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Sample records for factor receptor single-chain

  1. [Human single chain antibodies directed to tumor necrosis factor].

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    Vikhrova, M A; Batanova, T A; Lebedev, L R; Shingarova, L N; Frank, L A; Kirpichnikov, M P; Tikunova, N V

    2011-01-01

    Six unique phage antibodies to human TNF have been selected from a combinatorial library of human single chain fragment variable. ELISA and Western-blotting was used to study selected phage antibodies binding with TNF. The specificity of selected antibodies was determined by binding with interferon alpha and gamma, bovine serum albumin, ovalbumin and ubiquitin. Two antibodies, sA1 and sB3, were converted into a soluble single-chain antibody form and their affinity was 2.5 and 13.7 nM respectively.

  2. Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli.

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    Sun, Xue-Wen; Wang, Xiao-Hua; Yao, Yan-Bing

    2014-12-01

    Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb.

  3. Purification optimization for a recombinant single-chain variable fragment against type 1 insulin-like growth factor receptor (IGF-1R) by using design of experiment (DoE).

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    Song, Yong-Hong; Sun, Xue-Wen; Jiang, Bo; Liu, Ji-En; Su, Xian-Hui

    2015-12-01

    Design of experiment (DoE) is a statistics-based technique for experimental design that could overcome the shortcomings of traditional one-factor-at-a-time (OFAT) approach for protein purification optimization. In this study, a DoE approach was applied for optimizing purification of a recombinant single-chain variable fragment (scFv) against type 1 insulin-like growth factor receptor (IGF-1R) expressed in Escherichia coli. In first capture step using Capto L, a 2-level fractional factorial analysis and successively a central composite circumscribed (CCC) design were used to identify the optimal elution conditions. Two main effects, pH and trehalose, were identified, and high recovery (above 95%) and low aggregates ratio (below 10%) were achieved at the pH range from 2.9 to 3.0 with 32-35% (w/v) trehalose added. In the second step using cation exchange chromatography, an initial screening of media and elution pH and a following CCC design were performed, whereby the optimal selectivity of the scFv was obtained on Capto S at pH near 6.0, and the optimal conditions for fulfilling high DBC and purity were identified as pH range of 5.9-6.1 and loading conductivity range of 5-12.5 mS/cm. Upon a further gel filtration, the final purified scFv with a purity of 98% was obtained. Finally, the optimized conditions were verified by a 20-fold scale-up experiment. The purities and yields of intermediate and final products all fell within the regions predicted by DoE approach, suggesting the robustness of the optimized conditions. We proposed that the DoE approach described here is also applicable in production of other recombinant antibody constructs.

  4. Single chain human interleukin 5 and its asymmetric mutagenesis for mapping receptor binding sites.

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    Li, J; Cook, R; Dede, K; Chaiken, I

    1996-01-26

    Wild type human (h) interleukin 5 (wt IL5) is composed of two identical peptide chains linked by disulfide bonds. A gene encoding a single chain form of hIL5 dimer was constructed by linking the two hIL5 chain coding regions with Gly-Gly linker. Expression of this gene in COS cells yielded a single chain IL5 protein (sc IL5) having biological activity similar to that of wt IL5, as judged by stimulation of human cell proliferation. Single chain and wt IL5 also had similar binding affinity for soluble IL5 receptor alpha chain, the specificity subunit of the IL5 receptor, as measured kinetically with an optical biosensor. The design of functionally active sc IL5 molecule. Such mutagenesis was exemplified by changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor binding and bioactivity data obtained are consistent with a model in which residues from both IL5 monomers interact with the receptor alpha chain, while the interaction likely is asymmetric due to the intrinsic asymmetry of folded receptor. The results demonstrate a general route to the further mapping of receptor and other binding sites on the surface of human IL5.

  5. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  6. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  7. Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

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    Hojo, Keiko; Hossain, Mohammed Akhter; Tailhades, Julien; Shabanpoor, Fazel; Wong, Lilian L L; Ong-Pålsson, Emma E K; Kastman, Hanna E; Ma, Sherie; Gundlach, Andrew L; Rosengren, K Johan; Wade, John D; Bathgate, Ross A D

    2016-08-25

    Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  9. Single-chain VαVβ T-cell receptors function without mispairing with endogenous TCR chains.

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    Aggen, D H; Chervin, A S; Schmitt, T M; Engels, B; Stone, J D; Richman, S A; Piepenbrink, K H; Baker, B M; Greenberg, P D; Schreiber, H; Kranz, D M

    2012-04-01

    Transduction of exogenous T-cell receptor (TCR) genes into patients' activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These 'mixed' heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vβ single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T-cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.

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

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    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

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

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    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.

  12. Single-Chain VEGF/Cy5.5 Targeting VEGF Receptors to Indicate Atherosclerotic Plaque Instability

    NARCIS (Netherlands)

    Lam, Ming Kai; Al-Ansari, Sali; van Dam, Gooitzen M.; Tio, Rene A.; Breek, Jan-Cees; Slart, Riemer H. J. A.; Hillebrands, Jan-Luuk; Zeebregts, Clark J.

    2013-01-01

    Unstable plaques may cause clinical events. Plaque destabilization results from the synergy between intraplaque angiogenesis and inflammation. Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are considered to be involved in these processes. We investigated the efficacy of the a

  13. Generation of recombinant single-chain antibodies neutralizing the cytolytic activity of vaginolysin, the main virulence factor of Gardnerella vaginalis

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    Pleckaityte Milda

    2011-11-01

    Full Text Available Abstract Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance. Results Single-chain variable fragments of immunoglobulins (scFvs were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G4S4 were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent Kd values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells. Conclusions Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in

  14. Preparation and characterization of anti-tissue factor single-chain variable fragment antibody for cancer diagnosis.

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    Sato, Ryuta; Obonai, Toshifumi; Tsumura, Ryo; Tsumoto, Kouhei; Koga, Yoshikatsu; Yasunaga, Masahiro; Matsumura, Yasuhiro

    2014-12-01

    Tissue factor (TF), which serves as the initiator of the extrinsic blood coagulation cascade, has been found to be overexpressed in various solid tumors, especially brain tumors, pancreatic cancer, and gastric cancer. Overexpression of TF is considered to contribute to the high incidence of thrombotic complications and poor prognosis in patients with such cancers. Therefore, detection or targeting of TF may be a promising approach for the diagnosis and treatment of solid tumors that are known to overexpress the protein. Here, we used the recombinant DNA technology to develop an anti-TF single-chain Fv (scFv) of small size and high affinity for its target. The biochemical characteristics of the anti-TF scFv were evaluated using surface plasmon resonance (SPR) sensing and flow cytometry. The data obtained showed that the affinity of the anti-TF scFv was 2.04 × 10(-8) (KD), and that the protein showed significant binding to the cancer cells. Then, Alexa 647-labeled anti-TF scFv and anti-TF IgG were administered to mice bearing chemically induced spontaneous tumors. The maximum tumor to background ratios of anti-TF scFv and anti-TF IgG were obtained 3 and 24 h after the injections, respectively. This study indicates anti-TF scFv may be suitable as an imaging probe for the diagnosis of solid tumors.

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

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    Ye Qing

    2012-11-01

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

  16. Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor.

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    Sotiriadis, A; Keshavarz, T; Keshavarz-Moore, E

    2001-01-01

    A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1). A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1). A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1). Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment.

  17. A single chain variant of factor VIII Fc fusion protein retains normal in vivo efficacy but exhibits altered in vitro activity.

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    Yang Buyue

    Full Text Available Recombinant factor VIII Fc (rFVIIIFc is a fusion protein consisting of a single B-domain-deleted (BDD FVIII linked recombinantly to the Fc domain of human IgG1 to extend half-life. To determine if rFVIIIFc could be further improved by maintaining the heavy and light chains within a contiguous single chain (SC, we evaluated the activity and function of SC rFVIIIFc, an isoform that is not processed at residue R1648. SC rFVIIIFc showed equivalent activity in a chromogenic assay compared to rFVIIIFc, but approximately 40% activity by the one-stage clotting assay in the presence of von Willebrand Factor (VWF, with full activity in the absence of VWF. Moreover, SC rFVIIIFc demonstrated markedly delayed thrombin-mediated release from VWF, but an activity similar to that of rFVIIIFc upon activation in FXa generation assays. Therefore, the apparent reduction in specific activity in the aPTT assay appears to be primarily due to delayed release of FVIII from VWF. To assess whether stability and activity of SC rFVIIIFc were affected in vivo, a tail vein transection model in Hemophilia A mice was utilized. The results demonstrated similar pharmacokinetic profiles and comparable efficacy for SC rFVIIIFc and rFVIIIFc. Thus, while the single chain configuration did not promote enhanced half-life, it reduced the rate of release of FVIII from VWF required for activation. This impaired release may underlie the observed reduction in the one-stage clotting assay, but does not appear to affect the physiological activity of SC rFVIIIFc.

  18. Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

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    Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A

    2016-05-01

    Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation.

  19. Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

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    Tamura, Masaaki; Yan, Heping; Zegarra-Moro, Ofelia; Edl, Jennifer; Oursler, Stephanie; Chard-Bergstrom, Cindy; Andrews, Gordon; Kanehira, Tsutomu; Takekoshi, Susumu; Mernaugh, Ray

    2008-08-01

    To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

  20. Humanization of an agonistic anti-death receptor 4 single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity.

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    Lee, Seung-Hyun; Park, Dong-Woon; Sung, Eun-Sil; Park, Hye-Ran; Kim, Jin-Kyoo; Kim, Yong-Sung

    2010-01-01

    Development of agonistic monoclonal antibodies (mAbs) against the pro-apoptotic molecule death receptor 4 (DR4) [or tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor 1] is an attractive anti-cancer strategy because of their potential for inducing tumor-specific cell death. In this study, we humanized an agonistic anti-DR4 AY4 scFv raised in mice (mAY4) by grafting the complementarity-determining regions (CDRs) onto a fixed human framework, while preserving the so-called Vernier zone residues, a group of framework (FR) residues directly underneath the CDRs, with the murine residues in the humanized antibody, hAY4. The humanized hAY4 scFv maintained the antigen binding affinity and epitope specificity of mAY4. To investigate how the valence of hAY4 scFv affects DR4-mediated cell death, bivalent and trivalent forms of hAY4 scFv were generated by linking a hinge region to the coiled-coil domain of a dimerizing leucine zipper and trimerizing isoleucine zipper, respectively. Compared to the monovalent and bivalent forms, the trivalent hAY4 scFv induced more potent caspase-dependent apoptotic cell death as evidenced by increased activation of caspase-8 and downstream pro-apoptotic molecules. Our results suggest that like other TNF family receptors, avidity-mediated oligomerization of DR4 augments the receptor-mediated apoptotic cell death by promoting intracellular cell death signaling.

  1. A Mouse Model for Conditional Secretion of Specific Single-Chain Antibodies Provides Genetic Evidence for Regulation of Cortical Plasticity by a Non-cell Autonomous Homeoprotein Transcription Factor.

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    Clémence Bernard

    2016-05-01

    Full Text Available During postnatal life the cerebral cortex passes through critical periods of plasticity allowing its physiological adaptation to the environment. In the visual cortex, critical period onset and closure are influenced by the non-cell autonomous activity of the Otx2 homeoprotein transcription factor, which regulates the maturation of parvalbumin-expressing inhibitory interneurons (PV cells. In adult mice, the maintenance of a non-plastic adult state requires continuous Otx2 import by PV cells. An important source of extra-cortical Otx2 is the choroid plexus, which secretes Otx2 into the cerebrospinal fluid. Otx2 secretion and internalization requires two small peptidic domains that are part of the DNA-binding domain. Thus, mutating these "transfer" sequences also modifies cell autonomous transcription, precluding this approach to obtain a cell autonomous-only mouse. Here, we develop a mouse model with inducible secretion of an anti-Otx2 single-chain antibody to trap Otx2 in the extracellular milieu. Postnatal secretion of this single-chain antibody by PV cells delays PV maturation and reduces plasticity gene expression. Induced adult expression of this single-chain antibody in cerebrospinal fluid decreases Otx2 internalization by PV cells, strongly induces plasticity gene expression and reopens physiological plasticity. We provide the first mammalian genetic evidence for a signaling mechanism involving intercellular transfer of a homeoprotein transcription factor. Our single-chain antibody mouse model is a valid strategy for extracellular neutralization that could be applied to other homeoproteins and signaling molecules within and beyond the nervous system.

  2. An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

    NARCIS (Netherlands)

    Ait-Azzouzene, D.; Verkoczy, L.; Peters, J.H.; Gavin, A.; Skog, P.; Vela, J.L.; Nemazee, D.

    2005-01-01

    Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly

  3. Characterization of a novel single-chain bispecific antibody for retargeting of T cells to tumor cells via the TCR co-receptor CD8.

    Directory of Open Access Journals (Sweden)

    Irene Michalk

    Full Text Available There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs. Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells.

  4. Studies on Single Chain Structure of Konjac Glucomannan

    Institute of Scientific and Technical Information of China (English)

    PANG Jie; SUN Yu-Jing; SUN Yuan-Ming

    2006-01-01

    The simulation is carried out by employing the method of molecular dynamics with the single chain of konjac glucomannan (KGM) in vacuum as the structural model to discuss the factors that affect the single chain structure, the dynamic structure of the chain and the acting forces that maintain the chain structure. The results show that the shape and stability of the chain are affected by the degree of polymerization. As for the KGM with high degree of polymerization, its chain presents random coiling state and its stability declines. Both before and after deacetylation in the process of dynamic motion, the chain of KGM presents random coiling state with periodic variation of extension and coil and demonstrates favorable flexibility, indicating acetyl is not the main factor that affects the shape of chain, whereas dihedral angle and static actions are respectively the key bonding and nonbonding acting forces that influence the single chain conformations in vacuum.

  5. Engineered single chain antibody fragments for radioimmunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Huhalov, A.; Chester, K. A. [Cancer Research UK Imaging and Targeting Group Royal Free, London (United Kingdom). Department of Oncology; University College Medical School Royal Free Campus, London (United Kingdom)

    2004-12-01

    An ideal molecule to deliver radioimmunotherapy (RIT) would be target specific and have prolonged residence time at high concentrations in the tumour with rapid clearance from normal tissues. It would also be non-immunogenic. These features can be rationally introduced into recombinant antibody-based proteins using antibody engineering techniques. This reviews focuses on the use of antibody engineering in the design and development of RIT molecules which have single chain Fv (scFv) antibody fragments as building blocks.

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

    Institute of Scientific and Technical Information of China (English)

    黄一丁; 梁镇和; 冯佑民

    2001-01-01

    To investigate the relationship between the biological activity of recombined single chain insulin and the length of the connecting peptide, we designed and prepared three single chain insulin molecules, namely, PIP, [A]5PIP and [A]10PIP, by site-directed mutagenesis, in which B30 and A1 were linked through dipeptide A-K, heptapeptide A-A-A-A-A-A-K, and dodecapeptide A-A-A-A-A-A-A-A-A-A-A-K, respectively. Their receptor binding capacities were 0.14%, 14.3% and 11.1% of that of insulin respectively and their in vivo biological activities were in consistence with their receptor binding capacity; whereas their growth promoting activities were 17%, 116.3% and 38% of that of insulin. These results suggested the following conclusions. (i) The recombined single chain insulin could also possess the same metabolic and mitogenic function as insulin. (ii) The receptor binding capacity of recombined single chain insulin to insulin receptor was closely related to the length and amino acid composition of the connecting peptide and could change from 0 to 100% of insulin depending on the different connecting peptides. This result further illustrated the necessity of B chain C-terminus swaying away from A chain N-terminus when insulin binds to its receptor. (iii) The mitogenic activity of recombined single chain insulin also depended on the length and the amino acid composition of the connecting peptide and was higher than its metabolic activity.

  7. Single chain Fab (scFab fragment

    Directory of Open Access Journals (Sweden)

    Brenneis Mariam

    2007-03-01

    Full Text Available Abstract Background The connection of the variable part of the heavy chain (VH and and the variable part of the light chain (VL by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. Results Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd and the light chain (LC, resulting in the formation of a single chain Fab fragment (scFab, can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC connecting the constant part 1 of the heavy chain (CH1 and the constant part of the light chain (CL were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies as well as multimers were characterised. Conclusion A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of

  8. Design of an Active Ultrastable Single-chain Insulin Analog

    Science.gov (United States)

    Hua, Qing-xin; Nakagawa, Satoe H.; Jia, Wenhua; Huang, Kun; Phillips, Nelson B.; Hu, Shi-quan; Weiss, Michael A.

    2008-01-01

    Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 ± 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (ΔΔGu = 0.7 ± 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 ± 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts ValA3 at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 α-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world. PMID:18332129

  9. 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.

  10. Discovery of high potency, single-chain insulin analogs with a shortened B-chain and nonpeptide linker.

    Science.gov (United States)

    Kaur, Zachary P; Ochman, Alexander R; Mayer, John P; Gelfanov, Vasily M; DiMarchi, Richard D

    2013-08-16

    A series of novel, single chain insulin analogs containing polyethylene glycol based connecting segments were synthesized by native chemical ligation and tested for biological activity. While the full length single chain insulin analogs exhibited low potency, deletion of amino acids B26-B30 unexpectedly generated markedly higher activity. This observation is unprecedented in all previous studies of single chain insulin analogs and is consistent with the presumption that in the native hormone this sequence must translocate to achieve high potency insulin receptor interaction. Optimization of the sequence yielded an insulin analog with potency and selectivity comparable to that of native insulin. These results establish a basis for discovery of novel higher potency, single chain insulin analogs of shortened length.

  11. 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

  12. ELASTIC BEHAVIOR OF PROTEIN-LIKE SINGLE CHAIN

    Institute of Scientific and Technical Information of China (English)

    Wei-qi Yi; Lin-xi Zhang

    2005-01-01

    The conformational properties and elastic behaviors of protein-like single chains in the process of tensile elongation were investigated by means of Monte Carlo method. The sequences of protein-like single chains contain two types of residues: hydrophobic (H) and hydrophilic (P). The average conformations and thermodynamics statistical properties of protein-like single chains with various elongation ratio λ were calculated. It was found that the mean-square end-to-end distance r increases with elongation ratio,λ. The tensor eigenvalues ratio of : decreases with elongation ratio λ for short (HP)x protein-like polymers, however, the ratio of : increases with elongation ratioλ,especially for long (H)x sequence. Average energy per bond increases with elongation ratioλ, especially for(H)x protein-like single chains. Helmholtz free energy per bond also increases with elongation ratioλ. Elastic force (f), energy contribution to force (fU) and entropy contribution to force (fs) for different protein-like single chains were also calculated.These investigations may provide some insights into elastic behaviors of proteins.

  13. Fluobodies : green fluorescent single-chain Fv fusion proteins

    NARCIS (Netherlands)

    Griep, R.A.; Twisk, van C.; Wolf, van der J.M.; Schots, A.

    1999-01-01

    An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selec

  14. Expression, characterization, and evaluation of a RANK-binding single chain fraction variable: an osteoclast targeting drug delivery strategy.

    Science.gov (United States)

    Newa, Madhuri; Lam, Michael; Bhandari, Krishna Hari; Xu, Biwen; Doschak, Michael R

    2014-01-06

    A single chain Fraction variable (scFv) employs antibody-like target recognition specificity. Osteoclasts, responsible for bone resorption, express Receptor Activator of Nuclear factor Kappa B (RANK) receptors. This study aimed to express, characterize, and evaluate scFv against RANK receptors that may serve as a platform to target osteoclasts. Using phage display technology, scFv against RANK receptor was expressed and characterized by DNA sequencing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption-ionization time-of-flight (MALDI TOF), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunocytochemistry. The potential for cytotoxicity was evaluated using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, and its cross reactivity was evaluated using ELISA. Osteoclast-like cells were generated from RAW 264.7 cells, and the osteoclast targeting ability of scFv was evaluated using immunocytochemistry. ScFv's antiresorptive efficacy was studied using a tartrate-resistant acid phosphatase (TRAP) assay and resorption assay. Anti-RANK scFv was successfully expressed and characterized. No cross reactivity with other tumor necrosis factor receptor (TNFR) members and no cytotoxic effect on a non-RANK bearing cell line were observed. It showed specificity toward a RANK receptor and an inhibitory effect on osteoclast activity. With the increase in development trends for biologics as therapeutics and growing knowledge on the importance of osteoclast targeted therapy, this study may provide a drug delivery strategy to target osteoclasts, thereby leading to a promising therapy for resorptive bone diseases.

  15. De novo design of a single-chain diphenylporphyrin metalloprotein.

    Science.gov (United States)

    Bender, Gretchen M; Lehmann, Andreas; Zou, Hongling; Cheng, Hong; Fry, H Christopher; Engel, Don; Therien, Michael J; Blasie, J Kent; Roder, Heinrich; Saven, Jeffrey G; DeGrado, William F

    2007-09-01

    We describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV-vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization, and introduction of spectroscopic probes.

  16. Chiral imprinting of diblock copolymer single-chain particles.

    Science.gov (United States)

    Njikang, Gabriel; Liu, Guojun; Hong, Liangzhi

    2011-06-07

    This Article reports the molecular imprinting of polymer single-chain particles that have a radius ∼3.7 nm. For this, the template L-phenylalanine anilide or L-ΦAA and a diblock copolymer PtBA-b-P(CEMA-r-CA) were used. Here, PtBA denotes poly(tert-butyl acrylate), and P(CEMA-r-CA) denotes a random block consisting of cinnamoyloxyethyl methacrylate (CEMA) and carboxyl-bearing (CA) units. In CHCl(3)/cyclohexane (CHX) with 64 vol % of CHX or at f(CHX) = 64%, a block-selective solvent for PtBA, PtBA-b-P(CEMA-r-CA) formed spherical micelles. The core consisted of the insoluble P(CEMA-r-CA) block and L-ΦAA, which complexed with the CA groups. Pumping slowly this micellar solution into stirred CHCl(3)/(CHX) at f(CHX) = 64% triggered micelle dissociation into single-chain micelles, which comprised presumably a solubilized PtBA tail and a collapsed P(CEMA-r-CA)/L-ΦAA head. Because the solvent reservoir was under constant UV irradiation, the photo-cross-linkable units in the P(CEMA-r-CA) head cross-linked, and the single-chain micelles were converted into cross-linked single-chain micelles or tadpoles. Synchronizing the micelle addition and photoreaction rates allowed the preparation, from this protocol, of essentially pure tadpoles at high final polymer concentrations. Imprinted tadpoles were procured after L-ΦAA was extracted from the tadpole heads. Under optimized conditions, the produced imprinted tadpoles had exceptionally high binding capacity and high selectivity for L-ΦAA. In addition, the rates of L-ΦAA release from and rebinding by the particles were high.

  17. Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

    Science.gov (United States)

    Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

    2011-11-15

    Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that

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

    Science.gov (United States)

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

    2014-12-01

    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.

  19. Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

    Institute of Scientific and Technical Information of China (English)

    Fangfang Li; Fanping Meng; Quanxin Jin; Changyuan Sun; Yingxin Li; Honghua Li; Songzhu Jin

    2014-01-01

    Single-chain variable domain fragment (scFv) 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.

  20. The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells.

    Science.gov (United States)

    Siqin; Minagawa, Itaru; Okuno, Mitsutoshi; Yamada, Kimihiko; Sugawara, Yasushi; Nagura, Yoshio; Hamano, Koh-Ichi; Park, Enoch Y; Sasada, Hiroshi; Kohsaka, Tetsuya

    2013-09-01

    Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yoshikawa, Kenichi; Yoshinaga, Natsuhiko [Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502 (Japan)

    2005-08-10

    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.

  2. Single chain stochastic polymer modeling at high strain rates.

    Energy Technology Data Exchange (ETDEWEB)

    Harstad, E. N. (Eric N.); Harlow, Francis Harvey,; Schreyer, H. L.

    2001-01-01

    Our goal is to develop constitutive relations for the behavior of a solid polymer during high-strain-rate deformations. In contrast to the classic thermodynamic techniques for deriving stress-strain response in static (equilibrium) circumstances, we employ a statistical-mechanics approach, in which we evolve a probability distribution function (PDF) for the velocity fluctuations of the repeating units of the chain. We use a Langevin description for the dynamics of a single repeating unit and a Lioville equation to describe the variations of the PDF. Moments of the PDF give the conservation equations for a single polymer chain embedded in other similar chains. To extract single-chain analytical constitutive relations these equations have been solved for representative loading paths. By this process we discover that a measure of nonuniform chain link displacement serves this purpose very well. We then derive an evolution equation for the descriptor function, with the result being a history-dependent constitutive relation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Surinder Batra, Ph D

    2006-02-27

    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

  4. 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.

  5. CXCR4 and CXCL12 (SDF-1) in prostate cancer: inhibitory effects of human single chain Fv antibodies.

    Science.gov (United States)

    Vaday, Gayle G; Hua, Shao-Bing; Peehl, Donna M; Pauling, Michelle H; Lin, Yu-Huei; Zhu, Li; Lawrence, Diana M; Foda, Hussein D; Zucker, Stanley

    2004-08-15

    Metastasis is a major cause of morbidity in prostate cancer (PCa). Several studies have shown that the chemokine receptor CXCR4 and its ligand, CXCL12 (stromal cell-derived factor-1), regulate tumor cell metastasis to specific organs. Recently, it was demonstrated that CXCL12 enhances PCa cell adhesion, migration, and invasion, implicating CXCR4 in PCa metastasis. In this study, we examined the inhibitory effects of anti-CXCR4 antibodies on CXCL12-mediated PCa cell activities. We developed fully human single chain Fv antibodies (scFv), Ab124 and Ab125, against CXCR4 using the yeast two-hybrid system. We performed immunofluorescent staining, flow cytometry, and ELISA-binding assays to measure scFv binding to PCa cells. We also examined the effects of scFv on CXCL12-mediated calcium mobilization, cell migration, and invasion. Our results confirmed that PCa cell lines express cell-surface CXCR4. Real-time quantitative reverse transcription-PCR and immunohistochemical staining also verified that CXCR4 is expressed in primary cultures of prostate epithelial cells from adenocarcinomas and in human prostate tissues. Ab124 and Ab125 demonstrated specific binding to PCa cell lines by flow cytometry and in binding assays. Preincubation with scFv resulted in significant reduction of CXCL12-induced calcium mobilization in PC-3 and LNCaP cells. Ab124 and Ab125 also inhibited PCa cell migration toward CXCL12, as well as invasion through extracellular matrix gels. Ab124 and Ab125 inhibit CXCL12-mediated cellular activities by binding the receptor CXCR4. Recombinant scFv are an efficient mode of targeting tumor antigens. Considering the high incidence of PCa, the development of fully human scFv may be a useful therapeutic approach in the prevention and treatment of PCa metastasis.

  6. Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

    Science.gov (United States)

    Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

    2017-01-01

    Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

  7. Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications.

    Science.gov (United States)

    Hua, Qing-xin; Nakagawa, Satoe H; Jia, Wenhua; Huang, Kun; Phillips, Nelson B; Hu, Shi-quan; Weiss, Michael A

    2008-05-23

    Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 +/- 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (DeltaDeltaG(u) = 0.7 +/- 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 +/- 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts Val(A3) at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 alpha-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.

  8. Targeted elimination of activated hepatic stellate cells by an anti-epidermal growth factor-receptor single chain fragment variable antibody-tumor necrosis factor-related apoptosis-inducing ligand (scFv425-sTRAIL)

    NARCIS (Netherlands)

    Arabpour, Mohammad; Poelstra, Klaas; Helfrich, Wijnand; Bremer, Edwin; Haisma, Hidde J.

    2014-01-01

    BackgroundProgressive liver fibrosis is the result of chronic liver injury and is characterized by the excessive accumulation of extracellular matrix that may result in liver failure. Activated hepatic stellate cells are known to play a central role in this process and their elimination is a crucial

  9. A single-chain Fv reactive with the Goodpasture antigen.

    Science.gov (United States)

    Ross, C N; Turner, N; Savage, P; Cashman, S J; Spooner, R A; Pusey, C D

    1996-06-01

    Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

  10. Secretory expression of α single-chain insulin precursor in yeast and its conversion into human insulin

    Institute of Scientific and Technical Information of China (English)

    张友尚; 胡红明; 蔡若蓉; 冯佑民; 朱尚权; 贺潜斌; 唐月华; 徐明华; 许英镐; 张新堂; 刘滨; 梁镇和

    1996-01-01

    A synthetic single-chain porcine insulin precursor (PIP) gene and an α-mating factor leader sequence (αMFL) gene obtained by the PCR method are inserted between the promoter and 3’-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/α MFL-PIP. The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cererisiae transformed by pVT102-U/αMFL-PIP. The precursor is purified and converted into human insulin by tryptic transpeptidation. The purified human insulin is fully active and can be crystallized. The overall yield of human insulin is 25 mg per liter of culture medium.

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

    OpenAIRE

    Oliinyk O. S.; Kaberniuk A. A.; Kolibo D. V.; Komisarenko S. V.

    2014-01-01

    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, an...

  12. Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies.

    Science.gov (United States)

    Prechl, J; Tchorbanov, A; Horváth, A; Baiu, D C; Hazenbos, W; Rajnavölgyi, E; Kurucz, I; Capel, P J; Erdei, A

    1999-05-01

    Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

  13. Generation and characterization of a single-chain anti-EphA2 antibody.

    Science.gov (United States)

    Goldgur, Yehuda; Susi, Petri; Karelehto, Eveliina; Sanmark, Hanna; Lamminmäki, Urpo; Oricchio, Elisa; Wendel, Hans-Guido; Nikolov, Dimitar B; Himanen, Juha P

    2014-12-01

    Recombinant antibody phage library technology provides multiple advantages, including that human antibodies can be generated against proteins that are highly conserved between species. We used this technology to isolate and characterize an anti-EphA2 single-chain antibody. We show that the antibody binds the antigen with 1:1 stoichiometry and has high specificity for EphA2. The crystal structure of the complex reveals that the antibody targets the same receptor surface cavity as the ephrin ligand. Specifically, a lengthy CDR-H3 loop protrudes deep into the ligand-binding cavity, with several hydrophobic residues at its tip forming an anchor-like structure buried within the hydrophobic Eph pocket, in a way similar to the ephrin receptor-binding loop in the Eph/ephrin structures. Consequently, the antibody blocks ephrin binding to EphA2. Furthermore, it induces apoptosis and reduces cell proliferation in lymphoma cells lines. Since Ephs are important mediators of tumorigenesis, such antibodies could have applications both in research and therapy.

  14. Characterization of single chain antibody targets through yeast two hybrid

    Directory of Open Access Journals (Sweden)

    Vielemeyer Ole

    2010-08-01

    Full Text Available Abstract Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv, are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID, efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise

  15. MORPHOLOGICAL OBSERVATION OF SINGLE-CHAIN POLY(METHYL METHACRYLATE) PARTICLES

    Institute of Scientific and Technical Information of China (English)

    NI Ming; CHEN Liusheng; JIA Shijun; JIN Xigao; GE Shouren; Atsushi TAKAHARA; Tisato KAJIYAMA

    1997-01-01

    Tapping mode atomic force microscope has been applied to observe single-chain PMMA particles which were stored for six months at room temperature after sprayed from very dilute solutions in good solvents, good/poor mixed solvents, and a theta solvent. Monodisperse PMMA standards of molecular weights ranging from 7.90 × 104 to 1.3 × 106 were used to investigate the effect of molecular weight on the size of the single-chain PMMA particles. These single-chain particles showed close to spherical shapes. The morphology of single-chain PMMA particles of a given molecular weight was found to be identical in spite of different solvents used for solution spraying. Molecular weight dependence of the particle dimension was also found. The diameters of single-chain PMMA particles after correction of tip-geometry effect were compared to the values estimated from molecular weight and density.

  16. 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.

  17. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3.

    Science.gov (United States)

    Rauchenberger, Robert; Borges, Eric; Thomassen-Wolf, Elisabeth; Rom, Eran; Adar, Rivka; Yaniv, Yael; Malka, Michael; Chumakov, Irina; Kotzer, Sarit; Resnitzky, Dalia; Knappik, Achim; Reiffert, Silke; Prassler, Josef; Jury, Karin; Waldherr, Dirk; Bauer, Susanne; Kretzschmar, Titus; Yayon, Avner; Rothe, Christine

    2003-10-03

    The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wölle, J., Plückthun, A., and Virnekäs, B. (2000) J. Mol. Biol. 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments. The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes. HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far. A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence. Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry. Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity. This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications.

  18. Epidermal Growth Factor Receptor in Pancreatic Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira-Cunha, Melissa, E-mail: melissacunha@doctors.org.uk [Hepatobiliary Surgery Unit, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL (United Kingdom); Newman, William G. [Genetic Medicine, MAHSC, University of Manchester, St Mary' s Hospital, Oxford Road, Manchester, M13 9WL (United Kingdom); Siriwardena, Ajith K. [Hepatobiliary Surgery Unit, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL (United Kingdom)

    2011-03-24

    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.

  19. 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...... pigments, polycyclic aromatic hydrocarbons (PAHs) which can function as light harvesting systems1. Thus, mixed membranes formed by single chain amphiphile and PAHs could represent simple primitive energy uptake systems which transduce light into chemical energy that can power an internalized catalytic...

  20. Cellular signaling by fibroblast growth factor receptors.

    Science.gov (United States)

    Eswarakumar, V P; Lax, I; Schlessinger, J

    2005-04-01

    The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.

  1. Fibroblast growth factor 23 and its receptors.

    Science.gov (United States)

    Yu, Xijie; White, Kenneth E

    2005-08-01

    Fibroblast growth factor 23 (FGF23) is a circulating factor that plays critical roles in phosphate and vitamin D metabolism, as evidenced by the fact that FGF23 missense mutations cause autosomal dominant hypophosphatemic rickets (ADHR). Autosomal dominant hypophosphatemic rickets is characterized by hypophosphatemia with inappropriately normal 1,25-dihydroxyvitamin D concentrations, as well as bone pain, fracture and rickets. This phenotype parallels that of patients with tumor induced osteomalacia (TIO), X-linked hypophosphatemic rickets (XLH), and fibrous dysplasia (FD), in whom elevated serum FGF23 levels are often observed. The fibroblast growth factor receptors (FGFR1-4) play key roles in skeletal development, as well as in normal metabolic processes. Several FGFR isoforms that potentially mediate the activity of FGF23 have been implicated. In the short term, these findings will lead to further understanding of FGF23 function, and potentially in the long term, to targeted therapies in disorders of hypo- and hyperphosphatemia that involve FGF23.

  2. 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 amp...... network. References 1 Cape, J., et al. (2011) Chem. Sci., 2 (4), 661-667. 2 Maurer, S. E., et al. (2009) Astrobiology, 9, 979-987. 3 Caschera, F., et al. Langmuir, In press....

  3. Isoforms of receptors of fibroblast growth factors.

    Science.gov (United States)

    Gong, Siew-Ging

    2014-12-01

    The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases.

