized to the liver (62.5% vs. 32.2%; p = 0.001), than HER2-negative cancers. On binary regression analysis, hyperattenuation of the tumor (odds ratio [OR], 4.68; p < 0.001) and hepatic metastasis (OR, 4.43; p = 0.001) were significant independent factors that predict HER2-positive cancers. Median survival of HER2-positive cancers (13.7 months) was significantly longer than HER2-negative cancers (9.6 months) (p = 0.035). HER2-positive gastric cancers show less-advanced T stage, hyperattenuation on the portal phase, and frequently metastasize to the liver, as compared to HER2-negative cancers.

  19. Optical Molecular Imaging of Epidermal Growth Factor Receptor Expression to Improve Detection of Oral Neoplasia

    Directory of Open Access Journals (Sweden)

    Nitin Nitin

    2009-06-01

    Full Text Available Background: The development of noninvasive molecular imaging approaches has the potential to improve management of cancer. Methods: In this study, we demonstrate the potential of noninvasive topical delivery of an epidermal growth factor-Alexa 647 (EGF-Alexa 647 conjugate to image changes in epidermal growth factor receptor expression associated with oral neoplasia. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of EGF-Alexa 647 in a variety of model systems, including cells, three-dimensional tissue cultures, and intact human tissue specimens using wide-field and high-resolution fluorescence imaging. Data were collected from 17 different oral cancer patients: eight pairs of normal and abnormal biopsies and nine resected tumors were examined. Results: The EGF-dye conjugate can be uniformly delivered throughout the oral epithelium with a penetration depth exceeding 500 µm and incubation time of less than 30 minutes. After EGF-Alexa 647 incubation, the presence of oral neoplasia is associated with a 1.5- to 6.9-fold increase in fluorescence contrast compared with grossly normal mucosa from the same patient with both wide-field and high-resolution fluorescence imaging. Conclusions: Results illustrate the potential of EGF-targeted fluorescent agents for in vivo molecular imaging, a technique that may aid in the diagnosis and characterization of oral neoplasia and allow real-time detection of tumor margins.

  20. Involvement of Corticotropin-Releasing Factor and Receptors in Immune Cells in Irritable Bowel Syndrome

    Directory of Open Access Journals (Sweden)

    Mahanand Chatoo

    2018-02-01

    Full Text Available Irritable bowel syndrome (IBS is a common functional gastrointestinal disorder defined by ROME IV criteria as pain in the lower abdominal region, which is associated with altered bowel habit or defecation. The underlying mechanism of IBS is not completely understood. IBS seems to be a product of interactions between various factors with genetics, dietary/intestinal microbiota, low-grade inflammation, and stress playing a key role in the pathogenesis of this disease. The crosstalk between the immune system and stress in IBS mechanism is increasingly recognized. Corticotropin-releasing factor (CRF, a major mediator in the stress response, is involved in altered function in GI, including inflammatory processes, colonic transit time, contractile activity, defecation pattern, pain threshold, mucosal secretory function, and barrier functions. This mini review focuses on the recently establish local GI-CRF system, its involvement in modulating the immune response in IBS, and summarizes current IBS animal models and mapping of CRF, CRFR1, and CRFR2 expression in colon tissues. CRF and receptors might be a key molecule involving the immune and movement function via brain–gut axis in IBS.

  1. Relationship between serum response factor and androgen receptor in prostate cancer.

    Science.gov (United States)

    Prencipe, Maria; O'Neill, Amanda; O'Hurley, Gillian; Nguyen, Lan K; Fabre, Aurelie; Bjartell, Anders; Gallagher, William M; Morrissey, Colm; Kay, Elaine W; Watson, R William

    2015-11-01

    Serum response factor (SRF) is an important transcription factor in castrate-resistant prostate cancer (CRPC). Since CRPC is associated with androgen receptor (AR) hypersensitivity, we investigated the relationship between SRF and AR. Transcriptional activity was assessed by luciferase assay. Cell proliferation was measured by MTT and flow cytometry. Protein expression in patients was assessed by immunohistochemistry. To investigate AR involvement in SRF response to androgen, AR expression was down-regulated using siRNA. This resulted in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in AR-negative PC346 DCC cells, which was only restored following AR over-expression. Next, SRF expression was down-regulated by siRNA, resulting in AR increased transcriptional activity in castrate-resistant LNCaP Abl cells but not in the parental LNCaP. This negative feedback loop in the resistant cells was confirmed by immunohistochemistry which showed a negative correlation between AR and SRF expression in CRPC bone metastases and a positive correlation in androgen-naïve prostatectomies. Cell proliferation was next assessed following SRF inhibition, demonstrating that SRF inhibition is more effective than AR inhibition in castrate-resistant cells. Our data support SRF as a promising therapeutic target in combination with current treatments. © 2015 Wiley Periodicals, Inc.

  2. Human epidermal growth factor receptor2 expression in unresectable gastric cancers: Relationship with CT characteristics

    International Nuclear Information System (INIS)

    Lee, Jeong Sub; Kim, Se Hyung; Im, Seock Ah; Kim, Min A; Han, Joon Koo

    2017-01-01

    To retrospectively analyze the qualitative CT features that correlate with human epidermal growth factor receptor 2 (HER2)-expression in pathologically-proven gastric cancers. A total of 181 patients with pathologically-proven unresectable gastric cancers with HER2-expression (HER2-positive [n = 32] and negative [n = 149]) were included. CT features of primary gastric and metastatic tumors were reviewed. The prevalence of each CT finding was compared in both groups. Thereafter, binary logistic regression determined the most significant differential CT features. Clinical outcomes were compared using Kaplan-Meier method. HER2-postive cancers showed lower clinical T stage (21.9% vs. 8.1%; p = 0.015), hyperattenuation on portal phase (62.5% vs. 30.9%; p = 0.003), and was more frequently metastasized to the liver (62.5% vs. 32.2%; p = 0.001), than HER2-negative cancers. On binary regression analysis, hyperattenuation of the tumor (odds ratio [OR], 4.68; p < 0.001) and hepatic metastasis (OR, 4.43; p = 0.001) were significant independent factors that predict HER2-positive cancers. Median survival of HER2-positive cancers (13.7 months) was significantly longer than HER2-negative cancers (9.6 months) (p = 0.035). HER2-positive gastric cancers show less-advanced T stage, hyperattenuation on the portal phase, and frequently metastasize to the liver, as compared to HER2-negative cancers

  3. Fibroblast growth factor receptor 4 polymorphism is associated with liver cirrhosis in hepatocarcinoma.

    Directory of Open Access Journals (Sweden)

    Ming-Jen Sheu

    Full Text Available Fibroblast growth factor receptor 4 (FGFR4 polymorphisms are positively correlated with tumor progression in numerous malignant tumors. However, the association between FGFR4 genetic variants and the risk of hepatocellular carcinoma (HCC has not yet been determined. In this study, we investigated the potential associations of FGFR4 single nucleotide polymorphisms (SNPs with HCC susceptibility and its clinicopathological characteristics.Four SNPs in FGFR4 (rs1966265, rs351855, rs2011077, and rs7708357 were analyzed among 884 participants, including 595 controls and 289 patients with HCC. The samples were further analyzed to clarify the associations between these gene polymorphisms and the risk of HCC, and the impact of these SNPs on the susceptibility and clinicopathological characteristics of HCC. After adjusting for other covariants, HCC patients who carrying at least one A genotype (GA and AA at rs351855 were observed to have a higher risk of liver cirrhosis compared with those carrying the wild-type genotype (GG (OR: 2.113, 95% CI: 1.188-3.831. Moreover, the patients with at least one A genotype were particularly showed a high level of alpha-fetoprotein (AFP.Our findings suggest that genetic polymorphism in FGFR4 rs351855 may be associated with the risk of HCC coupled with liver cirrhosis and may markedly increase the AFP level in Taiwanese patients with HCC. In addition, this is the first study that evaluated the risk factors associated with FGFR4 polymorphism variants in Taiwanese patients with HCC.

  4. Treatment challenges for community oncologists treating postmenopausal women with endocrine-resistant, hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer

    International Nuclear Information System (INIS)

    Gradishar, William J

    2016-01-01

    Community-based oncologists are faced with challenges and opportunities when delivering quality patient care, including high patient volumes and diminished resources; however, there may be the potential to deliver increased patient education and subsequently improve outcomes. This review discusses the treatment of postmenopausal women with endocrine-resistant, hormone receptor-positive, human epidermal growth factor receptor 2- negative advanced breast cancer in order to illustrate considerations in the provision of pertinent quality education in the treatment of these patients and the management of therapy-related adverse events. An overview of endocrine-resistant breast cancer and subsequent treatment challenges is also provided. Approved treatment options for endocrine-resistant breast cancer include hormonal therapies and mammalian target of rapamycin inhibitors. Compounds under clinical investigation are also discussed

  5. Nutrition, anthropometry, gastrointestinal dysfunction, and circulating levels of tumour necrosis factor alpha receptor I and interleukin-1 receptor antagonist in children during stem cell transplantation

    DEFF Research Database (Denmark)

    Andreassen, B. U.; Pærregaard, Anders; Michaelsen, Kim F.