  4. Influence of backbone rigidness on single chain conformation of thiophene-based conjugated polymers.

    Science.gov (United States)

    Hu, Zhongjian; Liu, Jianhua; Simón-Bower, Lauren; Zhai, Lei; Gesquiere, Andre J

    2013-04-25

    Structural order of conjugated polymers at different length scales directs the optoelectronic properties of the corresponding materials; thus it is of critical importance to understand and control conjugated polymer morphology for successful application of these materials in organic optoelectronics. Herein, with the aim of probing the dependence of single chain folding properties on the chemical structure and rigidness of the polymer backbones, single molecule fluorescence spectroscopy was applied to four thiophene-based conjugated polymers. These include regioregular poly(3-hexylthiophene) (RR-P3HT), poly(2,5-bis(3-tetradecylthiophen-2-yl)thieno[3,2-b]thiophene) (PBTTT-14), poly(2,5-bis(3-tetradecylthiophen-2-yl)thiophene-2-yl)thiophen-2-ylthiazolo[5,4-d]thiazole) (PTzQT-12), and poly(3,3-didodecylquaterthiophene)] (PQT-12). Our previous work has shown that RR-P3HT and PBTTT-14 polymer chains fold in their nanostructures, whereas PQT-12 and PTzQT-12 do not fold in their nanostructures. At the single molecule level, it was found that RR-P3HT single chains almost exclusively fold into loosely and strongly aggregated conformations, analogous to the folding properties in nanostructures. PQT-12 displays significant chain folding as well, but only into loosely aggregated conformations, showing an absence of strongly aggregated polymer chains. PBTTT-14 exhibits a significant fraction of rigid polymer chain. The findings made for single molecules of PQT-12 and PBTTT-14 are thus in contrast with the observations made in their corresponding nanostructures. PTzQT-12 appears to be the most rigid and planar conjugated polymer of these four polymers. However, although the presumably nonfolding polymers PQT-12 and PTzQT-12 exhibit less folding than RR-P3HT, there is still a significant occurrence of chain folding for these polymers at the single molecule level. These results suggest that the folding properties of conjugated polymers can be influenced by the architecture of the

  5. Anti-Staphylococcus aureus single-chain variable region fragments provide protection against mastitis in mice.

    Science.gov (United States)

    Wang, Man; Zhang, Yan; Zhu, Jianguo

    2016-03-01

    Staphylococcus aureus is a leading causative agent of bovine mastitis, which can result in significant economic losses to the dairy industry. However, available vaccines against bovine mastitis do not confer adequate protection, although passive immunization with antibodies may be useful to prevent disease. Hence, we constructed a bovine single-chain variable region fragment (scFv) phage display library using cDNAs from peripheral blood lymphocytes of cows with S. aureus-induced mastitis. After four rounds of selection, eight scFvs that bound S. aureus antigens with high affinity were obtained. The framework regions of the variable domains (VH and VL) of the eight scFvs were highly conserved, and the complementarity-determining regions (CDRs) displayed significant diversity, especially CDR3 of the VH domain. All eight scFvs inhibited S. aureus growth in culture medium. Lactating mice were challenged by injecting S. aureus into the fourth mammary gland. Histopathological analysis showed that treatment with these scFvs prior to bacterial challenge maintained the structure of the mammary acini, decreased infiltration of polymorphonuclear neutrophils, increased levels of interferon-gamma and interleukin-4, and reduced tumor necrosis factor-alpha levels in mammary tissues, as compared with mice treatment with physiological saline (P < 0.05). These novel bovine scFvs may be suitable candidates for therapeutic agents for the prevention of S. aureus-induced bovine mastitis.

  6. 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

  7. 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; Pabinger, Ingrid

    2016-08-04

    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.

  8. 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.

  9. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    Directory of Open Access Journals (Sweden)

    Benevolo Maria

    2006-09-01

    Full Text Available Abstract Background Aberrant signaling by ErbB-2 (HER 2, Neu, a member of the human Epidermal Growth Factor (EGF receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs to ErbB-2 that involves their functional expression in (a bacteria, (b transient as well as stable transgenic tobacco plants, and (c a newly developed cell-free transcription-translation system. Results An ScFv (ScFv800E6 was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T, displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1 only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. Conclusion ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology

  10. Soluble and cell surface receptors for tumor necrosis factor

    DEFF Research Database (Denmark)

    Wallach, D; Engelmann, H; Nophar, Y

    1991-01-01

    Tumor necrosis factor (TNF) initiates its multiple effects on cell function by binding at a high affinity to specific cell surface receptors. Two different molecular species of these receptors, which are expressed differentially in different cells, have been identified. The cDNAs of both receptor...... have recently been cloned. Antibodies to one of these receptor species (the p55, type I receptor) can trigger a variety of TNF like effects by cross-linking of the receptor molecules. Thus, it is not TNF itself but its receptors that provide the signal for the response to this cytokine...... in certain pathological situations. Release of the soluble receptors from the cells seems to occur by proteolytic cleavage of the cell surface forms and appears to be a way of down-regulating the cell response to TNF. Because of their ability to bind TNF, the soluble receptors exert an inhibitory effect...

  11. Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.

    Science.gov (United States)

    Hosseini, Hossien; Rajabibazl, Masoumeh; Ebrahimizadeh, Walead; Dehbidi, Gholamreza Rafiei

    2015-01-01

    Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10μg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.

  12. Cardiovascular risk factors regulate the expression of vascular endothelin receptors

    DEFF Research Database (Denmark)

    Xu, Cang-Bao; Sun, Yang; Edvinsson, Lars

    2010-01-01

    , cigarette smoking and hypertension (both strongly related to arterial wall injury), inflammation and atherosclerosis. The vascular endothelin receptors are a protein family that belongs to the larger family of G-protein coupled receptors. They mediate vascular smooth muscle contraction, proliferation......-activated protein kinase pathways and downstream transcription factors such as nuclear factor-kappaB. Understanding the mechanisms involved in vascular endothelin receptor upregulation during cardiovascular disease may provide novel therapeutic approaches....

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

    African Journals Online (AJOL)

    2015-02-05

    Feb 5, 2015 ... Key words: Breast cancer, human epidermal growth factor receptor 2/neu, immunohistochemistry, ... therapy.[6‑8] Of all these prognostic and predictive factors, ... one of the biggest private medical laboratories in Nigeria.

  14. Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus

    Science.gov (United States)

    Transgenic plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were developed that contain single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or suga...

  15. Molecular dynamics simulation of secondary sorption behavior of montmorillonite modified by single chain quaternary ammonium cations.

    Science.gov (United States)

    Zhao, Qian; Burns, Susan E

    2012-04-03

    Organoclays synthesized from single chain quaternary ammonium cations (QAC) ((CH(3))(3)NR(+)) exhibit different mechanisms for the sorption of nonpolar organic compounds as the length of the carbon chain is increased. The interaction between a nonpolar sorbate and an organoclay intercalated with small QACs has been demonstrated to be surface adsorption, while partitioning is the dominant mechanism in clays intercalated with long chain surfactants. This study presents the results of a molecular dynamics (MD) simulation performed to examine the sorption mechanisms of benzene in the interlayer of three organoclays with chain lengths ranging from 1 to 16 carbons: tetramethylammonium (TMA) clay; decyltrimethylammonium (DTMA) clay; and hexadecyltrimethylammonium (HDTMA) clay. The basis of the overall simulation was a combined force field of ClayFF and CVFF. In the simulations, organic cations were intercalated and benzene molecules were introduced to the interlayer, followed by whole system NPT and NVT time integration. Trajectories of all the species were recorded after the system reached equilibrium and subsequently analyzed. Simulation results confirmed that the arrangement of the surfactants controlled the sorption mechanism of organoclays. Benzene molecules were observed to interact directly with the clay surface in the presence of TMA cations, but tended to interact with the aliphatic chain of the HDTMA cation in the interlayer. The simulation provided insight into the nature of the adsorption/partitioning mechanisms in organoclays, and explained experimental observations of decreased versus increased uptake capacities as a function of increasing total organic carbon (TOC) for TMA clay and HDTMA clay, respectively. The transition of sorption mechanisms was also quantified with simulation of DTMA clay, with a chain length between that of TMA and HDTMA. Furthermore, this study suggested that at the molecular level, the controlling factor for the ultimate sorption

  16. Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library

    Indian Academy of Sciences (India)

    Ming Ni; Bing Yu; Y U Huang; Zhenjie Tang; Ping Lei; Xin Shen; Wei Xin; Huifen Zhu; Guanxin Shen

    2008-12-01

    We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naïve phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was refined using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting. SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.

  17. Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts

    OpenAIRE

    1984-01-01

    The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to th...

  18. Methods of preparing and using single chain anti-tumor antibodies

    Science.gov (United States)

    Cheung, Nai-Kong; Guo, Hong-Fen

    2010-02-23

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  19. 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.

  20. the significance of epidermal growth factor receptor and survivin ...

    African Journals Online (AJOL)

    2013-01-01

    Jan 1, 2013 ... SURVIVIN EXPRESSION IN BLADDER CANCER TISSUE AND URINE ... Objective: To assess whether epidermal growth factor receptor (EGFR) and survivin ..... lung cancer by the FDA in 2003 (28) and is currently.

  1. Single chain folding of synthetic polymers by covalent and non-covalent interactions: current status and future perspectives.

    Science.gov (United States)

    Altintas, Ozcan; Barner-Kowollik, Christopher

    2012-06-14

    The present feature article highlights the preparation of polymeric nanoparticles and initial attempts towards mimicking the structure of natural biomacromolecules by single chain folding of well-defined linear polymers through covalent and non-covalent interactions. Initially, the discussion focuses on the synthesis and characterization of single chain self-folded structures by non-covalent interactions. The second part of the article summarizes the folding of single chain polymers by means of covalent interactions into nanoparticle systems. The current state of the art in the field of single chain folding indicates that covalent-bond-driven nanoparticle preparation is well advanced, while the first encouraging steps towards building reversible single chain folding systems by the use of mutually orthogonal hydrogen-bonding motifs have been made. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Construction of Large Human Single-chain Antibody Phage Display Library

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A large human naive single chain antibody (scFv) library is constructed from 60 healthy donors via phage display technique. During the period, some methods are employed to optimize the diversity, such as multi donors, different annealing temperature, half-nest PCR, and assembly by two-way fusion PCR. In this stud y, 78 electroporations resulted in 1010 library, diversity of which is assayed by enzyme fingerprint. The efficiency and diversity are all better than other rese arches.

  3. Effects of interlinker sequences on the biological properties of bispecific single-chain antibodies

    Institute of Scientific and Technical Information of China (English)

    FANG Min; JIANG Xin; YANG Zhi; YIN Changcheng; LI Hua; ZHAO Rui; ZHANG Zhong; LIN Qing; HUANG Hualiang

    2003-01-01

    Single-chain bispecific antibody (scBsAb) is one of the promising genetic engineering antibody formats for clinical application. But the effects of interlinker sequences on the biological properties of bispecific single-chain antibodies have not been studied in detail. Three interlinker sequences were designed and synthesized, and denominated as Fc, HSA, 205C′, respectively. Universal vectors with these different interlinker sequences for scBsAb expression in E. coli were constructed. A model scBsAb based on a reshaped single-chain antibody (scFv) against human CD3 and a scFv directed against human ovarian carcinoma were generated and expressed in E. coli. The results of SDS-PAGE and Western blot showed that the different interlinker sequences did not affect the expression levelof scBsAb. However, as demonstrated by ELISA and pharmacokinetics studies performed in mice, scBsAbs with different interlinker sequences had difference in the antigen-binding activities and terminal half-life time (T1/2β) in vivo, the interlinker HSA could remarkably prolong the retention time of scBsAb in blood. These results indicated that the peptide sequence of interlinker could affect important biological properties of scBsAb, such as antigen-binding properties and stability in vivo. So, selection of an appropriate interlinker sequence is very important for scBsAb construction. Optimal interlinker can bring scBsAb biologicalproperties more suitable for clinical application.

  4. Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor β receptor expression.

    Science.gov (United States)

    Vasefi, Maryam S; Kruk, Jeff S; Liu, Hui; Heikkila, John J; Beazely, Michael A

    2012-03-09

    Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)β receptors. The increased PDGFβ receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to Gα(s) in primary neurons. Interestingly, up-regulated PDGFβ receptors display an increased basal phosphorylation state at the phospholipase Cγ-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons.

  5. I. Enabling Single-Chain Surfactants to Form Vesicles by Nonamphiphilic Liquid Crystals in Water II. Controlling Attachment and Ligand-Mediated Adherence of Candida albicans on Monolayers

    Science.gov (United States)

    Varghese, Nisha

    This dissertation describes a fundamental study of weak noncovalent interactions and surface forces that exist at the interfaces of various interacting moieties (small molecules or microbes), and its relevance to colloidal and material chemistry. Chapter 1 presents an emulsion system that enables single-chain anionic or nonionic surfactants to sequester and encapsulate certain water-soluble organic salts, leading to the formation of vesicles in water. The water-soluble organic salt in the system comprises of disodium cromoglycate crystals that are emulsified by surfactants in water to form stable liquid crystal droplets. The work provides an exception to the rule of geometric packing factor that dictates formation of micelles by the surfactants in water. Chapter 2 shows that the odd or even number of carbon atoms present in the aliphatic chain of surfactants affect the ability of surfactants to emulsify aqueous-based liquid crystals of disodium cromoglycate. Such an odd-even effect is frequently observed for solid state properties like melting point, heat of fusion and refractive index but is rarely observed for molecules present in solution. When mixed in water, anionic single-chain surfactants with odd number of carbon atoms emulsifies disodium cromoglycate to form liquid crystal droplets, while surfactants with even number of carbon atoms fail to emulsify disodium cromoglycate. Chapter 3 Bolaamphiphiles usually form vesicles only in extreme conditions or in the presence of surfactants. Here, we explore the co-assembly system of synthesized bolaamphiphiles and disodium cromoglycate in water. The combination of the self-assembly forces of the bolaamphiphile and self-associating property of disodium cromoglycate liquid crystals act together at the interface form a unique microemulsion of liquid crystal droplets of disodium cromoglycate embedded in liquid crystal phase. Chapter 4 describes a key event (adhesion) that precedes infections caused by Candida albicans

  6. Synthesis and purification of a toxin-linked conjugate targeting epidermal growth factor receptor in Escherichia coli.

    Science.gov (United States)

    Ma, Chengyuan; Li, Yang; Li, Zhixin; Huang, Haiyan; Xu, Kan; Xu, Haiyang; Bai, Jieying; Li, Xiao; Zhao, Gang

    2012-05-01

    Aberrant epidermal growth factor receptor (EGFR) signaling is a common feature of multiple tumor types, including glioblastoma (GBM). As such, EGFR has emerged as an attractive target for antitumor therapy. In the present study, we sought to develop an immunotoxin capable of specifically targeting EGFR-expressing cells and mediating inhibition of cell growth and cell killing. The Luffin P1 (LP1) ribosome inactivating protein was chosen to generate a fusion protein, antiEGFR/LP1, based upon its potent protein synthesis inhibition and small size (5 kDa). LP1 was fused to the C-terminus of an anti-EGFR single-chain antibody (scFv). The recombinant antiEGFR/LP1 protein was expressed in Escherichia coli, and refolded and purified on an immobilized Ni(2+)-affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed that antiEGFR/LP1 was sufficiently expressed. Confocal microscopy and flow cytometry demonstrated that antiEGFR/LP1 bound specifically to EGFR-positive cells (U251), as almost no binding to EGFR-negative (Jurkat cells) was observed under identical time and dosage conditions. Finally, the MTT cell viability assay showed that antiEGFR/LP1 elicited obvious cytotoxicity toward EGFR-positive tumor cells. Collectively, these results suggest that antiEGFR/LP1 is biologically active and specific toward EGFR-positive tumor cells and may represent an effective EGFR-targeted cancer therapy.

  7. 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.

  8. ISOLATION OF ENDOTOXIN-SPECIFIC ANTIBODIES BY SELECTION OF AN SINGLE CHAIN PHAGE ANTIBODY LIBRARY

    Institute of Scientific and Technical Information of China (English)

    陈鸣; 俞丽丽; 张雪; 府伟灵

    2002-01-01

    Objective: To isolate murine anti endotoxin single chain phage antibody from a constructed library. Methods: Total RNA was firstly extracted from murine splenic cells and mRNA was reverse-transcribed into cDNA. Then the designed primers were used to amplify the variable region genes of the heavy and light chain (VH, VL) with polymerase chain reaction. The linker was used to assemble the VH and VL into ScFv, and the NotI and SfiI restriction enzymes were used to digest the ScFv in order to ligate into the pCANTAB5E phagemid vector that was already digested with the same restriction enzymes. The ligated vector was then introduced into competent E.coli TG1 cells to construct a single-chain phage antibody library. After rescued with M13KO7 helper phage, recombinant phages displaying ScFv fragments were harvested from the supernatant and selected with endotoxin. The enriched positive clones were reinfected into TG1 cells. Finally, 190 clones were randomly selected to detect the anti endotoxin antibody with indirect ELISA. Results: The titer of anti endotoxin in murine sera was 1:12,800. The concentration of total RNA was 12.38 μg/ml. 1.9×107 clones were obtained after transformed into TG1. 3×104 colonies were gotten after one round panning. Two positive colonies were confirmed with indirect ELISA among 190 randomly selected colonies. Conclusion: A 1.9×107 murine anti endotoxin single chain phage antibody library was successfully constructed. Two anti endotoxin antibodies were obtained from the library.

  9. Nano-magnetic materials: spin crossover compounds vs. single molecule magnets vs. single chain magnets.

    Science.gov (United States)

    Brooker, Sally; Kitchen, Jonathan A

    2009-09-28

    Brief introductions to spin crossover (SCO), single molecule magnetism (SMM) and single chain magnetism (SCM) are provided. Each section is illustrated by selected examples that have contributed significantly to the development of these fields, including recent efforts to produce materials (films, attachment to surfaces etc.). The advantages and disadvantages of each class of magnetically interesting compound are considered, along with the key challenges that remain to be overcome before such compounds can be used commercially as nanocomponents. This invited perspective article is intended to be easily comprehensible to non-specialists in the field.

  10. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    OpenAIRE

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2010-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Bece...

  11. 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.

  12. Modulation of the NMDA Receptor Through Secreted Soluble Factors.

    Science.gov (United States)

    Cerpa, Waldo; Ramos-Fernández, Eva; Inestrosa, Nibaldo C

    2016-01-01

    Synaptic activity is a critical determinant in the formation and development of excitatory synapses in the central nervous system (CNS). The excitatory current is produced and regulated by several ionotropic receptors, including those that respond to glutamate. These channels are in turn regulated through several secreted factors that function as synaptic organizers. Specifically, Wnt, brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), and transforming growth factor (TGF) particularly regulate the N-methyl-D-aspartate receptor (NMDAR) glutamatergic channel. These factors likely regulate early embryonic development and directly control key proteins in the function of important glutamatergic channels. Here, we review the secreted molecules that participate in synaptic organization and discuss the cell signaling behind of this fine regulation. Additionally, we discuss how these factors are dysregulated in some neuropathologies associated with glutamatergic synaptic transmission in the CNS.

  13. 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

  14. Placental growth factor and vascular endothelial growth factor receptor-2 in human lung development.

    Science.gov (United States)

    Janér, Joakim; Andersson, Sture; Haglund, Caj; Karikoski, Riitta; Lassus, Patrik

    2008-08-01

    We examined the pulmonary expression of 2 proangiogenic factors, namely, placental growth factor and vascular endothelial growth factor receptor-2, during lung development and acute and chronic lung injury in newborn infants. Six groups were included in an immunohistochemical study of placental growth factor and vascular endothelial growth factor receptor-2, that is, 9 fetuses, 4 preterm and 8 term infants without lung injury who died soon after birth, 5 preterm infants with respiratory distress syndrome of 10 days, and 6 with bronchopulmonary dysplasia. Placental growth factor concentrations in tracheal aspirate fluid were measured in 70 samples from 20 preterm infants during the first postnatal week. In immunohistochemical analyses, placental growth factor staining was seen in bronchial epithelium and macrophages in all groups. Distal airway epithelium positivity was observed mostly in fetuses and in preterm infants who died soon after birth. Vascular endothelial growth factor receptor-2 staining was seen in vascular endothelium in all groups and also in lymphatic endothelium in fetuses. Vascular endothelial growth factor receptor-2 staining in arterial endothelium was associated with higher and staining in venous endothelium with lower gestational age. In capillaries, less vascular endothelial growth factor receptor-2 staining was seen in bronchopulmonary dysplasia. The mean placental growth factor protein concentration in tracheal aspirate fluid during the first postnatal week was 0.64 +/- 0.42 pg/mL per IgA-secretory component unit. Concentrations during the first postnatal week were stable. Lower placental growth factor concentrations correlated with chorioamnionitis and lactosyl ceramide positivity. The vascular endothelial growth factor receptor-2 staining pattern seems to reflect ongoing differentiation and activity of different endothelia. Lower vascular endothelial growth factor receptor-2 expression in capillary endothelium in bronchopulmonary dysplasia

  15. Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates.

    Science.gov (United States)

    Neumann-Schaal, Meina; Messerschmidt, Katrin; Grenz, Nicole; Heilmann, Katja

    2013-03-01

    Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.

  16. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease.

    Science.gov (United States)

    Grizot, Sylvestre; Smith, Julianne; Daboussi, Fayza; Prieto, Jesús; Redondo, Pilar; Merino, Nekane; Villate, Maider; Thomas, Séverine; Lemaire, Laetitia; Montoya, Guillermo; Blanco, Francisco J; Pâques, Frédéric; Duchateau, Philippe

    2009-09-01

    Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lin-Xu [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Mellon, Michael [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Bowder, Dane [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Quinn, Meghan [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States); Shea, Danielle; Wood, Charles [Nebraska Center for Virology, Lincoln, NE (United States); School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583 (United States); Xiang, Shi-Hua, E-mail: sxiang2@unl.edu [Nebraska Center for Virology, Lincoln, NE (United States); School of Veterinary Medicine and Biomedical Sciences, Lincoln, NE (United States)

    2015-01-15

    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.

  18. Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells.

    Science.gov (United States)

    Zuhaida, A A; Ali, A M; Tamilselvan, S; Alitheen, N B; Hamid, M; Noor, A M; Yeap, S K

    2013-11-18

    A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications.

  19. 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.

  20. Creation and Evaluation of a Single-chain Antibody Tetramer that Targets Brain Endothelial Cells.

    Science.gov (United States)

    Zhang, Xiaobin; Wang, Xin Xiang; Shusta, Eric V

    2014-04-01

    Antibodies that target and internalize into blood-brain barrier (BBB) endothelial cells offer promise as drug delivery agents. Previously, we identified a single-chain antibody (scFvA) capable of binding to the BBB. In an attempt to improve the binding and internalization properties of the single chain antibody (scFvA), a biotinylation tag (Avitag) was fused to scFvA and the protein secreted by yeast. The scFvA-Avitag could be biotinylated by yeast-displayed BirA enzyme and biotinylated scFvA-Avitag could be used to create scFv tetramers. Tetramerization of scFvA improved the internalization of scFvA into BBB endothelial cells, and biotinylated scFvA-Avitag could also be used to target streptavidin-coated quantum dots for BBB endothelial cell internalization. Perfusing the rat brain with scFvA-tetramer confirmed that the antigen targeted by scFvA is distributed on blood side of the BBB, suggesting the potential for downstream application of scFvA in brain-targeted drug delivery.

  1. Preparative crystallization of a single chain antibody using an aqueous two-phase system.

    Science.gov (United States)

    Huettmann, Hauke; Berkemeyer, Matthias; Buchinger, Wolfgang; Jungbauer, Alois

    2014-11-01

    A simultaneous crystallization and aqueous two-phase extraction of a single chain antibody was developed, demonstrating process integration. The process conditions were designed to form an aqueous two-phase system, and to favor crystallization, using sodium sulfate and PEG-2000. At sufficiently high concentrations of PEG, a second phase was generated in which the protein crystallization occurred simultaneously. The single chain antibody crystals were partitioned to the top, polyethylene glycol-rich phase. The crystal nucleation took place in the sodium sulfate-rich phase and at the phase boundary, whereas crystal growth was progressing mainly in the polyethylene glycol-rich phase. The crystals in the polyethylene glycol-rich phase grew to a size of >50 µm. Additionally, polyethylene glycol acted as an anti-solvent, thus, it influenced the crystallization yield. A phase diagram with an undersaturation zone, crystallization area, and amorphous precipitation zone was established. Only small differences in polyethylene glycol concentration caused significant shifts of the crystallization yield. An increase of the polyethylene glycol content from 2% (w/v) to 4% (w/v) increased the yield from approximately 63-87%, respectively. Our results show that crystallization in aqueous two-phase systems is an opportunity to foster process integration.

  2. Y-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

    Institute of Scientific and Technical Information of China (English)

    王字玲; 荣康泰; 杨日芳; 恽榴红

    2000-01-01

    A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitro-phenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P6 to the carrier proteins BSA (bovine serum albumin) and LPH (Limulus polyphenus hemocyanin). Mice were immunized with P6-LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P6-BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP6) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number kcatt= 198 min-1 and the rate enhancement kcatkuncat = 122 419. When 0.16 mg · mL-1

  3. Cell and molecular biology of epidermal growth factor receptor.

    Science.gov (United States)

    Ceresa, Brian P; Peterson, Joanne L

    2014-01-01

    The epidermal growth factor receptor (EGFR) has been one of the most intensely studied cell surface receptors due to its well-established roles in developmental biology, tissue homeostasis, and cancer biology. The EGFR has been critical for creating paradigms for numerous aspects of cell biology, such as ligand binding, signal transduction, and membrane trafficking. Despite this history of discovery, there is a continual stream of evidence that only the surface has been scratched. New ways of receptor regulation continue to be identified, each of which is a potential molecular target for manipulating EGFR signaling and the resultant changes in cell and tissue biology. This chapter is an update on EGFR-mediated signaling, and describes some recent developments in the regulation of receptor biology.

  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. Nerve growth factor receptor molecules in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Taniuchi, M.; Schweitzer, J.B.; Johnson, E.M. Jr.

    1986-03-01

    The authors have developed a method to immunoprecipitate rat nerve growth factor (NGF) receptor proteins and have applied the method to detect NGF receptor molecules in the rat brain. Crosslinking /sup 125/I-labeled NGF to either PC12 cells or cultured rat sympathetic neurons yielded two radiolabeled molecules (90 kDa and 220 kDa) that were immunoprecipitated by monoclonal antibody 192-IgG. Further, 192-IgG precipitated two radiolabeled proteins, with the expected sizes (80 kDa and 210 kDa) of noncrosslinked NGF receptor components, from among numerous surface-iodinated PC12 cell proteins. These results demonstrate the specific immunoprecipitation of NGF receptor molecules by 192-IgG. They applied the /sup 125/I-NGF crosslinking and 192-IgG-mediated immunoprecipitation procedures to plasma membrane preparations of rat brain: NGF receptor molecules of the same molecular masses as the peripheral receptor components were consistently detected in all regions and in preparations from whole brains. Removal of the peripheral sympathetic innervation of the brain did not eliminate these NGF receptor proteins, indicating that the receptor is endogenous to central nervous system tissues. They also observed retrograde transport of /sup 125/I-labeled 192-IgG from the parietal cortex to the nucleus basalis and from the hippocampus to the nucleus of the diagonal band of Broca and the medial septal nucleus. These findings demonstrate the presence in brain of NGF receptor molecules indistinguishable from those of the peripheral nervous system.

  6. Advances in Variations of Estrogen Receptor, Progesterone Receptor and Human Epidermal Growth Factor Receptor-2 Status in Metastatic Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Yuan Yuan; Zhang Lili

    2013-01-01

    Chemotherapy, endocrine therapy and molecular targeted therapy are vital means in the treatment of metastatic breast cancer (MBC), whose reasonable and standard applications are of great importance to prolong patients’ survival and improve the quality of life. The expressions of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) present signiifcant differences between primary and metastatic breast cancer. However, these differences may affect the selection of MBC patients for therapeutic strategies and judgment on the prognosis. Hence, the relevant researches on variations of hormone receptors and HER-2 in primary and metastatic breast cancer, discordant causes of ER, PR and HER-2 expression in primary and metastatic lesions and clinical value of biopsy to the metastases are reviewed in the study.

  7. Characterization of the receptor for endothelial cell growth factor (ECGF) by affinity cross-linking

    Energy Technology Data Exchange (ETDEWEB)

    Friesel, R.; Burgess, W.H.; Mehlman, T.; Maciag, T.

    1986-05-01

    The authors have demonstrated high affinity receptors for ECGF on endothelial cells by covalent cross-linking of (/sup 125/I)-ECGF with disuccinimidyl suberate and observe a single chain cross-linked polypeptide species with an apparent M/sub r/ of 170K. The M/sub r/ 170K species represents (/sup 125/I)-ECGF bound to its receptor since (i) excess unlabeled ECGF inhibits the cross-linking of (/sup 125/I)-ECGF, (ii) labeling of the M/sub r/170K species does not take place in the absence of cross-linker, (iii) cells previously shown to be refractory to ECGF and lack ECGF receptors do not yield a cross-linked species, (iv) the cross-linked species can be immunoprecipitated with anti-ECGF antibodies, and (v) preincubation of cells with ECGF at 37/sup 0/C significantly reduces cross-linking while incubation at 4/sup 0/C does not. These data demonstrate that ECGF induced cell proliferation occurs through the occupancy of a specific cell surface polypeptide receptor with an apparent M/sub r/ of 150K, and suggests that internalization of the receptor-ligand complex may be relevant to ECGF-induced signal transduction.

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

    DEFF Research Database (Denmark)

    Dengjel, Joern; Akimov, Vyacheslav; Blagoev, Blagoy;

    2007-01-01

    -out by mass spectrometry-based proteomics has allowed exciting views on the very early events in signal transduction. Activation profiles of regulated phosphorylation sites on epidermal growth factor receptor and downstream signal transducers showed different kinetics within the first ten seconds...

  9. The epidermal growth factor receptor pathway in chronic kidney diseases

    NARCIS (Netherlands)

    Harskamp, Laura R.; Gansevoort, Ron T.; Goor, van Harry; Meijer, Esther

    2016-01-01

    The epidermal growth factor receptor (EGFR) pathway has a critical role in renal development, tissue repair and electrolyte handling. Numerous studies have reported an association between dysregulation of this pathway and the initiation and progression of various chronic kidney diseases such as diab

  10. Epidermal growth factor receptor: Target for delivery and silencing

    NARCIS (Netherlands)

    Santos Oliveira, S.

    2008-01-01

    Epidermal growth factor receptor in cancer therapy Recently, cancer research has been able to identify molecular targets that are specific for (or highly expressed by) cancer cells. These molecular targets serve as models for the development of rationally designed anticancer drugs that target import

  11. Fibroblast growth factor receptors, developmental corruption and malignant disease.

    Science.gov (United States)

    Kelleher, Fergal C; O'Sullivan, Hazel; Smyth, Elizabeth; McDermott, Ray; Viterbo, Antonella

    2013-10-01

    Fibroblast growth factors (FGF) are a family of ligands that bind to four different types of cell surface receptor entitled, FGFR1, FGFR2, FGFR3 and FGFR4. These receptors differ in their ligand binding affinity and tissue distribution. The prototypical receptor structure is that of an extracellular region comprising three immunoglobulin (Ig)-like domains, a hydrophobic transmembrane segment and a split intracellular tyrosine kinase domain. Alternative gene splicing affecting the extracellular third Ig loop also creates different receptor isoforms entitled FGFRIIIb and FGFRIIIc. Somatic fibroblast growth factor receptor (FGFR) mutations are implicated in different types of cancer and germline FGFR mutations occur in developmental syndromes particularly those in which craniosynostosis is a feature. The mutations found in both conditions are often identical. Many somatic FGFR mutations in cancer are gain-of-function mutations of established preclinical oncogenic potential. Gene amplification can also occur with 19-22% of squamous cell lung cancers for example having amplification of FGFR1. Ontologic comparators can be informative such as aberrant spermatogenesis being implicated in both spermatocytic seminomas and Apert syndrome. The former arises from somatic FGFR3 mutations and Apert syndrome arises from germline FGFR2 mutations. Finally, therapeutics directed at inhibiting the FGF/FGFR interaction are a promising subject for clinical trials.

  12. 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

    The epidermal growth factor receptor (EGFR) is over-expressed, as well as mutated, in many types of cancers. In particular, the EGFR variant type III mutant (EGFRvIII) has attracted much attention as it is frequently and exclusively found on many tumor cells, and hence both EGFR and EGFRvIII have...... 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...

  13. Induced crystallization of single-chain polyethylene on a graphite surface: molecular dynamics simulation.

    Science.gov (United States)

    Yang, Hua; Zhao, Xiao Jun; Sun, Miao

    2011-07-01

    Molecular dynamics (MD) simulations have been carried out on the crystallization of single-chain polyethylene (PE) which was adsorbed on a graphite (001) surface on one side and exposed to vacuum on the other at different temperatures. The MD simulation data have been analyzed to provide information about the crystallization process of polymer adsorbed on the solid substrate. The isothermal crystallization of PE proceeds in two steps: (1) adsorption and (2) orientation. The results detail the radial density distribution function, ordered parameters, local bond-orientational order parameters, and the local properties displayed in layers of the polymer parallel to the graphite and vacuum interfaces. It was also shown that the film thickness affected the critical crystallization temperature of the adsorbed polymer on the substrate surface. Furthermore, the influence of the graphite surface area on the crystallization of PE is discussed by comparing the crystallinity evolution of PE on graphite with different coverage.

  14. Engineered single-chain variable fragment antibody for immunodiagnosis of groundnut bud necrosis virus infection.

    Science.gov (United States)

    Maheshwari, Yogita; Vijayanandraj, S; Jain, R K; Mandal, Bikash

    2015-05-01

    Few studies have been done on engineered antibodies for diagnosis of tospovirus infections. The present study was undertaken to develop a single-chain variable fragment (scFv) for specific diagnosis of infection by groundnut bud necrosis virus (GBNV), the most prevalent serogroup IV tospovirus in India. Heavy chain (372 nucleotide [nt]) and light chain (363 nt) variable region clones obtained from a hybridoma were used to make an scFv construct that expressed a ~29-kDa protein in E. coli. The scFv specifically detected GBNV in field samples of cowpea, groundnut, mung bean, and tomato, and it did not recognize watermelon bud necrosis virus, a close relative of GBNV belonging to tospovirus serogroup IV. This study for the first time demonstrated the application of a functional scFv against a serogroup-IV tospovirus.

  15. Magnetic Relaxation and Coercivity of Finite-size Single Chain Magnets

    Science.gov (United States)

    Gredig, Thomas; Byrne, Matthew; Vindigni, Alessandro

    2015-03-01

    The magnetic coercivity of hysteresis loops for iron phthalocyanine thin films depends on the iron chain length and the measurement sweep speed below 5 K. The average one-dimensional (1D) iron chain length in samples is controlled during deposition. These 1D iron chains can be tuned over one order of magnitude with the shortest chain having 100 elements. We show that the coercivity strongly increases with the average length of the iron chains, which self-assemble parallel to the substrate surface. Magnetic relaxation and sweep speed data suggest spin dynamics play an important role. Implementing Glauber dynamics with a finite-sized 1D Ising model provides qualitative agreement with experimental data. This suggests that iron phthalocyanine thin films act as single chain magnets and provide a solid test system for tunable finite-sized magnetic chains. This research has been supported with the NSF-DMR 0847552 grant.

  16. 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.

  17. Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv against human ICAM-1

    Directory of Open Access Journals (Sweden)

    H. Sun

    2014-07-01

    Full Text Available Intercellular adhesion molecule-1 (ICAM-1 is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

  18. An improved single-chain Fab platform for efficient display and recombinant expression.

    Science.gov (United States)

    Koerber, James T; Hornsby, Michael J; Wells, James A

    2015-01-30

    Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however, many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain fragment antigen binding (Fab) (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain fragment variable with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3 mg/L. Furthermore, rerouting of the scFab to the co-translational signal recognition particle pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (Tm of 81 °C), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.

  19. Production of single chain Fab (scFab fragments in Bacillus megaterium

    Directory of Open Access Journals (Sweden)

    Dübel Stefan

    2007-11-01

    Full Text Available Abstract Background The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab antibody format combining properties of single chain Fv (scFv and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli. Results The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli. Conclusion B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.