    2008-01-01

    To evaluate anthropometry, nutrition and gastrointestinal dysfunction, and to characterize the relation between these parameters and the inflammatory activity evaluated by plasma levels of soluble tumour necrosis factor alpha receptor I (sTNFRI) and interleukin-1 receptor antagonist (IL-1Ra) levels...... during stem cell transplantation (SCT) in children. Clinical assessments and blood sampling were performed on days -3, 0, +7, +15 and +31 in eight children undergoing SCT. Energy intake, anthropometry, gastrointestinal dysfunction (WHO toxicity score) and sTNFRI and IL-1Ra were evaluated. The energy...... intake was below recommended levels. There was a loss of lean body mass (arm muscle area)(median, 2031 mm(2) (day -3) vs 1477 mm(2) (day 31); p = 0.04), and of fat mass (arm fat area) (791 mm(2) (day -3) vs 648 mm(2) (day +31); p = 0.04). sTNFRI was elevated throughout the course of transplantation...

  6. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

    1988-01-01

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe -1 , Val 1 , Asn 2 , Gln 3 , His 4 , Ser 8 , His 9 , Glu 12 , Tyr 15 , Leu 16 ]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln 3 , Ala 4 ] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr 15 , Leu 16 ] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln 3 , Ala 4 , Tyr 15 ,Leu 16 ]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

  7. Tumor necrosis factor α sensitizes spinal cord TRPV1 receptors to the endogenous agonist N-oleoyldopamine

    Directory of Open Access Journals (Sweden)

    Spicarova Diana

    2010-08-01

    Full Text Available Abstract Modulation of synaptic transmission in the spinal cord dorsal horn is thought to be involved in the development and maintenance of different pathological pain states. The proinflamatory cytokine, tumor necrosis factor α (TNFα, is an established pain modulator in both the peripheral and the central nervous system. Up-regulation of TNFα and its receptors (TNFR in dorsal root ganglion (DRG cells and in the spinal cord has been shown to play an important role in neuropathic and inflammatory pain conditions. Transient receptor potential vanilloid 1 (TRPV1 receptors are known as molecular integrators of nociceptive stimuli in the periphery, but their role on the spinal endings of nociceptive DRG neurons is unclear. The endogenous TRPV1 receptor agonist N-oleoyldopamine (OLDA was shown previously to activate spinal TRPV1 receptors. In our experiments the possible influence of TNFα on presynaptic spinal cord TRPV1 receptor function was investigated. Using the patch-clamp technique, miniature excitatory postsynaptic currents (mEPSCs were recorded in superficial dorsal horn neurons in acute slices after incubation with 60 nM TNFα. A population of dorsal horn neurons with capsaicin sensitive primary afferent input recorded after the TNFα pretreatment had a basal mEPSC frequency of 1.35 ± 0.20 Hz (n = 13, which was significantly higher when compared to a similar population of neurons in control slices (0.76 ± 0.08 Hz; n = 53; P

  8. Cell-cell adhesion mediated by binding of membrane-anchored transforming growth factor α to epidermal growth factor receptors promotes cell proliferation

    International Nuclear Information System (INIS)

    Anklesaria, P.; Greenberger, J.S.; Teixido, J.; Laiho, M.; Massague, J.; Pierce, J.H.

    1990-01-01

    The precursor for transforming growth factor α, pro-TGF-α, is a cell surface glycoprotein that can establish contact with epidermal growth factor (EGF) receptors on adjacent cells. To examine whether the pro-TGF-α/EGF receptor pair can simultaneously mediate cell adhesion and promote cell proliferation, the authors have expressed pro-TGF-α in a bone marrow stromal cell line labeled with [ 35 S] cysteine. Expression of pro-TGF-α allows these cells to support long-term attachment of an EGF/interleukin-3-dependent hematopoietic progenitor cell line that expresses EGF receptors but is unable to adhere to normal stroma. This interaction is inhibited by soluble EGF receptor ligands. Further, the hematopoietic progenitor cells replicate their DNA while they are attached to the stromal cell layer and become foci of sustained cell proliferation. Thus, pro-TGF-α and the EGF receptor can function as mediators of intercellular adhesion and this interaction may promote a mitogenic response. They propose the term juxtacrine to designate this form of stimulation between adjacent cells

  9. Direct observation of the discrete energy spectrum of two lanthanide-based single-chain magnets by far-infrared spectroscopy

    Science.gov (United States)

    Haas, Sabrina; Heintze, Eric; Zapf, Sina; Gorshunov, Boris; Dressel, Martin; Bogani, Lapo

    2014-05-01

    The far-infrared optical transmission has been studied for two lanthanide-based single-chain magnets DyPhOPh and TbPhOPh in the frequency range between 3 and 80 cm-1. The spectra were acquired at temperatures between 2 and 80 K and magnetic fields up to 6 T. Based on their magnetic field dependence in DyPhOPh two of the observed absorption lines are identified as transitions inside the crystal field split Dy3+ ground multiplet 6H15/2, coupled to the neighboring spins. In TbPhOPh one transition was observed inside the crystal-field-split Tb3+ ground multiplet 7F6. The results allow a spectroscopic investigation of the role of single-ion anisotropy and exchange in Glauber dynamics.

  10. Radiotherapy and receptor of epidermal growth factor; Radiotherapie et recepteur de l'Epidermal Growth Factor

    Energy Technology Data Exchange (ETDEWEB)

    Deberne, M. [Institut Gustave-Roussy, 94 - Villejuif (France)

    2009-10-15

    The expression level of the receptor of the epidermal growth factor is in correlation with the tumor cells radiosensitivity. An overexpression of the E.G.F.R. is often present in the bronchi cancer, epidermoid carcinomas of the O.R.L. sphere, esophagus, uterine cervix, and anal duct but also in the rectum cancers and glioblastomas. At the clinical level, the E.G.F.R. expression is in correlation with an unfavourable prognosis after radiotherapy in numerous tumoral localizations. In the rectum cancers it is an independent prognosis factor found in multifactorial analysis: increase of the rate of nodes and local recurrence when the E.G.F.R. is over expressed. In the uterine cervix cancers, the survival is is negatively affected in multifactorial analysis by the E.G.F.R. membranes expression level. At the therapy level, the development of anti E.G.F.R. targeted therapies (tyrosine kinase inhibitors and monoclonal antibodies) opens a new therapy field at radio-sensitivity potentiality. The irradiation makes an activation of the E.G.F.R. way that would be partially responsible of the post irradiation tumoral repopulation. This activation leads the phosphorylation of the PI3 kinase ways and M.A.P. kinase ones, then the Akt protein one that acts an apoptotic modulator part. It has been shown that blocking the E.G.F.R. way acts on three levels: accumulation of ells in phase G1, reduction of the cell repair and increasing of apoptosis. he inhibition of post irradiation action of the E.G.F.R. signal way is a factor explaining the ionizing radiation - anti E.G.F.R. synergy. The preclinical data suggest that the E.G.F.R. blocking by the monoclonal antibodies is more important than the use of tyrosine kinase inhibitors. A first positive randomized study with the cetuximab, published in 2006 in the epidermoid carcinomas of the O.R.L. sphere lead to its authorization on the market with the radiotherapy for this localization. The use of cetuximab in other indication with or in

  11. Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

    Science.gov (United States)

    Gao, Wei; Cai, Liting; Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

    2014-01-01

    Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

  12. Intraoperative fluorescence delineation of head and neck cancer with a fluorescent Anti-epidermal growth factor receptor nanobody

    NARCIS (Netherlands)

    Van Driel, P.B.A.A.; Van Der Vorst, J.R.; Verbeek, F.P.R.; Oliveira, S.|info:eu-repo/dai/nl/304841455; Snoeks, T.J.A.; Keereweer, S.; Chan, B.; Boonstra, M.C.; Frangioni, J.V.; Van Bergen En Henegouwen, P.M.P.|info:eu-repo/dai/nl/071919481; Vahrmeijer, A.L.; Lowik, C.W.G.M.

    2014-01-01

    Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for

  13. Resveratrol prevents angiotensin II-induced hypertrophy of vascular smooth muscle cells through the transactivation of growth factor receptors.

    Science.gov (United States)

    Hossain, Ekhtear; Anand-Srivastava, Madhu B

    2017-08-01

    We previously showed that augmented levels of endogenous angiotensin II (AngII) contribute to vascular smooth muscle cell (VSMC) hypertrophy through the transactivation of growth factor receptors in spontaneously hypertensive rats. Resveratrol (RV), a polyphenolic component of red wine, has also been shown to attenuate AngII-evoked VSMC hypertrophy; however, the molecular mechanism mediating this response is obscure. The present study was therefore undertaken to examine whether RV could prevent AngII-induced VSMC hypertrophy through the transactivation of growth factor receptor and associated signaling pathways. AngII treatment of VSMC enhanced the protein synthesis that was attenuated towards control levels by RV pretreatment as well as by the inhibitors of NADPH oxidase, c-Src, and growth factor receptors. Furthermore, RV pretreatment also inhibited enhanced levels of superoxide anion, NADPH oxidase activity, increased expression of NADPH oxidase subunits, and phosphorylation of c-Src, EGF-R, PDGE-R, ERK1/2, and AKT1/2. In conclusion, these results indicate that RV attenuates AngII-induced VSMC hypertrophy through the inhibition of enhanced oxidative stress and activation of c-Src, growth factor receptors, and MAPK/AKT signaling. We suggest that RV could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension and hypertrophy.

  14. Radiolabeled cetuximab: dose optimization for epidermal growth factor receptor imaging in a head-and-neck squamous cell carcinoma model

    NARCIS (Netherlands)

    Hoeben, B.A.W.; Molkenboer-Kuenen, J.D.M.; Oyen, W.J.G.; Peeters, W.J.M.; Kaanders, J.H.A.M.; Bussink, J.; Boerman, O.C.

    2011-01-01

    Noninvasive imaging of the epidermal growth factor receptor (EGFR) in head-and-neck squamous cell carcinoma could be of value to select patients for EGFR-targeted therapy. We assessed dose optimization of (111) Indium-DTPA-cetuximab ((111) In-cetuximab) for EGFR imaging in a head-and-neck squamous

  15. Limited human epidermal growth factor receptor 2 discordance in metastatic breast cancer patients treated with trastuzumab, a population based study

    NARCIS (Netherlands)

    van Rooijen, J.M.; de Munck, L.; de Graaf, J.C.; Siesling, Sabine; de Vries, Erik G.; Boers, J.E.

    2014-01-01

    Background Accurate assessment of the human epidermal growth factor receptor 2 (HER2) in breast cancer is essential for proper treatment decisions. HER2 positivity confirmation rates in breast cancer trials by central testing pathology laboratories were reported to be approximately 85%. The aim of

  16. Molecular cloning and functional characterization of the human platelet-derived growth factor alpha receptor gene promoter

    NARCIS (Netherlands)

    Afink, G. B.; Nistér, M.; Stassen, B. H.; Joosten, P. H.; Rademakers, P. J.; Bongcam-Rudloff, E.; van Zoelen, E. J.; Mosselman, S.

    1995-01-01

    Expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) is strictly regulated during mammalian development and tumorigenesis. The molecular mechanisms involved in the specific regulation of PDGF alpha R expression are unknown, but transcriptional regulation of the PDGF alpha R

  17. Expression of hepatocyte growth factor and the proto-oncogenic receptor c-Met in canine osteosarcoma

    NARCIS (Netherlands)

    Fieten, H; Spee, B; Ijzer, J; Kik, M J; Penning, L C; Kirpensteijn, J

    Hepatocyte growth factor (HGF) and the proto-oncogenic receptor c-Met are implicated in growth, invasion, and metastasis in human cancer. Little information is available on the expression and role of both gene products in canine osteosarcoma. We hypothesized that the expression of c-Met is

  18. Positive fibroblast growth factor receptor 3 immunoreactivity is associated with low-grade non-invasive urothelial bladder cancer

    NARCIS (Netherlands)

    C. Poyet (Cédric); T. Hermanns (Thomas); Q. Zhong (Qing); E. Drescher (Eva); D. Eberli (Daniel); M. Burger (Maximilian); F. Hofstaedter (Ferdinand); A. Hartmann (Arndt); R. Stöhr (Robert); E.C. Zwarthoff (Ellen); T. Sulser (Tullio); P.J. Wild (Peter J.)

    2015-01-01

    textabstractIn addition to conventional clinicopathological parameters, molecular markers are also required in order to predict the course of disease in patients with urothelial bladder cancer (BC). Little is known about fibroblast growth factor receptor 3 (FGFR3) immunoreactivity and the clinical

  19. Limited human epidermal growth factor receptor 2 discordance in metastatic breast cancer patients treated with trastuzumab, a population based study

    NARCIS (Netherlands)

    van Rooijen, J. M.; de Munck, L.; de Graaf, J. C.; Siesling, S.; de Vries, E. G.; Boers, J. E.

    Background: Accurate assessment of the human epidermal growth factor receptor 2 (HER2) in breast cancer is essential for proper treatment decisions. HER2 positivity confirmation rates in breast cancer trials by central testing pathology laboratories were reported to be approximately 85%. The aim of

  20. 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

    2017-01-01

    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...

  1. A nomogram for predicting pathological complete response in patients with human epidermal growth factor receptor 2 negative breast cancer

    International Nuclear Information System (INIS)

    Jin, Xi; Jiang, Yi-Zhou; Chen, Sheng; Yu, Ke-Da; Ma, Ding; Sun, Wei; Shao, Zhi-Min; Di, Gen-Hong

    2016-01-01

    The response to neoadjuvant chemotherapy has been proven to predict long-term clinical benefits for patients. Our research is to construct a nomogram to predict pathological complete response of human epidermal growth factor receptor 2 negative breast cancer patients. We enrolled 815 patients who received neoadjuvant chemotherapy from 2003 to 2015 and divided them into a training set and a validation set. Univariate logistic regression was performed to screen for predictors and construct the nomogram; multivariate logistic regression was performed to identify independent predictors. After performing the univariate logistic regression analysis in the training set, tumor size, hormone receptor status, regimens of neoadjuvant chemotherapy and cycles of neoadjuvant chemotherapy were the final predictors for the construction of the nomogram. The multivariate logistic regression analysis demonstrated that T4 status, hormone receptor status and receiving regimen of paclitaxel and carboplatin were independent predictors of pathological complete response. The area under the receiver operating characteristic curve of the training set and the validation set was 0.779 and 0.701, respectively. We constructed and validated a nomogram to predict pathological complete response in human epidermal growth factor receptor 2 negative breast cancer patients. We also identified tumor size, hormone receptor status and paclitaxel and carboplatin regimen as independent predictors of pathological complete response. The online version of this article (doi:10.1186/s12885-016-2652-z) contains supplementary material, which is available to authorized users

  2. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  3. Decreased numbers of chemotactic factor receptors in chronic neutropenia with defective chemotaxis: spontaneous recovery from the neutrophil abnormalities during early childhood

    International Nuclear Information System (INIS)

    Yasui, K.; Yamazaki, M.; Miyagawa, Y.; Komiyama, A.; Akabane, T.

    1987-01-01

    Childhood chronic neutropenia with decreased numbers of chemotactic factor receptors as well as defective chemotaxis was first demonstrated in an 8-month-old girl. Chemotactic factor receptors on neutrophils were assayed using tritiated N-formyl-methionyl-leucyl-phenylalanine ( 3 H-FMLP). The patient's neutrophils had decreased numbers of the receptors: numbers of the receptors were 20,000 (less than 3 SD) as compared with those of control cells of 52,000 +/- 6000 (mean +/- SD) (n = 10). The neutropenia disappeared spontaneously by 28 months of age parallel with the improvement of chemotaxis and increase in numbers of chemotactic factor receptors. These results demonstrate a transient decrease of neutrophil chemotactic factor receptors as one of the pathophysiological bases of a transient defect of neutrophil chemotaxis in this disorder

  4. Experimental diabetes increases insulin-like growth factor I and II receptor concentration and gene expression in kidney

    International Nuclear Information System (INIS)

    Werner, H.; Shen-Orr, Z.; Stannard, B.; Burguera, B.; Roberts, C.T. Jr.; LeRoith, D.

    1990-01-01

    Insulinlike growth factor I (IGF-I) is a mitogenic hormone with important regulatory roles in growth and development. One of the target organs for IGF-I action is the kidney, which synthesizes abundant IGF-I receptors and IGF-I itself. To study the involvement of IGF-I and the IGF-I receptor in the development of nephropathy, one of the major complications of diabetes mellitus, we measured the expression of these genes in the kidney and in other tissues of the streptozocin-induced diabetic rat. The binding of 125I-labeled IGF-I to crude membranes was measured in the same tissues. We observed a 2.5-fold increase in the steady-state level of IGF-I-receptor mRNA in the diabetic kidney, which was accompanied by a 2.3-fold increase in IGF-I binding. In addition to this increase in IGF-I binding to the IGF-I receptor, there was also binding to a lower-molecular-weight material that may represent an IGF-binding protein. No change was detected in the level of IGF-I-peptide mRNA. Similarly, IGF-II-receptor mRNA levels and IGF-II binding were significantly increased in the diabetic kidney. IGF-I- and IGF-II-receptor mRNA levels and IGF-I and IGF-II binding returned to control values after insulin treatment. Because the IGF-I receptor is able to transduce mitogenic signals on activation of its tyrosine kinase domain, we hypothesize that, among other factors, high levels of receptor in the diabetic kidney may also be involved in the development of diabetic nephropathy. Increased IGF-II-receptor expression in the diabetic kidney may be important for the intracellular transport and packaging of lysosomal enzymes, although a role for this receptor in signal transduction cannot be excluded. Finally, the possible role of IGF-binding proteins requires further study

  5. Reproductive factors and risk of hormone receptor positive and negative breast cancer: a cohort study

    International Nuclear Information System (INIS)

    Ritte, Rebecca; Grote, Verena; Boeing, Heiner; Aleksandrova, Krasimira; Trichopoulou, Antonia; Lagiou, Pagona; Trichopoulos, Dimitrios; Palli, Domenico; Berrino, Franco; Mattiello, Amalia; Tumino, Rosario; Tikk, Kaja; Sacerdote, Carlotta; Quirós, José Ramón; Buckland, Genevieve; Molina-Montes, Esther; Chirlaque, María-Dolores; Ardanaz, Eva; Amiano, Pilar; Bueno-de-Mesquita, H Bas; Gils, Carla H van; Peeters, Petra HM; Lukanova, Annekatrin; Wareham, Nick; Khaw, Kay-Tee; Key, Timothy J; Travis, Ruth C; Weiderpass, Elisabete; Dumeaux, Vanessa; Lund, Eliv; Sund, Malin; Andersson, Anne; Romieu, Isabelle; Tjønneland, Anne; Rinaldi, Sabina; Vineis, Paulo; Merritt, Melissa A; Riboli, Elio; Kaaks, Rudolf; Olsen, Anja; Overvad, Kim; Dossus, Laure; Fournier, Agnès; Clavel-Chapelon, Françoise