  20. γ-Hydroxyphosphonate inducing single-chain antibodies capable of catalyzing soman hydrolysis

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A γ-hydroxyphosphonate P6 (O1-methyl-O2-(1, 2, 2-trimethylpropyl)-2-hydroxy-5-nitrophenyl methylphosphonic acid) which is proposed to be an analog of the transition state in hydrolysis of soman was synthesized. Artificial antigens were obtained by conjugating P6 to the carrier proteins BSA (bovine serum albumin) and LPH (Limulus polyphenus hemocyanin). Mice were immunized with P6-LPH and recombinant single-chain antibody phage display library was constructed. After 4 rounds of panning against P6-BSA and competitive inhibition enzyme immunoassay, more than 70 strains of phage antibodies capable of binding soman were obtained and 11 of them can accelerate the hydrolysis reaction of soman. One of them (EP6) was studied further. Soluble single-chain antibody was prepared and purification was performed by gel filtration and ion exchange chromatography. The kinetic experiment was carried out showing that the turnover number kcat = 198 minγ1 and the rate enhancement kcat/kuncat = 122 419. When 0.16 mg·mLγ1 EP6 was preincubated in vitro with 0.132 mmol·Lγ1 (220 g·kgγ1 = 1.1×LD95) of soman prior to the administration to mice by subcutaneous route, all animals (19 mice) survived whereas all the control mice (14) treated with PBS and soman died within 30 min. Furthermore, EP6 could prolong the latent time of spasm and death when mice were passively immunized with EP6 intravenously 15 min before 1×LD95 of soman challenge. These results demonstrate that EP6 is able to increase the rate of soman degradation and protect against soman's toxicity, especially in vitro.

  1. Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.

    Science.gov (United States)

    Kumano-Kuramochi, Miyuki; Fujimura, Takashi; Komba, Shiro; Maeda-Yamamoto, Mari; Machida, Sachiko

    2016-09-01

    Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (γ+κ and μ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1-2 μg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 μg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations.

  2. Expression and characterization of a single-chain variable fragment against human LOX-1 in Escherichia coli and Brevibacillus choshinensis.

    Science.gov (United States)

    Hu, Wei; Xiang, Jun-Yan; Kong, Ping; Liu, Ling; Xie, Qiuhong; Xiang, Hongyu

    2017-03-09

    The single-chain variable fragment (scFv) against lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a promising molecule for its potential use in the diagnosis and immunotherapy of atherosclerosis. Producing this scFv in several milligram amounts could be the starting point for further engineering and application of the scFv. In this study, the abundant expression of the anti-LOX-1 scFv was attempted using Escherichia coli (E. coli) and Brevibacillus choshinensis (B. choshinensis). The scFv had limited soluble yield in E. coli, but it was efficiently secreted by B. choshinensis. The optimized fermentation was determined using the Plackett-Burman screening design and the response surface methodology (RSM), under which the yield reached up to 1.5 g/L in a 5-L fermentor. Moreover, the properties of the scFvs obtained from the two expression systems were different. The antigen affinity, transition temperature and particle diameter size is 1.01E-07 M, 55.2 ± 0.3°C and 9.388 nm for the scFv expressed by B. choshinensis and 4.53E-07 M, 52.5 ± 0.3°C and 13.54 nm for the scFv expressed by E. coli. This study established an efficient scale-up production methodology for the anti-LOX-1 scFv, which will boost its use in LOX-1-based therapy.

  3. Cloning and expression of an anti-LDL(-) single-chain variable fragment, and its inhibitory effect on experimental atherosclerosis.

    Science.gov (United States)

    Kazuma, Soraya M; Cavalcante, Marcela F; Telles, Andréia E R; Maranhão, Andrea Queiroz; Abdalla, Dulcineia S P

    2013-01-01

    The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr(-/-) mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr(-/-) mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).

  4. Synthesis and Preliminary Investigations of the siRNA Delivery Potential of Novel, Single-Chain Rigid Cationic Carotenoid Lipids

    Directory of Open Access Journals (Sweden)

    Philip L. Leopold

    2012-03-01

    Full Text Available The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethanecarbamoyl]-cholesterol (DC-Chol and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC as positive controls. All cationic lipids (controls and single-chain lipids were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE. Cationic lipid-siRNA complexes of varying (+/− molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/− molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.

  5. Single chain fragment variable antibodies developed by using as target the 3rd fibronectin type III homologous repeat fragment of human neural cell adhesion molecule L1 promote cell migration and neuritogenesis.

    Science.gov (United States)

    Tang, Dan-Yang; Yu, Yang; Zhao, Xuan-Jun; Schachner, Melitta; Zhao, Wei-Jiang

    2015-01-15

    L1CAM plays important roles during ontogeny, including promotion of neuronal cell migration and neuritogenesis, and stimulation of axonal outgrowth, fasciculation and myelination. These functions are at least partially exerted through a 16-mer amino acid sequence in the third fibronectin type III-like repeat of L1, which associates with several interaction partners, including integrins, other adhesion molecules and growth factor receptors. Here, using the Tomlinson I library for phage display, we obtained two single-chain variable fragment antibodies (scFvs) against this peptide sequence of human L1, hereafter called H3 peptide. Both scFvs recognize the H3 peptide and the extracellular domain of L1, as tested by enzyme-linked immunosorbent assay (ELISA), Western blot analysis and immunofluorescence staining of L1 expresssing cells. Furthermore, both scFvs reduce U-87 MG cell adhesion to fibronectin, while stimulating cell migration. Application of scFvs to human neuroblastoma SK-N-SH cells promote process outgrowth. Similar to triggering of endogenous L1 functions at the cell surface, both scFvs activate the signal transducers Erk and Src in these cells. Our results indicate that scFvs against a functionally pivotal domain in L1 trigger its regeneration-beneficial functions in vitro, encouraging thoughts on therapy of neurodegenerative diseases in the hope to ameliorate human nervous system diseases.

  6. 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.

  7. Production of recombinant orange-spotted grouper (Epinephelus coioides) follicle-stimulating hormone (FSH) in single-chain form and dimer form by Pichia pastoris and their biological activities.

    Science.gov (United States)

    Chen, Jun; Zhang, Yanhong; Tang, Zhiguo; Mao, Jiewei; Kuang, Zhonglei; Qin, Chaobin; Li, Wensheng

    2012-09-01

    FSH is a key regulator of steroidogenesis and gonadal growth in teleosts. However, function of FSH is elusive in grouper due to the lack of purified and native FSH. In the present study, we reported production of bioactive orange-spotted grouper (Epinephelus coioides) FSH in dimer form and single-chain form by Pichia pastoris. Dimer form of recombinant grouper FSH (rgFSHba) was accomplished by co-expressing mature FSHb-subunit and a-subunit genes. Fusion of mature FSHb-subunit and a-subunit genes together linking with a polypeptide (4×(Gly-Ser)-Gly-Thr) gene generated single-chain form of recombinant grouper FSH (rgFSHb-a). Recombinant grouper common α-subunit (rgCga) and FSHb-subunit (rgFSHb) were also separately produced. Recombinant proteins were verified by Western blot and mass spectrometry assays, and characterized by deglycosylation analysis. Deglycosylation assay suggested that glycosylation of recombinant FSH mainly occurred on common a-subunit. Bioactivities of recombinant proteins were initially evaluated by activating grouper FSH receptor, and further demonstrated by incubating ovarian fragments of adult grouper and intraperitoneal injection in juvenile female grouper. Two forms of recombinant FSH presented similar biological activities of activating FSH receptor and stimulating in vitro testosterone (T) and estradiol-17β (E2) secretion, though the dimer form functioned slightly weaker than the single-chain form. However, injections of rgFSHb-a or rgFSHba could significantly increase serum T and E2 levels, induce early ovarian development, reduce hypothalamic gnrh1 mRNA level, and increase hypothalamic cyp19a1b mRNA level. Data in this study suggested that recombinant gonadotropin could be produced in dimer form or single-chain form by P. pastoris, and FSH could regulate steroidogenesis and early ovarian development in juvenile grouper.

  8. Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

    Science.gov (United States)

    Read, Jolene; Ingram, Alistair; Al Saleh, Hassan A; Platko, Khrystyna; Gabriel, Kathleen; Kapoor, Anil; Pinthus, Jehonathan; Majeed, Fadwa; Qureshi, Talha; Al-Nedawi, Khalid

    2017-01-01

    Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.

  9. 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.

  10. Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

    OpenAIRE

    Hyde, Caroline A. C.; Giese, Alexandra; Stuttfeld, Edward; Abram Saliba, Johan; Villemagne, Denis; Schleier, Thomas; Binz, H. Kaspar; Ballmer-Hofer, Kurt

    2012-01-01

    Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron...

  11. Beclin 1 regulates growth factor receptor signaling in breast cancer.

    Science.gov (United States)

    Rohatgi, R A; Janusis, J; Leonard, D; Bellvé, K D; Fogarty, K E; Baehrecke, E H; Corvera, S; Shaw, L M

    2015-10-16

    Beclin 1 is a haploinsufficient tumor suppressor that is decreased in many human tumors. The function of beclin 1 in cancer has been attributed primarily to its role in the degradative process of macroautophagy. However, beclin 1 is a core component of the vacuolar protein sorting 34 (Vps34)/class III phosphatidylinositoI-3 kinase (PI3KC3) and Vps15/p150 complex that regulates multiple membrane-trafficking events. In the current study, we describe an alternative mechanism of action for beclin 1 in breast cancer involving its control of growth factor receptor signaling. We identify a specific stage of early endosome maturation that is regulated by beclin 1, the transition of APPL1-containing phosphatidyIinositol 3-phosphate-negative (PI3P(-)) endosomes to PI3P(+) endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P(-)/APPL(+)-signaling-competent compartment. As a result, suppression of BECN1 sustains growth factor-stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Our data identify a novel role for beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of beclin 1 expression would enhance breast cancer progression.

  12. Cheiradone: a vascular endothelial cell growth factor receptor antagonist

    Directory of Open Access Journals (Sweden)

    Ahmed Nessar

    2008-01-01

    Full Text Available Abstract Background Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing and pathological conditions (tumour development. Vascular endothelial growth factor (VEGF, fibroblast growth factor-2 (FGF-2 and epidermal growth factor (EGF are the major angiogenic regulators. We have identified a natural product (cheiradone isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation and in vivo (the chick chorioallantoic membrane models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC50 was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated. Results Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC50 values in the range 5.20–7.50 μM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC50 values of 2.9 and 0.61 μM respectively. Conclusion Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.

  13. Upregulation of epidermal growth factor receptor 4 in ora leukoplakia

    Institute of Scientific and Technical Information of China (English)

    Hiroshi Kobayashi; Kenichi Kumagai; Akito Gotoh; Takanori Eguchi; Hiroyuki Yamada; Yoshiki Hamada; Satsuki Suzuki; Ryuji Suzuki

    2013-01-01

    In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ ErbB 1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP), The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP.

  14. 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 ...

  15. 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.

  16. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments.

    Science.gov (United States)

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C; Johnson, Jennifer L; Entzminger, Kevin; Jain, Avni; Heaner, David P; Morales, Ivan A; Truskett, Thomas M; Maynard, Jennifer A; Lieberman, Raquel L

    2014-09-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three-dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts.

  17. Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.

    Science.gov (United States)

    Sato, Yusui; Tanaka, Yusuke; Inaba, Satomi; Sekiguchi, Hiroshi; Maruno, Takahiro; Sasaki, Yuji C; Fukada, Harumi; Kobayashi, Yuji; Azuma, Takachika; Oda, Masayuki

    2016-10-01

    Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.

  18. Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Imanaka Tadayuki

    2002-09-01

    Full Text Available Abstract Background Hep27 monoclonal (Hep27 Mab is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102. We attempted to produce a single-chain fragment (scFv, a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques. Results The sequences encoding the variable regions of heavy (VH and light (VL chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser3 and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa. Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab. Conclusion This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.

  19. Molecular Recognition-Mediated Transformation of Single-Chain Polymer Nanoparticles into Crosslinked Polymer Films.

    Science.gov (United States)

    Mahon, Clare S; McGurk, Christopher J; Watson, Scott M D; Fascione, Martin A; Sakonsinsiri, Chadamas; Turnbull, W Bruce; Fulton, David A

    2017-08-14

    We describe single-chain polymer nanoparticles (SCNPs) possessing intramolecular dynamic covalent crosslinks that can transform into polymer films through a molecular recognition-mediated crosslinking process. The SCNPs utilise molecular recognition with surface-immobilised proteins to concentrate upon a substrate, bringing the SCNPs into close spatial proximity with one another and allowing their dynamic covalent crosslinkers to undergo intra- to interpolymer chain crosslinking leading to the formation of polymeric film. SCNPs must possess both the capacity for specific molecular recognition and a dynamic nature to their intramolecular crosslinkers to form polymer films, and an investigation of the initial phase of film formation indicates it proceeds from features which form upon the surface then grow predominantly in the xy directions. This approach to polymer film formation presents a potential method to "wrap" surfaces displaying molecular recognition motifs-which could potentially include viral, cellular and bacterial surfaces or artificial surfaces displaying multivalent recognition motifs-within a layer of polymer film. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  20. Selection and characterization of single-chain recombinant antibodies against phosphoprotein of Newcastle disease virus.

    Science.gov (United States)

    Li, Benqiang; Ye, Jiaxin; Lin, Yuan; Wang, Man; Jia, Rui; Zhu, Jianguo

    2014-09-01

    Phosphoprotein (P), involved in virus RNA replication and transcription, had become a new target for the research on treating Newcastle disease virus (NDV). Here we described the cloning and expression of phosphoprotein from NDV, and then screened the anti-P antibodies from the chicken single chain fragment variable (scFv) library, which were generated from chickens immunized with the ND vaccines. As a first step, the recombinant expression vector pET28a-P was successfully constructed. In a following step, two anti-P positive scFv clones from the scFv library were selected by indirect enzyme-linked immunosorbent assay (ELISA) method. The sequence analysis of two positive clones showed that there were more variation in complementary determine region (CDR) of VH and VL, and the CDR3 in VH exhibited a significant change in amino acid number and type. In another experiment, the purified scFv antibodies used in the assay was shown to be specific for NDV-P by western blot. The results indicated that the strategy we used in this experiment proved to be convenient way for screening scFv antibody, which paved a new way for the immunization diagnosis and the exploration of integrated control of NDV.

  1. Selection of single chain variable fragments specific for the human-inducible costimulator using ribosome display.

    Science.gov (United States)

    Pan, Yangbin; Mao, Weiping; Liu, Xuanxuan; Xu, Chong; He, Zhijuan; Wang, Wenqian; Yan, Hao

    2012-11-01

    We applied a ribosome display technique to a mouse single chain variable fragment (scFv) library to select scFvs specific for the inducible costimulator (ICOS). mRNA was isolated from the spleens of BALB/c mice immunized with ICOS protein. Heavy and κ chain genes (VH and κ) were amplified separately by reverse transcriptase polymerase chain reaction, and the anti-ICOS VH/κ chain ribosome display library was constructed with a special flexible linker by overlap extension PCR. The VH/κ chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. Then, antibody-ribosome-mRNA complexes were produced and panned against ICOS protein under appropriate conditions. However, in order to isolate specific scFvs for ICOS, negative selection using CD28 was carried out before three rounds of positive selection on ICOS. After three rounds of panning, the selected scFv DNAs were cloned into pET43.1a and detected by SDS-PAGE. Then, enzyme-linked immunosorbent assay showed that we successfully constructed a native ribosome display library, and among seven clones, clone 5 had the highest affinity for the ICOS and low for the CD28. Anti-ICOS scFvs are assessed for binding specificity and affinity and may provide the potential for development of the humanized and acute and chronic allograft rejection.

  2. Duplex microfluidic SERS detection of pathogen antigens with nanoyeast single-chain variable fragments.

    Science.gov (United States)

    Wang, Yuling; Rauf, Sakandar; Grewal, Yadveer S; Spadafora, Lauren J; Shiddiky, Muhammad J A; Cangelosi, Gerard A; Schlücker, Sebastian; Trau, Matt

    2014-10-07

    Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.

  3. Construction of an antimyoglobin single-chain variable fragment with rapid reaction kinetics.

    Science.gov (United States)

    Jang, Jun-Hyuck; Kim, Dong-Hyung; Paek, Se-Hwan; Woo, Eui-Jeon; Kim, Young-Wan

    2016-01-01

    Antibodies with rapid reaction kinetics (high association and dissociation rates), named reversible antibodies, are used to perform continuous monitoring of sensitive disease biomarkers. In cases of acute myocardial infarction (AMI), continuous monitoring and early diagnosis are important. Human myoglobin (Myo) is a useful biomarker for AMI during the early stage after the onset of symptoms. In this study, a single-chain variable fragment (scFv) specific to Myo was derived from an IgG antibody that has rapid reaction kinetics. Enzyme-linked immunosorbent assay revealed that recombinant scFv exhibited 3.8-fold reduced affinity compared with the parent IgG antibody based on the antibody concentration necessary for 50% of the maximum signal. The scFv retained the rapid reaction kinetic mode with average kon and koff of 2.63 × 10(5) M(-1) Sec(-1) and 3.25 × 10(-3) Sec(-1) , respectively, which were reduced to 10- and 2.3-fold compared with those of the parent antibody. The equilibrium constant for the association of the scFv (KA = 8.09 × 10(7) M(-1) ) was 4.6-fold lower than that of its parent IgG antibody. This scFv may be a starting point for further mutagenesis/kinetic and structural analyses providing valuable insight into the mechanism of reversible antibodies.

  4. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    Science.gov (United States)

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1.

  5. Molecular Dynamics Simulations of a Single Chain Pentablock Ionomer in Dilute Solutions

    Science.gov (United States)

    Aryal, Dipak; Perahia, Dvora; Grest, Gary S.

    2013-03-01

    Co-polymers are in the core of many applications such as fuel cells, batteries and purification membranes that require transport across membranes. The challenge remains however that under the condition that transport is optimized, the stability of the membranes is compromised. To surmount this challenge, co-polymers with blocks targeting specific roles have been designed. Using molecular dynamics simulations we have studies the structure and dynamics of ionic single chain pentablock copolymer (A-B-C-B-A) containing randomly sulfonated polystyrene in the center, tethered to poly-ethylene-r-propylene end-capped by poly - t - butyl styrene. The ionic block facilitates transport while the A and B componenet are incorporated for mechanical stability. The conformation and dynamics of single pentablock ionomer of molecular weight Mw = 50,000g/mol in an implicit poor solvent with dielectric constant of 1 and 77 .7, water, and mixture (1:1) of cyclohexane and n-heptane at 300K and 500K will be presented. The effect of solvents on conformation of a single molecule of pentablock was determined and compared with experiment, providing a stepping stone to the understanding phase behavior of this polymer. This work is partially supported by DOE DE-FG02-12ER46843.

  6. Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development.

    Science.gov (United States)

    Isaacs, Alison T; Jasinskiene, Nijole; Tretiakov, Mikhail; Thiery, Isabelle; Zettor, Agnès; Bourgouin, Catherine; James, Anthony A

    2012-07-10

    Anopheles stephensi mosquitoes expressing m1C3, m4B7, or m2A10 single-chain antibodies (scFvs) have significantly lower levels of infection compared to controls when challenged with Plasmodium falciparum, a human malaria pathogen. These scFvs are derived from antibodies specific to a parasite chitinase, the 25 kDa protein and the circumsporozoite protein, respectively. Transgenes comprising m2A10 in combination with either m1C3 or m4B7 were inserted into previously-characterized mosquito chromosomal "docking" sites using site-specific recombination. Transgene expression was evaluated at four different genomic locations and a docking site that permitted tissue- and sex-specific expression was researched further. Fitness studies of docking site and dual scFv transgene strains detected only one significant fitness cost: adult docking-site males displayed a late-onset reduction in survival. The m4B7/m2A10 mosquitoes challenged with P. falciparum had few or no sporozoites, the parasite stage infective to humans, in three of four experiments. No sporozoites were detected in m1C3/m2A10 mosquitoes in challenge experiments when both genes were induced at developmentally relevant times. These studies support the conclusion that expression of a single copy of a dual scFv transgene can completely inhibit parasite development without imposing a fitness cost on the mosquito.

  7. Isolation of BNYVV coat protein-specific single chain Fv from a mouse phage library antibody.

    Science.gov (United States)

    Jahromi, Zahra Moghaddassi; Salmanian, Ali Hatef; Rastgoo, Nasrin; Arbabi, Mehdi

    2009-10-01

    Beet necrotic yellow vein virus (BNYVV) infects sugar beet plants worldwide and is responsible for the rhizomania disease and severe economic losses. Disease severity and lack of naturally occurring resistant plants make it very difficult to control the virus, both from epidemiological and economic standpoints. Therefore, early detection is vital to impose hygiene restrictions and prevent further spread of the virus in the field. Immunoassays are one of the most popular methodologies for the primary identification of plant pathogens including BNYVV since they are robust, sensitive, fast, and inexpensive. In this study, the major coat protein (CP21) of BNYVV was cloned and expressed in Escherichia coli. Thereafter, mice were immunized with purified CP21 and a phage antibody library was constructed from their PCR-amplified immunoglobulin repertoire. Following filamentous phage rescue of the library and four rounds of panning against recombinant CP21 antigen, several specific single chain Fv fragments were isolated and characterized. This approach may pave the way to develop novel immunoassays for a rapid detection of viral infection. Moreover, it will likely provide essential tools to establish antibody-mediated resistant transgenic technology in sugar beet plants.

  8. Antioxidant Effect of Human Selenium-containing Single-chain Fv in Rat Cardiac Myocytes

    Institute of Scientific and Technical Information of China (English)

    HUO Rui; SHI Yi; XU Jun-jie; YAN Fei; L(U) Shao-wu; SU Jia-ming; DUAN Yu-jing; FAN Jia; NING Bo; CONG Deng-li; YAN Gang-lin; LUO Gui-min; WEI Jing-yan

    2009-01-01

    Reactive oxygen species(ROS) plays a key role in human heart diseases.Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide.In order to investigate the antioxidant effect of human selenium-containing single-chain Fv(Se-scFv-B3),a new mimic of GPX,a model system of hydrogen peroxide(H2O2)-induced rat cardiac myocyte damage was established.The cardiac myocyte damage was characterized in terms of cell viability,lipid peroxidation,cell membrane integrity,and intracellular H2O2 level.The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability,the decline of malondialdehyde(MDA) production,lactate dehydrogenase(LDH) release,and intracellular H2O2 level.So Se-scFvB3 may have a great potential in the treatment of human heart diseases induced by ROS.

  9. Construction and Expression of a Single Chain Antibody Mimicing Human Ovarian Cancer Antigen CA125

    Institute of Scientific and Technical Information of China (English)

    Aidong Li; Zheng Li; Yinghong Wang; Yongming Zhang; Jie Ma

    2006-01-01

    One concept for immune therapy of cancer involves induction of antigen mimic antibodies to trigger the immune response against tumor cells. Anti-idiotypic antibodies directed against the antigen-binding site of antibodies specific for tumor antigen may functionally and even structurally mimic antigen and induce anti-anti-idiotypic immune response. Monoclonal antibody WJ02 is one of such anti-idiotypic antibodies, which contains internal image of CA125. In order to improve the immunospecificity of mAb WJ02, we constructed a single chain of mAb WJ02 in Vl-linker-Vh orientation. The scFv-WJ02 could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein with a molecular weight of 30 kD retained the biological activity of mAb WJ02, which was proved by a direct binding assay and inhibition experiment. Our results indicated that the scFv-WJ02 could be used as a possible tool for idiotypic therapy against ovarian cancer, which might enhance the possibility of eliminating nonspecific responses induced by mAb WJ02.

  10. Single-chain self-folding of synthetic polymers induced by metal-ligand complexation.

    Science.gov (United States)

    Willenbacher, Johannes; Altintas, Ozcan; Roesky, Peter W; Barner-Kowollik, Christopher

    2014-01-01

    The controlled folding of a single polymer chain is for the first time realized by metal- complexation. α,ω-Bromine functional linear polymers are prepared via activators regenerated by electron transfer (ARGET) ATRP (M¯n,SEC = 5900 g mol(-1) , Đ = 1.07 and 12 000 g mol(-1) , Đ = 1.06) and the end groups of the polymers are subsequently converted to azide functionalities. A copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is carried out in the presence of a novel triphenylphosphine ligand and the polymers to afford homotelechelic bis-triphenylphosphine polymeric-macroligands (MLs) (M¯n,SEC = 6600 g mol(-1) , Đ = 1.07, and 12 800 g mol(-1) , Đ = 1.06). Single-chain metal complexes (SCMCs) are formed in the presence of Pd(II) ions in highly diluted solution at ambient temperature. The results derived via (1) H and (31) P{(1) H} NMR experiments, SEC, and DLS unambiguously evidence the efficient formation of SCMCs via metal ligand complexation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. New approach for designing single-chain magnets: organization of chains via hydrogen bonding between nucleobases.

    Science.gov (United States)

    Zhang, Wei-Xiong; Shiga, Takuya; Miyasaka, Hitoshi; Yamashita, Masahiro

    2012-04-25

    Two one-dimensional (1D) manganese complexes, [Mn(2)(naphtmen)(2)(L)](ClO(4))·2Et(2)O·2MeOH·H(2)O (1) and [Mn(2)(naphtmen)(2)(HL)](ClO(4))(2)·MeOH (2), were synthesized by using a bridging ligand with a nucleobase moiety, 6-amino-9-β-carboxyethylpurine, and a salen-type manganese(III) dinuclear complex, [Mn(2)(naphtmen)(2)(H(2)O)(2)](ClO(4))(2) (naphtmen(2-) = N,N'-(1,1,2,2-tetramethylethylene)bis(naphthylideneiminato) dianion). In 1 and 2, the carboxylate-bridged Mn(III) dinuclear units are alternately linked by two kinds of weak Mn···O interactions into 1D chains. As a result, canted antiferromagnetic and ferromagnetic interactions are alternately present along the chains, leading to a 1D chain with non-cancellation of anisotropic spins. Since the chains connected via H-bonds between nucleobase moieties are magnetically isolated, both 1 and 2 act as single-chain magnets (SCMs). More importantly, this result shows the smaller canting angles hinder long-range ordering in favor of SCM dynamics.

  12. Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

    Institute of Scientific and Technical Information of China (English)

    LIANG; Tiebing

    2001-01-01

    [1]Aggarwal, A. K., Rodgers, D. W., Drottar, M. et al., Recognition of a DNA operator by the repressor of phage 434: A view at high resolution, Science, 1988, 242: 899-907.[2]Anderson, J. E., Ptashne, M., Harrison, S. C., Structure of the repressor-operator complex of bacteriophage 434, Nature, 1987, 326: 846-852.[3]Bushman, F. D., The Bacteriophage 434 right operator roles of OR1, OR2 and OR3, J. Mol. Biol., 1993, 230: 28-40.[4]Bell, A. C., Koudelka, G. B., How 434 repressor discriminates between OR1 and OR3, J. Biological Chemistry, 1995, 270: 1205-1212.[5]Bell, A. C., Koudelka, G. B., Operator sequence context influences amino acid-base-pair interaction in 434 repressor-operator complexes, J. Mol. Biol., 1993, 234: 542-553.[6]Wharton, R. P., Ptashne, M., A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact, Na-ture, 1987, 326: 888-891.[7]Wharton, R. P., Brown, E. L., Ptashne, M., Substituting an α-helix switches the sequence-specific DNA interaction of a repressor, Cell., 1984, 38: 361-369.[8]Hollis, M., Valenzuela, D., Pioli, D. et al., A repressor heterodimer binds to a chimeric operator, Proc. Natl. Acad. Sci. USA, 1988, 85: 5834-5838.[9]Huang, L. -X., Sera, T., Schultz, P. G., A permutational approach toward protein-DNA recognition, Proc. Natl. Acad. Sci. USA, 1994, 91: 3969-3973.[10]Percipalle, P., Simoncsits, A., Zakhariev, S. et al., Rationally designed helix-turn-helix proteins and their conformational changes upon DNA binding, EMBO J., 1995, 14: 3200-3205.[11]Simoncsits, A., Chen, J. -Q., Percipalle, P. et al., Single-chain repressors containing engineered DNA-binding domains of the phage 434 repressor recognize symmetric or asymmetric DNA operators, J. Mol. Biol., 1997, 267: 118-131.[12]Gates, C. M., Stemmer, W. P. C., Kaptein, R. et al., Affinity selective isolation of ligands from peptide libraries through display on a lac repressor "headpiece dimmer", J. Mol. Biol

  13. Topography of human placental receptors for epidermal growth factor.

    Science.gov (United States)

    Rao, C V; Ramani, N; Chegini, N; Stadig, B K; Carman, F R; Woost, P G; Schultz, G S; Cook, C L

    1985-02-10

    These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Berasain, Carmen, E-mail: cberasain@unav.es; Latasa, María Ujue; Urtasun, Raquel; Goñi, Saioa; Elizalde, María; Garcia-Irigoyen, Oihane; Azcona, María [Division of Hepatology and Gene Therapy, CIMA, University of Navarra, Pamplona 31008 (Spain); Prieto, Jesús [Division of Hepatology and Gene Therapy, CIMA, University of Navarra, Pamplona 31008 (Spain); CIBERehd, University Clinic, University of Navarra, Pamplona 31080 (Spain); Ávila, Matías A. [Division of Hepatology and Gene Therapy, CIMA, University of Navarra, Pamplona 31008 (Spain)

    2011-05-18

    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.

  15. Redox-dependent regulation of epidermal growth factor receptor signaling

    Directory of Open Access Journals (Sweden)

    David E. Heppner

    2016-08-01

    Full Text Available Tyrosine phosphorylation-dependent cell signaling represents a unique feature of multicellular organisms, and is important in regulation of cell differentiation and specialized cell functions. Multicellular organisms also contain a diverse family of NADPH oxidases (NOXs that have been closely linked with tyrosine kinase-based cell signaling and regulate tyrosine phosphorylation via reversible oxidation of cysteine residues that are highly conserved within many proteins involved in this signaling pathway. An example of redox-regulated tyrosine kinase signaling involves the epidermal growth factor receptor (EGFR, a widely studied receptor system with diverse functions in normal cell biology as well as pathologies associated with oxidative stress such as cancer. The purpose of this Graphical Redox Review is to highlight recently emerged concepts with respect to NOX-dependent regulation of this important signaling pathway.

  16. Lamellar self-assemblies of single-chain amphiphiles and sterols and their derived liposomes: distinct compositions and distinct properties.

    Science.gov (United States)

    Cui, Zhong-Kai; Lafleur, Michel

    2014-02-01

    Typically, single-chain amphiphiles and sterols do not form fluid lamellar phases once hydrated individually. Most of the single-chain amphiphiles form actually micelles in aqueous environments, while sterols display a very limited solubility in water. However, under certain conditions, mixtures of single-chain amphiphiles and sterols lead to the formation of stable fluid bilayers. Over the past decade, several of these systems leading to fluid lamellar self-assemblies have been identified and this article reviews the current knowledge relative to these non-phospholipid bilayers made of single-chain amphiphiles and sterols. It presents an integrated view about the molecular features that are required for their stability, the properties they share, and the origin of these characteristics. It was also shown that these lamellar systems could lead to the formation of unilamellar vesicles, similar to phospholipid based liposomes. These vesicles display distinct properties that make them potentially appealing for technological applications; they display a limited permeability, they are stable, they are formed with molecules that are relatively chemically inert (and relatively cheap), and they can be readily functionalized. The features of these distinct liposomes and their technological applications are reviewed. Finally, the putative biological implications of these non-phospholipid fluid bilayers are also discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  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

    2002-01-01

    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 s

  18. Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin

    NARCIS (Netherlands)

    Munk, G.A.W. de; Groeneveld, E.; Rijken, D.C.

    1991-01-01

    The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50%

  19. Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Jiong Cai; Fang Li; Shizhen Wang

    2008-01-01

    BACKGROUND: Studies have shown that monoclonal or polyclonal antibody injections ofamyloid β peptide arc effective in removing amyloid β peptide overload in the brain.OBJECTIVE: Based on successful screening of a human single-chain fragment variable antibody specific to amyloid β peptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide.DESIGN, TIME AND SETTING: A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital (Beijing, China) from January to July 2006.MATERIALS: Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library.METHODS: Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a Teasy plasmid for pT-seFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvA β was cut by EcoRl and Notl endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvA β expression vector, which was confirmed by gene sequencing. Linearized pPICgK-scFvA β was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5 % methanol to express human single-chain fragment variable antibody specific to amyloid β peptide.MAIN OUTCOME MEASURES: Protein electrophoresis was used to identify PCR products, gene sequencing was uscd to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris.RESULTS: Gene sequencing confirmed pPICgK-scFvA β orientation. Rccomhinants were obtained by lineadzed pPIC9K-scFvA β transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size

  20. 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.

  1. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

    Science.gov (United States)

    Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

    2015-12-23

    Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

  2. Structural and Functional Characterization of a Single-Chain Form of the Recognition Domain of Complement Protein C1q.

    Science.gov (United States)

    Moreau, Christophe; Bally, Isabelle; Chouquet, Anne; Bottazzi, Barbara; Ghebrehiwet, Berhane; Gaboriaud, Christine; Thielens, Nicole

    2016-01-01

    Complement C1q is a soluble pattern recognition molecule comprising six heterotrimeric subunits assembled from three polypeptide chains (A-C). Each heterotrimer forms a collagen-like stem prolonged by a globular recognition domain. These recognition domains sense a wide variety of ligands, including pathogens and altered-self components. Ligand recognition is either direct or mediated by immunoglobulins or pentraxins. Multivalent binding of C1q to its targets triggers immune effector mechanisms mediated via its collagen-like stems. The induced immune response includes activation of the classical complement pathway and enhancement of the phagocytosis of the recognized target. We report here, the first production of a single-chain recombinant form of human C1q globular region (C1q-scGR). The three monomers have been linked in tandem to generate a single continuous polypeptide, based on a strategy previously used for adiponectin, a protein structurally related to C1q. The resulting C1q-scGR protein was produced at high yield in stably transfected 293-F mammalian cells. Recombinant C1q-scGR was correctly folded, as demonstrated by its X-ray crystal structure solved at a resolution of 1.35 Å. Its interaction properties were assessed by surface plasmon resonance analysis using the following physiological C1q ligands: the receptor for C1q globular heads, the long pentraxin PTX3, calreticulin, and heparin. The 3D structure and the binding properties of C1q-scGR were similar to those of the three-chain fragment generated by collagenase digestion of serum-derived C1q. Comparison of the interaction properties of the fragments with those of native C1q provided insights into the avidity component associated with the hexameric assembly of C1q. The interest of this functional recombinant form of the recognition domains of C1q in basic research and its potential biomedical applications are discussed.

  3. Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi.

    Science.gov (United States)

    Dobhal, S; Chaudhary, V K; Singh, A; Pandey, D; Kumar, A; Agrawal, S

    2013-12-01

    Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.

  4. Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses.

    Science.gov (United States)

    Hurwitz, Amy M; Huang, Wanzhi; Kou, Baijun; Estes, Mary K; Atmar, Robert L; Palzkill, Timothy

    2017-01-01

    Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)-named NJT-R3-A2 and NJT-R3-A3-that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.

  5. Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D; Becerril, Baltazar

    2011-02-25

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

  6. 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.

  7. Construction of humanized carcinoembryonic antigen specific single chain variable fragment and mitomycin conjugate

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To construct a new target-oriented conjugate of humanized carcinoembryonic antigen (CEA) specific single chain variable fragment (scFv) and mitomycin (MMC) against colorectal cancer, and to investigate its influence on the growth and apoptosis of colorectal cancer cells.METHODS: The primer was designed according to the gene sequence described in reference 16, which respectively contains restriction enzyme cleavage sites BamH Ⅰ and EcoR Ⅰ in its upstream and downstream.PCR was performed with the plasmid as template containing genes of humanized anti-CEA scFv. The product was digested by BamH Ⅰ and EcoR Ⅰ, and connected to an expression vector which also has the restriction enzyme cleavage sites BamH Ⅰ and EcoR.Expression of the reaction was induced by isopropy-β-D-thiogalactoside (IPTG). Then the expression product was covalently coupled with MMC by dextran T-40. The immunoreactivity of the conjugate against colorectal cancer cells as well as CEA was measured by enzyme linked immunosorbent assay (ELISA). The inhibiting ratio of conjugate on the growth of colorectal cancer cells was also measured by ELISA. The effect of conjugate on the apoptosis of colorectal cancer cells was determined by flow cytometry (FCM).RESULTS: Restriction endonuclease cleavage and gene sequencing confirmed that the expression vector was successfully constructed. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-VAGE) confirmed that this vector correctly expressed the fusion protein.ELISA confirmed that the conjugate had quite a strong immunoreactivity against colorectal cancer cells and CEA. The conjugate had inhibitory effects on colorectal cancer cells in a concentration-dependent manner and could induce apoptosis of colorectal cancer cells in a concentration-dependent manner.CONCLUSION: The CEA-scFv-MMC conjugate can be successfully constructed and is able to inhibit the growth and induce apoptosis of colorectal cancer cells.