    2013-01-01

    The association of reproductive factors with hormone receptor (HR)-negative breast tumors remains uncertain. Within the EPIC cohort, Cox proportional hazards models were used to describe the relationships of reproductive factors (menarcheal age, time between menarche and first pregnancy, parity, number of children, age at first and last pregnancies, time since last full-term childbirth, breastfeeding, age at menopause, ever having an abortion and use of oral contraceptives [OC]) with risk of ER-PR- (n = 998) and ER+PR+ (n = 3,567) breast tumors. A later first full-term childbirth was associated with increased risk of ER+PR+ tumors but not with risk of ER-PR- tumors (≥35 vs. ≤19 years HR: 1.47 [95% CI 1.15-1.88] p trend < 0.001 for ER+PR+ tumors; ≥35 vs. ≤19 years HR: 0.93 [95% CI 0.53-1.65] p trend = 0.96 for ER-PR- tumors; P het = 0.03). The risk associations of menarcheal age, and time period between menarche and first full-term childbirth with ER-PR-tumors were in the similar direction with risk of ER+PR+ tumors (p het = 0.50), although weaker in magnitude and statistically only borderline significant. Other parity related factors such as ever a full-term birth, number of births, age- and time since last birth were associated only with ER+PR+ malignancies, however no statistical heterogeneity between breast cancer subtypes was observed. Breastfeeding and OC use were generally not associated with breast cancer subtype risk. Our study provides possible evidence that age at menarche, and time between menarche and first full-term childbirth may be associated with the etiology of both HR-negative and HR-positive malignancies, although the associations with HR-negative breast cancer were only borderline significant

  6. Human Epidermal Growth Factor Receptor 2 Overexpression in Micropapillary and Other Variants of Urothelial Carcinoma.

    Science.gov (United States)

    Behzatoğlu, Kemal; Yörükoğlu, Kutsal; Demir, Hale; Bal, Nebil

    2016-06-21

    Human epidermal growth factor receptor 2 (HER2) protein overexpression or gene amplification has been shown in urothelial bladder cancer. This could be helpful when using targeted anti-HER2 therapy on these tumors. To evaluate HER2 immunohistochemical expression in conventional urothelial carcinoma (UC), in situ UC, and UC variants primarily in micropapillary urothelial carcinoma (MPUC). The study evaluated 60 MPUC cases; 25 invasive, 20 low-grade noninvasive, and 10 high-grade noninvasive UC cases; 8 in situ UC cases; and 69 UC variant cases. The immunohistochemistry staining was scored according to recommendations of the American Society of Clinical Oncology/College of American Pathologists 2013 HER2 test guideline established for breast cancer and only 3+ staining was considered HER2 overexpression. HER2 overexpression was determined by 3+ staining. 34 of 60 MPUC cases (56%) showed HER2 overexpression (3+ staining). We observed 3+ staining HER2 overexpression in nine of 25 conventional invasive UC cases (36%), four of eight in situ UC cases (50%), and three of six lipid cell variant cases (50%). 3+ staining HER2 overexpression was not seen in eight glandular, six small cell, and five sarcomatoid variant cases. HER2 overexpression was negative in the 20 low-grade noninvasive UC cases but positive in two of the 10 high-grade noninvasive UC cases (20%). We observed HER2 overexpression most commonly in MPUC cases. We also found HER2 overexpression in conventional invasive and in situ UC cases. Pure in situ UC and conventional invasive UC, especially MPUC, could be candidate tumors for treatment with anti-HER2 antibody (trastuzumab therapy). Targeted therapy has a limited place in treatment of bladder cancer. In this study, human epidermal growth factor receptor 2 (HER2) overexpression in bladder carcinomas was evaluated in a large number of cases. Anti-HER2 therapy could be used in bladder cancers, as in breast and gastric cancers. Copyright © 2016 European

  7. Significance of epidermal growth factor receptor signaling for acquisition of meiotic and developmental competence in mammalian oocytes

    Czech Academy of Sciences Publication Activity Database

    Procházka, Radek; Blaha, Milan; Němcová, Lucie

    2017-01-01

    Roč. 97, č. 4 (2017), s. 537-549 ISSN 0006-3363 R&D Projects: GA MZe(CZ) QJ1510138; GA MŠk EF15_003/0000460 Institutional support: RVO:67985904 Keywords : amphiregulin * cumulus cells * epidermal growth factor receptor Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Reproductive biology (medical aspects to be 3) Impact factor: 3.432, year: 2016

  8. Enhancement of insulin-like growth factor 2 receptors in glioblastoma

    International Nuclear Information System (INIS)

    Sara, V.; Prisell, Per; Sjoegren, Barbro; Enberg, Goesta

    1986-01-01

    The somatomedins (IGF-1/IGF-2) are a family of growth-promoting hormones which have been identified in the human central nervous system where their specific receptors are distributed. The present study identified somatomedin receptors in glioblastoma and compared them with those found in normal brain. A significant enhancement in the binding of 125 1-IGF-2 but not 125 1-IGF-1 to glioblastoma membranes was found. A fourfold increase in IGF-2 receptor concentration was observed. These findings indicate enhanced expression of the IGF-2 receptor in glioblastoma. (author)

  9. Enhancement of insulin-like growth factor 2 receptors in glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Sara, V; Prisell, P; Sjoegren, B; Persson, L; Boethius, J; Enberg, G

    1986-09-01

    The somatomedins (IGF-1/IGF-2) are a family of growth-promoting hormones which have been identified in the human central nervous system where their specific receptors are distributed. The present study identified somatomedin receptors in glioblastoma and compared them with those found in normal brain. A significant enhancement in the binding of /sup 125/1-IGF-2 but not /sup 125/1-IGF-1 to glioblastoma membranes was found. A fourfold increase in IGF-2 receptor concentration was observed. These findings indicate enhanced expression of the IGF-2 receptor in glioblastoma. 14 refs.

  10. Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice

    Directory of Open Access Journals (Sweden)

    Takamitsu Sasaki

    2007-12-01

    Full Text Available The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR and vascular endothelial growth factor receptor (VEGFR signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α and vascular endothelial growth factor (VEGF but were negative for EGFR, human epidermal growth factor receptor 2 (HER2, VEGFR. Double immunofluorescence staining revealed that tumorassociated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR, phosphorylated VEGFR (pVEGFR. Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01; this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001. AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, increased the level of apoptosis in both tumorassociated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer.

  11. A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

    Science.gov (United States)

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

    2014-10-31

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity

    DEFF Research Database (Denmark)

    Engelmann, H; Holtmann, H; Brakebusch, C

    1990-01-01

    Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ....... These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies...

  13. Overexpression of Insulin-like Growth Factor-1 Receptor Is Associated With Penile Cancer Progression.

    Science.gov (United States)

    Ball, Mark W; Bezerra, Stephania M; Chaux, Alcides; Faraj, Sheila F; Gonzalez-Roibon, Nilda; Munari, Enrico; Sharma, Rajni; Bivalacqua, Trinity J; Netto, George J; Burnett, Arthur L

    2016-06-01

    To evaluate insulin-like growth factor-1 receptor (IGF1R) expression in penile cancer and its association with oncologic outcomes. Tissue microarrays were constructed from 53 patients treated at our institution. Expression of IGF1R was evaluated using a Her2-like scoring system. Overexpression was defined as 1+ or greater membranous staining. Association of IGF1R expression with pathologic features was assessed with comparative statistics, and association with local recurrence, progression to nodal or distance metastases, or death was assessed with Kaplan-Meier survival analysis and Cox proportional hazard regression models. Overall, IGF1R overexpression was seen in 33 (62%) cases. With a median follow-up of 27.8 months, IGF1R overexpression was associated with inferior progression-free survival (PFS) (P  =  .003). In a multivariable model controlling for grade, T stage, perineural invasion, and lymphovascular invasion, IGF1R expression was independently associated with disease progression (hazard ratio 2.3, 95% confidence interval 1.1-5.1, P  =  .03. Comparing patients without IGF1R overexpression to those with overexpression, 5-year PFS was 94.1% vs 45.8%. IGF1R overexpression was associated with inferior PFS in penile cancer. Drugs that target IGF1R and downstream messengers may have a therapeutic benefit in patients that exhibit IGF1R overexpression. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Critical role of tumor necrosis factor receptor 1 in the pathogenesis of pulmonary emphysema in mice.

    Science.gov (United States)

    Fujita, Masaki; Ouchi, Hiroshi; Ikegame, Satoshi; Harada, Eiji; Matsumoto, Takemasa; Uchino, Junji; Nakanishi, Yoichi; Watanabe, Kentaro

    2016-01-01

    COPD is a major cause of chronic morbidity and mortality throughout the world. Although tumor necrosis factor-α (TNF-α) has a critical role in the development of COPD, the role of different TNF receptors (TNFRs) in pulmonary emphysema has not been resolved. We aimed to clarify the role of TNFRs in the development of pulmonary emphysema. TNF-α transgenic mice, a murine model of COPD in which the mice spontaneously develop emphysema with a large increase in lung volume and pulmonary hypertension, were crossed with either TNFR1-deficient mice or TNFR2-deficient mice. After 6 months, the gross appearance of the lung, lung histology, and pulmonary and cardiac physiology were determined. In addition, the relationship between apoptosis and emphysema was investigated. Pulmonary emphysema-like changes disappeared with deletion of TNFR1. However, slight improvements were attained with deletion of TNFR2. Apoptotic cells in the interstitium of the lung were observed in TNF-α transgenic mice. The apoptotic signals through TNFR1 appear critical for the pathogenesis of pulmonary emphysema. In contrast, the inflammatory process has a less important role for the development of emphysema.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

    2005-09-01

    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.

  16. Leukemia inhibitory factor promote trophoblast invasion via urokinase-type plasminogen activator receptor in preeclampsia.