  8. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2011-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity. PMID:21156801

  9. Identification of internalizing human single-chain antibodies targeting brain tumor sphere cells.

    Science.gov (United States)

    Zhu, Xiaodong; Bidlingmaier, Scott; Hashizume, Rintaro; James, C David; Berger, Mitchel S; Liu, Bin

    2010-07-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv, and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together, these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. (c)2010 AACR.

  10. Identification of internalizing human single chain antibodies targeting brain tumor sphere cells

    Science.gov (United States)

    Zhu, Xiaodong; Bidlingmaier, Scott; Hashizume, Rintaro; James, C. David; Berger, Mitchel S.; Liu, Bin

    2010-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor and there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive media, and exhibit enhanced tumor initiating ability and resistance to therapy. We report here the identification of internalizing human single chain antibodies (scFvs) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133 positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular non-selective media. Taken together, these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. PMID:20587664

  11. 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.

  12. EXPRESSION OF GROWTH-FACTORS AND GROWTH-FACTOR RECEPTORS IN NORMAL AND TUMOROUS HUMAN THYROID TISSUES

    NARCIS (Netherlands)

    van der Laan, B.F.A.M.; FREEMAN, JL; ASA, SL

    1995-01-01

    A number of growth factors have been implicated as stimuli of thyroid cell proliferation; overexpression of these growth factors and/or their receptors may play a role in the growth of thyroid tumors. To determine if immunohistochemical detection of growth factors and/or their receptors correlates w

  13. Augmentation of radiation response by motesanib, a multikinase inhibitor that targets vascular endothelial growth factor receptors.

    NARCIS (Netherlands)

    Kruser, T.J.; Wheeler, D.L.; Armstrong, E.A.; Iida, M.; Kozak, K.R.; Kogel, A.J. van der; Bussink, J.; Coxon, A.; Polverino, A.; Harari, P.M.

    2010-01-01

    BACKGROUND: Motesanib is a potent inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3, platelet-derived growth factor receptor, and Kit receptors. In this report we examine the interaction between motesanib and radiation in vitro and in head and neck squamous cell carcinoma

  14. Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1.

    Science.gov (United States)

    Meza-Carmen, Victor; Pacheco-Rodriguez, Gustavo; Kang, Gi Soo; Kato, Jiro; Donati, Chiara; Zhang, Chun-Yi; Vichi, Alessandro; Payne, D Michael; El-Chemaly, Souheil; Stylianou, Mario; Moss, Joel; Vaughan, Martha

    2011-06-28

    ADP-ribosylation factor domain protein 1 (ARD1) is a 64-kDa protein containing a functional ADP-ribosylation factor (GTP hydrolase, GTPase), GTPase-activating protein, and E3 ubiquitin ligase domains. ARD1 activation by the guanine nucleotide-exchange factor cytohesin-1 was known. GTPase and E3 ligase activities of ARD1 suggest roles in protein transport and turnover. To explore this hypothesis, we used mouse embryo fibroblasts (MEFs) from ARD1-/- mice stably transfected with plasmids for inducible expression of wild-type ARD1 protein (KO-WT), or ARD1 protein with inactivating mutations in E3 ligase domain (KO-E3), or containing persistently active GTP-bound (KO-GTP), or inactive GDP-bound (KO-GDP) GTPase domains. Inhibition of proteasomal proteases in mifepristone-induced KO-WT, KO-GDP, or KO-GTP MEFs resulted in accumulation of these ARD1 proteins, whereas KO-E3 accumulated without inhibitors. All data were consistent with the conclusion that ARD1 regulates its own steady-state levels in cells by autoubiquitination. Based on reported growth factor receptor-cytohesin interactions, EGF receptor (EGFR) was investigated in induced MEFs. Amounts of cell-surface and total EGFR were higher in KO-GDP and lower in KO-GTP than in KO-WT MEFs, with levels in both mutants greater (p = 0.001) after proteasomal inhibition. Significant differences among MEF lines in content of TGF-β receptor III were similar to those in EGFR, albeit not as large. Differences in amounts of insulin receptor mirrored those in EGFR, but did not reach statistical significance. Overall, the capacity of ARD1 GTPase to cycle between active and inactive forms and its autoubiquitination both appear to be necessary for the appropriate turnover of EGFR and perhaps additional growth factor receptors.

  15. Targeting of adenoviral vectors through a bispecific single-chain antibody

    NARCIS (Netherlands)

    Haisma, HJ; Grill, J; Curiel, DT; Hoogeland, S; van Beusechem, VW; Pinedo, HM; Gerritsen, WR

    2000-01-01

    Recombinant adenoviral vectors are attractive in the context of cancer gene therapy because they are capable of delivering genes to a wide variety of tissues. The utility of adenoviruses is limited by their lack of specificity and by the absence of the receptor(s) for these viruses on many tumor cel

  16. Polychlorinated Biphenyls Disrupt Hepatic Epidermal Growth Factor Receptor Signaling.

    Science.gov (United States)

    Hardesty, Josiah E; Wahlang, Banrida; Falkner, K Cameron; Clair, Heather B; Clark, Barbara J; Ceresa, Brian P; Prough, Russell A; Cave, Matthew C

    2016-07-26

    1. Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that disrupt hepatic xenobiotic and intermediary metabolism, leading to metabolic syndrome and nonalcoholic steatohepatitis (NASH). 2. Since phenobarbital indirectly activates Constitutive Androstane Receptor (CAR) by antagonizing growth factor binding to the epidermal growth factor receptor (EGFR), we hypothesised that PCBs may also diminish EGFR signaling. 3. The effects of the PCB mixture Aroclor 1260 on the protein phosphorylation cascade triggered by EGFR activation were determined in murine (in vitro and in vivo) and human models (in vitro). EGFR tyrosine residue phosphorylation was decreased by PCBs in all models tested. 4. The IC50 values for Aroclor 1260 concentrations that decreased Y1173 phosphorylation of EGFR were similar in murine AML-12 and human HepG2 cells (∼2-4 μg/mL). Both dioxin and non-dioxin-like PCB congeners decreased EGFR phosphorylation in cell culture. 5. PCB treatment reduced phosphorylation of downstream EGFR effectors including Akt and mTOR, as well as other phosphoprotein targets including STAT3 and c-RAF in vivo. 6. PCBs diminish EGFR signaling in human and murine hepatocyte models and may dysregulate critical phosphoprotein regulators of energy metabolism and nutrition, providing a new mechanism of action in environmental diseases.

  17. Preparation of single chain variable fragment of MG7 mAb by phage display technology

    Institute of Scientific and Technical Information of China (English)

    Zhao-Cai Yu; Jie Ding; Yong-Zhan Nie; Dai-Ming Fan; Xue-Yong Zhang

    2001-01-01

    AIM To develop the single chain variable fragment of MG7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS mRNA was isolated from MG7-producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOⅢ of highly expressing MG7binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG7 ScFv. ELISA assay was used to detect the antigenbinding affinity of the soluble MG7 ScFv. Finally, the relative molecular mass of soluble MG7 ScFv was measured by SDS-PAGE. RESULTS The VH, VL and ScFv DNAs were about 340bp,320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG7 antibody for binding to the antigen expressed on KATO Ⅲ cells. Within 2 strong positive phage clones, the soluble MG7 ScFv from one clone was found to have the binding activity with KATO Ⅲ cells.SDS-PAGE showed that the relative molecular weight of soluble MG7 ScFv was 32. CONCLUSION The MG7 ScFv was successfully produced by phage antibody technology, which may

  18. Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses

    Science.gov (United States)

    Hurwitz, Amy M.; Huang, Wanzhi; Kou, Baijun; Estes, Mary K.; Atmar, Robert L.; Palzkill, Timothy

    2017-01-01

    Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)—named NJT-R3-A2 and NJT-R3-A3—that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (KD) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics. PMID:28095447

  19. Modular and dynamic approaches to the formation of single-chain polymer nanoparticles

    Science.gov (United States)

    Tuten, Bryan Tyler

    The methodology towards the creation of nanoscale polymeric objects by way of the folding of single polymer chains has been enjoying success in the field of polymer chemistry and materials science. By synthesizing polymer chains with built in functionality either through functional side groups, or direct incorporation into the polymer backbone, polymer chemists are able to fold single polymer chains onto themselves through a broad range of covalent and non-covalent interactions in dilute solution. These compact, nano-sized objects can now be used in a wide arrange of functions and applications. The aim of this dissertation is to provide first, a comprehensive overview of the recent advances and success enjoyed by this field and second, to showcase some of the various routes towards the dynamic and modular creation of these single-chain polymer nanoparticles (SCNPs). Chapter 2 of this work discusses the use of dynamic covalent cross-linking chemistry via reversible disulfide bridges in the folding and unfolding of SCNPs. Through the use of triple detection size-exclusion chromatography (SEC) it was shown through changes in retention time, a phenomena indicative of hydrodynamic volume, a polymer was being folded into compact SCNPs and then unfolded and refolded via redox chemistry. Chapter 3 explores the design of polymers that had various different cross-linkable moieties incorporated into the monomer side units. By having cross-linkable moieties that can undergo different chemical cross-linking reactions (i.e thiol-yne click reactions, epoxide ring-opening reactions, activated esters), a modular approach towards the folding and subsequent functionalization of SCNPs is created. Looking to design a system with a greater degree of control over the modular functionality, chapter 4 investigates the use of norbornene imide monomers containing pentafluorophenyl activated esters with varying methylene spacer unites between the polymerizable olefin and the activated ester

  20. Cloning,expression and characterization of a single-chain antibody PS-9 targeted to pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴; 田波; 李力; 袁玫

    1995-01-01

    Genes encoding single-chain antibodies have been first constructed,which consist of the heavyand light chain variable domains of antibody PS-9 joined together by a flexible peptide linker.The geneswere cloned into coat protein g3p genes of pCANTAB5 phagemids,and expressed as fusion proteins on thephage tips.Immunological assay demonstrated that the engineered antibodies specifically bound to cancer cellsLS-174-T as well as to pure bovine submaxillary gland mucin.Their specificity and affinity appeared the sameas their parent antibodies.Our results supposed that the single-chain antibodies will be a target for thediagnosis and treatment of cancer.

  1. Poly(ethylene glycol)-graft-poly(vinyl acetate) single-chain nanoparticles for the encapsulation of small molecules.

    Science.gov (United States)

    Bartolini, Arianna; Tempesti, Paolo; Resta, Claudio; Berti, Debora; Smets, Johan; Aouad, Yousef G; Baglioni, Piero

    2017-02-08

    Amphiphilic poly(ethylene glycol)-graft-poly(vinyl acetate) copolymers with a low degree of grafting undergo self-folding in water driven by hydrophobic interactions, resulting in single-chain nanoparticles (SCNPs) possessing a hydrodynamic radius of about 10 nm. A temperature scan revealed a lower critical solution temperature (LCST)-type phase behavior. In addition, SAXS data collected close to the LCST showed that these SCNPs aggregate into one-dimensional elongated objects, preferentially. With respect to the typical linear complex-structured polymer chains, this material is ideally suited for industrial and/or biomedical applications because of its simple molecular architecture and persistence of SCNPs up to 100 mg mL(-1). The so-obtained single-chain globular particles are able to swell upon loading with small hydrophobic molecules therefore promoting solubilization of flavors or drugs, which could be of interest in the food and pharmaceutical industry.

  2. Thyroid hormones regulate fibroblast growth factor receptor signaling during chondrogenesis.

    Science.gov (United States)

    Barnard, Joanna C; Williams, Allan J; Rabier, Bénédicte; Chassande, Olivier; Samarut, Jacques; Cheng, Sheue-Yann; Bassett, J H Duncan; Williams, Graham R

    2005-12-01

    Childhood hypothyroidism causes growth arrest with delayed ossification and growth-plate dysgenesis, whereas thyrotoxicosis accelerates ossification and growth. Thyroid hormone (T(3)) regulates chondrocyte proliferation and is essential for hypertrophic differentiation. Fibroblast growth factors (FGFs) are also important regulators of chondrocyte proliferation and differentiation, and activating mutations of FGF receptor-3 (FGFR3) cause achondroplasia. We investigated the hypothesis that T(3) regulates chondrogenesis via FGFR3 in ATDC5 cells, which undergo a defined program of chondrogenesis. ATDC5 cells expressed two FGFR1, four FGFR2, and one FGFR3 mRNA splice variants throughout chondrogenesis, and expression of each isoform was stimulated by T(3) during the first 6-12 d of culture, when T(3) inhibited proliferation by 50%. FGFR3 expression was also increased in cells treated with T(3) for 21 d, when T(3) induced an earlier onset of hypertrophic differentiation and collagen X expression. FGFR3 expression was reduced in growth plates from T(3) receptor alpha-null mice, which exhibit skeletal hypothyroidism, but was increased in T(3) receptor beta(PV/PV) mice, which display skeletal thyrotoxicosis. These findings indicate that FGFR3 is a T(3)-target gene in chondrocytes. In further experiments, T(3) enhanced FGF2 and FGF18 activation of the MAPK-signaling pathway but inhibited their activation of signal transducer and activator of transcription-1. FGF9 did not activate MAPK or signal transducer and activator of transcription-1 pathways in the absence or presence of T(3). Thus, T(3) exerted differing effects on FGFR activation during chondrogenesis depending on which FGF ligand stimulated the FGFR and which downstream signaling pathway was activated. These studies identify novel interactions between T(3) and FGFs that regulate chondrocyte proliferation and differentiation during chondrogenesis.

  3. Epidermal Growth Factor Receptor Cell Survival Signaling Requires Phosphatidylcholine Biosynthesis

    Directory of Open Access Journals (Sweden)

    Matt Crook

    2016-11-01

    Full Text Available Identification of pro-cell survival signaling pathways has implications for cancer, cardiovascular, and neurodegenerative disease. We show that the Caenorhabditis elegans epidermal growth factor receptor LET-23 (LET-23 EGFR has a prosurvival function in counteracting excitotoxicity, and we identify novel molecular players required for this prosurvival signaling. uv1 sensory cells in the C. elegans uterus undergo excitotoxic death in response to activation of the OSM-9/OCR-4 TRPV channel by the endogenous agonist nicotinamide. Activation of LET-23 EGFR can effectively prevent this excitotoxic death. We investigate the roles of signaling pathways known to act downstream of LET-23 EGFR in C. elegans and find that the LET-60 Ras/MAPK pathway, but not the IP3 receptor pathway, is required for efficient LET-23 EGFR activity in its prosurvival function. However, activation of LET-60 Ras/MAPK pathway does not appear to be sufficient to fully mimic LET-23 EGFR activity. We screen for genes that are required for EGFR prosurvival function and uncover a role for phosphatidylcholine biosynthetic enzymes in EGFR prosurvival function. Finally, we show that exogenous application of phosphatidylcholine is sufficient to prevent some deaths in this excitotoxicity model. Our work implicates regulation of lipid synthesis downstream of EGFR in cell survival and death decisions.

  4. Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes.

    Directory of Open Access Journals (Sweden)

    Geetanjali Kharmate

    Full Text Available Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa. However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa.

  5. Glucocorticoids and atrial natriuretic factor receptors on vascular smooth muscle.

    Science.gov (United States)

    Yasunari, K; Kohno, M; Murakawa, K; Yokokawa, K; Takeda, T

    1990-11-01

    The effect of glucocorticoids on the atrial natriuretic factor (ANF)-mediated formation of cyclic guanosine monophosphate (cGMP) by intact vascular smooth muscle cells (VSMC) was studied in rats. Cultured VSMC were obtained from the renal arteries of 14-week-old Wistar rats by the explant method. Micromolar concentrations of dexamethasone, given as pretreatment for 48 hours, suppressed the ANF-mediated response. The dexamethasone-induced suppression was detectable at 6 hours and reached a maximum 24 hours after administration in a dose-dependent manner. Inhibitors of protein synthesis blocked this effect of the glucocorticoid. The basal activity of guanylate cyclase in the dexamethasone-treated cells was lower than in the control cells. Other steroids having glucocorticoid action mimicked this suppression of the ANF-mediated response. This suppression was blocked by a glucocorticoid receptor antagonist. The results suggest that glucocorticoids suppress ANF-mediated cGMP formation by VSMC through glucocorticoid type II receptors and the induction of protein synthesis. Suppression of the ANF-mediated response may play a role in glucocorticoid-induced hypertension.

  6. Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1.

    Science.gov (United States)

    Pasello, Michela; Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Picci, Piero; Cianfriglia, Maurizio; Scotlandi, Katia

    2016-04-20

    Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×10(9) individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

  7. Force spectroscopy of hyaluronan by atomic force microscopy: from hydrogen-bonded networks toward single-chain behavior.

    Science.gov (United States)

    Giannotti, Marina I; Rinaudo, Marguerite; Vancso, G Julius

    2007-09-01

    The conformational behavior of hyaluronan (HA) polysaccharide chains in aqueous NaCl solution was characterized directly at the single-molecule level. This communication reports on one of the first single-chain atomic force microscopy (AFM) experiments performed at variable temperatures, investigating the influence of the temperature on the stability of the HA single-chain conformation. Through AFM single-molecule force spectroscopy, the temperature destabilization of a local structure was proven. This structure involved a hydrogen-bonded network along the polymeric chain, with hydrogen bonds between the polar groups of HA and possibly water, and a change from a nonrandom coil to a random coil behavior was observed when increasing the temperature from 29 +/- 1 to 46 +/- 1 degrees C. As a result of the applied force, this superstructure was found to break progressively at room temperature. The use of a hydrogen-bonding breaker solvent demonstrated the hydrogen-bonded water-bridged nature of the network structure of HA single chains in aqueous NaCl solution.

  8. 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....../inactive endometria and seven of 13 (54%) endometria with adenomatous hyperplasia were EGF-R positive, with an immunostaining pattern rather similar to that of the carcinomas....

  9. Interferon gamma-dependent transactivation of epidermal growth factor receptor.

    Science.gov (United States)

    Burova, Elena; Vassilenko, Konstantin; Dorosh, Victoria; Gonchar, Ilya; Nikolsky, Nikolai

    2007-04-03

    The present report provides evidence that, in A431 cells, interferon gamma (IFNgamma) induces the rapid (within 5 min), and reversible, tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). IFNgamma-induced EGFR transactivation requires EGFR kinase activity, as well as activity of the Src-family tyrosine kinases and JAK2. Here, we show that IFNgamma-induced STAT1 activation in A431 and HeLa cells partially depends on the kinase activity of both EGFR and Src. Furthermore, in these cells, EGFR kinase activity is essential for IFNgamma-induced ERK1,2 activation. This study is the first to demonstrate that EGFR is implicated in IFNgamma-dependent signaling pathways.

  10. Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv

    Directory of Open Access Journals (Sweden)

    Elias Waldir P

    2011-02-01

    Full Text Available Abstract Background Intimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC and enterohemorrhagic Escherichia coli (EHEC. Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667. In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv. Findings Anti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction, was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with

  11. Nod factor receptors form heteromeric complexes and are essential for intracellular infection in Medicago nodules

    NARCIS (Netherlands)

    Moling, S.; Pietraszewska-Bogiel, A.; Postma, M.; Fedorova, E.E.; Hink, M.A.; Limpens, E.H.M.; Gadella, T.W.J.; Bisseling, T.

    2014-01-01

    Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It wa

  12. Nod factor receptors form heteromeric complexes and are essential for intracellular infection in medicago nodules

    NARCIS (Netherlands)

    Moling, S.; Pietraszewska-Bogiel, A.; Postma, M.; Fedorova, E.; Hink, M.A.; Limpens, E.; Gadella, T.W.J.; Bisseling, T.

    2014-01-01

    Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It wa

  13. 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

  14. 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.

  15. Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment.

    Science.gov (United States)

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D; Torres-Larios, Alfredo

    2011-06-10

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na(+) channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  16. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment*

    Science.gov (United States)

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-01-01

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs. PMID:21489992

  17. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    Energy Technology Data Exchange (ETDEWEB)

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo (U. NAM)

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Gustavo M S G Moreira

    Full Text Available 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.

  20. A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes

    Directory of Open Access Journals (Sweden)

    Jordaan Frances

    2004-04-01

    Full Text Available Abstract Background Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers. Results With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA. Conclusion The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naïve phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.

  1. LysM domain receptor kinases regulating rhizobial Nod factor-induced infection

    NARCIS (Netherlands)

    Limpens, E.H.M.; Franken, C.L.; Smit, P.E.J.; Willemse, J.J.; Bisseling, T.; Geurts, R.

    2003-01-01

    The rhizobial infection of legumes has the most stringent demand toward Nod factor structure of all host responses, and therefore a specific Nod factor entry receptor has been proposed. The SYM2 gene identfied in certain ecotypes of pea (Pisum sativum) is a good candidate for such an entry receptor.

  2. Fibroblast growth factor receptor 3 protein is overexpressed in oral and oropharyngeal squamous cell carcinoma

    NARCIS (Netherlands)

    Koole, Koos; van Kempen, Pauline M W; Swartz, Justin E; Peeters, Ton; van Diest, Paul J; Koole, Ron; van Es, Robert J. J.; Willems, Stefan M

    Fibroblast growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor receptor tyrosine kinase family. It has been identified as a promising therapeutic target in multiple types of cancer. We have investigated FGFR3 protein expression and FGFR3 gene copy-numbers in a single

  3. Epidermal growth factor receptor-targeted antibody therapy - Mechanisms of action and modulators of therapeutic efficacy

    NARCIS (Netherlands)

    Lammerts van Bueren, Jeroen Jilles

    2008-01-01

    Cancer is an increasing disease in the world population, and in recent years there has been substantial interest in the development of novel therapeutic agents specifically targeting growth factor receptors on tumor cells. The epidermal growth factor receptor (EGFR) represents a tyrosine kinase cell

  4. Evidence for biosynthesis of lactase-phlorizin hydrolase as a single-chain high-molecular weight precursor

    DEFF Research Database (Denmark)

    Skovbjerg, H; Danielsen, E M; Noren, Ove

    1984-01-01

    Precursor forms of lactase-phlorizin hydrolase, sucrase-isomaltase and aminopeptidase N were studied by pulse-labelling of organ-cultured human intestinal biopsies. After labelling the biopsies were fractionated by the Ca2+-precipitation method and the enzymes isolated by immunoprecipitation....... The results indicate that the lactase-phlorizin hydrolase is synthesized as a Mr 245 000 polypeptide, which is intracellularly cleaved into its mature Mr 160 000 form. Sucrase-isomaltase is shown to be synthesized as a single chain precursor (Mr 245 000 and 265 000) while the precursor of aminopeptidase N...

  5. 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...

  6. Expression of epidermal growth factor receptors in human brain tumors.

    Science.gov (United States)

    Libermann, T A; Razon, N; Bartal, A D; Yarden, Y; Schlessinger, J; Soreq, H

    1984-02-01

    The expression of receptors for epidermal growth factor (EGF-R) was determined in 29 samples of brain tumors from 22 patients. Primary gliogenous tumors, of various degrees of cancer, five meningiomas, and two neuroblastomas were examined. Tissue samples were frozen in liquid nitrogen immediately after the operation and stored at -70 degrees until use. Cerebral tissue samples from 11 patients who died from diseases not related to the central nervous system served as controls. Immunoprecipitation of functional EGF-R-kinase complexes revealed high levels of EGF-R in all of the brain tumors of nonneuronal origin that were examined. The level of EGF-R varied between tumors from different patients and also between specimens prelevated from different areas of the same tumor. In contrast, the levels of EGF-R from control specimens were invariably low. The biochemical properties of EGF-R in brain tumor specimens were found to be indistinguishable from those of the well-characterized EGF-R from the A-431 cell line, derived from human epidermoid carcinomas. Human brain EGF-R displays a molecular weight of 170,000 by polyacrylamide-sodium dodecyl sulfate gel electrophoresis. It is phosphorylated mainly in tyrosine residues and shows a 2-dimensional phosphopeptide map similar to that obtained with the phosphorylated EGF-R from membranes of A-431 cells. Our observations suggest that induction of EGF-R expression may accompany the malignant transformation of human brain cells of nonneuronal origin.

  7. Indoxyl Sulfate Downregulates Mas Receptor via Aryl Hydrocarbon Receptor/Nuclear Factor-kappa B, and Induces Cell Proliferation and Tissue Factor Expression in Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Ng, Hwee-Yeong; Bolati, Wulaer; Lee, Chien-Te; Chien, Yu-Shu; Yisireyili, Maimaiti; Saito, Shinichi; Pei, Sung-Nan; Nishijima, Fuyuhiko; Niwa, Toshimitsu

    2016-01-01

    Angiotensin converting enzyme-related carboxypeptidase 2/angiotensin (Ang)-(1-7)/Mas receptor axis is protective in the development of chronic kidney disease and cardiovascular disease. This study is aimed at investigating whether indoxyl sulfate (IS) affects Mas receptor expression, cell proliferation and tissue factor expression in vascular smooth muscle cells, and if Ang-(1-7), an activator of Mas receptor, counteracts the IS-induced effects. IS was administered to normotensive and hypertensive rats. Human aortic smooth muscle cells (HASMCs) were cultured with IS. IS reduced the expression of Mas receptor in the aorta of normotensive and hypertensive rats. IS downregulated the Mas receptor expression in a time- and dose-dependent manner in HASMCs. Knockdown of aryl hydrocarbon receptor (AhR) and nuclear factor-kappa B (NF-x03BA;B) inhibited IS-induced downregulation of Mas receptor. Further, IS stimulated cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) attenuated IS-induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) suppressed phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-x03BA;B in HASMCs. IS downregulated the expression of Mas receptor via AhR/NF-x03BA;B, and induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) inhibited IS-induced cell proliferation and tissue factor expression by suppressing the phosphorylation of ERK1/2 and NF-x03BA;B p65. © 2016 S. Karger AG, Basel.

  8. Specific antibodies and sensitive immunoassays for the human epidermal growth factor receptors (HER2, HER3, and HER4).

    Science.gov (United States)

    Broughton, Marianne Nordlund; Westgaard, Arne; Paus, Elisabeth; Øijordsbakken, Miriam; Henanger, Karoline J; Naume, Bjørn; Bjøro, Trine

    2017-06-01

    The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, human epidermal growth factor receptor 3, human epidermal growth factor receptor 4, human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2-positive and tissue human epidermal growth factor receptor 2-negative ( p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 ( p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with

  9. Dynamic tracing for epidermal growth factor receptor mutations in urinary circulating DNA in gastric cancer patients.

    Science.gov (United States)

    Shi, Xiu-Qin; Xue, Wen-Hua; Zhao, Song-Feng; Zhang, Xiao-Jian; Sun, Wukong

    2017-02-01

    The mutations of epidermal growth factor receptor are detected in gastric cancer, indicating its suitability as a target for receptor tyrosine kinase inhibitors, as well as a marker for clinical outcome of chemotherapeutic treatments. However, extraction of quality tumor tissue for molecular processes remains challenging. Here, we aimed to examine the clinical relevance of urinary cell-free DNA as an alternative tumor material source used specifically for monitoring epidermal growth factor receptor mutations. Therefore, 120 gastric cancer patients with epidermal growth factor receptor mutations and 100 healthy controls were recruited for the study. The gastric patients also received epidermal growth factor receptor inhibitor treatment for a serial monitoring study. Paired primary tumor specimens were obtained with blood and urine samples, which were taken at a 1-month interval for a duration of 12 months. We found that urinary cell-free DNA yielded a close agreement of 92% on epidermal growth factor receptor mutation status when compared to primary tissue at baseline, and of 99% epidermal growth factor receptor mutation status when compared to plasma samples at different time points. Thus, our data suggest that urinary cell-free DNA may be a reliable source for screening and monitoring epidermal growth factor receptor mutations in the primary gastric cancer.

  10. Potent, selective inhibitors of fibroblast growth factor receptor define fibroblast growth factor dependence in preclinical cancer models.

    Science.gov (United States)

    Squires, Matthew; Ward, George; Saxty, Gordan; Berdini, Valerio; Cleasby, Anne; King, Peter; Angibaud, Patrick; Perera, Tim; Fazal, Lynsey; Ross, Douglas; Jones, Charlotte Griffiths; Madin, Andrew; Benning, Rajdeep K; Vickerstaffe, Emma; O'Brien, Alistair; Frederickson, Martyn; Reader, Michael; Hamlett, Christopher; Batey, Michael A; Rich, Sharna; Carr, Maria; Miller, Darcey; Feltell, Ruth; Thiru, Abarna; Bethell, Susanne; Devine, Lindsay A; Graham, Brent L; Pike, Andrew; Cosme, Jose; Lewis, Edward J; Freyne, Eddy; Lyons, John; Irving, Julie; Murray, Christopher; Newell, David R; Thompson, Neil T

    2011-09-01

    We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role.

  11. Oncogenic fingerprint of epidermal growth factor receptor pathway and emerging epidermal growth factor receptor blockade resistance in colorectal cancer

    Science.gov (United States)

    Sobani, Zain A; Sawant, Ashwin; Jafri, Mikram; Correa, Amit Keith; Sahin, Ibrahim Halil

    2016-01-01

    Epidermal growth factor receptor (EGFR) has been an attractive target for treatment of epithelial cancers, including colorectal cancer (CRC). Evidence from clinical trials indicates that cetuximab and panitumumab (anti-EGFR monoclonal antibodies) have clinical activity in patients with metastatic CRC. The discovery of intrinsic EGFR blockade resistance in Kirsten RAS (KRAS)-mutant patients led to the restriction of anti-EGFR antibodies to KRAS wild-type patients by Food and Drug Administration and European Medicine Agency. Studies have since focused on the evaluation of biomarkers to identify appropriate patient populations that may benefit from EGFR blockade. Accumulating evidence suggests that patients with mutations in EGFR downstream signaling pathways including KRAS, BRAF, PIK3CA and PTEN could be intrinsically resistant to EGFR blockade. Recent whole genome studies also suggest that dynamic alterations in signaling pathways downstream of EGFR leads to distinct oncogenic signatures and subclones which might have some impact on emerging resistance in KRAS wild-type patients. While anti-EGFR monoclonal antibodies have a clear potential in the management of a subset of patients with metastatic CRC, further studies are warranted to uncover exact mechanisms related to acquired resistance to EGFR blockade. PMID:27777877

  12. Mn(III)(tetra-biphenyl-porphyrin)-TCNE single-chain magnet via suppression of the interchain interactions.

    Science.gov (United States)

    Ishikawa, Ryuta; Katoh, Keiichi; Breedlove, Brian K; Yamashita, Masahiro

    2012-08-20

    A single-chain magnet (SCM) of [Mn(TBPP)(TCNE)]·4m-PhCl(2) (1), where TBPP(2-) = meso-tetra(4-biphenyl)porphyrinate; TCNE(•-) = tetracyanoethenide radical anion; m-PhCl(2) = meta-dichlorobenzene, was prepared via suppression of interchain interactions. 1 has a one-dimensional alternating Mn(III)(porphrin)-TCNE(•-)chain structure similar to those of a family of complexes reported by Miller and co-workers. From a comparison of the static magnetic properties of 1 with other Mn(III)(porphyrin)-TCNE(•-) chains, a magneto-structural correlation between the intrachain magnetic exchange and both the dihedral angle between the mean plane on [Mn(TBPP)(TCNE)] and Mn-N≡C was observed. The ac magnetic susceptibility data of 1 could be fit with the Arrhenius law, indicating that slow magnetic relaxation and ruling out three-dimensional long-range ordering and spin-glass-like behavior. The Cole-Cole plot for 1 was semicircular, verifying that it is an SCM. Therefore, 1 is an ideal single-chain magnet with significantly strong intrachain magnetic exchange interactions beyond the Ising limit.

  13. Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry

    Institute of Scientific and Technical Information of China (English)

    Yan-Wei Zhong; Jun Cheng; Gang Wang; Shuang-Shuang Shi; Li Li; Ling-Xia Zhang; Ju-Mei Chen

    2002-01-01

    AIM: To screen human single chain Fv antibody (scFv)against hepatitis C virus E2 antigen and identify its applicationin immunohistochemistry.METHODS: The phage antibody library was panned by HCVE2 antigen, which was coated in microtiter plate. After fiverounds of biopanning,56 phage clones were identified specificto HCV E2 antigen. The selected scFv clones were digestedby SfiI/NotI and DNA was sequenced. Then it was subclonedinto the vector pCANTABSE for expression as E-taggedsoluble scFv. The liver tissue sections from normal personand patients with chronic hepatitis B and chronic hepatitis Cwere immunostained with HCV E2 scFv antibody.RESULTS: The data of scFv-E2 DNA digestion and DNAsequencing showed that the scFv gene is composed of 750bp. ELISA and immunohistochemistry demonstrated that thehuman single chain Fy antibody against hepatitis C E2 antigenhas a specific binding character with hepatitis virus E2 antigenand paraffin-embedded tissue, but did not react with liver tissuesfrom healthy persons or patients with chronic hepatitis B.CONCLUSION: We have successfully screened andidentified HCV E2 scFv and the scFv could be used in theimmunostaining of liver tissue sections from patients withchronic hepatitis C.

  14. Chemical Chaperones Increasing Expression Level of Soluble Single-chain Fv Antibody(scFv2F3)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Glutathione peroxidase (GPX) is a selenoenzyme that protects the biomembrane and other cellular components against oxidative damage. The selenium-containing single chain Fv fragment of monoclonal antibody 2F3 (SescFv2F3 ) is a kind of GPX mimic and it has a wide clinical applications because of its high activity and low antigenicity. Se-scFv2F3 is generated by the chemical modification of the single chain Fy fragment of monoclonal antibody 2F3 ( scFv2F3 ), which can be expressed in E. coli. In this article, the effect of chemical chaperones, such as glycerol, glucose, and β-cyclodextrin added to the culture medium, on the expression of soluble scFv2F3 was investigated. The expression level was evaluated by the determination of soluble scFv2F3 contents in the whole cell lysates.The results suggest that both glycerol and β-cyclodextrin greatly increase the expression level of soluble scFv2F3, and β-cyclodextrin is found to be more effective compared with glycerol. Glucose has a slight effect on the expression level of soluble scFv2F3. This is the first example, wherein β-cyclodextrin has been used as a chemical chaperone during the cell culture to improve the expression level of recombinant proteins. In addition, chemical chaperones are found to decrease the toxic effect of IPTG on cells.

  15. Basic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor-1in Human Meningiomas

    Institute of Scientific and Technical Information of China (English)

    YI Wei; CHEN Jian; Filimon H. Golwa; XUE Delin

    2005-01-01

    The expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) in human meningiomas and the relationships between their expression and the tumors' histological features and angiogenesis were investigated by means of immunohistochemical technique. The expression of bFGF and FGFR-1 was detected by antibody of bFGF or FGFR-1.The tumors' angiogenesis was evaluated by microvascular density (MVD) and, which was observed by use of CD34-antibody immunohistochemically. The results showed that there were varied degrees of the expression of bFGF and FGFR-1 proteins in meningiomas. The expression was correlated with the tumors' histological characters and angiogenesis. It was concluded that bFGF and FGFR-1 might play important roles in meningiomas' angiogenesis and proliferation. The expression positive rate of bFGF and FGFR-1 may provide an indication of evaluating the histological and malignant degree of the tumor.

  16. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3

    Energy Technology Data Exchange (ETDEWEB)

    Keegan, K.; Hayman, M.J. (State Univ. of New York, Stony Brook (United States)); Johnson, D.E.; Williams, L.T. (Univ. of California, San Francisco (United States))

    1991-02-15

    The fibroblast growth factors are a family of polypeptide growth factors involved in a variety of activities including mitogenesis, angiogenesis, and wound healing. Fibroblast growth factor receptors (FGFRs) have previously been identified in chicken, mouse, and human and have been shown to contain an extracellular domain with either two or three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. The authors have isolated a human cDNA for another tyrosine kinase receptor that is highly homologous to the previously described FGFR. Expression of this receptor cDNA in COS cells directs the expression of a 125-kDa glycoprotein. They demonstrate that this cDNA encodes a biologically active receptor by showing that human acidic and basic fibroblast growth factors activate this receptor as measured by {sup 45}Ca{sup 2+} efflux assays. These data establish the existence of an additional member of the FGFR family that they have named FGFR-3.

  17. 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.

  18. Tumor necrosis factor downregulates granulocyte-colony-stimulating factor receptor expression on human acute myeloid leukemia cells and granulocytes.

    OpenAIRE

    Elbaz, O; Budel, L M; Hoogerbrugge, H; Touw, I P; Delwel, R.; Mahmoud, L A; Löwenberg, B. (Bernward)

    1991-01-01

    Tumor necrosis factor (TNF) inhibits granulocyte-colony-stimulating factor (G-CSF)-induced human acute myeloid leukemia (AML) growth in vitro. Incubation of blasts from three patients with AML in serum-free medium with TNF (10(3) U/ml), and subsequent binding studies using 125I-G-CSF reveal that TNF downregulates the numbers of G-CSF receptors by approximately 70%. G-CSF receptor numbers on purified blood granulocytes are also downmodulated by TNF. Downregulation of G-CSF receptor expression ...

  19. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    Science.gov (United States)

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  20. Epidermal growth factor receptor transactivation by intracellular prostaglandin E2-activated prostaglandin E2 receptors. Role in retinoic acid receptor-β up-regulation.

    Science.gov (United States)

    Fernández-Martínez, Ana B; Lucio Cazaña, Francisco J

    2013-09-01

    The pharmacological modulation of renoprotective factor vascular endothelial growth factor-A (VEGF-A) in the proximal tubule has therapeutic interest. In human proximal tubular HK-2 cells, treatment with all-trans retinoic acid or prostaglandin E2 (PGE2) triggers the production of VEGF-A. The pathway involves an initial increase in intracellular PGE2, followed by activation of EP receptors (PGE2 receptors, most likely an intracellular subset) and increase in retinoic acid receptor-β (RARβ) expression. RARβ then up-regulates transcription factor hypoxia-inducible factor-1α (HIF-1α), which increases the transcription and production of VEGF-A. Here we studied the role in this pathway of epidermal growth factor receptor (EGFR) transactivation by EP receptors. We found that EGFR inhibitor AG1478 prevented the increase in VEGF-A production induced by PGE2- and all-trans retinoic acid. This effect was due to the inhibition of the transcriptional up-regulation of RARβ, which resulted in loss of the RARβ-dependent transcriptional up-regulation of HIF-1α. PGE2 and all-trans retinoic acid also increased EGFR phosphorylation and this effect was sensitive to antagonists of EP receptors. The role of intracellular PGE2 was indicated by two facts; i) PGE2-induced EGFR phosphorylation was substantially prevented by inhibitor of prostaglandin uptake transporter bromocresol green and ii) all-trans retinoic acid treatment, which enhanced intracellular but not extracellular PGE2, had lower effect on EGFR phosphorylation upon pre-treatment with cyclooxygenase inhibitor diclofenac. Thus, EGFR transactivation by intracellular PGE2-activated EP receptors results in the sequential activation of RARβ and HIF-1α leading to increased production of VEGF-A and it may be a target for the therapeutic modulation of HIF-1α/VEGF-A. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant Ⅲ single chain antibody%抗表皮生长因子受体Ⅲ型突变体噬菌体单链抗体库的构建与初步筛选

    Institute of Scientific and Technical Information of China (English)

    韩东刚; 段小艺; 郭佑民; 周琦; 王全颖; 杨广笑

    2010-01-01

    目的 构建抗表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)特异性单链抗体库,从中筛选抗EGFRvⅢ特异性单链抗体.方法 用pep-3偶联载体蛋白OVA,然后免疫BALB/c小鼠从脾淋巴细胞中提取总RNA,采用RT-PCR方法扩增莺链(VH)和轻链(VL)的可变区基因片段.将两基因片段由编码(Gly4Ser)3的linker连在一起,并克隆人噬菌体展示表达载体pCANTAB5E中,电转化至E. Coli TG1,经辅助噬菌体超感染,构建噬菌体单链抗体库.以pep-3-BSA为抗原对所建抗体库进行4轮亲和筛选,用ELISA鉴定抗体与EGFRvⅢ结合的特异性.结果 经琼脂糖凝胶电泳分析,RT-PCR方法扩增的抗体VH和VL基因片段大小约350和320 bp,与预期理论值相符.经重组PCR得到大小约780 bp的ScFv基因片段.ScFv基因片段经双酶切后成功定向克隆到噬菌粒载体,回收后构建成库容量为5.0×10~6的噬菌体抗体库,噬菌体滴度为3.0×10~4pfu/ml.通过亲和筛选使抗EGFRvⅢ噬菌体单链抗体得到富集,第4轮为第1轮的56倍.在E. Coli HB2151中实现了单链抗体的可溶性表达.SDS-PAGE结果显示抗体相对分子质量为28 000左右.ELISA测定结果显示该单链抗体具有结合EGFRvⅢ的特异性.结论 成功构建了噬菌体单链抗体库,筛选得到了抗EGFRvⅢ特异性噬菌体单链抗体.

  2. Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

    OpenAIRE

    Bodine, P V; Litwack, G

    1988-01-01

    We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Mo...

  3. Regulation of ciliary neurotrophic factor receptor alpha in sciatic motor neurons following axotomy.

    Science.gov (United States)

    MacLennan, A J; Devlin, B K; Neitzel, K L; McLaurin, D L; Anderson, K J; Lee, N

    1999-01-01

    Spinal motor neurons are one of the few classes of neurons capable of regenerating axons following axotomy. Injury-induced expression of neurotrophic factors and corresponding receptors may play an important role in this rare ability. A wide variety of indirect data suggests that ciliary neurotrophic factor receptor alpha may critically contribute to the regeneration of injured spinal motor neurons. We used immunohistochemistry, in situ hybridization and retrograde tracing techniques to study the regulation of ciliary neurotrophic factor receptor alpha in axotomized sciatic motor neurons. Ciliary neurotrophic factor receptor alpha immunoreactivity, detected with two independent antisera, is increased in a subpopulation of caudal sciatic motor neuron soma one, two and six weeks after sciatic nerve transection and reattachment, while no changes are detected at one day and 15 weeks post-lesion. Ciliary neurotrophic factor receptor alpha messenger RNA levels are augmented in the same classes of neurons following an identical lesion, suggesting that increased synthesis contributes, at least in part, to the additional ciliary neurotrophic factor receptor alpha protein. Separating the proximal and distal nerve stumps with a plastic barrier does not noticeably affect the injury-induced change in ciliary neurotrophic factor receptor alpha regulation, thereby indicating that this injury response is not dependent on signals distal to the lesion traveling retrogradely through the nerve or signals generated by axonal growth through the distal nerve. The prolonged increases in ciliary neurotrophic factor receptor alpha protein and messenger RNA found in regenerating sciatic motor neurons contrast with the responses of non-regenerating central neurons, which are reported to display, at most, a short-lived increase in ciliary neurotrophic factor receptor alpha messenger RNA expression following injury. The present data are the first to demonstrate, in vivo, neuronal regulation of

  4. LysM domain receptor kinases regulating rhizobial Nod factor-induced infection.

    Science.gov (United States)

    Limpens, Erik; Franken, Carolien; Smit, Patrick; Willemse, Joost; Bisseling, Ton; Geurts, René

    2003-10-24

    The rhizobial infection of legumes has the most stringent demand toward Nod factor structure of all host responses, and therefore a specific Nod factor entry receptor has been proposed. The SYM2 gene identified in certain ecotypes of pea (Pisum sativum) is a good candidate for such an entry receptor. We exploited the close phylogenetic relationship of pea and the model legume Medicago truncatula to identify genes specifically involved in rhizobial infection. The SYM2 orthologous region of M. truncatula contains 15 putative receptor-like genes, of which 7 are LysM domain-containing receptor-like kinases (LYKs). Using reverse genetics in M. truncatula, we show that two LYK genes are specifically involved in infection thread formation. This, as well as the properties of the LysM domains, strongly suggests that they are Nod factor entry receptors.

  5. Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Single-chain repressor RRTRES is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain RTRES by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of RTRES at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic, in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RRTRES. The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1×10-12 mol/L-1×10-11mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1×10-9 mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5′ position. We constructed a new homodimeric single-chain repressor RTRESRTRES and its DNA-binding specificity was tested by using a series of new operators designed according to the recog-nition properties previously determined for the RTRES domain. These operators containing the con-sensus sequence GTAAGAAARNTTACN or GGAAGAAARNTTCCN (R is A or G) were recognized by RTRESRTRES specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA inter-actions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Pangburn Heather A

    2005-09-01

    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

  8. Neural stem cells express melatonin receptors and neurotrophic factors: colocalization of the MT1 receptor with neuronal and glial markers

    Directory of Open Access Journals (Sweden)

    McMillan Catherine R

    2004-10-01

    Full Text Available Abstract Background In order to optimize the potential benefits of neural stem cell (NSC transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different in vitro conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT1 and MT2 receptors are expressed in NSCs. Results RT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF, brain-derived neurotrophic factor (BDNF and nerve growth factor (NGF in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT1 receptor mRNA was detected in NSCs maintained for two days in culture, but the MT2 receptor was not seen. An immature MT1 receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 – 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT1 receptor is expressed in both neural (β-tubulin III positive and glial (GFAP positive progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours. Conclusions The phenotypic characteristics of C17.2 cells suggest that they are

  9. Expression and purification of Nod factor receptors - Initial characterization of ligand binding

    DEFF Research Database (Denmark)

    Broghammer, Angelique

    . Lipochitooligosaccharides also serve as signals in the mutually beneficial interactions between arbuscular mycorrhiza (AM) and most land plants. In the model legume Lotus japonicus the Nod factor receptors, LjNFR1 and LjNFR5, two LysM receptor like kinases (LysM-RLK), are responsible for perceiving the rhizobial...

  10. Targeting hepatocyte growth factor receptor (Met) positive tumor cells using internalizing nanobody-decorated albumin nanoparticles

    NARCIS (Netherlands)

    Heukers, Raimond|info:eu-repo/dai/nl/325788103; Altintas, Isil|info:eu-repo/dai/nl/341537160; Raghoenath, Smiriti; De Zan, Erica; Pepermans, Richard; Roovers, Rob C.|info:eu-repo/dai/nl/205435599; Haselberg, Rob|info:eu-repo/dai/nl/304822647; Hennink, Wim E.|info:eu-repo/dai/nl/070880409; Schiffelers, Raymond M.|info:eu-repo/dai/nl/212909509; Kok, Robbert J.|info:eu-repo/dai/nl/170678326; Van Bergen en Henegouwen, Paul M P|info:eu-repo/dai/nl/071919481

    2014-01-01

    The hepatocyte growth factor receptor (HGFR, c-Met or Met) is a receptor tyrosine kinase that is involved in embryogenesis, tissue regeneration and wound healing. Abnormal activation of this proto-oncogene product is implicated in the development, progression and metastasis of many cancers. Current

  11. Regulation of Epidermal Growth Factor Receptor Trafficking by Lysine Deacetylase HDAC6

    DEFF Research Database (Denmark)

    Lissanu Deribe, Yonathan; Wild, Philipp; Chandrashaker, Akhila;

    2009-01-01

    Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated...

  12. 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 in

  13. 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

  14. Tumor necrosis factor receptor-associated factor 3 is a positive regulator of pathological cardiac hypertrophy.

    Science.gov (United States)

    Jiang, Xi; Deng, Ke-Qiong; Luo, Yuxuan; Jiang, Ding-Sheng; Gao, Lu; Zhang, Xiao-Fei; Zhang, Peng; Zhao, Guang-Nian; Zhu, Xueyong; Li, Hongliang

    2015-08-01

    Cardiac hypertrophy, a common early symptom of heart failure, is regulated by numerous signaling pathways. Here, we identified tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein in tumor necrosis factor-related signaling cascades, as a key regulator of cardiac hypertrophy in response to pressure overload. TRAF3 expression was upregulated in hypertrophied mice hearts and failing human hearts. Four weeks after aortic banding, cardiac-specific conditional TRAF3-knockout mice exhibited significantly reduced cardiac hypertrophy, fibrosis, and dysfunction. Conversely, transgenic mice overexpressing TRAF3 in the heart developed exaggerated cardiac hypertrophy in response to pressure overload. TRAF3 also promoted an angiotensin II- or phenylephrine-induced hypertrophic response in isolated cardiomyocytes. Mechanistically, TRAF3 directly bound to TANK-binding kinase 1 (TBK1), causing increased TBK1 phosphorylation in response to hypertrophic stimuli. This interaction between TRAF3 and TBK1 further activated AKT signaling, which ultimately promoted the development of cardiac hypertrophy. Our findings not only reveal a key role of TRAF3 in regulating the hypertrophic response but also uncover TRAF3-TBK1-AKT as a novel signaling pathway in the development of cardiac hypertrophy and heart failure. This pathway may represent a potential therapeutic target for this pathological process.

  15. Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-single-chain Fv fusion protein.

    Science.gov (United States)

    Kapelski, Stephanie; de Almeida, Melanie; Fischer, Rainer; Barth, Stefan; Fendel, Rolf

    2015-01-01

    We present here the first evidence that granzyme B acts against Plasmodium falciparum (50% inhibitory concentration [IC50], 1,590 nM; 95% confidence interval [95% CI], 1,197 to 2,112 nM). We created a novel antimalarial fusion protein consisting of granzyme B fused to a merozoite surface protein 4 (MSP4)-specific single-chain Fv protein (scFv), which targets the enzyme to infected erythrocytes, with up to an 8-fold reduction in the IC50 (176 nM; 95% CI, 154 to 202 nM). This study confirms the therapeutic efficacies of recombinant antibody-mediated antimalarial immunotherapeutics based on granzyme B.

  16. Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y.

    Science.gov (United States)

    Rouis, Souad; Lafaye, Pierre; Jaoua-Aydi, Leila; Sghaier, Zidani; Ayadi, Hammadi; Gargouri-Bouzid, Radhia

    2006-10-01

    Three single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies.

  17. Development of competitive ELISA for the detection of bovine serum albumin using single-chain variable fragments.

    Science.gov (United States)

    Liu, Yuan; Lin, Manman; Zhang, Xifeng; Hu, Xiaodan; Lin, Jieru; Hao, Jia; He, Dan; Zhang, Xiao; Xu, Chongxin; Zhong, Jianfeng; Xie, Yajing; Zhang, Cunzheng; Liu, Xianjin

    2017-05-15

    Soluble anti-bovine serum albumin (BSA) single-chain variable fragments (scFvs) were expressed in E. Coli. HB2151. The antigen-binding equilibrium dissociation constant of the scFvs was determined to be 2.9 × 10(-8) M by surface plasmon resonance analysis. A competitive ELISA for the detection of BSA was developed using the antibody fragment above. The limits of detection (I10) and I50 were 0.002 and 0.74 μg/ml respectively, with a recovery between 87.8 and 119.2% in spiked milk samples. The assay has the potential to be used to detect concentration of BSA in milk or other matrix instead of the ELISA based on traditional antibodies.

  18. Molecular characterization of a single-chain antibody variable fragment (scFv) specific for PspA from Streptococcus pneumoniae.

    Science.gov (United States)

    Jang, ShinA; Kim, Gyuhee; Oh, Jihye; Lee, Seungyeop; Kim, Dongho; Kim, Kook-Han; Kim, Yong Ho; Rhee, Dong-Kwon; Lee, Sangho

    2017-01-01

    Streptococcus pneumoniae is a major infectious agent responsible for pneumonia, otitis media, sepsis and meningitis. Pneumococcal surface protein A (PspA) is a well-characterized virulence factor localized on the surface and a target for vaccine development. In this study, we screened a single-chain antibody variable fragment (scFv) using phage display from a human synthetic library to select a clone 2B11. Affinity (Kd) of 2B11 was measured to be 5 nM using biolayer interferometry. 2B11 exhibited a dose-dependent recognition of recombinant PspA with no cross-reactivity towards pneumococcal antigens. The epitope on PspA was defined to residues 231-242 by mutational analysis. Molecular docking analysis supported the experimentally determined epitope, suggesting that the helix spanning residues 231-242 can bind to 2B11 with residues in the CDR-H3 (complementarity determining region 3 in the heavy chain) actively participating in the molecular contacts. Comparison of 2B11 with a commercial PspA antibody revealed that 2B11 exhibited a better specificity towards recombinant PspA antigen. 2B11 was capable of detecting endogenous PspA from pneumococcal lysates with affinity similar to that of the commercial antibody. Our study provides a molecular tool for biosensors detecting pneumococcal diseases.

  19. Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously.

    Science.gov (United States)

    Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kontermann, Roland E; Rüger, Ronny

    2015-03-10

    Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification.

  20. Equine insulin receptor and insulin-like growth factor-1 receptor expression in digital lamellar tissue and insulin target tissues.

    Science.gov (United States)

    Kullmann, A; Weber, P S; Bishop, J B; Roux, T M; Norby, B; Burns, T A; McCutcheon, L J; Belknap, J K; Geor, R J

    2016-09-01

    Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horses and examine the effect of dietary nonstructural carbohydrate (NSC) on mRNA expression of these receptors in LT, skeletal muscle, liver and two adipose tissue (AT) depots of lean and obese ponies. In vivo experiment. Lamellar tissue samples were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for receptor mRNA expression (n = 8) and immunoblotting for protein expression (n = 3). Archived LT, skeletal muscle, liver and AT from lean and obese mixed-breed ponies fed either a low (~7% NSC as dry matter; 5 lean, 5 obese) or high NSC diet (~42% NSC as dry matter; 6 lean, 6 obese) for 7 days were evaluated by RT-qPCR to determine the effect of body condition and diet on expression of the receptors in different tissues. Significance was set at P≤0.05. Lamellar tissue expresses both InsR isoforms, IGF-1R and Hybrid. LT IGF-1R gene expression was greater than either InsR isoform and InsR-A expression was greater than InsR-B (P≤0.05). Obesity significantly lowered IGF-1R, InsR-A and InsR-B mRNA expression in LT and InsR-A in tailhead AT. High NSC diet lowered expression of all three receptor types in liver; IGF-1R and InsR-A in LT and InsR-A in tailhead AT. Lamellar tissue expresses IGF-1R, InsR isoforms and Hybrids. The functional characteristics of these receptors and their role in endocrinopathic laminitis warrants further investigation. © 2015 EVJ

  1. EXPRESSION OF EPIDERMAL GROWTH FACTOR, TRANSFORMING GROWTH FACTOR-a AND THEIR RECEPTOR IN HUMAN PITUITARY TUMORS

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Long

    2001-01-01

    [1]LIU Xu-wen, FU Pei-yu, GAO Zhi-xian. Expression of epidermal growth factor receptors in human glioma [J]. Chin J Neurosurgery 1998; 14:71.[2]Wong AJ, Ruppert JM, Bigner SH, et al. Structural alterations of the epidermal growth factor receptor gene in human gliomas [J]. Proc Natl Acad Sci USA 1992; 89:4309.[3]Webster J, Ham J, Bevan JS. Preliminary characterization of growth factors secreted by human pituitary tumors [J]. J Clin Endocrinol Metab 1991; 72:687.[4]Klibanski A. Nonsecreting pituitary tumors [J]. Endocrinol Metab Clin North Am 1987; 16:793.[5]LeRiche VK, Asa SL, Ezzat S. Epidermal growth factor and its receptor (EGF-R) in human pituitary adenomas: EGF-R correlates with tumor aggressiveness [J]. J Clin Endocrinol Metab 1996; 81:656.

  2. Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics

    DEFF Research Database (Denmark)

    Akimov, Vyacheslav; Rigbolt, Kristoffer T G; Nielsen, Mogens M;

    2011-01-01

    ) for selectively decoding ubiquitination-driven processes involved in the regulation of cellular signaling networks. We applied this approach to characterize the temporal dynamics of ubiquitination events accompanying epidermal growth factor receptor (EGFR) signal transduction. We used recombinant UBDs derived...

  3. New factors influencing G protein coupled receptors’ system ...

    African Journals Online (AJOL)

    Abdelaziz Ghanemi

    2012-11-24

    Nov 24, 2012 ... system functions. Abdelaziz .... systems. Such factors will be added to those already known including ..... crossroad between cell biology and physics. Nat Cell ... classification of receptors for 5-hydroxytryptamine (Serotonin).

  4. Gene expression of growth factors and growth factor receptors for potential targeted therapy of canine hepatocellular carcinoma.

    Science.gov (United States)

    Iida, Gentoku; Asano, Kazushi; Seki, Mamiko; Sakai, Manabu; Kutara, Kenji; Ishigaki, Kumiko; Kagawa, Yumiko; Yoshida, Orie; Teshima, Kenji; Edamura, Kazuya; Watari, Toshihiro

    2014-03-01

    The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-α, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC.

  5. Nerve growth factor and receptor expression in rheumatoid arthritis and spondyloarthritis

    NARCIS (Netherlands)

    Barthel, C.; Yeremenko, N.; Jacobs, R.; Schmidt, R.E.; Bernateck, M.; Zeidler, H.; Tak, P.P.; Baeten, D.; Rihl, M.

    2009-01-01

    Introduction We previously described the presence of nerve growth factor receptors in the inflamed synovial compartment. Here we investigated the presence of the corresponding nerve growth factors, with special focus on nerve growth factor (NGF). Methods mRNA expression levels of four ligands (NGF,

  6. 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

    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......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...... noticed that components of 136 and 97 kDa bound to a peptide from this region of the receptor in a phosphorylation-independent manner. These components have now been purified and identified as alpha- and beta'-coatomer proteins (COPs), respectively. COPs are a family of proteins involved in the regulation...

  7. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    Science.gov (United States)

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  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 intracellular region of the receptor. This tyrosine kinase phosphorylates a number of intracellular substrates that activates pathways leading to cell growth, DNA synthesis and the expression of oncogenes such as fos and jun. EGFR is thought to be involved the development of cancer, as the EGFR gene is often...... amplified, and/or mutated in cancer cells. In this review we will focus on: (I) the structure and function of EGFR, (II) implications of receptor/ligand coexpression and EGFR mutations or overexpression, (III) its effect on cancer cells, (IV) the development of the malignant phenotype and (V) the clinical...

  9. Troglitazone inhibits cell proliferation by attenuation of epidermal growth factor receptor signaling independent of peroxisome proliferator-activated receptor γ

    Institute of Scientific and Technical Information of China (English)

    Xiaoqi Li; Xuanming Yang; Youli Xu; Xuejun Jiang; Xin Li; Fajun Nan; Hong Tang

    2009-01-01

    Peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily of ligand-dependent transcription factors. Recent results have shown that agonists of PPARy, such as troglitazone (TGZ), can inhibit cell proliferation and promote cell differentiation independent of PPARγ. In the present study, we provide evidence that TGZ may bind directly to EGFR and trigger its signaling and internalization independent of PPARγ. Detailed studies revealed that prolonged incubation with TGZ effectively attenuated EGFR signaling by target-ing the receptor to the endo-lysosomal degradation machinery. Although the extracellular signal-regulated kinase-signaling pathway was transiently activated by TGZ in EGFR overexpressing cancer cells, inhibition of EGF-induced Akt phosphorylation most likely accounted for the growth arrest of tumor cells caused by TGZ at pharmacologically achievable concentrations. Therefore, we have provided a new line of evidence indicating that TGZ inhibits cell pro-liferation by promoting EGFR degradation and attenuating Akt phosphorylation.

  10. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research

    Directory of Open Access Journals (Sweden)

    Zhixiang Wang

    2016-01-01

    Full Text Available Both G protein-coupled receptors (GPCRs and receptor-tyrosine kinases (RTKs regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR, a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges.

  11. DETERMINATION OF SERUM SOLUBLE MACROPHAGE COLONY- STIMULATING FACTOR RECEPTOR LEVELS IN PATIENTS with hematological diseases

    Institute of Scientific and Technical Information of China (English)

    RAO; Qing

    2001-01-01

    [1]Heaney MK, Golde DW. Soluble receptors in human disease [J]. J Leukoc Biol 1998; 61:135.[2]Fix P, Praloram V. M-CSF: Haematopoietic growth factor or inflammatory cytokine [J]? Cytokine 1998; 10:32.[3]Sherr C. Colony-stimulating factor ? 1 receptor [J]. Blood 1990; 75:1.[4]Downing JR, Roussel MF, Sherr CJ. Ligand and protein kinase C down modulate the colony-stimulating factor 1 receptor by independent mechanisms [J]. Mol Cell Biol 1989; 9:2890.[5]Baker AH, Cachia PG, Tennant GB, et al. A novel CSF-1 binding factor in a patient in complete remission following cytotoxic therapy for lymphoma [J]. Br J Haematol 1995; 89:219.[6]Wu KF, Zheng GG, Rao Q, et al. Cellular macrophage colony-stimulating factor and its role [J]. Hematologica 1999; 84:951.[7]Rao Q, Han JS, Geng YQ, et al. Antigen association of J6-1 cell membrane associated factor receptor with macrophage colony-stimulating factor receptor [J]. Chin J Cancer Res 1999; 11:235.[8]Rao Q, Han JS, Geng YQ, et al. Quantitation of human soluble macrophage colony stimulating factor receptor in human serum by ELISA assay [J]. Exp Hematol 1999; 27:105.[9]Luo SQ, Zheng DX, Liu YX, et al. Analysis of the ligand-binding domain of macrophage colony- stimulating factor receptor [J]. Chin Sci Bull 2000; 45:1191.[10]Wypych J, Bennett LG, Schwartz MG, et al. Soluble Kit receptor in human serum [J]. Blood 1995; 85:66.[11]Tiesman J, Hart CE. Identification of a soluble receptor for platelet-derived growth factor in cell-conditioned medium and human plasma [J]. J Biol Chem 1993; 269:9621.[12]Zhang Q, Xue YP, Song YH, et al. Expression of cellular M-CSF and M-CSFR in hematopoietic cells [J]. Chin J Hematol 1999; 20:249.[13]Tang SS, Liu HZ, Chen GB, et al. Internalization mediated by membrane-bound macrophage colony- stimulating factor and half-life of cell associated macrophage colony-stimulating factor and its receptor [J]. Chin Sci Bull 2000; 45:627.[14]Zeigler ZR

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-01

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

  13. The nerve growth factor and its receptors in airway inflammatory diseases.

    Science.gov (United States)

    Freund-Michel, V; Frossard, N

    2008-01-01

    The nerve growth factor (NGF) belongs to the neurotrophin family and induces its effects through activation of 2 distinct receptor types: the tropomyosin-related kinase A (TrkA) receptor, carrying an intrinsic tyrosine kinase activity in its intracellular domain, and the receptor p75 for neurotrophins (p75NTR), belonging to the death receptor family. Through activation of its TrkA receptor, NGF activates signalling pathways, including phospholipase Cgamma (PLCgamma), phosphatidyl-inositol 3-kinase (PI3K), the small G protein Ras, and mitogen-activated protein kinases (MAPK). Through its p75NTR receptor, NGF activates proapoptotic signalling pathways including the MAPK c-Jun N-terminal kinase (JNK), ceramides, and the small G protein Rac, but also activates pathways promoting cell survival through the transcription factor nuclear factor-kappaB (NF-kappaB). NGF was first described by Rita Levi-Montalcini and collaborators as an important factor involved in nerve differentiation and survival. Another role for NGF has since been established in inflammation, in particular of the airways, with increased NGF levels in chronic inflammatory diseases. In this review, we will first describe NGF structure and synthesis and NGF receptors and their signalling pathways. We will then provide information about NGF in the airways, describing its expression and regulation, as well as pointing out its potential role in inflammation, hyperresponsiveness, and remodelling process observed in airway inflammatory diseases, in particular in asthma.

  14. Expression of tumor necrosis factor related apoptosis inducing ligand receptor in glioblastoma

    Institute of Scientific and Technical Information of China (English)

    Dongling Gao; Zhongwei Zhao; Hongxin Zhang; Lan Zhang; Kuisheng Chen; Yunhan Zhang

    2008-01-01

    BACKGROUND: Receptors for tumor necrosis factor related apoptosis inducing ligand (TRAIL) include death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2. Activation of death receptor 4 and 5 selectively kills tumor cells.OBJECTIVE: To detect TRAIL receptor expression in glioblastoma by immunohistochemistry and RT-PCR and to compare this expression to that in normal brain tissue.DESIGN: Observational analysis.SETTING: Department of Pathology, the First Affiliated Hospital of Zhengzhou University; Henan Tumor Pathology Key Laboratory.PARTICIPANTS: Twenty-five patients (17 males and 8 females) who received glioblastoma resection were selected from the Fifth Affiliated Hospital of Zhengzhou University, between September 2003 to June 2004. All glioblastoma samples were diagnosed pathologically. Twenty patients (12 males and 8 females) with craniocerebral injury who received normal brain tissue resection were selected in the same time period. There were no significant differences in sex and age between glioblastoma patients or between craniocerebral injury patients (P>0.05). All patients and appropriate relatives provided informed consent, and this study was approved by the local research ethics committee.METHODS: Polyclonal antibody against TRAIL receptors and an immunohistochemical kit (batch number: 200502) were purchased from Boster Company, Wuhan. Immunohistochemistry: Expression of death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2 were observed in both glioblastoma and normal brain tissue. The experiment was performed according to the kit instructions, and positive staining was brown-yellow. Assessment: There were no positive signals (-); weakly positive signals, positive cells75% (++++). Evaluation: Expression levels of TRAIL receptors were estimated in both normal brain tissue and glioblastoma. Expression of decoy receptor 1 and decoy receptor 2 mRNA in glioblastoma were detected by reverse transcription polymerase

  15. Epidermal growth factor receptor mutation enhances expression of vascular endothelial growth factor in lung cancer.

    Science.gov (United States)

    Hung, Ming-Szu; Chen, I-Chuan; Lin, Paul-Yann; Lung, Jr-Hau; Li, Ya-Chin; Lin, Yu-Ching; Yang, Cheng-Ta; Tsai, Ying-Huang

    2016-12-01

    Epidermal growth factor receptor (EGFR) activation has been demonstrated to have a critical role in tumor angiogenesis. In the present study, the correlation between EGFR mutations and vascular endothelial growth factor (VEGF) was investigated in lung cancer cell lines and non-small-cell lung cancer (NSCLC) tumor tissues. VEGF levels were significantly increased in culture medium of lung cancer cells and NSCLC tissues with EGFR mutations (H1650 vs. A549, P=0.0399; H1975 vs. A549, Plung cancer cell lines expressing mutant (exon 19 deletion, E746-A750; exon 21 missense mutation, L858R) and wild-type EGFR genes were established. Significantly increased expression of VEGF and stronger inhibitory effects of gefitinib to VEGF expression were observed in exon 19 deletion stable lung cancer cells (exon 19 deletion vs. wild-type EGFR, P=0.0005). The results of the present study may provide an insight into the association of mutant EGFR and VEGF expression in lung cancer, and may assist with further development of targeted therapy for NSCLC in the future.

  16. 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.

  17. The oncogenic epidermal growth factor receptor variant Xiphophorus melanoma receptor kinase induces motility in melanocytes by modulation of focal adhesions.

    Science.gov (United States)

    Meierjohann, Svenja; Wende, Elisabeth; Kraiss, Anita; Wellbrock, Claudia; Schartl, Manfred

    2006-03-15

    One of the most prominent features of malignant melanoma is the fast generation of metastasizing cells, resulting in the poor prognosis of patients with this tumor type. For this process, cells must gain the ability to migrate. The oncogenic receptor Xmrk (Xiphophorus melanoma receptor kinase) from the Xiphophorus melanoma system is a mutationally activated version of the epidermal growth factor receptor that induces the malignant transformation of pigment cells. Here, we show that the activation of Xmrk leads to a clear increase of pigment cell motility in a fyn-dependent manner. Stimulation of Xmrk induces its interaction with the focal adhesion kinase (FAK) and the interaction of active, receptor-bound fyn with FAK. This results in changes in FAK activity and induces the modulation of stress fibers and focal adhesions. Overexpression of dominant-negative FAK shows that the activity of innate FAK and a receptor-induced focal adhesion turnover are a prerequisite for pigment cell migration. Our findings show that in our system, Xmrk is sufficient for the induction of pigment cell motility and underlines a role of the src family protein tyrosine kinase fyn in melanoma development and progression.

  18. Optimization of production of the anti-keratin 8 single-chain Fv TS1-218 in Pichia pastoris using design of experiments

    Directory of Open Access Journals (Sweden)

    Sundström Birgitta E

    2011-05-01

    Full Text Available Abstract Background Optimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions. Results In this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-keratin 8 scFv TS1-218, during expression in P. pastoris in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2% suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization. Conclusion The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.

  19. [Dependence of EGF receptor and STAT factor activation on redox of A431 cells].

    Science.gov (United States)

    Gonchar, I V; Burova, E B; Dorosh, V N; Gamaleĭ, I A; Nikol'skiĭ, N N

    2003-01-01

    Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.

  20. Blocking transforming growth factor- receptor signaling down-regulates transforming growth factor-β1 autoproduction in keloid fibroblasts

    Institute of Scientific and Technical Information of China (English)

    刘伟; 蔡泽浩; 王丹茹; 武小莉; 崔磊; 商庆新; 钱云良; 曹谊林

    2002-01-01

    Objective: To study transforming growth factor-β1(TGF-β1) autoproduction in keloid fibroblasts and theregulation effect of blocking TGF-β intracellular signalingon rhTGF-β1 autoproduction.Methods: Keloid fibroblasts cultured in vitro weretreated with either rhTGF-β1 (5 ng/ml ) or recombinantadenovirus containing a truncated type II TGF-β receptorgene (50 pfu/cell ). Their effects of regulating geneexpression of TGF-β1 and its receptor I and II wereobserved with Northern blot.Results: rhTGF-β1 up-regulated the gene expressionof TGF-β1 and receptor I, but not receptor II. Over-expression of the truncated receptor II down-regulated thegene expression of TGF-β1 and its receptor I, but notreceptor II.Conclusions: TGF-β1 autoproduction was observed inkeloid fibroblasts. Over-expression of the truncated TGF-βreceptor H decreased TGF-β1 autoproduction via blockingTGF-β receptor signaling.

  1. 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

  2. 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.

  3. 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.

  4. 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

    , 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...... and the mannose-6-phosphate (Man-6-P)/IGF-II receptor. There is consensus that the cellular effects of IGF-II are mediated by the IGF-I receptor via activation of its intrinsic tyrosine kinase. The Man-6-P/IGF-II receptor is involved in endocytosis of lysosomal enzymes and IGF-II. In selected cell types, however...... complex suggesting that its biological actions can be regulated at different levels including the transcription, translation, posttranslational processing, receptor binding and intracellular signalling....

  5. Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

    NARCIS (Netherlands)

    Kuznetsova, T.; Wang, S.Y.; Rao, N.A.; Mandoli, A.; Martens, J.H.; Rother, N; Aartse, A.; Groh, L.; Janssen-Megens, E.M.; Li, G.; Ruan, Y.; Logie, C.; Stunnenberg, H.G.

    2015-01-01

    BACKGROUND: The impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by activ

  6. The transforming growth factor-beta receptor genes and the risk of intracranial aneurysms

    NARCIS (Netherlands)

    Ruigrok, Ynte M.; Baas, Annette F.; Medic, Jelena; Wijmenga, Cisca; Rinkel, Gabriel J. E.