    Science.gov (United States)

    Zheng, Qin; Dai, Kuixing; Cui, Xinyuan; Yu, Ming; Yang, Xuesong; Yan, Bin; Liu, Shuai; Yan, Qiu

    2016-05-01

    Preeclampsia is a pregnancy-related syndrome which can cause perinatal mortality and morbidity. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta, even result in preeclampsia. Understanding the molecular mechanisms underlying placentation facilitates the better intervention of preeclampsia. Urokinase-type plasminogen activator receptor (uPAR) is involved in the physiological and pathological processes. Leukemia inhibitory factor (LIF) is an important regulator in the establishment of pregnancy. However, the expression of uPAR in preeclamptic patients and its relationship with LIF remains unclear. In the current study, we found that the level of uPAR was relatively lower in the placentas from preeclamptic patients as compared with normal pregnant women. LIF promoted trophoblast cell outgrowth by upregulating uPAR in an explants culture, and LIF also enhanced migration and invasion potential through uPAR in trophoblast JAR and JEG-3 cell lines, and with increased gelatinolytic activities of matrix metalloproteinase 2 (MMP-2). The effect of LIF and uPAR on trophoblast migration and invasion was mediated by PI3K/AKT signaling pathway. Our data indicates the roles of LIF in promoting trophoblast migration and invasion through uPAR and suggest that abnormal expression of uPAR might be associated with the etiology of preeclampsia. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Receptor activator of nuclear factor kappa B (RANK as a determinant of peri-implantitis

    Directory of Open Access Journals (Sweden)

    Rakić Mia

    2013-01-01

    Full Text Available Background/Aim. Peri-implantitis presents inflammatory process that affects soft and hard supporting tissues of osseointegrated implant based on inflammatory osteoclastogenesis. The aim of this study was to investigate whether receptor activator of nuclear factor kappa B (RANK concentrations in peri-implant crevicular fluid could be associated with clinical parameters that reflect inflammatory nature of peri-implantitis. Methods. The study included 67 patients, 22 with diagnosed peri-implantitis, 22 persons with healthy peri-implant tissues and 23 patients with periodontitis. Clinical parameters from each patient were recorded and samples of peri-implant/gingival crevicular fluid were collected for the enzyme-linked immunosorbent assay (ELISA analysis. Results. RANK concentration was significantly increased in samples from the patients with periimplantitis when compared to healthy implants (p < 0.0001, where the average levels were 9 times higher. At the same time RANK concentration was significantly higher in periimplantitis than in periodontitis sites (p < 0.0001. In implant patients pocket depths and bleeding on probing values were positively associated with high RANK concentrations (p < 0.0001. Conclusion. These results revealed association of increased RANK concentration in samples of periimplant/ gingival crevicular fluid with peri-implant inflammation and suggests that RANK could be a pathologic determinant of peri-implantitis, thereby a potential parameter in assessment of peri-implant tissue inflammation and a potential target in designing treatment strategies.

  18. Inhibitors of the epidermal growth factor receptor in apple juice extract.

    Science.gov (United States)

    Kern, Melanie; Tjaden, Zeina; Ngiewih, Yufanyi; Puppel, Nicole; Will, Frank; Dietrich, Helmut; Pahlke, Gudrun; Marko, Doris

    2005-04-01

    The polyphenol-rich extract of a consumer-relevant apple juice blend was found to potently inhibit the growth of the human colon cancer cell line HT29 in vitro. The epidermal growth factor receptor (EGFR) and its subsequent signaling cascade play an important role in the regulation of cell proliferation in HT29 cells. The protein tyrosine kinase activity of an EGFR preparation was effectively inhibited by the polyphenol-rich apple juice extract. Treatment of intact cells with this extract resulted in the suppression of the subsequent mitogen-activated protein kinase cascade. Amongst the so far identified apple juice constituents, the proanthocyanidins B1 and B2 as well as quercetin-3-glc (isoquercitrin) and quercetin-3-gal (hyperoside) were found to possess substantial EGFR-inhibitory properties. However, as to be expected from the final concentration of these potential EGFR inhibitors in the original polyphenol-rich extract, a synthetic mixture of the apple juice constituents identified and available so far, including both proanthocyanidins and the quercetin glycosides, showed only marginal inhibitory effects on the EGFR. These results permit the assumption that yet unknown constituents contribute substantially to the potent EGFR-inhibitory properties of polyphenol-rich apple juice extract. In summary, the polyphenol composition of apple juice possesses promising growth-inhibitory properties, affecting proliferation-associated signaling cascades in colon tumor cells.

  19. Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

    Energy Technology Data Exchange (ETDEWEB)

    Jorge, S.E.D.C.; Kobayashi, S.S.; Costa, D.B. [Harvard Medical School, Beth Israel Deaconess Medical Center, Department of Medicine, Division of Hematology/Oncology, Boston, MA (United States)

    2014-09-05

    Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.

  20. Expression of granulocyte colony-stimulating factor receptor correlates with prognosis in oral and mesopharyngeal carcinoma.

    Science.gov (United States)

    Tsuzuki, H; Fujieda, S; Sunaga, H; Noda, I; Saito, H

    1998-02-15

    Granulocyte colony-stimulating factor receptors (G-CSFRs) have been observed on the surface of not only hematopoietic cells but also several cancer cells. The stimulation of G-CSF has been demonstrated to induce proliferation and activation of G-CSFR-positive cells. In this study, we investigated the expression of G-CSFR on the surface of tumor cells and G-CSF production in oral and mesopharyngeal squamous cell carcinoma (SCC) by an immunohistochemical approach. Of 58 oral and mesopharyngeal SCCs, 31 cases (53.4%) and 36 cases (62.1%) were positive for G-CSFR and G-CSF, respectively. There was no association between G-CSFR expression and G-CSF staining. In the group positive for G-CSFR expression, relapse was significantly more likely after primary treatment (P = 0.0069), whereas there was no association between G-CSFR expression and age, sex, tumor size, lymph node metastasis, and clinical stage. Also, the G-CSFR-positive groups had a significantly lower disease-free and overall survival rate than the G-CSFR-negative groups (P = 0.0172 and 0.0188, respectively). However, none of the clinical markers correlated significantly with G-CSF staining, nor did the status of G-CSF production influence the overall survival. The results imply that assessment of G-CSFR may prove valuable in selecting patients with oral and mesopharyngeal SCC for aggressive therapy.

  1. Epidermal growth factor receptor expression in different subtypes of oral lichenoid disease.

    Science.gov (United States)

    Cortés-Ramírez, Dionisio-Alejandro; Rodríguez-Tojo, María-Jose; Coca-Meneses, Juan-Carlos; Marichalar-Mendia, Xabier; Aguirre-Urizar, José-Manuel

    2014-09-01

    The oral lichenoid disease (OLD) includes different chronic inflammatory processes such as oral lichen planus (OLP) and oral lichenoid lesions (OLL), both entities with controversial diagnosis and malignant potential. Epidermal growth factor receptor (EFGR) is an important oral carcinogenesis biomarker and overexpressed in several oral potentially malignant disorders. To analyze the EGFR expression in the OLD to find differences between OLP and OLL, and to correlate it with the main clinical and pathological features. Forty-four OLD cases were studied and classified according to their clinical (Group C1: only papular lesions / Group C2: papular and other lesions) and histopathological features (Group HT: OLP-typical / Group HC: OLP-compatible) based in previous published criteria. Standard immunohistochemical identification of EGFR protein was performed. Comparative and descriptive statistical analyses were performed. Thirty-five cases (79.5%) showed EGFR overexpression without significant differences between clinical and histopathological groups (p<0.05). Histological groups showed significant differences in the EGFR expression pattern (p=0.016). Conlusions: All OLD samples showed high EGFR expression. The type of clinical lesion was not related with EGFR expression; however, there are differences in the EGFR expression pattern between histological groups that may be related with a different biological profile and malignant risk.

  2. Involvement of Fibroblast Growth Factor Receptor Genes in Benign Prostate Hyperplasia in a Korean Population

    Directory of Open Access Journals (Sweden)

    Hae Jeong Park

    2013-01-01

    Full Text Available Fibroblast growth factors (FGFs and their receptors (FGFRs have been implicated in prostate growth and are overexpressed in benign prostatic hyperplasia (BPH. In this study, we investigated whether single nucleotide polymorphisms (SNPs of the FGFR genes (FGFR1 and FGFR2 were associated with BPH and its clinical phenotypes in a population of Korean men. We genotyped four SNPs in the exons of FGFR1 and FGFR2 (rs13317 in FGFR1; rs755793, rs1047100, and rs3135831 in FGFR2 using direct sequencing in 218 BPH patients and 213 control subjects. No SNPs of FGFR1 or FGFR2 genes were associated with BPH. However, analysis according to clinical phenotypes showed that rs1047100 of FGFR2 was associated with prostate volume in BPH in the dominant model (GA/AA versus GG, P = 0.010. In addition, a significant association was observed between rs13317 of FGFR1 and international prostate symptom score (IPSS in the additive (TC versus CC versus TT, P = 0.0022 and dominant models (TC/CC versus TT, P = 0.005. Allele frequency analysis also showed significant association between rs13317 and IPSS (P = 0.005. These results suggested that FGFR genes could be related to progression of BPH.

  3. Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

    International Nuclear Information System (INIS)

    Jorge, S.E.D.C.; Kobayashi, S.S.; Costa, D.B.

    2014-01-01

    Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC

  4. Systemic Hypoxia Changes the Organ-Specific Distribution of Vascular Endothelial Growth Factor and Its Receptors

    Science.gov (United States)

    Marti, Hugo H.; Risau, Werner

    1998-12-01

    Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.