    2012-01-01

    Background Mutations in the receptor genes of the transforming growth factor beta pathway, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysms, while genetic variants in TGFBR1 and TGFBR2 are associated with abdominal aortic aneurysms. The transforming growth factor-beta pathway may be

  7. Glucocorticoid receptor and nuclear factor kappa-b affect three-dimensional chromatin organization

    NARCIS (Netherlands)

    Kuznetsova, T.; Wang, S.Y.; Rao, N.A.; Mandoli, A.; Martens, J.H.; Rother, N; Aartse, A.; Groh, L.; Janssen-Megens, E.M.; Li, G.; Ruan, Y.; Logie, C.; Stunnenberg, H.G.

    2015-01-01

    BACKGROUND: The impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by

  8. Epidermal growth factor receptor signalling contributes to house dust mite-induced epithelial barrier dysfunction

    NARCIS (Netherlands)

    Heijink, I H; van Oosterhout, A; Kapus, A

    2010-01-01

    Impaired airway epithelial barrier function has emerged as a key factor in the pathogenesis of allergic asthma. We aimed to discern the involvement of the epidermal growth factor receptor (EGFR) in allergen-induced epithelial barrier impairment, as we previously observed that house dust mite (HDM) s

  9. Requirement for Tumor Necrosis Factor Receptor 2 Expression on Vascular Cells To Induce Experimental Cerebral Malaria

    OpenAIRE

    Stoelcker, Benjamin; Hehlgans, Thomas; Weigl, Karin; Bluethmann, Horst; Grau, Georges E.; Männel, Daniela N

    2002-01-01

    Using tumor necrosis factor receptor type 2 (TNFR2)-deficient mice and generating bone marrow chimeras which express TNFR2 on either hematopoietic or nonhematopoietic cells, we demonstrated the requirement for TNFR2 expression on tissue cells to induce lethal cerebral malaria. Thus, TNFR2 on the brain vasculature mediates tumor necrosis factor-induced neurovascular lesions in experimental cerebral malaria.

  10. Pharmacological targeting of the KIT growth factor receptor: a therapeutic consideration for mast cell disorders

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Akin, C; Gilfillan, A M

    2008-01-01

    KIT is a member of the tyrosine kinase family of growth factor receptors which is expressed on a variety of haematopoietic cells including mast cells. Stem cell factor (SCF)-dependent activation of KIT is critical for mast cell homeostasis and function. However, when KIT is inappropriately activa...

  11. Neurosteroid dehydroepiandrosterone interacts with nerve growth factor (NGF receptors, preventing neuronal apoptosis.

    Directory of Open Access Journals (Sweden)

    Iakovos Lazaridis

    2011-04-01

    Full Text Available The neurosteroid dehydroepiandrosterone (DHEA, produced by neurons and glia, affects multiple processes in the brain, including neuronal survival and neurogenesis during development and in aging. We provide evidence that DHEA interacts with pro-survival TrkA and pro-death p75(NTR membrane receptors of neurotrophin nerve growth factor (NGF, acting as a neurotrophic factor: (1 the anti-apoptotic effects of DHEA were reversed by siRNA against TrkA or by a specific TrkA inhibitor; (2 [(3H]-DHEA binding assays showed that it bound to membranes isolated from HEK293 cells transfected with the cDNAs of TrkA and p75(NTR receptors (K(D: 7.4 ± 1.75 nM and 5.6 ± 0.55 nM, respectively; (3 immobilized DHEA pulled down recombinant and naturally expressed TrkA and p75(NTR receptors; (4 DHEA induced TrkA phosphorylation and NGF receptor-mediated signaling; Shc, Akt, and ERK1/2 kinases down-stream to TrkA receptors and TRAF6, RIP2, and RhoGDI interactors of p75(NTR receptors; and (5 DHEA rescued from apoptosis TrkA receptor positive sensory neurons of dorsal root ganglia in NGF null embryos and compensated NGF in rescuing from apoptosis NGF receptor positive sympathetic neurons of embryonic superior cervical ganglia. Phylogenetic findings on the evolution of neurotrophins, their receptors, and CYP17, the enzyme responsible for DHEA biosynthesis, combined with our data support the hypothesis that DHEA served as a phylogenetically ancient neurotrophic factor.

  12. Changes of expression of estrogen and progestrone receptors, human epithelial growth factor receptor 2 and Ki-67 after neoadjuvant chemotherapy in the treatment of breast cancer.

    Science.gov (United States)

    Li, M L; Dong, Y; Luan, S L; Zhao, Z H; Ning, F L

    2016-01-01

    Recent studies suggest that the development and prognosis of breast cancer is in close correlation to molecular subtype of breast cancer. Neoadjuvant chemotherapy has been extensively applied in the treatment of local breast cancer in advanced stage. In order to verify the correlation between expression changes of estrogen receptor, progestrone receptor, human epithelial growth factor receptor 2 and Ki-67 after neoadjuvant chemotherapy and neoadjuvant chemotherapy, we studied 120 patients with stage IIAIIIC breast cancer who underwent neoadjuvant chemotherapy in Binzhou Medical University Hospital, Shandong, China from February 2011 to February 2015. Clinical characteristics were retrospectively analyzed. The expression of estrogen receptor, progesterone receptor, human epithelial growth factor receptor 2 and Ki-67 of patients were detected using the immunohistochemical method before and after neoadjuvant chemotherapy. The results suggest that the overall remission rate of neoadjuvant chemotherapy was 76.7% (92/120) of which 16.7% (20/120) of cases had complete remission, 60% (72/120) had partial remission and 23.3% (28/120) were stable. There were no cases of progressive disease. The property of estrogen receptor and the expression of Ki-67 of primary tumor were correlated to the remission rate of neoadjuvant chemotherapy (P less than 0.05). The expression of Ki-67 had a significant decline after neoadjuvant chemotherapy, and the difference had statistical significance (P less than 0.05). The difference in expression of estrogen receptor, progesterone receptor and human epithelial growth factor receptor 2 before and after neoadjuvant chemotherapy had statistical significance (P > 0.05). Hence, it can be concluded that breast cancer patients with negative estrogen receptor expression and high Ki-67 expression before neoadjuvant chemotherapy can achieve better curative effects. Neoadjuvant chemotherapy cannot change the expression states of estrogen receptor

  13. Antiproliferative and Apoptotic Effects of a Specific Antiprostate Stem Cell Single Chain Antibody on Human Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Foroogh Nejatollahi

    2013-01-01

    Full Text Available Prostate stem cell antigen (PSCA is a highly glycosylated cell surface protein which is overexpressed in several malignancies including prostate, pancreas, and urinary bladder cancers. Tumor suppression has been reported by anti-PSCA antibody. Small and high affinity single chain antibodies (scFv have been introduced as effective agents for cancer immunotargeting approaches. In the present study, we used a phage antibody display library of scFv and selected two antibodies against two immunodominant epitopes of PSCA by panning process. The reactivity of the scFvs for the corresponding epitopes was determined by phage ELISA. The binding specificity of antibodies to PSCA-expressing prostate cancer cell line, DU-145, was analyzed by flow cytometry. The antiproliferative and apoptotic induction effects were evaluated by MTT and Annexin-V assays, respectively. Results represented functional scFv C5-II which could bind specifically to DU-145 cells and significantly inhibited the proliferation of these cells (61% with no effect on PSCA-negative cells. The antibody also induced apoptosis in the PSCA expressing cells. The percentage of the apoptotic cells after 24 hrs of exposure to 500 scFv/cell was 33.80%. These results demonstrate that the functional anti-PSCA scFv C5-II has the potential to be considered as a new agent for targeted therapy of prostate cancer.

  14. 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.

  15. wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2.

    Science.gov (United States)

    Rideout, Marc C; Naili, Ilham; Boldt, Jeffrey L; Flores-Fujimoto, America; Patra, Sukanya; Rostron, Jason E; Segall, Anca M

    2013-02-01

    DNA repair pathways in bacteria that use homologous recombination involve the formation and subsequent resolution of Holliday junction (HJ) intermediates. We have previously identified several hexameric peptides that bind to HJs and interfere with HJ processing enzymes in vitro. The peptide WRWYCR and its D-amino acid stereoisomer wrwycr, are potent antibacterial agents. These hexapeptides must form homodimers in order to interact stably with HJs, and inhibit bacterial growth, and this represents a potential limitation. Herein we describe a disulfide bond-independent inhibitor, WRWYRGGRYWRW and its D-stereoisomer wrwyrggrywrw. We have characterized these single-chain, linear analogs of the hexapeptides, and show that in addition to effectively binding to HJs, and inhibiting the activity of DNA repair enzymes that process HJs, they have equal or greater potency against Gram-positive and Gram-negative bacterial growth. The analogs were also shown to cause DNA damage in bacteria, and disrupt the integrity of the bacterial cytoplasmic membrane. Finally, we found that they have little toxicity toward several eukaryotic cell types at concentrations needed to inhibit bacterial growth. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

    Science.gov (United States)

    Thatikonda, Naresh; Delfani, Payam; Jansson, Ronnie; Petersson, Linn; Lindberg, Diana; Wingren, Christer; Hedhammar, My

    2016-03-01

    Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant were examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle.

    Science.gov (United States)

    Zhang, Kathy; Geddie, Melissa L; Kohli, Neeraj; Kornaga, Tad; Kirpotin, Dmitri B; Jiao, Yang; Rennard, Rachel; Drummond, Daryl C; Nielsen, Ulrik B; Xu, Lihui; Lugovskoy, Alexey A

    2015-01-01

    Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

  18. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    Science.gov (United States)

    Lin, Yuan; Li, Benqiang; Ye, Jiaxin; Wang, Man; Wang, Jianhua; Zhang, Ying; Zhu, Jianguo

    2015-09-01

    Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression.

  19. Phase analysis in single-chain variable fragment production by recombinant Pichia pastoris based on proteomics combined with multivariate statistics.

    Science.gov (United States)

    Fujiki, Yuya; Kumada, Yoichi; Kishimoto, Michimasa

    2015-08-01

    The proteomics technique, which consists of two-dimensional gel electrophoresis (2-DE), peptide mass fingerprinting (PMF), gel image analysis, and multivariate statistics, was applied to the phase analysis of a fed-batch culture for the production of a single-chain variable fragment (scFv) of an anti-C-reactive protein (CRP) antibody by Pichia pastoris. The time courses of the fed-batch culture were separated into three distinct phases: the growth phase of the batch process, the growth phase of the fed-batch process, and the production phase of the fed-batch process. Multivariate statistical analysis using 2-DE gel image analysis data clearly showed the change in the culture phase and provided information concerning the protein expression, which suggested a metabolic change related to cell growth and production during the fed-batch culture. Furthermore, specific proteins, such as alcohol oxidase, which is strongly related to scFv expression, and proteinase A, which could biodegrade scFv in the latter phases of production, were identified via the PMF method. The proteomics technique provided valuable information about the effect of the methanol concentration on scFv production.

  20. Development of an Immunoassay for Chloramphenicol Based on the Preparation of a Specific Single-Chain Variable Fragment Antibody.

    Science.gov (United States)

    Du, Xin-jun; Zhou, Xiao-nan; Li, Ping; Sheng, Wei; Ducancel, Frédéric; Wang, Shuo

    2016-04-13

    Specific antibodies are essential for the immune detection of small molecule contaminants. In the present study, the heavy and light variable regions (V(H )and V(L)) of the immunoglobulin genes from a hybridoma secreting a chloramphenicol (CAP)-specific monoclonal antibody (mAb) were cloned and sequenced. In addition, the light and heavy chains obtained from the monoclonal antibody were separated using SDS-PAGE and analyzed using Orbitrap mass spectrometry. The results of DNA sequencing and mass spectrometry analysis were compared, and the V(H) and V(L) chains specific for CAP were determined and used to construct a single-chain variable fragment (scFv). This fragment was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein and used to develop a direct competitive ELISA. Compared with the parent mAb, scFv exhibits lower sensitivity but better food matrix resistance. This work highlights the application of engineered antibodies for CAP detection.

  1. Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display.

    Science.gov (United States)

    Min, Won-Ki; Kim, Sung-Gun; Seo, Jin-Ho

    2015-12-01

    As aflatoxin B1 is one of the most toxic mycotoxins, it is important to detect and to quantify aflatoxin B1 accurately by immunological methods. To enhance aflatoxin B1-binding affinity of the single-chain variable fragment, yeast surface display technique combined with fluorescence-activated cell sorting was applied. A randomly mutated scFv library was subjected to 4 rounds of fluorescence-activated cell sorting, resulting in isolation of 5 scFv variants showing an affinity improvement compared to the parental wild type scFv. The best scFv with a 9-fold improvement in affinity for aflatoxin B1 exhibited similar specificity to the monoclonal antibody. Most of the mutations in scFv-M37 were located outside of the canonical antigen-contact loops, suggesting that its affinity improvement might be driven by an allosteric effect inducing scFv-M37 to form a more favorable binding pocket for aflatoxin B1 than the wild type scFv.

  2. Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli.

    Science.gov (United States)

    Chen, Weifeng; Hu, Li; Liu, Aiping; Li, Jinquan; Chen, Fusheng; Wang, Xiaohong

    2014-11-01

    The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10(-8) mol·L(-1), its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10(-7) mol·L(-1). The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L(-1).

  3. Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

    Institute of Scientific and Technical Information of China (English)

    WANG Cheng; MU Ying; L(U) Shao-wu; LUO Gui-min; WAN Pei; GONG Ping-sheng; L(U) Li-min; XU Ya-wei; ZHAO Yang; HE Bo; ZHAO Gang; YAN Gang-lin

    2011-01-01

    In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv),a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis,three-dimensional(3D)model building,binding-site analysis and docking.The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+),then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC).The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method,thus,the human Se-scFv with GPX activity was obtained.The GPX activity of the Se-scFv protein was characterized.Compared with other Se-scFv,the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione.It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mechanism.

  4. Disulfide-stabilized single-chain antibody-targeted superantigen: Construction of a prokaryotic expression system and its functional analysis

    Institute of Scientific and Technical Information of China (English)

    Jian-Li Wang; Yu-Ling Zheng; Ru Ma; Bao-Li Wang; Ai-Guang Guo; Yong-Qiang Jiang

    2005-01-01

    AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

  5. Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue

    Science.gov (United States)

    Lee, Hyun Chul; Kim, Su-Jin; Kim, Kyung-Sup; Shin, Hang-Cheol; Yoon, Ji-Won

    2000-11-01

    A cure for diabetes has long been sought using several different approaches, including islet transplantation, regeneration of β cells and insulin gene therapy. However, permanent remission of type 1 diabetes has not yet been satisfactorily achieved. The development of type 1 diabetes results from the almost total destruction of insulin-producing pancreatic β cells by autoimmune responses specific to β cells. Standard insulin therapy may not maintain blood glucose concentrations within the relatively narrow range that occurs in the presence of normal pancreatic β cells. We used a recombinant adeno-associated virus (rAAV) that expresses a single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, under the control of hepatocyte-specific L-type pyruvate kinase (LPK) promoter, which regulates SIA expression in response to blood glucose levels. Here we show that SIA produced from the gene construct rAAV-LPK-SIA caused remission of diabetes in streptozotocin-induced diabetic rats and autoimmune diabetic mice for a prolonged time without any apparent side effects. This new SIA gene therapy may have potential therapeutic value for the cure of autoimmune diabetes in humans.

  6. Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3

    Institute of Scientific and Technical Information of China (English)

    刘喜富; 萧飒; 顾征; 王勇; 张卫国; 陈艾; 林晴; 黄华梁; 孙健; 陈润生; 沈倍奋; 陈兴

    1997-01-01

    Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.

  7. Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors

    OpenAIRE

    1992-01-01

    The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell- associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on...

  8. Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation

    OpenAIRE

    Zhao, Haotian; Yang, Tianyu; Madakashira, Bhavani P.; Thiels, Cornelius A.; Bechtle, Chad A.; Garcia, Claudia M.; Zhang, Huiming; Yu, Kai; Ornitz, David M.; Beebe, David C.; Robinson, Michael L.

    2008-01-01

    The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens dev...

  9. Corticotropin-releasing factor receptors and stress-related alterations of gut motor function.

    OpenAIRE

    2007-01-01

    International audience; Over the past few decades, corticotropin-releasing factor (CRF) signaling pathways have been shown to be the main coordinators of the endocrine, behavioral, and immune responses to stress. Emerging evidence also links the activation of CRF receptors type 1 and type 2 with stress-related alterations of gut motor function. Here, we review the role of CRF receptors in both the brain and the gut as part of key mechanisms through which various stressors impact propulsive ac...

  10. 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....

  11. Colony stimulating factor 1 receptor inhibition eliminates microglia and attenuates brain injury after intracerebral hemorrhage.

    Science.gov (United States)

    Li, Minshu; Li, Zhiguo; Ren, Honglei; Jin, Wei-Na; Wood, Kristofer; Liu, Qiang; Sheth, Kevin N; Shi, Fu-Dong

    2017-07-01

    Microglia are the first responders to intracerebral hemorrhage, but their precise role in intracerebral hemorrhage remains to be defined. Microglia are the only type of brain cells expressing the colony-stimulating factor 1 receptor, a key regulator for myeloid lineage cells. Here, we determined the effects of a colony-stimulating factor 1 receptor inhibitor (PLX3397) on microglia and the outcome in the context of experimental mouse intracerebral hemorrhage. We show that PLX3397 effectively depleted microglia, and the depletion of microglia was sustained after intracerebral hemorrhage. Importantly, colony-stimulating factor 1 receptor inhibition attenuated neurodeficits and brain edema in two experimental models of intracerebral hemorrhage induced by injection of collagenase or autologous blood. The benefit of colony-stimulating factor 1 receptor inhibition was associated with reduced leukocyte infiltration in the brain and improved blood-brain barrier integrity after intracerebral hemorrhage, and each observation was independent of lesion size or hematoma volume. These results demonstrate that suppression of colony-stimulating factor 1 receptor signaling ablates microglia and confers protection after intracerebral hemorrhage.

  12. Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells

    Institute of Scientific and Technical Information of China (English)

    Wei Song; Haixing Xuan; Qishui Lin

    2008-01-01

    The epidermal growth factor receptor (EGFR) is a cyto-skeleton-binding protein. Although purified EGFR can interact with actins in vitro and normally at least 10% of EGFR exist in the insoluble cytoskeleton fraction of A431 cells, interaction of cytosolic EGFR with actin can only be visualized by fluorescence resonance energy transfer when epidermal growth factor presents in the cell medium. Results indicate that the correct orientation between EGFR and actin is important in the signal transduction process.

  13. Aberrant expression of leukemia inhibitory factor receptor (LIFR) and leukemia inhibitory factor (LIF) is associated with tubal pregnancy occurrence

    OpenAIRE

    Li, Yong; Sun, Lizhou; ZHAO, Denmei; Ouyang, Jun; Xiang, Mei

    2015-01-01

    Tubal pregnancy is a major cause of maternal death in the first trimester and exploration of its underlying molecular mechanism is of great importance. This study aimed to explore the association of tubal pregnancy with leukemia inhibitory factor (LIF) and leukemia inhibitory factor receptor (LIFR) expression in oviduct tissues. Materials and methods: Immunohistochemistry was performed to probe the differential expression of LIF and LIFR in oviduct tissues among a control group (including NP...

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

    Directory of Open Access Journals (Sweden)

    Jiing-Chyuan Luo

    Full Text Available OBJECTIVES: 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. METHODS: 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. RESULTS: 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 (p0.5, p<0.001. CONCLUSIONS: Our findings implied that diminished activity of proangiogenic factors and their receptors may contribute to the pathogenesis of gastric ulcers in cirrhotic patients.

  15. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  16. Characterization of the interaction between human complement protein C4 and a single-chain variable fragment antibody by capillary electrophoresis and surface plasmon resonance

    NARCIS (Netherlands)

    Seifar, R.M.; Cool, Robbert; Quax, Wim; Bischoff, Rainer

    2004-01-01

    Immunoaffinity capillary electrophoresis and surface plasmon resonance have been used for the characterization of the interaction between two large-sized proteins, the human complement protein C4 and the single-chain variable fragment C43. The rather high kinetic rate constants as determined by surf

  17. Application of phage display in selecting Tomato spotted wilt virus - specific single-chain antibodies (scFvs) for sensitive diagnosis in ELISA

    NARCIS (Netherlands)

    Griep, R.A.; Prins, M.; Twisk, van C.; Keller, H.J.H.G.; Kerschbaumer, R.J.; Kormelink, R.; Goldbach, R.W.; Schots, A.

    2000-01-01

    A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S,

  18. Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants.

    Science.gov (United States)

    Brar, Hargeet K; Bhattacharyya, Madan K

    2012-06-01

    Plants do not produce antibodies. However, plants can correctly assemble functional antibody molecules encoded by mammalian antibody genes. Many plant diseases are caused by pathogen toxins. One such disease is the soybean sudden death syndrome (SDS). SDS is a serious disease caused by the fungal pathogen Fusarium virguliforme. The pathogen, however, has never been isolated from diseased foliar tissues. Thus, one or more toxins produced by the pathogen have been considered to cause foliar SDS. One of these possible toxins, FvTox1, was recently identified. We investigated whether expression of anti-FvTox1 single-chain variable-fragment (scFv) antibody in transgenic soybean can confer resistance to foliar SDS. We have created two scFv antibody genes, Anti-FvTox1-1 and Anti-FvTox1-2, encoding anti-FvTox1 scFv antibodies from RNAs of a hybridoma cell line that expresses mouse monoclonal anti-FvTox1 7E8 antibody. Both anti-FvTox1 scFv antibodies interacted with an antigenic site of FvTox1 that binds to mouse monoclonal anti-FvTox1 7E8 antibody. Binding of FvTox1 by the anti-FvTox1 scFv antibodies, expressed in either Escherichia coli or transgenic soybean roots, was initially verified on nitrocellulose membranes. Expression of anti-FvTox1-1 in stable transgenic soybean plants resulted in enhanced foliar SDS resistance compared with that in nontransgenic control plants. Our results suggest that i) FvTox1 is an important pathogenicity factor for foliar SDS development and ii) expression of scFv antibodies against pathogen toxins could be a suitable biotechnology approach for protecting crop plants from toxin-induced diseases.

  19. Virotherapy of canine tumors with oncolytic vaccinia virus GLV-1h109 expressing an anti-VEGF single-chain antibody.

    Directory of Open Access Journals (Sweden)

    Sandeep S Patil

    Full Text Available Virotherapy using oncolytic vaccinia virus (VACV strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor single-chain antibody (scAb GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.

  20. Vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 expression in non-small cell lung cancer patients: relation to prognosis

    DEFF Research Database (Denmark)

    Bonnesen, Barbara; Pappot, Helle; Holmstav, Julie;

    2009-01-01

    elements in neoplastic cells and their microenvironment have recently been and are continuously developed including drugs inhibiting the angiogenic system. Angiogenic factor vascular endothelial growth factor (VEGF) and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) seem to play key...

  1. Deregulation of Flk-1/vascular endothelial growth factor receptor-2 in fibroblast growth factor receptor-1-deficient vascular stem cell development.

    Science.gov (United States)

    Magnusson, Peetra; Rolny, Charlotte; Jakobsson, Lars; Wikner, Charlotte; Wu, Yan; Hicklin, Daniel J; Claesson-Welsh, Lena

    2004-03-15

    We have employed embryoid bodies derived from murine embryonal stem cells to study effects on vascular development induced by fibroblast growth factor (FGF)-2 and FGF receptor-1, in comparison to the established angiogenic factor vascular endothelial growth factor (VEGF)-A and its receptor VEGF receptor-2. Exogenous FGF-2 promoted formation of morphologically distinct, long slender vessels in the embryoid bodies, whereas VEGF-A-treated bodies displayed a compact plexus of capillaries. FGF-2 stimulation of embryonal stem cells under conditions where VEGF-A/VEGFR-2 function was blocked, led to formation of endothelial cell clusters, which failed to develop into vessels. FGFR-1(-/-) embryoid bodies responded to VEGF-A by establishment of the characteristic vascular plexus, but FGF-2 had no effect on vascular development in the absence of FGFR-1. The FGFR-1(-/-) embryoid bodies displayed considerably increased basal level of vessel formation, detected by immunohistochemical staining for platelet-endothelial cell adhesion molecule (PECAM)/CD31. This basal vascularization was blocked by neutralizing antibodies against VEGFR-2 or VEGF-A and biochemical analyses indicated changes in regulation of VEGFR-2 in the absence of FGFR-1 expression. We conclude that VEGF-A/VEGFR-2-dependent vessel formation occurs in the absence of FGF-2/FGFR-1, which, however, serve to modulate vascular development.

  2. 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.

  3. Dual silencing of insulin-like growth factor-I receptor and epidermal growth factor receptor in colorectal cancer cells is associated with decreased proliferation and enhanced apoptosis.

    Science.gov (United States)

    Kaulfuss, Silke; Burfeind, Peter; Gaedcke, Jochen; Scharf, Jens-Gerd

    2009-04-01

    Overexpression and activation of tyrosine kinase receptors are common features of colorectal cancer. Using the human colorectal cancer cell lines DLD-1 and Caco-2, we evaluated the role of the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and epidermal growth factor receptor (EGFR) in cellular functions of these cells. We used the small interfering RNA (siRNA) technology to specifically down-regulate IGF-IR and EGFR expression. Knockdown of IGF-IR and EGFR resulted in inhibition of cell proliferation of DLD-1 and Caco-2 cells. An increased rate of apoptosis was associated with siRNA-mediated silencing of IGF-IR and EGFR as assessed by activation of caspase-3/caspase-7. The combined knockdown of both EGFR and IGF-IR decreased cell proliferation and induced cell apoptosis more effectively than did silencing of either receptor alone. Comparable effects on cell proliferation and apoptosis were observed after single and combinational treatment of cells by the IGF-IR tyrosine kinase inhibitor NVP-AEW541 and/or the EGFR tyrosine kinase inhibitor erlotinib. Combined IGF-IR and EGFR silencing by either siRNAs or tyrosine kinase inhibitors diminished the phosphorylation of downstream signaling pathways AKT and extracellular signal-regulated kinase (ERK)-1/2 more effectively than did the single receptor knockdown. Single IGF-IR knockdown inhibited IGF-I-dependent phosphorylation of AKT but had no effect on IGF-I- or EGF-dependent phosphorylation of ERK1/2, indicating a role of EGFR in ligand-dependent ERK1/2 phosphorylation. The present data show that inhibition of the IGF-IR transduction cascade augments the antipoliferative and proapoptotic effects of EGFR inhibition in colorectal cancer cells. A clinical application of combination therapy targeting both EGFR and IGF-IR could be a promising therapeutic strategy.

  4. An Integrated Model of Epidermal Growth Factor Receptor Trafficking and Signal Transduction

    Energy Technology Data Exchange (ETDEWEB)

    Resat, Haluk; Ewald, Jonathan A.; Dixon, David A.; Wiley, H. S.

    2003-08-01

    Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and about 13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multi-compartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physio-chemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor- alpha (TGF-a). Our simulations predict that when EGFR is activated with TGF-a, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias towards the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor down-regulation induced by either EGF or TGF-a. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis.

  5. Growth factor receptors and related signalling pathways as targets for novel treatment strategies of hepatocellular cancer

    Institute of Scientific and Technical Information of China (English)

    Michael Hopfner; Detlef Schuppan; Hans Scherübl

    2008-01-01

    Growth factors and their corresponding receptors are commonly overexpressed and/or dysregulated in many cancers including hepatocellular cancer (HCC). Clinical trials indicate that growth factor receptors and their related signalling pathways play important roles in HCC cancer etiology and progression, thus providing rational targets for innovative cancer therapies. A number of strategies including monoclonal antibodies, tyrosine kinase inhibitors ("small molecule inhibitors") and antisense oligonucleotides have already been evaluated for their potency to inhibit the activity and downstream signalling cascades of these receptors in HCC. First clinical trials have also shown that multi-kinase inhibition is an effective novel treatment strategy in HCC. In this respect sorafenib, an inhibitor of Raf-, VEGF- and PDGF-signalling, is the first multi-kinase inhibitor that has been approved by the FDA for the treatment of advanced HCC. Moreover, the serine-threonine kinase of mammalian target of rapamycin (mTOR) upon which the signalling of several growth factor receptors converge plays a central role in cancer cell proliferation, mTOR inhibition of HCC is currently also being studied in preclinical trials. As HCCs represent hypervascularized neoplasms, inhibition of tumour vessel formation via interfering with the VEGF/VEGFR system is another promising approach in HCC treatment. This review will summarize the current status of the various growth factor receptor-based treatment strategies and in view of the multitude of novel targeted approaches, the rationale for combination therapies for advanced HCC treatment will also be taken into account.

  6. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis

    Directory of Open Access Journals (Sweden)

    Xin Gao

    2016-03-01

    Full Text Available Hematopoietic stem cells (HSCs originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5 enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis.

  7. Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

    Science.gov (United States)

    Kyakumoto, S; Kurokawa, R; Ota, M

    1990-07-12

    Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor

  8. 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

    2016-04-19

    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.

  9. Effects of (-)-Epigallocatechin gallate on some protein factors involved in the epidermal growth factor receptor signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Yinjiu Huang; Ruiqing Xu; Baoan Song; Song Yang; Li Zhao; Shouwei Wua

    2009-01-01

    (-)-Epigallocatechin gallate (EGCG), a major polyphenolic constituent of green tea, can inhibit activity of specific receptor tyrosine kinases (RTKs) and related downstream signal transduction pathways, resulting in the control of unwanted cell proliferation. The epidermal growth factor receptor (EGFR) signaling pathway is one of the most important pathways that regulates growth, survival, proliferation and differentiation in mammalian cells. This review addresses the effects of EGCG on some protein factors involved in the EGFR signaling pathway in a direct or indirect manner. Based on our understanding of the interaction between EGCG and these factors, and based on their structures, EGCG could be used as a lead compound for designing and synthesizing novel drugs with significant biological activity.

  10. Receptors for T cell-replacing factor/interleukin 5. Specificity, quantitation, and its implication

    OpenAIRE

    1988-01-01

    T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically [35S]methionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1- B20 (in vitro line) (a murine chronic B cell leukemic cell line previou...

  11. 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.

  12. Developmentally Regulated Expression of the Nerve Growth Factor Receptor Gene in the Periphery and Brain

    Science.gov (United States)

    Buck, C. R.; Martinez, Humberto J.; Black, Ira B.; Chao, Moses V.

    1987-05-01

    Nerve growth factor (NGF) regulates development and maintenance of function of peripheral sympathetic and sensory neurons. A potential role for the trophic factor in brain has been detected only recently. The ability of a cell to respond to NGF is due, in part, to expression of specific receptors on the cell surface. To study tissue-specific expression of the NGF receptor gene, we have used sensitive cRNA probes for detection of NGF receptor mRNA. Our studies indicate that the receptor gene is selectively and specifically expressed in sympathetic (superior cervical) and sensory (dorsal root) ganglia in the periphery, and by the septum-basal forebrain centrally, in the neonatal rat in vivo. Moreover, examination of tissues from neonatal and adult rats reveals a marked reduction in steady-state NGF receptor mRNA levels in sensory ganglia. In contrast, a 2- to 4-fold increase was observed in the basal forebrain and in the sympathetic ganglia over the same time period. Our observations suggest that NGF receptor mRNA expression is developmentally regulated in specific areas of the nervous system in a differential fashion.

  13. 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

    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...... 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...

  14. 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.

  15. Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxycytidine.

    Science.gov (United States)

    Ohshima, Motohiro; Tadakuma, Tomomi; Hayashi, Hideki; Inoue, Kazuyuki; Itoh, Kunihiko

    2010-01-01

    We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly(4)-Ser)(3). The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m(5)dCyd and weakly with 5-methylcytidine (m(5)Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC(50) 0.054 microg/ml) was approximately 80 times higher than that of the parent MoAb (IC(50) 4.27 microg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m(5)dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained approximately 1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

  16. Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in Sf9 insect cell.

    Science.gov (United States)

    Sakamoto, Seiichi; Taura, Futoshi; Tsuchihashi, Ryota; Putalun, Waraporn; Kinjo, Junei; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-12-01

    Plumbagin (PL; 5-hydroxy-2-methyl-1, 4-naphthoquinone) is an important secondary metabolite, mainly produced in the Plumbago zeylanica L. (Plumbaginaceae). A single-chain variable fragment (scFv) antibody, fusion of the variable regions of the heavy chain and light chain of immunoglobulin against PL (PL-scFv) was expressed by Bac-to-Bac Baculovirus Expression System using Spodoptera frugiperda (Sf9) insect cells and characterized to investigate potential use of PL-scFv as a tool for plant immunomodulation. Functional PL-scFv expressed in the Sf9 insect cells were purified using cation exchange chromatography followed by immobilized metal ion affinity chromatography (IMAC). The yields of the purified PL-scFv in the culture supernatant and Sf9 insect cells were 2.0 mg and 5.2 mg per 1 liter of Sf9 culture medium, respectively. Recombinant purified PL-scFv was then characterized by the indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivity and sensitivity of PL-scFv expressed in Sf9 insect cells were compared with PL-scFv expressed in Escherichia coli and its parental anti-plumbagin monoclonal antibody (MAb 3A3) secreted from hybridoma cells. Intriguingly, the specificity of the PL-scFv expressed in Sf9 insect cells was found to be different from that expressed in E. coli and parental MAb 3A3, although the detectable level (0.2-25 μg/mL) was the same in ELISA using each antibody. Even more interestingly, the characteristics of PL-scFv, which have wide cross-reactivity against 1,4-napththoquinone, suggest its potential use as a tool for plant immunomodulation not only for breeding Plumbaginacea family containing PL but also for breeding other medicinal plants containing bioactive naphthoquinones.

  17. Modular Synthetic Platform for the Construction of Functional Single-Chain Polymeric Nanoparticles: From Aqueous Catalysis to Photosensitization.

    Science.gov (United States)

    Liu, Yiliu; Pauloehrl, Thomas; Presolski, Stanislav I; Albertazzi, Lorenzo; Palmans, Anja R A; Meijer, E W

    2015-10-14

    Single-chain polymeric nanoparticles (SCPNs) are intriguing systems for multiple applications. In order to arrive at a controlled, but random, positioning of the different side groups to the polymer backbone, alternative synthetic routes have to be developed. Here, a general postpolymerization modification strategy of poly(pentafluorophenyl acrylate) (pPFPA) is presented as a versatile method to rapidly access functional SCPNs. We first show that the sequential addition of a benzene-1,3,5-tricarboxamide-based amine, acting as the supramolecular recognition motif, and water-soluble polyetheramine (Jeffamine) to pPFPA affords random copolymers that fold in water into SCPNs. The scope of the modular platform is illustrated by preparing two types of functional SCPNs. First, we prepared SCPNs designed for bio-orthogonal catalysis by attaching pendant mono(benzimidazoylmethyl)-bis(pyridylmethyl) (Bimpy), phenanthroline (Phen), or 2,2'-bipyridine (BiPy), ligands capable of binding either Cu(I) or Pd(II). The Bimpy- and Phen-containing SCPNs ligated to Cu(I) significantly accelerate azide-alkyne cycloaddition reactions while Bipy-containing SCPNs ligated to Pd(II) efficiently catalyze depropargylation reactions. In all cases, reactions proceeded efficiently in phosphate buffer at a physiological pH and at low substrate concentrations. Next, the potential of SCPNs for photodynamic therapy was evaluated. Introducing porphyrins in SCPNs leads to novel photosensitizers that can produce singlet oxygen ((1)O2) upon photoirradiation. Additionally, by attaching both porphyrins and prodrug models, attached via (1)O2-cleavable amino-acrylate linker, to the SCPNs, we show that irradiation of the SCPNs results in a cascade reaction of (1)O2 generation followed by cleavage of the amino-acrylate linkers, releasing the drug model. The modular synthesis strategy reported here provides rapid and controlled access to SCPNs with tunable amounts of active units that fulfill different

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

    Directory of Open Access Journals (Sweden)

    Shuli Zhao

    2012-03-01

    Full Text Available 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(Dex1.3(DOX20. 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.