  5. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

    Science.gov (United States)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Optimization of 1H-indazol-3-amine derivatives as potent fibroblast growth factor receptor inhibitors.

    Science.gov (United States)

    Cui, Jing; Peng, Xia; Gao, Dingding; Dai, Yang; Ai, Jing; Li, Yingxia

    2017-08-15

    Fibroblast growth factor receptor (FGFR) is a potential target for cancer therapy because of its critical role in promoting cancer formation and progression. In a continuing effort to improve the cellular activity of hit compound 7r bearing an indazole scaffold, which was previously discovered by our group, several compounds harnessing fluorine substituents were designed, synthesized and biological evaluated. Besides, the region extended out to the ATP binding pocket toward solvent was also explored. Among them, compound 2a containing 2,6-difluoro-3-methoxyphenyl residue exhibited the most potent activities (FGFR1: less than 4.1nM, FGFR2: 2.0±0.8nM). More importantly, compound 2a showed an improved antiproliferative effect against KG1 cell lines and SNU16 cell lines with IC 50 values of 25.3±4.6nM and 77.4±6.2nM respectively. Copyright © 2017. Published by Elsevier Ltd.

  7. Catalase reverses tumorigenicity in a malignant cell line by an epidermal growth factor receptor pathway.

    Science.gov (United States)

    Finch, Joanne S; Tome, Margaret E; Kwei, Kevin A; Bowden, G Tim

    2006-03-01

    We have used a keratinocyte in vivo/in vitro cell model to test the hypothesis that hydrogen peroxide acts as a signaling molecule, contributing to proliferation and tumorigenesis. A cell line, 6M90, that produces squamous cell carcinoma (SCC), has high levels of ROS and low levels of catalase. A new cell line, MTOC2, generated from parental 6M90 cells by introduction of a Tet-responsive catalase transgene, effectively expressed higher peroxisomal catalase. Increased catalase expression diminished constitutive ROS and enhanced viability after treatment with hydrogen peroxide. Protein tyrosine phosphatase activity was higher in the MTOC2 cells with high catalase, consistent with detection of a lower level of phosphorylation at tyrosine 1068 of the epidermal growth factor receptor (EGF-R). Transcription of downstream c-fos, AP-1 transactivation and cell proliferation were higher in the low catalase cells. An EGF-R inhibitor, AG1478, blocks the higher AP-1 transactivation and cell proliferation of the low catalase 6M90 cells. Tumorigenesis in SCID mice was greatly diminished in the high catalase cells. Our data suggest that hydrogen peroxide functions as a signaling molecule that can modulate activity of a protein tyrosine phosphatase/(s) resulting in phosphorylation of tryrosine/(s) on the EGF-R. Therefore, catalase acts as a tumor-suppressor gene in part by decreasing EGF-R signaling.

  8. Molecular Imaging of Hepatocellular Carcinoma Xenografts with Epidermal Growth Factor Receptor Targeted Affibody Probes

    Directory of Open Access Journals (Sweden)

    Ping Zhao

    2013-01-01

    Full Text Available Hepatocellular carcinoma (HCC is a highly aggressive and lethal cancer. It is typically asymptomatic at the early stage, with only 10%–20% of HCC patients being diagnosed early enough for appropriate surgical treatment. The delayed diagnosis of HCC is associated with limited treatment options and much lower survival rates. Therefore, the early and accurate detection of HCC is crucial to improve its currently dismal prognosis. The epidermal growth factor receptor (EGFR has been reported to be involved in HCC tumorigenesis and to represent an attractive target for HCC imaging and therapy. In this study, an affibody molecule, Ac-Cys-ZEGFR:1907, targeting the extracellular domain of EGFR, was used for the first time to assess its potential to detect HCC xenografts. By evaluating radio- or fluorescent-labeled Ac-Cys-ZEGFR:1907 as a probe for positron emission tomography (PET or optical imaging of HCC, subcutaneous EGFR-positive HCC xenografts were found to be successfully imaged by the PET probe. Thus, affibody-based PET imaging of EGFR provides a promising approach for detecting HCC in vivo.

  9. Epidermal Growth Factor Receptor Is Related to Poor Survival in Glioblastomas: Single-Institution Experience

    Science.gov (United States)

    Choi, Youngmin; Lee, Hyung-Sik; Hur, Won-Joo; Sung, Ki-Han; Kim, Ki-Uk; Choi, Sun-Seob; Kim, Su-Jin; Kim, Dae-Cheol

    2013-01-01

    Purpose There are conflicting results surrounding the prognostic significance of epidermal growth factor receptor (EGFR) status in glioblastoma (GBM) patients. Accordingly, we attempted to assess the influence of EGFR expression on the survival of GBM patients receiving postoperative radiotherapy. Materials and Methods Thirty three GBM patients who had received surgery and postoperative radiotherapy at our institute, between March 1997 and February 2006, were included. The evaluation of EGFR expression with immunohistochemistry was available for 30 patients. Kaplan-Meier survival analysis and Cox regression were used for statistical analysis. Results EGFR was expressed in 23 patients (76.7%), and not expressed in seven (23.3%). Survival in EGFR expressing GBM patients was significantly less than that in non-expressing patients (median survival: 12.5 versus 17.5 months, p=0.013). Patients who received more than 60 Gy showed improved survival over those who received up to 60 Gy (median survival: 17.0 versus 9.0 months, p=0.000). Negative EGFR expression and a higher radiation dose were significantly correlated with improved survival on multivariate analysis. Survival rates showed no differences according to age, sex, and surgical extent. Conclusion The expression of EGFR demonstrated a significantly deleterious effect on the survival of GBM patients. Therefore, approaches targeting EGFR should be considered in potential treatment methods for GBM patients, in addition to current management strategies. PMID:23225805

  10. Radiogenomic correlation in lung adenocarcinoma with epidermal growth factor receptor mutations: Imaging features and histological subtypes

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Su Jin [Seoul National University Bundang Hospital, Department of Radiology, Seongnam-si, Gyeonggi-do (Korea, Republic of); Hanyang University, Department of Radiology, School of Medicine, Seoul (Korea, Republic of); Kim, Tae Jung [Seoul National University Bundang Hospital, Department of Radiology, Seongnam-si, Gyeonggi-do (Korea, Republic of); Samsung Medical Center, Department of Radiology, Seoul (Korea, Republic of); Choi, Yo Won [Hanyang University, Department of Radiology, School of Medicine, Seoul (Korea, Republic of); Park, Jeong-Soo [Dankook Universicity, Department of Biochemistry, College of Medicine, Cheonan (Korea, Republic of); Chung, Jin-Haeng [Seoul National University Bundang Hospital, Department of Pathology, Seongnam-si, Gyeonggi-do (Korea, Republic of); Lee, Kyung Won [Seoul National University Bundang Hospital, Department of Radiology, Seongnam-si, Gyeonggi-do (Korea, Republic of)

    2016-10-15

    To correlate imaging features of resected lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutation and the IASLC/ATS/ERS classification histological subtypes. In 250 consecutive patients with resected lung adenocarcinoma, EGFR mutation status was correlated with demographics, imaging features including ground-glass opacity (GGO) proportion and the IASLC/ATS/ERS classification histological subtypes. EGFR mutations were significantly more frequent in women (54.5 % vs. 38.1 %, p = 0.011) and in never-smokers (54.7 % vs. 35.3 %, p = 0.003). GGO proportion was significantly higher in tumours with EGFR mutation than in those without (30.3 ± 33.8 % vs. 19.0 ± 29.3 %, p = 0.005). EGFR mutation was significantly more frequent in tumours with GGO ≥ 50 % and tumours with any GGO (p = 0.026 and 0.008, respectively). Adenocarcinomas with exon 19 or 21 mutation showed significantly higher GGO proportion than that in EGFR wild-type tumours (p = 0.009 and 0.029, respectively). Absence of GGO was an independent predictor of negative EGFR mutation (odds ratio, 1.81; 95 % confidence interval, 1.16-3.04; p = 0.018). GGO proportion in adenocarcinomas with EGFR mutation was significantly higher than that in EGFR wild-type tumours, and the absence of GGO on CT was an independent predictor of negative EGFR mutation. (orig.)

  11. Dialkoxyquinazolines: Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

    International Nuclear Information System (INIS)

    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.

    2005-01-01

    The epidermal growth factor receptor (EGFR), a long-standing drug development target, is also a desirable target for imaging. Sixteen dialkoxyquinazoline analogs, suitable for labeling with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro assays to ascertain their chemical and biological properties. These characteristics provided the basis for the adoption of a selection schema to identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of the compounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFR tyrosine kinase. All of the analogs inhibited ligand-induced EGFR tyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimated octanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline as well as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the best combination of characteristics that warrant radioisotope labeling and further evaluation in tumor-bearing mice

  12. Prognostic significance of epidermal growth factor receptor (EGFR) over expression in urothelial carcinoma of urinary bladder.

    Science.gov (United States)

    Hashmi, Atif Ali; Hussain, Zubaida Fida; Irfan, Muhammad; Khan, Erum Yousuf; Faridi, Naveen; Naqvi, Hanna; Khan, Amir; Edhi, Muhammad Muzzammil

    2018-06-07

    Epidermal growth factor receptor (EGFR) has been shown to have abnormal expression in many human cancers and is considered as a marker of poor prognosis. Frequency of over expression in bladder cancer has not been studied in our population; therefore we aimed to evaluate the frequency and prognostic significance of EGFR immunohistochemical expression in locoregional population. We performed EGFR immunohistochemistry on 126 cases of bladder cancer and association of EGFR expression with tumor grade, lamina propria invasion, deep muscle invasion and recurrence of disease was evaluated. High EGFR expression was noted in 26.2% (33 cases), 15.1% (19 cases) and 58.7% (74 cases) revealed low and no EGFR expression respectively. Significant association of EGFR expression was noted with tumor grade, lamina propria invasion, deep muscle invasion and recurrence status while no significant association was seen with age, gender and overall survival. Kaplan- Meier curves revealed significant association of EGFR expression with recurrence while no significant association was seen with overall survival. Significant association of EGFR overexpression with tumor grade, muscularis propria invasion and recurrence signifies its prognostic value; therefore EGFR can be used as a prognostic biomarker in Urothelial bladder carcinoma.

  13. Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Jeffrey C Lee

    2006-12-01

    Full Text Available Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy.Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132 of glioblastomas and 12.5% (1/8 of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors.Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

  14. Global Developmental Gene Programing Involves a Nuclear Form of Fibroblast Growth Factor Receptor-1 (FGFR1.

    Directory of Open Access Journals (Sweden)

    Christopher Terranova

    Full Text Available Genetic studies have placed the Fgfr1 gene at the top of major ontogenic pathways that enable gastrulation, tissue development and organogenesis. Using genome-wide sequencing and loss and gain of function experiments the present investigation reveals a mechanism that underlies global and direct gene regulation by the nuclear form of FGFR1, ensuring that pluripotent Embryonic Stem Cells differentiate into Neuronal Cells in response to Retinoic Acid. Nuclear FGFR1, both alone and with its partner nuclear receptors RXR and Nur77, targets thousands of active genes and controls the expression of pluripotency, homeobox, neuronal and mesodermal genes. Nuclear FGFR1 targets genes in developmental pathways represented by Wnt/β-catenin, CREB, BMP, the cell cycle and cancer-related TP53 pathway, neuroectodermal and mesodermal programing networks, axonal growth and synaptic plasticity pathways. Nuclear FGFR1 targets the consensus sequences of transcription factors known to engage CREB-binding protein, a common coregulator of transcription and established binding partner of nuclear FGFR1. This investigation reveals the role of nuclear FGFR1 as a global genomic programmer of cell, neural and muscle development.

  15. Brain metastasis in human epidermal growth factor receptor 2-positive breast cancer: from biology to treatment

    Energy Technology Data Exchange (ETDEWEB)

    Koo, Tae Ryool [Dept. of Radiation Oncology, Hallym University Chuncheon Sacred Heart Hospital, Chuncheon (Korea, Republic of); Kim, In Ah [Dept. of Radiation Oncology, Seoul National University Bundang Hospital, Seongnam (Korea, Republic of)

    2016-03-15

    Overexpression of human epidermal growth factor receptor 2 (HER2) is found in about 20% of breast cancer patients. With treatment using trastuzumab, an anti-HER2 monoclonal antibody, systemic control is improved. Nonetheless, the incidence of brain metastasis does not be improved, rather seems to be increased in HER2-positive breast cancer. The mainstay treatment for brain metastases is radiotherapy. According to the number of metastatic lesions and performance status of patients, radiosurgery or whole brain radiotherapy can be performed. The concurrent use of a radiosensitizer further improves intracranial control. Due to its large molecular weight, trastuzumab has a limited ability to cross the blood-brain barrier. However, small tyrosine kinase inhibitors such as lapatinib, has been noted to be a promising agent that can be used as a radiosensitizer to affect HER2-positive breast cancer. This review will outline general management of brain metastases and will focus on preclinical findings regarding the radiosensitizing effect of small molecule HER2 targeting agents.

  16. Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity.

    Science.gov (United States)

    Yao, Yongxue; Harrison, Kathleen A; Al-Hassani, Mohammed; Murphy, Robert C; Rezania, Samin; Konger, Raymond L; Travers, Jeffrey B

    2012-03-16

    To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.

  17. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-02-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

  18. Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

    2000-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 1 ), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 μg/100 μCi of 99m Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of 99m Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14±2.50 %ID/g, 5.06±2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy

  19. Biologic effects of platelet-derived growth factor receptor α blockade in uterine cancer.

    Science.gov (United States)

    Roh, Ju-Won; Huang, Jie; Hu, Wei; Yang, XiaoYun; Jennings, Nicholas B; Sehgal, Vasudha; Sohn, Bo Hwa; Han, Hee Dong; Lee, Sun Joo; Thanapprapasr, Duangmani; Bottsford-Miller, Justin; Zand, Behrouz; Dalton, Heather J; Previs, Rebecca A; Davis, Ashley N; Matsuo, Koji; Lee, Ju-Seog; Ram, Prahlad; Coleman, Robert L; Sood, Anil K

    2014-05-15

    Platelet-derived growth factor receptor α (PDGFRα) expression is frequently observed in many kinds of cancer and is a candidate for therapeutic targeting. This preclinical study evaluated the biologic significance of PDGFRα and PDGFRα blockade (using a fully humanized monoclonal antibody, 3G3) in uterine cancer. Expression of PDGFRα was examined in uterine cancer clinical samples and cell lines, and biologic effects of PDGFRα inhibition were evaluated using in vitro (cell viability, apoptosis, and invasion) and in vivo (orthotopic) models of uterine cancer. PDGFRα was highly expressed and activated in uterine cancer samples and cell lines. Treatment with 3G3 resulted in substantial inhibition of PDGFRα phosphorylation and of downstream signaling molecules AKT and mitogen-activated protein kinase (MAPK). Cell viability and invasive potential of uterine cancer cells were also inhibited by 3G3 treatment. In orthotopic mouse models of uterine cancer, 3G3 monotherapy had significant antitumor effects in the PDGFRα-positive models (Hec-1A, Ishikawa, Spec-2) but not in the PDGFRα-negative model (OVCA432). Greater therapeutic effects were observed for 3G3 in combination with chemotherapy than for either drug alone in the PDGFRα-positive models. The antitumor effects of therapy were related to increased apoptosis and decreased proliferation and angiogenesis. These findings identify PDGFRα as an attractive target for therapeutic development in uterine cancer. ©2014 American Association for Cancer Research.

  20. Targeting the Insulin-Like Growth Factor 1 Receptor in Ewing's Sarcoma: Reality and Expectations

    Directory of Open Access Journals (Sweden)

    David Olmos

    2011-01-01

    Full Text Available Ewing's sarcoma family of tumours comprises a group of very aggressive diseases that are potentially curable with multimodality treatment. Despite the undoubted success of current treatment, approximately 30% of patients will relapse and ultimately die of disease. The insulin-like growth factor 1 receptor (IGF-1R has been implicated in the genesis, growth, proliferation, and the development of metastatic disease in Ewing's sarcoma. In addition, IGF1-R has been validated, both in vitro and in vivo, as a potential therapeutic target in Ewing's sarcoma. Phase I studies of IGF-1R monoclonal antibodies reported several radiological and clinical responses in Ewing's sarcoma patients, and initial reports of several Phase II studies suggest that about a fourth of the patients would benefit from IGF-1R monoclonal antibodies as single therapy, with approximately 10% of patients achieving objective responses. Furthermore, these therapies are well tolerated, and thus far severe toxicity has been rare. Other studies assessing IGF-1R monoclonal antibodies in combination with traditional cytotoxics or other targeted therapies are expected. Despite, the initial promising results, not all patients benefit from IGF-1R inhibition, and consequently, there is an urgent need for the identification of predictive markers of response.

  1. Targeting the Insulin-Like Growth Factor 1 Receptor in Ewing's Sarcoma: Reality and Expectations

    Science.gov (United States)

    Olmos, David; Martins, Ana Sofia; Jones, Robin L.; Alam, Salma; Scurr, Michelle; Judson, Ian R.

    2011-01-01

    Ewing's sarcoma family of tumours comprises a group of very aggressive diseases that are potentially curable with multimodality treatment. Despite the undoubted success of current treatment, approximately 30% of patients will relapse and ultimately die of disease. The insulin-like growth factor 1 receptor (IGF-1R) has been implicated in the genesis, growth, proliferation, and the development of metastatic disease in Ewing's sarcoma. In addition, IGF1-R has been validated, both in vitro and in vivo, as a potential therapeutic target in Ewing's sarcoma. Phase I studies of IGF-1R monoclonal antibodies reported several radiological and clinical responses in Ewing's sarcoma patients, and initial reports of several Phase II studies suggest that about a fourth of the patients would benefit from IGF-1R monoclonal antibodies as single therapy, with approximately 10% of patients achieving objective responses. Furthermore, these therapies are well tolerated, and thus far severe toxicity has been rare. Other studies assessing IGF-1R monoclonal antibodies in combination with traditional cytotoxics or other targeted therapies are expected. Despite, the initial promising results, not all patients benefit from IGF-1R inhibition, and consequently, there is an urgent need for the identification of predictive markers of response. PMID:21647361

  2. Tumor necrosis factor-alpha and its receptors in epithelial ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Jacek Nikliński

    2010-05-01

    Full Text Available The aim of the present study was to characterize the expression pattern of tumor necrosis factor (TNF-alpha and its receptors (TNF-Rs in the epithelial ovarian cancer (EOC and compare these results with the outcome of 126 patients. Presence of TNF-alpha, TNFR-1 and TNFR-2 were studied by Western blotting and immunohistochemistry. The proportion of samples positive for TNF-alpha and TNF-R2 was higher in epithelial ovarian cancer patients than in benign ovarian diseases (p<0.001 and p=0.016, respectively. Immunostaining intensity of TNF-R2 were correlated with tumor stage (p<0.001 and with reduced mean survival time (MST (p=0.002. The results of the present study suggested that tissue expression of TNF-R2 in epithelial ovarian cancer was correlated with the highest risk of cancer progression. Thus, the clinical value of activated TNF system in epithelial ovarian cancer needs to be further investigated.