  19. Generation and characterization of a human single-chain fragment variable (scFv antibody against cytosine deaminase from Yeast

    Directory of Open Access Journals (Sweden)

    Tombesi Marina

    2008-09-01

    Full Text Available Abstract Background The ability of cytosine deaminase (CD to convert the antifungal agent 5-fluorocytosine (5-FC into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT aiming to improve the therapeutic ratio (benefit versus toxic side-effects of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies. Results An enzymatic active recombinant CD from yeast (yCD was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU. Conclusion The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

  20. The effect of internalizing human single chain antibody fragment on liposome targeting to epithelioid and sarcomatoid mesothelioma.

    Science.gov (United States)

    Iyer, Arun K; Su, Yang; Feng, Jinjin; Lan, Xiaoli; Zhu, Xiaodong; Liu, Yue; Gao, Dongwei; Seo, Youngho; Vanbrocklin, Henry F; Courtney Broaddus, V; Liu, Bin; He, Jiang

    2011-04-01

    Immunoliposomes (ILs) anchored with internalizing human antibodies capable of targeting all subtypes of mesothelioma can be useful for targeted imaging and therapy of this malignant disease. The objectives of this study were to evaluate both the in vitro and in vivo tumor targeted internalization of novel internalizing human single chain antibody (scFv) anchored ILs on both epithelioid (M28) and sarcomatoid (VAMT-1) subtypes of human mesothelioma. ILs were prepared by post-insertion of mesothelioma-targeting human scFv (M1) onto preformed liposomes and radiolabeled with (111)In ((111)In-IL-M1), along with control non-targeted liposomes ((111)In-CL). Incubation of (111)In-IL-M1 with M28, VAMT-1, and a control non-tumorigenic cell line (BPH-1) at 37 °C for 24 h revealed efficient binding and rapid internalization of ILs into both subtypes of tumor cells but not into the BPH-1 cells; internalization accounted for approximately 81-94% of total cell accumulation in mesothelioma cells compared to 37-55% in control cells. In tumor-bearing mice intravenous (i.v.) injection of (111)In-IL-M1 led to remarkable tumor accumulation: 4% and 4.7% injected dose per gram (% ID/g) for M28 and VAMT-1 tumors, respectively, 48 h after injection. Furthermore, tumor uptake of (111)In-IL-M1 in live xenograft animal models was verified by single photon emission computed tomography (SPECT/CT). In contrast, i.v. injection of (111)In-CL in tumor-bearing mice revealed very low uptake in both subtypes of mesothelioma, 48 h after injection. In conclusion, M1 scFv-anchored ILs showed selective tumor targeting and rapid internalization into both epithelioid and sarcomatoid subtypes of human mesothelioma, demonstrating its potential as a promising vector for enhanced tumor drug targeting.

  1. Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

    Science.gov (United States)

    He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

    2016-06-15

    Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection.

  2. Construction of a human functional single-chain variable fragment (scFv) antibody recognizing the malaria parasite Plasmodium falciparum.

    Science.gov (United States)

    Wajanarogana, Sumet; Prasomrothanakul, Teerawat; Udomsangpetch, Rachanee; Tungpradabkul, Sumalee

    2006-04-01

    Falciparum malaria is one of the most deadly and profound human health problems around the tropical world. Antimalarial drugs are now considered to be a powerful treatment; however, there are drugs currently being used that are resistant to Plasmodium falciparum parasites spreading in different parts of the world. Although the protective immune response against intraerythrocytic stages of the falciparum malaria parasite is still not fully understood, immune antibodies have been shown to be associated with reduced parasite prevalence. Therefore antibodies of the right specificity present in adequate concentrations and affinity are reasonably effective in providing protection. In the present study, VH (variable domain of heavy chain) and VL (variable domain of light chain) were isolated from human blood lymphocytes of P. falciparum in one person who had high serum titre to RESA (ring-infected erythrocyte surface antigen). Equal amounts of VH and VL were assembled together with universal linker (G4S)3 to generate scFvs (single-chain variable fragments). The scFv antibodies were expressed with a phage system for the selection process. Exclusively, an expressed scFv against asynchronous culture of P. falciparum-infected erythrocytes was selected and characterized. Sequence analysis of selected scFv revealed that this clone could be classified into a VH family-derived germline gene (VH1) and Vkappa family segment (Vkappa1). Using an indirect immunofluorescence assay, we could show that soluble expressed scFv reacted with falciparum-infected erythrocytes. The results encourage the further study of scFvs for development as a potential immunotherapeutic agent.

  3. 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; Chang, Ya-Chun

    2015-10-01

    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.

  4. Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.

    Science.gov (United States)

    Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Minenkova, Olga; Hartung, John

    2016-03-01

    'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.

  5. The protective effects and underlying mechanism of an anti-oligomeric Aβ42 single-chain variable fragment antibody.

    Science.gov (United States)

    Zhang, Yuan; Chen, Xu; Liu, Jinyu; Zhang, Yingjiu

    2015-12-01

    Oligomeric Aβ42 aggregates have been identified as one of the major neurotoxic components of Alzheimer's disease (AD). Immunotherapy targeted against these Aβ42 aggregates has been proposed as an appropriate therapeutic approach for the treatment of AD. Here, we report an anti-oligomeric Aβ42 single-chain variable fragment (scFv) antibody, named MO6, obtained from the human antibody library of a healthy donor. ScFv MO6 specifically recognized and bound to the oligomeric Aβ42 (Aβ42 oligomers and immature protofibrils; 18-37 kDa), and reduced their levels mainly by blocking their formation, although scFv MO6 also induced disaggregation of Aβ42 aggregates. More importantly, scFv MO6 ameliorated or attenuated Aβ42-induced cytotoxicity and increased cell viability by up to 33%. Furthermore, scFv MO6 efficiently passed through an in vitro blood-brain barrier (BBB) model with a delivery efficiency of 66% after 60 min post-administration. ScFv MO6 is a monovalent antibody with an affinity constant (KD) of 5.2×10(-6) M for Aβ42 oligomers. Molecular docking simulations of Aβ42 to scFv MO6 revealed that the approach and specific binding of scFv MO6 to oligomeric Aβ42 aggregates was achieved by conformational recognition and directed induction, which resulted in a more dynamic adaptation of Aβ42 to scFv MO6, occurring mainly in the N-terminal (3-4), middle (12-19) and C-terminal (34-42) regions of Aβ42. This binding mode of scFv MO6 to Aβ42 explains its protective effects against oligomeric Aβ42. Our findings may be applied for the design of a smaller antibody specific for Aβ42 oligermers.

  6. 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.

  7. Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2).

    Science.gov (United States)

    Li, Tai-Wei; Cheng, Shu-Fang; Tseng, Yen-Tzu; Yang, Yu-Chih; Liu, Wen-Chun; Wang, Sheng-Cyuan; Chou, Mei-Ju; Lin, Yu-Jen; Wang, Yueh; Hsiao, Pei-Wen; Wu, Suh-Chin; Chang, Ding-Kwo

    2016-01-01

    Influenza A viruses (IAV) are widespread in birds and domestic poultry, occasionally causing severe epidemics in humans and posing health threats. Hence, the need to develop a strategy for prophylaxis or therapy, such as a broadly neutralizing antibody against IAV, is urgent. In this study, single-chain variable fragment (scFv) phage display technology was used to select scFv fragments recognizing influenza envelope proteins. The Tomlinson I and J scFv phage display libraries were screened against the recombinant HA2 protein (rHA2) for three rounds. Only the third-round elution sample of the Tomlinson J library showed high binding affinity to rHA2, from which three clones (3JA18, 3JA62, and 3JA78) were chosen for preparative-scale production as soluble antibody by E. coli. The clone 3JA18 was selected for further tests due to its broad affinity for influenza H1N1, H3N2 and H5N1. Simulations of the scFv 3JA18-HA trimer complex revealed that the complementarity-determining region of the variable heavy chain (VH-CDR2) bound the stem region of HA. Neutralization assays using a peptide derived from VH-CDR2 also supported the simulation model. Both the selected antibody and its derived peptide were shown to suppress infection with H5N1 and H1N1 viruses, but not H3N2 viruses. The results also suggested that the scFvs selected from rHA2 could have neutralizing activity by interfering with the function of the HA stem region during virus entry into target cells.

  8. Single chain variable fragment against aβ expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

    Directory of Open Access Journals (Sweden)

    Ying Zhang

    Full Text Available Alzheimer's disease (AD is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb, A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ "on-pathway" aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM and thioflavin T (ThT binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of "on-pathway" aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free "on-pathway" Aβ aggregation and will assist in the development of therapeutic strategies for AD.

  9. Expression of vascular endothelial growth factor and its two receptors in normal human endometrium

    Institute of Scientific and Technical Information of China (English)

    王海燕; 陈贵安

    2003-01-01

    Objectives: We try to demonstrate the expression of vascular endothelial growthfactor (VEGF) and its receptors, flt-1 and KDR, in normal human emdometrium duringthe menstrual cycle.Methods: Immunohistochemical method was used to observe the expression ofVEGF and its two receptors in emdometrium throughout the normal menstrual cyclemeanwhile the isoforms of VEGF were also detected by Western blot analysis. The en-dothelial cells of micro-vessels were marked with Ⅷ factor antibody.Results: VEGF and its receptors existed in endometrial glandular, stromal and vas-cular endothelial cells of human endometrium. Their expressions were higher in the mid-secretory phase of menstrual cycle and highest at menstruation. VEGF121 and VEGF165were the predominant isoforms in normal human endometrium.Conclusion: The expression of VEGF and its two receptors showed cycle-dependentin human endometrium, probably involved in embryonic implantation and endometrialproliferation and differentiation.

  10. Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Tomlins, Scott A.; Bollinger, Nikki; Creim, Jeffrey A.; Rodland, Karin D.

    2005-08-15

    The calcium-sensing receptor (CaR) is a G-protein coupled receptor that is activated by extracellular calcium (Ca2+o). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Ca2+o. Further, we show that AG1478 acts downstream or separately from G-protein subunit activation of phospholipase C. AG1478 significantly inhibits Ca2+o-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Ca2+o. This is consistent with the known expression of TGFa by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR mediated response to increased Ca2+o in Rat-1 fibroblasts, and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.

  11. Epidermal Growth Factor Receptor Expression in Triple Negative and Nontriple Negative Breast Carcinomas.

    Science.gov (United States)

    Changavi, Arathi A; Shashikala, Arundhathi; Ramji, Ashwini S

    2015-01-01

    The panel of markers used for molecular classification include estrogen receptors (ER), progesterone receptors (PR), human epidermal growth factor receptor (HER)-2/neu, p53, Bcl-2 and basal markers like cytokeratin 5/6 or epidermal growth factor receptor (EGFR). Among these, EGFR plays an important role and is associated with bad prognosis. To study EGFR expression in triple negative breast carcinoma (TNBC) and non-TNBCs (NTNBCs). Fifty cases of breast carcinomas were classified and graded according to World Health Organization and Nottingham modification of Scarff-Bloom-Richardson (SBR) system, respectively. The age of the patients ranged from 28 to 69 years. Histological features such as necrosis, pushing borders, lymphocytic infiltrate and periductal elastosis were noted. The panel of markers used in our study included ER, PR, HER-2/neu and EGFR. EGFR expression was assessed based on membrane staining. Chi-square test was applied for statistical analysis to compare EGFR expression with hormonal status and prognostic factors. P triple negative and strongly expressed EGFR. EGFR expression was inversely associated with ER status and showed strong association with necrosis and lymphocytic infiltrate, but not with pushing border and periductal elastosis. EGFR is an important marker to stratify patients with breast cancer according to molecular classification. Its expression correlated positively with young age, higher SBR grade, necrosis, lymphocytic infiltrate and inversely with hormonal receptor expression.

  12. Expression of type 1 corticotropin-releasing factor receptor in the guinea pig enteric nervous system.

    Science.gov (United States)

    Liu, Sumei; Gao, Xiang; Gao, Na; Wang, Xiyu; Fang, Xiucai; Hu, Hong-Zhen; Wang, Guo-Du; Xia, Yun; Wood, Jackie D

    2005-01-17

    Reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiological recording, and intraneuronal injection of the neuronal tracer biocytin were integrated in a study of the functional expression of corticotropin-releasing factor (CRF) receptors in the guinea pig enteric nervous system. RT-PCR revealed expression of CRF1 receptor mRNA, but not CRF2, in both myenteric and submucosal plexuses. Immunoreactivity for the CRF1 receptor was distributed widely in the myenteric plexus of the stomach and small and large intestine and in the submucosal plexus of the small and large intestine. CRF1 receptor immunoreactivity was coexpressed with calbindin, choline acetyltransferase, and substance P in the myenteric plexus. In the submucosal plexus, CRF1 receptor immunoreactivity was found in neurons that expressed calbindin, substance P, choline acetyltransferase, or neuropeptide Y. Application of CRF evoked slowly activating depolarizing responses associated with elevated excitability in both myenteric and submucosal neurons. Histological analysis of biocytin-filled neurons revealed that both uniaxonal neurons with S-type electrophysiological behavior and neurons with AH-type electrophysiological behavior and Dogiel II morphology responded to CRF. The CRF-evoked depolarizing responses were suppressed by the CRF1/CRF2 receptor antagonist astressin and the selective CRF1 receptor antagonist NBI27914 and were unaffected by the selective CRF2 receptor antagonist antisauvagine-30. The findings support the hypothesis that the CRF1 receptor mediates the excitatory actions of CRF on neurons in the enteric nervous system. Actions on enteric neurons might underlie the neural mechanisms by which stress-related release of CRF in the periphery alters intestinal propulsive motor function, mucosal secretion, and barrier functions.

  13. Platelet-derived growth factor receptor-β in myocyte was upregulated by angiotensin II

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To observe the regulation of platelet-derived growth factor (PDGF) receptor-βin myocyte stimulated by angiotensin II (AngII) at both integrated and cellular levels and reveal the signal transduction mechanism in cell, two kidneys, one clip (2K1C) renal hypertension were performed by placing a sliver clip around the left renal artery. Blood pressure and the ratio of left ventricular weight to body weight were measured at 4 and 8 weeks after operation. The content of AngII in heart was detected by radioimmunology assay; the protein level of PDGF receptor-βin heart was measured by Western blot analysis. The alteration of PDGF receptor-βstimulated by AngII and several inhibitors was observed on cultured neonatal rat ventricular myocyte (NRVM). The content of AngII in heart of 2K1C renal hypertensive rat at 4 and 8 weeks after operation was increased. Compared with sham group, 4 and 8 weeks after operation, PDGF receptor-βin heart of 2K1C group was upregulated by 100.3% and 127.1% (P < 0.05), respectively. This upregulation could be inhibited by captopril. For cultured myocyte, PDGF receptor-βwas increased by 47.1% after being stimulated by AngII and this upregulation could be inhibited by losartan which was an inhibitor of AT1 receptor. PLC inhibitor (U73122) and MEK inhibitor (PD98059) could partly inhibit PDGF receptor-βupregulation induced by AngII. These results suggested that AngII could upregulate PDGF receptor-βin myocyte by its AT1 receptor and this effect was at least partly dependent on PLC and extracellular signal-regulated kinase (ERK).

  14. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1.

    Science.gov (United States)

    Finetti, Federica; Solito, Raffaella; Morbidelli, Lucia; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2008-01-25

    Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth.

  15. Kinetics of the Attachment of Intrinsic Factor-Bound Cobamides to Ileal Receptors

    Science.gov (United States)

    Mathan, V. I.; Babior, Bernard M.; Donaldson, Robert M.

    1974-01-01

    To determine whether the molecular configuration of vitamin B12 influences the attachment of intrinsic factor-vitamin B12 complex to ileal microvillous membrane receptor sites, we have examined the kinetics of uptake of intrinsic factor-bound cyanocobalamin by brush borders and microvillous membranes isolated from guinea pig ileum, and have compared this uptake with that of intrinsic factor alone and with that of intrinsic factor complexed with various analogs of cyanocobalamin. We first studied the kinetics of binding of cyanocobalamin and other cobamides to human gastric intrinsic factor. The binding of cyanocobalamin showed saturation kinetics and, at relatively high concentrations of cyanocobalamin, a Scatchard plot of binding was linear. The dissociation constant for the intrinsic factor-cyanocobalamin complex was 0.066 nM. When the binding of various vitamin B12 analogs to intrinsic factor was determined by competition experiments, the analogs could be separated into two categories: those with affinities similar to that of cyanocobalamin and those with affinities much lower than that of cyanocobalamin. The affinity of cyanocobalamin for intrinsic factor was not altered by various substitutions at the -CN position, while removal of a single amido group on the corrin ring of substitution of the dimethylbenzimidazole base greatly reduced affinity. Removal of the base totally abolished binding. These findings, confirming those reported by others, are consistent with the concept that the cyanocobalamin molecule fits into a “pocket” in the intrinsic factor molecule, with the nucleotide base facing inward and the -CN side of the planar corrin ring facing outward. We then investigated the attachment of intrinsic factor-bound cyanocobalamin to ileal receptor. Attachment to microvillous membranes showed saturation kinetics with a dissociation constant of 0.25 nM. Attachment was rapid and was 70% complete within 5 min; the second-order rate constant for attachment

  16. Differential requirement of the epidermal growth factor receptor for G protein-mediated activation of transcription factors by lysophosphatidic acid

    Directory of Open Access Journals (Sweden)

    Dent Paul

    2010-01-01

    Full Text Available Abstract Background The role of the epidermal growth factor receptor (EGFR and other receptor tyrosine kinases (RTKs in provoking biological actions of G protein-coupled receptors (GPCRs has been one of the most disputed subjects in the field of GPCR signal transduction. The purpose of the current study is to identify EGFR-mediated mechanisms involved in activation of G protein cascades and the downstream transcription factors by lysophosphatidic acid (LPA. Results In ovarian cancer cells highly responsive to LPA, activation of AP-1 by LPA was suppressed by inhibition of EGFR, an effect that could be reversed by co-stimulation of another receptor tyrosine kinase c-Met with hepatocyte growth factor, indicating that LPA-mediated activation of AP-1 requires activity of a RTK, not necessarily EGFR. Induction of AP-1 components by LPA lied downstream of Gi, G12/13, and Gq. Activation of the effectors of Gi, but not Gq or G12/13 was sensitive to inhibition of EGFR. In contrast, LPA stimulated another prominent transcription factor NF-κB via the Gq-PKC pathway in an EGFR-independent manner. Consistent with the importance of Gi-elicited signals in a plethora of biological processes, LPA-induced cytokine production, cell proliferation, migration and invasion require intact EGFR. Conclusions An RTK activity is required for activation of the AP-1 transcription factor and other Gi-dependent cellular responses to LPA. In contrast, activation of G12/13, Gq and Gq-elicited NF-κB by LPA is independent of such an input. These results provide a novel insight into the role of RTK in GPCR signal transduction and biological functions.

  17. The heparan sulfate co-receptor and the concentration of fibroblast growth factor-2 independently elicit different signalling patterns from the fibroblast growth factor receptor

    Directory of Open Access Journals (Sweden)

    Duchesne Laurence

    2010-06-01

    Full Text Available Abstract Background The fibroblast growth factor receptor (FGFR interprets concentration gradients of FGF ligands and structural changes in the heparan sulfate (HS co-receptor to generate different cellular responses. However, whether the FGFR generates different signals is not known. Results We have previously shown in rat mammary fibroblasts that in cells deficient in sulfation, and so in HS co-receptor, FGF-2 can only stimulate a transient phosphorylation of p42/44 MAPK and so cannot stimulate DNA synthesis. Here we demonstrate that this is because in the absence of HS, FGF-2 fails to stimulate the phosphorylation of the adaptor FGFR substrate 2 (FRS2. In cells possessing the HS co-receptor, FGF-2 elicits a bell-shaped dose response: optimal concentrations stimulate DNA synthesis, but supramaximal concentrations (≥ 100 ng/mL have little effect. At optimal concentrations (300 pg/mL FGF-2 stimulates a sustained dual phosphorylation of p42/44 MAPK and tyrosine phosphorylation of FRS2. In contrast, 100 ng/mL FGF-2 only stimulates a transient early peak of p42/44 MAPK phosphorylation and fails to stimulate appreciably the phosphorylation of FRS2 on tyrosine. Conclusions These results suggest that the nature of the FGFR signal produced is determined by a combination of the HS co-receptor and the concentration of FGF ligand. Both the phosphorylation of the adaptor FRS2, the kinetics (sustained or transient of phosphorylation of p42/44(MAPK are varied, and so differing cellular responses are produced.

  18. Modified epidermal growth factor receptor (EGFR-bearing liposomes (MRBLs are sensitive to EGF in solution.

    Directory of Open Access Journals (Sweden)

    Albert Wong

    Full Text Available Cancers often overexpress EGF and other growth factors to promote cell replication and migration. Previous work has not produced targeted drug carriers sensitive to abnormal amounts of growth factors. This work demonstrates that liposomes bearing EGF receptors covalently crosslinked to p-toluic acid or methyl-PEO(4-NHS ester (or, in short, MRBLs exhibit an increased rate of release of encapsulated drug compounds when EGF is present in solution. Furthermore, the modified EGF receptors retain the abilities to form dimers in the presence of EGF and bind specifically to EGF. These results demonstrate that MRBLs are sensitive to EGF in solution and indicate that MRBL-reconstituted modified EGF receptors, in the presence of EGF in solution, form dimers which increase MRBL permeability to encapsulated compounds.

  19. Interleukin-10 and soluble tumor necrosis factor receptors in cerebrospinal fluid of children with bacterial meningitis

    NARCIS (Netherlands)

    Kornelisse, R.F.; Savelkoul, H.F.J.; Mulder, P.H.G.; Suur, M.H.; Straaten, van der P.J.C.; Heijden, van der A.J.; Sukhai, R.N.; Hählen, K.; Neijens, H.J.; Groot, de R.

    1996-01-01

    The antiinflammatory mediators interleukin (IL)-10 and soluble tumor necrosis factor (TNF) receptors p55 (sTNFR-55) and sTNFR-75 in cerebrospinal fluid (CSF) from 37 children with bacterial meningitis were studied. CSF concentrations of IL-10, sTNFR-55, and sTNFR-75 and of the proinflammatory cytoki

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

    DEFF Research Database (Denmark)

    Harper, Peter; El-Hariry, Iman; Powles, Thomas;

    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...

  1. The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

    DEFF Research Database (Denmark)

    Christensen, Claus; Lauridsen, Jes B; Berezin, Vladimir;

    2006-01-01

    The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface...

  2. Endoglin structure and function - Determinants of endoglin phosphorylation by transforming growth factor-beta receptors

    NARCIS (Netherlands)

    Koleva, Rositsa I.; Conley, Barbara A.; Romero, Diana; Riley, Kristin S.; Marto, Jarrod A.; Lux, Andreas; Vary, Calvin P. H.

    2006-01-01

    Determination of the functional relationship between the transforming growth factor-beta(TGF beta) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGF beta 1 caused recruitment of ALK1 into a complex with end

  3. The insulin-like growth factor 1 receptor in cancer : Old focus, new future

    NARCIS (Netherlands)

    Hartog, Hermien; Wesseling, Jelle; Boezen, H. Marike; van der Graaf, Winette T. A.

    2007-01-01

    The importance of insulin-like growth factor 1 receptor (IGF-1R) signalling in malignant behaviour of tumour cells is well established. Currently, development of drugs targeting the IGF-1R as anticancer treatment is emerging. Several IGF-1R targeting strategies are being investigated in phases I and

  4. The insulin-like growth factor 1 receptor in cancer : Old focus, new future

    NARCIS (Netherlands)

    Hartog, Hermien; Wesseling, Jelle; Boezen, H. Marike; van der Graaf, Winette T. A.

    2007-01-01

    The importance of insulin-like growth factor 1 receptor (IGF-1R) signalling in malignant behaviour of tumour cells is well established. Currently, development of drugs targeting the IGF-1R as anticancer treatment is emerging. Several IGF-1R targeting strategies are being investigated in phases I and

  5. The insulin-like growth factor 1 receptor in cancer: old focus, new future.

    NARCIS (Netherlands)

    Hartog, H. de; Wesseling, J.; Boezen, H.M.; Graaf, W.T.A. van der

    2007-01-01

    The importance of insulin-like growth factor 1 receptor (IGF-1R) signalling in malignant behaviour of tumour cells is well established. Currently, development of drugs targeting the IGF-1R as anticancer treatment is emerging. Several IGF-1R targeting strategies are being investigated in phases I and

  6. Effect of fluoride on expression of insulinlike growth factor-1 and its receptor of rat osteoblasts

    Institute of Scientific and Technical Information of China (English)

    喻茂娟

    2013-01-01

    Objective To explore the influence of fluorine on mRNA and protein expression of the insulin-like growth factor-1 (IGF-1) and its receptor of rat osteoblasts.Methods Osteoblasts were isolated from rat bone by enzyme digestion.Different fluorine concentration[0 (con-

  7. 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...

  8. 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...

  9. 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,

  10. 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.

    2008-01-01

    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 cellula

  11. Epidermal growth factor receptor expression in pancreatic lesions induced in the rat by azaserine

    NARCIS (Netherlands)

    Visser, C.J.T.; Weger, R.A. de; Blokland, W.T.M. van; Seifert-Bock, I.; Kobrin, M.S.; Korc, M.; Woutersen, R.A.

    1996-01-01

    In the present study, the expression of the epidermal growth factor receptor (EGFR) was investigated in putative preneoplastic and neoplastic acinar cell lesions induced in the rat pancreas by azaserine, using Northern blotting, in situ hybridisation (ISH) and immunohistochemistry. EGFR protein leve

  12. Solubilization of nerve growth factor receptors of rabbit superior cervical ganglia.

    Science.gov (United States)

    Banerjee, S P; Cuatrecasas, P; Snyder, S H

    1976-09-25

    Nerve growth factor (NGF) receptors of rabbit superior cervical ganglia can be solubilized by treatment with detergents and readily assayed in the soluble state. Triton X-100 and deoxycholate reduce specific binding of NGF to ganglia membranes. In membranes treated with Triton X-100 (0.5 to 2.0%) the reduction in NGF binding by membranes is accompanied by a corresponding increase in binding in the supernatant fluid. NGF binding in soluble preparations can be rapidly assayed by precipitating NGF bound to receptors with polyethylene glycol under conditions in which unbound NGF is not precipitated. NGF binding to soluble preparations is saturable whether evaluated by the binding of 125I-NGF or by diluting 125I-NGF with native NGF. Using both techniques, the dissociation constant for NGF binding to soluble receptors is about 0.2 nM, the same as its dissociation constant from receptor sites in intact membranes. NGF binding to soluble receptors displays a high degree of peptide specificity, similar to receptor sites in intact membranes of superior cervical ganglia. A method of labeling NGF with 125I-3(4-hydroxyphenyl) propionic acid N-hydroxysuccinimide ester is described which leads to binding properties that are superior to those obtained with previously described 125I-NGF preparations.

  13. Nerve growth factor receptor from rabbit sympathetic ganglia membranes. Relationship between subforms.

    Science.gov (United States)

    Kouchalakos, R N; Bradshaw, R A

    1986-12-05

    The receptor for nerve growth factor (NGF) was purified from Triton X-100 extracts of sympathetic ganglia membranes by affinity chromatography on NGF-Sepharose. Elution of purified receptor was accomplished at pH 5 in the presence of 1 M NaCl. Sodium dodecyl sulfate gel electrophoresis of the purified iodinated receptor showed three major bands at Mr = 126,000, Mr = 105,000, and Mr = 81,000. Affinity labeling of the purified receptor using 125I-NGF and the photoreactive agent N-hydroxysuccinimidyl-p-azidobenzoate resulted in two major cross-linked complexes corresponding to Mr = 135,000 and Mr = 110,000. This labeling pattern is similar to that observed with sympathetic ganglia membranes (Massague, J., Guillette, B. J., Czech, M. P., Morgan, C. J., and Bradshaw, R. A. (1981) J. Biol. Chem. 256, 9419-9424) and indicates that these two forms do not arise from the cross-linking procedure. Reaction of the photoaffinity labeled NGF receptors with increasing amounts of trypsin resulted in a progressive decrease in the high molecular weight complex with a concomitant increase in the low molecular weight form. When the larger complex was isolated by electroelution from a sodium dodecyl sulfate gel and treated with trypsin, a species corresponding to Mr = 100,000 was generated. These observations are best explained by a precursor-product relationship for the two NGF receptor species of sympathetic neurons.

  14. miR-29a suppresses MCF-7 cell growth by downregulating tumor necrosis factor receptor 1.

    Science.gov (United States)

    Zhao, Yiling; Yang, Fenghua; Li, Wenyuan; Xu, Chunyan; Li, Li; Chen, Lifei; Liu, Yancui; Sun, Ping

    2017-02-01

    Tumor necrosis factor receptor 1 is the main receptor mediating many tumor necrosis factor-alpha-induced cellular events. Some studies have shown that tumor necrosis factor receptor 1 promotes tumorigenesis by activating nuclear factor-kappa B signaling pathway, while other studies have confirmed that tumor necrosis factor receptor 1 plays an inhibitory role in tumors growth by inducing apoptosis in breast cancer. Therefore, the function of tumor necrosis factor receptor 1 in breast cancer requires clarification. In this study, we first found that tumor necrosis factor receptor 1 was significantly increased in human breast cancer tissues and cell lines, and knockdown of tumor necrosis factor receptor 1 by small interfering RNA inhibited cell proliferation by arresting the cell cycle and inducing apoptosis. In addition, miR-29a was predicted as a regulator of tumor necrosis factor receptor 1 by TargetScan and was shown to be inversely correlated with tumor necrosis factor receptor 1 expression in human breast cancer tissues and cell lines. Luciferase reporter assay further confirmed that miR-29a negatively regulated tumor necrosis factor receptor 1 expression by binding to the 3' untranslated region. In our functional study, miR-29a overexpression remarkably suppressed cell proliferation and colony formation, arrested the cell cycle, and induced apoptosis in MCF-7 cell. Furthermore, in combination with tumor necrosis factor receptor 1 transfection, miR-29a significantly reversed the oncogenic role caused by tumor necrosis factor receptor 1 in MCF-7 cell. In addition, we demonstrated that miR-29a suppressed MCF-7 cell growth by inactivating the nuclear factor-kappa B signaling pathway and by decreasing cyclinD1 and Bcl-2/Bax protein levels. Taken together, our results suggest that miR-29a is an important regulator of tumor necrosis factor receptor 1 expression in breast cancer and functions as a tumor suppressor by targeting tumor necrosis factor receptor 1 to

  15. 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...... factor, subsequently initiating a signal cascade leading to symbiotic nodule development. Similar genes have also been identified in other plants: seven LysM-domain containing receptor-like kinases (LYKs) were found in the model legume Medicago truncatula, two of them, LYK3 and LYK4 playing a role...

  16. THE INFLUENCE OF HUMAN SINGLE CHAIN INTELEUKIN-12 GENE TRANSDUCTION ON THE BIOLOGICAL BEHAVIOR OF HEPATOMA 7721 CELLS

    Institute of Scientific and Technical Information of China (English)

    金莉; 来保长; 耿宜萍; 王一理; 司履生

    2001-01-01

    Objective. To investigate the anti-tumor effects of human single chain interleukin-12 (hscIL-12). Method. pcDNA/hscIL-12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL-12 cells which secrete hscIL-12 stably, were obtained via G418 selection, and in vitro the influence of hscIL-12 gene transduction on the growth of tumor cells was evaluated by cellcycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL-12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice, respectively. 7721/pcDNA and 7721/hscIL-12 groups were divided into two sub-groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equalvolume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. Results. hscIL-12 produced stably by 7721/hscIL-12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the7721/hscIL-12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL-12 cells. In ani-mal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5 -7mm,while those of 7721/hscIL-12 group were 2 -4mm. When treated with hPBL, the tumor of 7721/hscIL-12 groupdisappeared completely. Histologically, the tumors from 7721/hscIL-12 without hPBL treatment had numerouslymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis, whereas the tumors of 7721 and 772l/pcDNA groups grew thrivingly.Conclusion. hsclL-12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL-12 has certain effects on mice immune system. These findings suggest that hscIL-12 and hscIL-12 gene therapy might have promising prospects in clinical application.

  17. Three-dimensional antiferromagnetic order of single-chain magnets: a new approach to design molecule-based magnets.

    Science.gov (United States)

    Miyasaka, Hitoshi; Takayama, Karin; Saitoh, Ayumi; Furukawa, Sachie; Yamashita, Masahiro; Clérac, Rodolphe

    2010-03-22

    Two one-dimensional compounds composed of a 1:1 ratio of Mn(III) salen-type complex and Ni(II) oximato moiety with different counter anions, PF(6)(-) and BPh(4)(-), were synthesized: [Mn(3,5-Cl(2)saltmen)Ni(pao)(2)(phen)]PF(6) (1) and [Mn(5-Clsaltmen)Ni(pao)(2)(phen)]BPh(4) (2), where 3,5-Cl(2)saltmen(2-) = N,N'-(1,1,2,2-tetramethylethylene)bis(3,5-dichlorosalicylideneiminate); 5-Clsaltmen(2-) = N,N'-(1,1,2,2-tetramethylethylene)bis(5-chlorosalicylideneiminate); pao(-) = pyridine-2-aldoximate; and phen = 1,10-phenanthroline. Single-crystal X-ray diffraction study was carried out for both compounds. In 1 and 2, the chain topology is very similar forming an alternating linear chain with a [-Mn(III)-ON-Ni(II)-NO-] repeating motif (where -ON- is the oximate bridge). The use of a bulky counteranion, such as BPh(4)(-), located between the chains in 2 rather than PF(6)(-) in 1, successfully led to the magnetic isolation of the chains in 2. This minimization of the interchain interactions allows the study of the intrinsic magnetic properties of the chains present in 1 and 2. While 1 and 2 possess, as expected, very similar paramagnetic properties above 15 K, their ground state is antiferromagnetic below 9.4 K and paramagnetic down to 1.8 K, respectively. Nevertheless, both compounds exhibit a magnet-type behavior at temperatures below 6 K. While for 2, the observed magnetism is well explained by a Single-Chain Magnet (SCM) behavior, the magnet properties for 1 are induced by the presence in the material of SCM building units that order antiferromagnetically. By controlling both intra- and interchain magnetic interactions in this new [Mn(III)Ni(II)] SCM system, a remarkable AF phase with a magnet-type behavior has been stabilized in relation with the intrinsic SCM properties of the chains present in 1. This result suggests that the simultaneous enhancement of both intrachain (J) and interchain (J') magnetic interactions (with keeping J > J'), independently of the presence

  18. Tumor necrosis factor receptors support murine hematopoietic progenitor function in the early stages of engraftment.

    Science.gov (United States)

    Pearl-Yafe, Michal; Mizrahi, Keren; Stein, Jerry; Yolcu, Esma S; Kaplan, Ofer; Shirwan, Haval; Yaniv, Isaac; Askenasy, Nadir

    2010-07-01

    Tumor necrosis factor (TNF) family receptors/ligands are important participants in hematopoietic homeostasis, in particular as essential negative expansion regulators of differentiated clones. As a prominent injury cytokine, TNF-alpha has been traditionally considered to suppress donor hematopoietic stem and progenitor cell function after transplantation. We monitored the involvement of TNF receptors (TNF-R) 1 and 2 in murine hematopoietic cell engraftment and their inter-relationship with Fas. Transplantation of lineage-negative (lin(-)) bone marrow cells (BMC) from TNF receptor-deficient mice into wild-type recipients showed defective early engraftment and loss of durable hematopoietic contribution upon recovery of host hematopoiesis. Consistently, cells deficient in TNF receptors had reduced competitive capacity as compared to wild-type progenitors. The TNF receptors were acutely upregulated in bone marrow (BM)-homed donor cells (wild-type) early after transplantation, being expressed in 60%-75% of the donor cells after 6 days. Both TNF receptors were detected in fast cycling, early differentiating progenitors, and were ubiquitously expressed in the most primitive progenitors with long-term reconstituting potential (lin(-)c-kit(+) stem cell antigen (SCA)-1(+)). BM-homed donor cells were insensitive to apoptosis induced by TNF-alpha and Fas-ligand and their combination, despite reciprocal inductive cross talk between the TNF and Fas receptors. The engraftment supporting effect of TNF-alpha is attributed to stimulation of progenitors through TNF-R1, which involves activation of the caspase cascade. This stimulatory effect was not observed for TNF-R2, and this receptor did not assume redundant stimulatory function in TNFR1-deficient cells. It is concluded that TNF-alpha plays a tropic role early after transplantation, which is essential to successful progenitor engraftment.