  3. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule

    Directory of Open Access Journals (Sweden)

    Edyta Petters

    2015-08-01

    Full Text Available Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer.

  4. Aberrant Receptor Internalization and Enhanced FRS2-dependent Signaling Contribute to the Transforming Activity of the Fibroblast Growth Factor Receptor 2 IIIb C3 Isoform*

    Science.gov (United States)

    Cha, Jiyoung Y.; Maddileti, Savitri; Mitin, Natalia; Harden, T. Kendall; Der, Channing J.

    2009-01-01

    Alternative splice variants of fibroblast growth factor receptor 2 (FGFR2) IIIb, designated C1, C2, and C3, possess progressive reduction in their cytoplasmic carboxyl termini (822, 788, and 769 residues, respectively), with preferential expression of the C2 and C3 isoforms in human cancers. We determined that the progressive deletion of carboxyl-terminal sequences correlated with increasing transforming potency. The highly transforming C3 variant lacks five tyrosine residues present in C1, and we determined that the loss of Tyr-770 alone enhanced FGFR2 IIIb C1 transforming activity. Because Tyr-770 may compose a putative YXXL sorting motif, we hypothesized that loss of Tyr-770 in the 770YXXL motif may cause disruption of FGFR2 IIIb C1 internalization and enhance transforming activity. Surprisingly, we found that mutation of Leu-773 but not Tyr-770 impaired receptor internalization and increased receptor stability and activation. Interestingly, concurrent mutations of Tyr-770 and Leu-773 caused 2-fold higher transforming activity than caused by the Y770F or L773A single mutations, suggesting loss of Tyr and Leu residues of the 770YXXL773 motif enhances FGFR2 IIIb transforming activity by distinct mechanisms. We also determined that loss of Tyr-770 caused persistent activation of FRS2 by enhancing FRS2 binding to FGFR2 IIIb. Furthermore, we found that FRS2 binding to FGFR2 IIIb is required for increased FRS2 tyrosine phosphorylation and enhanced transforming activity by Y770F mutation. Our data support a dual mechanism where deletion of the 770YXXL773 motif promotes FGFR2 IIIb C3 transforming activity by causing aberrant receptor recycling and stability and persistent FRS2-dependent signaling. PMID:19103595

  5. The expression of receptors for estrogen and epithelial growth factor in the male rabbit prostate and prostatic urethra following castration

    DEFF Research Database (Denmark)

    Bødker, A; Balslev, E; Iversen, H G

    1997-01-01

    In the lower urinary tract of the male rabbit, estrogen receptors (ERs) are restricted to the urethra and the prostatic stroma. At present, the function of ERs in these tissues is not known. Epithelial growth factor (EGF) stimulates proliferation of epidermal and epithelial tissues, and several...... were included as controls. In the control group, ERs were found in the urothelial lining and lamina propria of the prostatic urethra, and in the prostatic stroma. EGF receptors were demonstrated in the epithelial lining of the prostatic urethra and the glandular epithelium of the prostate. Following...... castration, the expression of ERs, assessed as the increase in the number of positively stained specimens, increased significantly in the lamina propria of the prostatic urethra and the prostatic stroma. EGF receptor expression increased significantly in the epithelial lining of the prostatic urethra...

  6. Screening of the transcriptional regulatory regions of vascular endothelial growth factor receptor 2 (VEGFR2 in amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Hartley Judith

    2007-04-01

    Full Text Available Abstract Background Vascular endothelial growth factor (VEGF has neurotrophic activity which is mediated by its main agonist receptor, VEGFR2. Dysregulation of VEGF causes motor neurone degeneration in a mouse model of amyotrophic lateral sclerosis (ALS, and expression of VEGFR2 is reduced in motor neurones and spinal cord of patients with ALS. Methods We have screened the promoter region and 4 exonic regions of functional significance of the VEGFR2 gene in a UK population of patients with ALS, for mutations and polymorphisms that may affect expression or function of this VEGF receptor. Results No mutations were identified in the VEGFR2 gene. We found no association between polymorphisms in the regulatory regions of the VEGFR2 gene and ALS. Conclusion Mechanisms other than genetic variation may downregulate expression or function of the VEGFR2 receptor in patients with ALS.

  7. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    DEFF Research Database (Denmark)

    Green, M R; Couchman, J R

    1985-01-01

    , the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

  8. Insulin-like growth factor type-1 receptor down-regulation associated with dwarfism in Holstein calves.

    Science.gov (United States)

    Blum, J W; Elsasser, T H; Greger, D L; Wittenberg, S; de Vries, F; Distl, O

    2007-10-01

    Perturbations in endocrine functions can impact normal growth. Endocrine traits were studied in three dwarf calves exhibiting retarded but proportionate growth and four phenotypically normal half-siblings, sired by the same bull, and four unrelated control calves. Plasma 3,5,3'-triiodothyronine and thyroxine concentrations in dwarfs and half-siblings were in the physiological range and responded normally to injected thyroid-releasing hormone. Plasma glucagon concentrations were different (dwarfs, controls>half-siblings; Pcontrols, Pcontrols, P=0.08). Responses of GH to xylazine and to a GH-releasing-factor analogue were similar in dwarfs and half-siblings. Relative gene expression of IGF-1, IGF-2, GH receptor (GHR), insulin receptor, IGF-1 type-1 and -2 receptors (IGF-1R, IGF-2R), and IGF binding proteins were measured in liver and anconeus muscle. GHR mRNA levels were different in liver (dwarfsdwarfism in studied calves.

  9. Retrospective study of adjuvant icotinib in postoperative lung cancer patients harboring epidermal growth factor receptor mutations.

    Science.gov (United States)

    Yao, Shuyang; Zhi, Xiuyi; Wang, Ruotian; Qian, Kun; Hu, Mu; Zhang, Yi

    2016-09-01

    Epidermal growth factor receptor (EGFR) mutations occur in about 50% of Asian patients with non-small cell lung cancer (NSCLC). Patients with advanced NSCLC and EGFR mutations derive clinical benefit from treatment with EGFR-tyrosine kinase inhibitors (TKIs). This study assessed the efficacy and safety of adjuvant icotinib without chemotherapy in EGFR-mutated NSCLC patients undergoing resection of stage IB-IIIA. Our retrospective study enrolled 20 patients treated with icotinib as adjuvant therapy. Survival factors were evaluated by univariate and Cox regression analysis. The median follow-up time was 30 months (range 24-41). At the data cut-off, five patients (25%) had recurrence or metastasis and one patient had died of the disease. The two-year disease-free survival (DFS) rate was 85%. No recurrence occurred in the high-risk stage IB subgroup during the follow-up period. In univariate analysis, the micropapillary pattern had a statistically significant effect on DFS ( P = 0.040). Multivariate logistic regression analysis showed that there was no independent predictor. Drug related adverse events (AEs) occurred in nine patients (45.0%). The most common AEs were skin-related events and diarrhea, but were relatively mild. No grade 3 AEs or occurrences of intolerable toxicity were observed. Icotinib as adjuvant therapy is effective in patients harboring EGFR mutations after complete resection, with an acceptable AE profile. Further trials with larger sample sizes might confirm the efficiency of adjuvant TKI in selected patients. © 2016 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  10. Correlation between 18F Fluorodeoxyglucose uptake and epidermal growth factor receptor mutations in advanced lung cancer

    International Nuclear Information System (INIS)

    Choi, Yun Jung; Cho, Byoung Chul; Jeong, Youg Hyu; Seo, Hyo Jung; Kim, Hyun Jeong; Cho, Arthur; Lee, Jae Hoon; Yun, Mi Jin; Jeon, Tae Joo; Lee, Jong Doo; Kang, Won Jun

    2012-01-01

    Mutations in the epidermal growth factor receptor (EGFR)gene have been identified as potential targets for the treatment and prognostic factors for non small cell lung cancer (NSCLC). We assessed the correlation between fluorodeoxyglucose (FDG) uptake and EGFR mutations, as well as their prognostic implications. A total of 163 patients with pathologically confirmed NSCLC were enrolled (99 males and 64 females; median age, 60 years). All patients underwent FDG positron emission tomography before treatment, and genetic studies of EGFR mutations were performed. The maximum standardized uptake value (SUVmax)of the primary lung cancer was measured and normalized with regard to liver uptake. The SUVmax between the wild type and EGFR mutant groups was compared. Survival was evaluated according to SUVmax and EGFR mutation status. EGFR mutations were found in 57 patients (60.8%). The SUVmax tended to be higher in wild type than mutant tumors, but was not significantly different (11.1±5.7 vs. 9.8±4.4, P=0.103). The SUVmax was significantly lower in patients with an exon 19 mutation than in those with either an exon 21 mutation or wild type (P=0.003 and 0.009, respectively). The EGFR mutation showed prolonged overall survival (OS) compared to wild type tumors (P=0.004). There was no significant difference in survival according to SUVmax. Both OS and progression free survival of patients with a mutation in exon 19 were significant longer than in patients with wild type tumors. In patients with NSCLC, a mutation in exon 19 was associated with a lower SUVmax and is a reliable predictor for good survival

  11. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

    Science.gov (United States)

    Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

    2017-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.