  19. Growth Factor Receptor-Bound Protein 14 Undergoes Light-Dependent Intracellular Translocation in Rod Photoreceptors: Functional Role on Retinal Insulin Receptor Activation

    OpenAIRE

    Rajala, Ammaji; Roger J. Daly; Tanito, Masaki; Allen, Dustin T.; Lowenna J Holt; Lobanova, Ekaterina; Arshavsky, Vadim Y; Rajala, Raju V.S.

    2009-01-01

    Growth factor receptor-bound protein 14 (Grb14) is involved in growth factor receptor tyrosine kinase signaling. Here we report that light causes a major redistribution of Grb14 among the individual subcellular compartments of the retinal rod photoreceptor. Grb14 is localized predominantly to the inner segment, nuclear layer and synapse in dark-adapted rods, whereas in the light-adapted rods, Grb14 redistributed throughout the entire cell, including the outer segment. The translocation of Grb...

  20. Room temperature ferroelectricity in one-dimensional single chain molecular magnets [{M(Δ)M(Λ)}(ox)2(phen)2]n (M = Fe and Mn)

    Science.gov (United States)

    Bhatt, Pramod; Mukadam, M. D.; Meena, S. S.; Mishra, S. K.; Mittal, R.; Sastry, P. U.; Mandal, B. P.; Yusuf, S. M.

    2017-03-01

    The ferroelectric materials are mainly focused on pure inorganic oxides; however, the organic molecule based materials have recently attracted great attention because of their multifunctional properties. The mixing of oxalate and phenanthroline ligands with metal ions (Fe or Mn) at room temperature followed by hydrothermal treatment results in the formation of one-dimensional single chain molecular magnets which exhibit room temperature dielectric and ferroelectric behavior. The compounds are chiral in nature, and exhibit a ferroelectric behavior, attributed to the polar point group C2, in which they crystallized. The compounds are also associated with a dielectric loss and thus a relaxation process. The observed electric dipole moment, essential for a ferroelectricity, has been understood quantitatively in terms of lattice distortions at two different lattice sites within the crystal structure. The studied single chain molecular magnetic materials with room temperature ferroelectric and dielectric properties could be of great technological importance in non-volatile memory elements, and high-performance insulators.

  1. Corticotropin-releasing factor receptors and stress-related alterations of gut motor function.

    Science.gov (United States)

    Taché, Yvette; Bonaz, Bruno

    2007-01-01

    Over the past few decades, corticotropin-releasing factor (CRF) signaling pathways have been shown to be the main coordinators of the endocrine, behavioral, and immune responses to stress. Emerging evidence also links the activation of CRF receptors type 1 and type 2 with stress-related alterations of gut motor function. Here, we review the role of CRF receptors in both the brain and the gut as part of key mechanisms through which various stressors impact propulsive activity of the gastrointestinal system. We also examine how these mechanisms translate into the development of new approaches for irritable bowel syndrome, a multifactorial disorder for which stress has been implicated in the pathophysiology.

  2. Association between cadmium and breast cancer risk according to estrogen receptor and human epidermal growth factor receptor 2: epidemiological evidence.

    Science.gov (United States)

    Strumylaite, Loreta; Kregzdyte, Rima; Bogusevicius, Algirdas; Poskiene, Lina; Baranauskiene, Dale; Pranys, Darius

    2014-05-01

    The study aimed to examine the association between cadmium (Cd) and the risk of breast cancer according to estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). A hospital-based case-control study was carried out in 585 cases and 1,170 controls. Information on possible risk factors was collected via a structured questionnaire. Urinary Cd was determined by atomic absorption spectrometry. The ER and HER2 levels in tumor tissue were analyzed by immunohistochemistry. Logistic regression was used to calculate odds ratios (ORs) and 95 % confidence intervals (CIs) for breast cancer by creatinine-adjusted urinary Cd. Women with greater creatinine-adjusted urine Cd (3rd quartile: 0.241-0.399 μg/g and 4th quartile: ≥ 0.4 μg/g) experienced 1.6 times higher risk of breast cancer compared with those having Cd concentration lower than 0.147 μg/g (1st quartile) [OR = 1.6, (95 % CI 1.19, 2.17) and OR = 1.62 (95 % CI 1.19, 2.21), respectively, P trend = 0.001] after adjustment for age and other confounders. Both ER+ and HER2- cases from the highest quartile of urine Cd exhibited approximately twice the breast cancer risk of those in the lowest quartile [OR = 1.9, (95 % CI 1.31, 2.74) and OR = 1.87, (95 % CI 1.33, 2.62), respectively, P trend cadmium as a risk factor for breast cancer, especially for both ER+ and HER2- cancer patients.

  3. Internalization of exogenous cystatin F supresses cysteine proteases and induces the accumulation of single-chain cathepsin L by multiple mechanisms.

    Science.gov (United States)

    Colbert, Jeff D; Matthews, Stephen P; Kos, Janko; Watts, Colin

    2011-12-01

    Cystatin F is an unusual member of the cystatin family of protease inhibitors, which is made as an inactive dimer and becomes activated by proteolysis in the endo/lysosome pathway of the immune cells that produce it. However a proportion is secreted and can be taken up and activated by other cells. We show here that cystatin F acquired in this way induces a dramatic accumulation of the single-chain form of cathepsin L (CatL). Cystatin F was observed in the same cellular compartments as CatL and was tightly complexed with CatL as determined by co-precipitation studies. The observed accumulation of single-chain CatL was partly due to cystatin F-mediated inhibition of the putative single-chain to two-chain CatL convertase AEP/legumain and partly to general suppression of cathepsin activity. Thus, cystatin F stabilizes CatL leading to the dramatic accumulation of an inactive complex composed either of the single-chain or two-chain form depending on the capacity of cystatin F to inhibit AEP. Cross-transfer of cystatin F from one cell to another may therefore attenuate potentially harmful effects of excessive CatL activity while paradoxically, inducing accumulation of CatL protein. Finally, we confirmed earlier data (Beers, C., Honey, K., Fink, S., Forbush, K., and Rudensky, A. (2003) J. Exp. Med. 197, 169-179) showing a loss of CatL activity, but not of CatL protein, in macrophages activated with IFNγ. However, we found equivalent loss of CatL activity in wild type and cystatin F-null macrophages suggesting that an inhibitory activity other than cystatin F quenches CatL activity in activated macrophages.

  4. Regulation of Steroidogenesis, Development, and Cell Differentiation by Steroidogenic Factor-1 and Liver Receptor Homolog-1.

    Science.gov (United States)

    Yazawa, Takashi; Imamichi, Yoshitaka; Miyamoto, Kaoru; Khan, Md Rafiqul Islam; Uwada, Junsuke; Umezawa, Akihiro; Taniguchi, Takanobu

    2015-08-01

    Steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) belong to the nuclear receptor superfamily and are categorized as orphan receptors. In addition to other nuclear receptors, these play roles in various physiological phenomena by regulating the transcription of target genes. Both factors share very similar structures and exhibit common functions. Of these, the roles of SF-1 and LRH-1 in steroidogenesis are the most important, especially that of SF-1, which was originally discovered and named to reflect such roles. SF-1 and LRH-1 are essential for steroid hormone production in gonads and adrenal glands through the regulation of various steroidogenesis-related genes. As SF-1 is also necessary for the development of gonads and adrenal glands, it is also considered a master regulator of steroidogenesis. Recent studies have clearly demonstrated that LRH-1 also represents another master regulator of steroidogenesis, which similarly to SF-1, can induce differentiation of non-steroidogenic stem cells into steroidogenic cells. Here, we review the functions of both factors in these steroidogenesis-related phenomena.

  5. Tumor necrosis factor-alpha inhibits pre-osteoblast differentiation through its type-1 receptor.

    Science.gov (United States)

    Abbas, Sabiha; Zhang, Yan-Hong; Clohisy, John C; Abu-Amer, Yousef

    2003-04-01

    Tumor necrosis factor-alpha (TNF) is a pro-inflammatory cytokine with a profound role in many skeletal diseases. The cytokine has been described as a mediator of bone loss in osteolysis and other inflammatory bone diseases. In addition to its known bone resorptive action, TNF reduces bone formation by inhibiting osteoblast differentiation. Using primary and transformed osteoblastic cells, we first document that TNF inhibits expression of alkaline phosphatase and matrix deposition, both considered markers of osteoblast differentiation. The effects are dose- and time-dependent. Core-binding factor A1 (cbfa1) is a transcription factor critical for osteoblast differentiation, and we show here that it is activated by the osteoblast differentiation agent, beta-glycerophosphate. Therefore, we investigated whether the inhibitory effects of TNF were associated with altered activity of this transcription factor. Using retardation assays, we show that TNF significantly inhibits cbfal activation by beta-glycerophosphate, manifested by reduced DNA-binding activity. Next, we turned to determine the signaling pathway by which TNF inhibits osteoblast differentiation. Utilizing animals lacking individual TNF receptors, we document that TNFr1 is required for transmitting the cytokine's inhibitory effect. In the absence of this receptor, TNF failed to impact all osteoblast differentiation markers tested. In summary, TNF blocks expression of osteoblast differentiation markers and inhibits beta-glycerophosphate-induced activation of the osteoblast differentiation factor cbfa1. Importantly, these effects are mediated via a mechanism requiring the TNF type-1 receptor.

  6. Emulsion of aqueous-based nonspherical droplets in aqueous solutions by single-chain surfactants: templated assembly by nonamphiphilic lyotropic liquid crystals in water.

    Science.gov (United States)

    Varghese, Nisha; Shetye, Gauri S; Bandyopadhyay, Debjyoti; Gobalasingham, Nemal; Seo, JinAm; Wang, Jo-Han; Theiler, Barbara; Luk, Yan-Yeung

    2012-07-24

    Single-chain surfactants usually emulsify and stabilize oily substances into droplets in an aqueous solution. Here, we report a coassembly system, in which single types of anionic or non-ionic surfactants emulsify a class of water-soluble nonamphiphilic organic salts with fused aromatic rings in aqueous solutions. The nonamphiphilic organic salts are in turn promoted to form droplets of water-based liquid crystals (chromonic liquid crystals) encapsulated by single-chain surfactants. The droplets, stabilized against coalescence by encapsulated in a layer (or layers) of single chain surfactants, are of both nonspherical tactoid (elongated ellipsoid with pointy ends) and spherical shapes. The tactoids have an average long axis of ∼9 μm and a short axis of ∼3.5 μm with the liquid crystal aligning parallel to the droplet surface. The spherical droplets are 5-10 μm in diameter and have the liquid crystal aligning perpendicular to the droplet surface and a point defect in the center. Cationic and zwitterionic surfactants studied in this work did not promote the organic salt to form droplets. These results illustrate the complex interplay of self-association and thermodynamic incompatibility of molecules in water, which can cause new assembly behavior, including potential formation of vesicles or other assemblies, from surfactants that usually form only micelles. These unprecedented tactoidal shaped droplets also provide potential for the fabrication of new soft organic microcapsules.

  7. The ability of single-chain surfactants to emulsify an aqueous-based liquid crystal oscillates with odd-even parity of alkyl-chain length.

    Science.gov (United States)

    Varghese, Nisha; Shetye, Gauri S; Yang, Sijie; Wilkens, Stephan; Smith, Robert P; Luk, Yan-Yeung

    2013-12-15

    The physical properties of many organic molecules often oscillate when the number of carbons in their aliphatic chains changes from odd to even. This odd-even effect for single-chain surfactants in solution is rarely observed. Here, we report the ability of single-chain surfactants to emulsify a class of non-amphiphilic organic salts, disodium cromoglycate (5'DSCG) oscillates as a function of the odd or even number of the aliphatic carbons. This system provides a water-in-oil-in-water emulsion, in which aqueous droplets of 5'DSCG in liquid crystal phases are coated with single-chain surfactants in a bulk carrying aqueous solution. For both surfactants of [Formula: see text] and CH3(CH2)nCOO(-)Na(+), the ability to emulsify 5'DSCG molecules in water is stronger for surfactants with an odd number of sp(3)-hybridized carbon atoms in the aliphatic chains than those with an even number. This observed odd-even effect is consistent with the notion that conventional micelles possess a core of randomly arranged surfactant hydrocarbon tails. However, this water-in-oil-in-water resembles a vesicle system in which the surfactants assemble in a highly ordered structure that separates two aqueous systems. These new self-assembled phases have potential application in the formulation and design of new organic soft materials.

  8. Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening.

    Science.gov (United States)

    Sydor, Jens R; Mariano, Maria; Sideris, Steve; Nock, Steffen

    2002-01-01

    Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

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

    Energy Technology Data Exchange (ETDEWEB)

    Grob, P.M.; David, E.; Warren, T.C.; DeLeon, R.P.; Farina, P.R.; Homon, C.A. (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT (USA))

    1990-05-15

    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.

  10. Growth Factor Receptors and Apoptosis Regulators: Signaling Pathways, Prognosis, Chemosensitivity and Treatment Outcomes of Breast Cancer

    Directory of Open Access Journals (Sweden)

    Siddik Sarkar

    2009-01-01

    Full Text Available Biomarkers of breast cancer are necessary for prognosis and prediction to chemotherapy. Prognostic biomarkers provide information regarding outcome irrespective of therapy, while predictive biomarkers provide information regarding response to therapy. Candidate prognostic biomarkers for breast cancers are growth factor receptors, steroid receptors, Ki-67, cyclins, urokinase plasminogen activator, p53, p21, pro- and anti-apoptotic factors, BRCA1 and BRCA2. But currently, the predictive markers are Estrogen and Progesterone receptors responding to endocrine therapy, and HER-2 responding to herceptin. But there are numerous breast cancer cases, where tamoxifen is ineffective even after estrogen receptor positivity. This lead to search of new prognostic and predictive markers and the number of potential markers is constantly increasing due to proteomics and genomics studies. However, most biomarkers individually have poor sensitivity or specificity, or other clinical value. It can be resolved by studying various biomarkers simultaneously, which will help in better prognosis and increasing sensitivity for chemotherapeutic agents. This review is focusing on growth factor receptors, apoptosis markers, signaling cascades, and their correlation with other associated biomarkers in breast cancers. As our knowledge regarding molecular biomarkers for breast cancer increases, prognostic indices will be developed that combine the predictive power of individual molecular biomarkers with specific clinical and pathologic factors. Rigorous comparison of these existing as well as emerging markers with current treatment selection is likely to see an escalation in an era of personalized medicines to ensure the breast cancer patients receive optimal treatment. This will also solve the treatment modalities and complications related to chemotherapeutic regimens.

  11. Mammographic features of calcifications in DCIS: correlation with oestrogen receptor and human epidermal growth factor receptor 2 status

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Min Sun; Moon, Woo Kyung; Chang, Jung Min; Cho, Nariya [Seoul National University College of Medicine, Department of Radiology, Seoul (Korea, Republic of); Park, So Yeon; Won, Jae-Kyung; Jeon, Yoon-Kyung; Park, In Ae [Seoul National University College of Medicine, Department of Pathology, Seoul (Korea, Republic of); Moon, Hyeong-Gon; Han, Wonshik [Seoul National University College of Medicine, Department of Surgery, Seoul (Korea, Republic of)

    2013-08-15

    This study investigated the correlation of oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status with the probability of malignancy (POM) of mammographic calcifications in ductal carcinoma in situ (DCIS). A total of 101 women (age range, 27-83 years) with pure DCIS that presented as mammographic calcifications were included. Three radiologists independently reviewed mammograms according to the BI-RADS lexicon and provided 100-point POM scores and a BI-RADS category. ER, HER2 and breast cancer subtypes were determined using immunohistochemistry (IHC) and fluorescence in situ hybridisation. Pairwise correlations between POM and IHC biomarker scores were calculated, and mammographic features were compared between breast cancer subtypes. HER2 level positively correlated with the POM score (P < 0.0001) and BI-RADS category (P < 0.0001), and ER level inversely correlated with the POM score (P < 0.013) and BI-RADS category (P < 0.010). Fine linear branching (P = 0.004) and segmental (P = 0.014) calcifications were significantly associated with HER2-positive cancers, and clustered calcifications were more frequently observed in ER-positive cancers (P = 0.014). HER2 status in DCIS correlated positively with the POM of mammographic calcifications, as determined by radiologists on the basis of the BI-RADS lexicon. (orig.)

  12. Structural requirements for inducible shedding of the p55 tumor necrosis factor receptor

    DEFF Research Database (Denmark)

    Brakebusch, C; Varfolomeev, E E; Batkin, M

    1994-01-01

    Induced shedding of the p55 tumor necrosis factor receptor (p55-R) was previously shown to be independent of the amino acid sequence properties of the intracellular domain of this receptor. We now find it also independent of the sequence properties of the transmembrane domain and of the cysteine......-rich region that constitutes most of the extracellular domain of the receptor. The shedding is shown to depend solely on the sequence properties of a small region within the spacer that links the cysteine-rich region in the extracellular domain to the transmembrane domain. Detailed tests of effects......, however, by some mutations that seem to change the conformation of the spacer region. These findings suggest that a short amino acid sequence in the p55-R is essential and sufficient for its shedding and that the shedding is mediated either by a protease with limited sequence specificity or by several...

  13. Oxidative stress effect on progesterone-induced blocking factor (PIBF) binding to PIBF-receptor in lymphocytes.

    Science.gov (United States)

    de la Haba, Carlos; Palacio, José R; Palkovics, Tamas; Szekeres-Barthó, Júlia; Morros, Antoni; Martínez, Paz

    2014-01-01

    Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering.

  14. Interleukin-34:A new ligand for Colony-stimulating factor-1Receptor%Interleukin-34: A new ligand for Colony-stimulating factor-1Receptor

    Institute of Scientific and Technical Information of China (English)

    CHEN Yao; Gang-Qing Yao

    2011-01-01

    1 IntroductionColony-stimulating factor-1 ( CSF1 ) is a important hematopoietic growth factor that is involved in the proliferation,differentiation,and survival of monocytes, macrophages, and bone marrow progenitor cells[1].Its receptor (c-Fms) is known as the c-Fmsproto-oncoprotein[2].By far the most definitive studies demonstrating biologic functions for CSF-1 in vivo are those in the op/op mutant mouse.The deficiency results from a single base-pair insertion in the coding region of the gene to product defective CSF-1[3-4].Mice homozygous for this mutation have significant osteopetrosis,low growth rate,low body weight as well as a toothless phenotype because of a severe deficiency of osteoclasts and mononuclear phagocytes[5-6],and are devoid of serum and tissue CSF-1 activity[7].

  15. The Prostaglandin EP3 Receptor Is an Independent Negative Prognostic Factor for Cervical Cancer Patients

    Science.gov (United States)

    Heidegger, Helene; Dietlmeier, Sebastian; Kuhn, Christina; Vattai, Aurelia; Aberl, Caroline; Mahner, Sven; Kost, Bernd

    2017-01-01

    We know that one of the main risk factors for cervical cancer is an infection with high-risk human papillomavirus (HR-HPV). Prostaglandins and their receptors are very important for the tumour growth and tumour-associated angiogenesis. Little is known about the expression of the Prostaglandin E receptor type 3 (EP3) or the Prostaglandin (PG)E2-EP3 signalling in cervical cancer, so the aim of the study was to analyse the expression of the EP3 receptor in cervical cancer and find prognostic factors in relation to survival; EP3 immunohistological staining of 250 cervical cancer slides was performed and analysed with a semi-quantitative score. The statistical evaluation was performed with Statistical Package for the Social Sciences (SPSS) to evaluate the staining results and the survival analyses of the cervical cancer cases. A significant difference was observed in EP3 expression in Fédération Internationale de Gynécologie et d’Obstétrique (FIGO) stadium I versus FIGO stadium II–IV cases. High expression of EP3 (IRS ≥ 1.5) in cervical cancer patients was correlated with poor prognosis in overall survival rates. Survival in adenocarcinoma (AC) of the cervix was lower than in squamous cell carcinoma (SCC). Cox regression analysis shows that EP3 is an independent prognosticator. In this study we could show that the membrane-bound prostaglandin receptor EP3 is an independent prognosticator for cervical cancer patient survival. Targeting the EP3 receptor seems to be an interesting candidate for endocrine therapy. Therefore, more research is needed on the influence of the receptor system and its influence on cervical cancer growth. PMID:28753926

  16. Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors

    Science.gov (United States)

    Farroni, Jeffrey S; McCool, Brian A

    2004-01-01

    the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. PMID:15301692

  17. Induction of transforming growth factor beta receptors following focal ischemia in the rat brain.

    Directory of Open Access Journals (Sweden)

    Gabriella Pál

    Full Text Available Transforming growth factor-βs (TGF-βs regulate cellular proliferation, differentiation, and survival. TGF-βs bind to type I (TGF-βRI and II receptors (TGF-βRII, which are transmembrane kinase receptors, and an accessory type III receptor (TGF-βRIII. TGF-β may utilize another type I receptor, activin-like kinase receptor (Alk1. TGF-β is neuroprotective in the middle cerebral artery occlusion (MCAO model of stroke. Recently, we reported the expression pattern of TGF-β1-3 after MCAO. To establish how TGF-βs exert their actions following MCAO, the present study describes the induction of TGF-βRI, RII, RIII and Alk1 at 24 h, 72 h and 1 mo after transient 1 h MCAO as well as following 24 h permanent MCAO using in situ hybridization histochemistry. In intact brain, only TGF-βRI had significant expression: neurons in cortical layer IV contained TGF-βRI. At 24 h after the occlusion, no TGF-β receptors showed induction. At 72 h following MCAO, all four types of TGF-β receptors were induced in the infarct area, while TGF-βRI and RII also appeared in the penumbra. Most cells with elevated TGF-βRI mRNA levels were microglia. TGF-βRII co-localized with both microglial and endothelial markers while TGF-βRIII and Alk1 were present predominantly in endothels. All four TGF-β receptors were induced within the lesion 1 mo after the occlusion. In particular, TGF-βRIII was further induced as compared to 72 h after MCAO. At this time point, TGF-βRIII signal was predominantly not associated with blood vessels suggesting its microglial location. These data suggest that TGF-β receptors are induced after MCAO in a timely and spatially regulated fashion. TGF-β receptor expression is preceded by increased TGF-β expression. TGF-βRI and RII are likely to be co-expressed in microglial cells while Alk1, TGF-βRII, and RIII in endothels within the infarct where TGF-β1 may be their ligand. At later time points, TGF-βRIII may also appear in glial cells

  18. Altered Fibroblast Growth Factor Receptor 4 Stability Promotes Prostate Cancer Progression

    Directory of Open Access Journals (Sweden)

    Jianghua Wang

    2008-08-01

    Full Text Available Fibroblast growth factor receptor 4 (FGFR-4 is expressed at significant levels in almost all human prostate cancers, and expression of its ligands is ubiquitous. A common polymorphism of FGFR-4 in which arginine (Arg388 replaces glycine (Gly388 at amino acid 388 is associated with progression in human prostate cancer. We show that the FGFR-4 Arg388 polymorphism, which is present in most prostate cancer patients, results in increased receptor stability and sustained receptor activation. In patients bearing the FGFR-4 Gly388 variant, expression of Huntingtin-interacting protein 1 (HIP1, which occurs in more than half of human prostate cancers, also results in FGFR-4 stabilization. This is associated with enhanced proliferation and anchorage-independent growth in vitro. Our findings indicate that increased receptor stability and sustained FGFR-4 signaling occur in most human prostate cancers due to either the presence of a common genetic polymorphism or the expression of a protein that stabilizes FGFR-4. Both of these alterations are associated with clinical progression in patients with prostate cancer. Thus, FGFR-4 signaling and receptor turnover are important potential therapeutic targets in prostate cancer.

  19. Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway

    Energy Technology Data Exchange (ETDEWEB)

    De Angelis Campos, Ana Carolina; Rodrigues, Michele Angela; Andrade, Carolina de; Miranda de Goes, Alfredo [Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil); Nathanson, Michael H. [Department of Internal Medicine, Yale University School of Medicine, New Haven, CT (United States); Gomes, Dawidson A., E-mail: dawidson@icb.ufmg.br [Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG (Brazil)

    2011-08-26

    Highlights: {yields} EGF and its receptor translocates to the nucleus in liver cells. {yields} Real time imaging shows that EGF moves to the nucleus. {yields} EGF moves with its receptor to the nucleus. {yields} Dynamin and clathrin are necessary for EGFR nuclear translocation. -- Abstract: The epidermal growth factor (EGF) transduces its actions via the EGF receptor (EGFR), which can traffic from the plasma membrane to either the cytoplasm or the nucleus. However, the mechanism by which EGFR reaches the nucleus is unclear. To investigate these questions, liver cells were analyzed by immunoblot of cell fractions, confocal immunofluorescence and real time confocal imaging. Cell fractionation studies showed that EGFR was detectable in the nucleus after EGF stimulation with a peak in nuclear receptor after 10 min. Movement of EGFR to the nucleus was confirmed by confocal immunofluorescence and labeled EGF moved with the receptor to the nucleus. Small interference RNA (siRNA) was used to knockdown clathrin in order to assess the first endocytic steps of EGFR nuclear translocation in liver cells. A mutant dynamin (dynamin K44A) was also used to determine the pathways for this traffic. Movement of labeled EGF or EGFR to the nucleus depended upon dynamin and clathrin. This identifies the pathway that mediates the first steps for EGFR nuclear translocation in liver cells.

  20. Inhibition of epidermal growth factor receptor expression by RNA interference in A549 cells

    Institute of Scientific and Technical Information of China (English)

    MinZHANG; XinZHANG; Chun-xueBAI; JieCHEN; MinQWEI

    2004-01-01

    AIM: To investigate the biological features of A549 cells in which epidermal growth factor (EGF) receptors expression were suppressed by RNA interference (RNAi). METHODS: A549 cells were transfected using short small interfering RNAs (siRNAs) formulated with Lipofectamine 2000. The EGF receptor numbers were determined by Western blotting and flowcytometry. The antiproliferative effects of sequence specific double stranded RNA (dsRNA) were assessed using cell count, colony assay and scratch assay. The chemosensitivity of transfected cells to cisplatin was measured by MTT. RESULTS: Sequence specific dsRNA-EGFR down-regulated EGF receptor expression dramatically. Compared with the control group, dsRNA-EGFR reduced the cell number by 85.0 %, decreased the colonies by 63.3 %, inhibited the migration by 87.2 %, and increased the sensitivity of A549 to cisplatin by four-fold. CONCLUSION: Sequence specific dsRNA-EGFR were capable of suppressing EGF receptor expression, hence significantly inhibiting cellular proliferation and motility, and enhancing chemosensitivity of A549 cells to cisplatin. The successful application of dsRNA-EGFR for inhibition of proliferation in EGF receptor overexpressing cells can help extend the list of available therapeutic modalities in the treatment of non-small-cell lung carcinoma (NSCLC).

  1. Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease

    Institute of Scientific and Technical Information of China (English)

    Gui-Xin Du; Li-Hua Hou; Rong-Bin Guan; Yi-Gang Tong; Hai-Tao Wang

    2002-01-01

    AIM: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors. METHODS: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coliwas induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease.RESULTS: The single-chain recombinant protease was overexpressed as soluble protein when the E. coliwas induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15℃). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %).The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not.CONCLUSION: A simple

  2. Increased Stability and DNA Site Discrimination of Single Chain Variants of the Dimeric beta-Barrel DNA Binding Domain of the Human Papillomavirus E2 Transcriptional Regulator

    Energy Technology Data Exchange (ETDEWEB)

    Dellarole,M.; Sanchez, I.; Freire, E.; de Prat-Gay, G.

    2007-01-01

    Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric {beta}-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.

  3. Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

    Institute of Scientific and Technical Information of China (English)

    GENG De-chun; XU Yao-zeng; YANG Hui-lin; ZHU Guang-ming; WANG Xian-bin; ZHU Xue-song

    2011-01-01

    Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK),phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of >100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

  4. Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression.

    Directory of Open Access Journals (Sweden)

    Shadi Jafari

    Full Text Available The mechanism that specifies olfactory sensory neurons to express only one odorant receptor (OR from a large repertoire is critical for odor discrimination but poorly understood. Here, we describe the first comprehensive analysis of OR expression regulation in Drosophila. A systematic, RNAi-mediated knock down of most of the predicted transcription factors identified an essential function of acj6, E93, Fer1, onecut, sim, xbp1, and zf30c in the regulation of more than 30 ORs. These regulatory factors are differentially expressed in antennal sensory neuron classes and specifically required for the adult expression of ORs. A systematic analysis reveals not only that combinations of these seven factors are necessary for receptor gene expression but also a prominent role for transcriptional repression in preventing ectopic receptor expression. Such regulation is supported by bioinformatics and OR promoter analyses, which uncovered a common promoter structure with distal repressive and proximal activating regions. Thus, our data provide insight into how combinatorial activation and repression can allow a small number of transcription factors to specify a large repertoire of neuron classes in the olfactory system.

  5. Transforming growth factor β receptor type 1 is essential for female reproductive tract integrity and function.

    Directory of Open Access Journals (Sweden)

    Qinglei Li

    2011-10-01

    Full Text Available The transforming growth factor β (TGFβ superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1, also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1-mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9 signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1-mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.

  6. Transforming Growth Factor β Receptor Type 1 Is Essential for Female Reproductive Tract Integrity and Function

    Science.gov (United States)

    Li, Qinglei; Agno, Julio E.; Edson, Mark A.; Nagaraja, Ankur K.; Nagashima, Takashi; Matzuk, Martin M.

    2011-01-01

    The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1–mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1–mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus. PMID:22028666

  7. Fibroblast Growth Factor 10-Fibroblast Growth Factor Receptor 2b Mediated Signaling Is Not Required for Adult Glandular Stomach Homeostasis

    Science.gov (United States)

    Sala, Frederic G.; Ford, Henri R.; Bellusci, Saverio; Grikscheit, Tracy C.

    2012-01-01

    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. PMID:23133671

  8. 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.

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

    Science.gov (United States)

    Speer, Allison L; Al Alam, Denise; Sala, Frederic G; Ford, Henri R; Bellusci, Saverio; Grikscheit, Tracy C

    2012-01-01

    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.

  10. A novel fibroblast growth factor receptor family member promotes neuronal outgrowth and synaptic plasticity in aplysia.

    Science.gov (United States)

    Pollak, Daniela D; Minh, Bui Quang; Cicvaric, Ana; Monje, Francisco J

    2014-11-01

    Fibroblast Growth Factor (FGF) Receptors (FGFRs) regulate essential biological processes, including embryogenesis, angiogenesis, cellular growth and memory-related long-term synaptic plasticity. Whereas canonical FGFRs depend exclusively on extracellular Immunoglobulin (Ig)-like domains for ligand binding, other receptor types, including members of the tropomyosin-receptor-kinase (Trk) family, use either Ig-like or Leucine-Rich Repeat (LRR) motifs, or both. Little is known, however, about the evolutionary events leading to the differential incorporation of LRR domains into Ig-containing tyrosine kinase receptors. Moreover, although FGFRs have been identified in many vertebrate species, few reports describe their existence in invertebrates. Information about the biological relevance of invertebrate FGFRs and evolutionary divergences between them and their vertebrate counterparts is therefore limited. Here, we characterized ApLRRTK, a neuronal cell-surface protein recently identified in Aplysia. We unveiled ApLRRTK as the first member of the FGFRs family deprived of Ig-like domains that instead contains extracellular LRR domains. We describe that ApLRRTK exhibits properties typical of canonical vertebrate FGFRs, including promotion of FGF activity, enhancement of neuritic outgrowth and signaling via MAPK and the transcription factor CREB. ApLRRTK also enhanced the synaptic efficiency of neurons known to mediate in vivo memory-related defensive behaviors. These data reveal a novel molecular regulator of neuronal function in invertebrates, provide the first evolutionary linkage between LRR proteins and FGFRs and unveil an unprecedented mechanism of FGFR gene diversification in primeval central nervous systems.

  11. Type I insulin-like growth factor receptor signaling in skeletal muscle regeneration and hypertrophy.

    Science.gov (United States)

    Philippou, A; Halapas, A; Maridaki, M; Koutsilieris, M

    2007-01-01

    Skeletal muscle is able not only to increase its mass as an adaptation to mechanical loading generated by and imposed upon muscle but also to regenerate after damage, via its intrinsic regulation of gene transcription. Both cellular processes, muscle regeneration and hypertrophy, are mediated by the activation, proliferation and differentiation of muscle satellite cells and appear to be modulated by the mitotic and myogenic activity of locally produced insulin-like growth factor 1 (IGF-1), which functions in an autocrine/paracrine mode. Differentiation of satellite cells into myoblasts involves the regulation of skeletal muscle-specific proteins belonging to the family of myogenic regulatory factors (MRFs). The endocrine, autocrine and paracrine functions of IGF-1 are mediated through binding to the type I IGF receptor (IGF-1.R), which is a ligand-activated receptor tyrosine kinase. The binding of IGF-1 to IGF-1.R induces its autophosphorylation, which recruits specific cytoplasmic molecules containing the Insulin Receptor Substrate Proteins (IRS). The recruitment of IRS proteins by IGF-1/IGF-1.R binding is a critical level at which the proliferative and differentiative actions of IGF-1 diverge. Specific signaling pathways downstream of IGF-1, potentially involved in the mitogenic and myogenic responses and mediating skeletal muscle protein synthesis and hypertrophy following exercise-induced muscle overloading and damage, are discussed. A potential alternative activation of different signaling pathway(s) via a different receptor remains to be demonstrated.

  12. A Murine Fibroblast Growth Factor (FGF) Receptor Expressed in CHO Cells is Activated by Basic FGF and Kaposi FGF

    Science.gov (United States)

    Mansukhani, Alka; Moscatelli, David; Talarico, Daniela; Levytska, Vera; Basilico, Claudio

    1990-06-01

    We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.

  13. TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

    Science.gov (United States)

    Pype, S; Declercq, W; Ibrahimi, A; Michiels, C; Van Rietschoten, J G; Dewulf, N; de Boer, M; Vandenabeele, P; Huylebroeck, D; Remacle, J E

    2000-06-16

    CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

  14. Nanoconjugation prolongs endosomal signaling of the epidermal growth factor receptor and enhances apoptosis

    Science.gov (United States)

    Wu, L.; Xu, F.; Reinhard, B. M.

    2016-07-01

    It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of free EGF. Overall, these findings indicate nanoconjugation as a rational strategy for modifying signaling that acts by modulating the temporo-spatial distribution of the activated EGF-EGFR ligand-receptor complex.It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF

  15. Antioxidants for Healthy Skin: The Emerging Role of Aryl Hydrocarbon Receptors and Nuclear Factor-Erythroid 2-Related Factor-2.

    Science.gov (United States)

    Furue, Masutaka; Uchi, Hiroshi; Mitoma, Chikage; Hashimoto-Hachiya, Akiko; Chiba, Takahito; Ito, Takamichi; Nakahara, Takeshi; Tsuji, Gaku

    2017-03-03

    Skin is the outermost part of the body and is, thus, inevitably exposed to UV rays and environmental pollutants. Oxidative stress by these hazardous factors accelerates skin aging and induces skin inflammation and carcinogenesis. Aryl hydrocarbon receptors (AHRs) are chemical sensors that are abundantly expressed in epidermal keratinocytes and mediate the production of reactive oxygen species. To neutralize or minimize oxidative stress, the keratinocytes also express nuclear factor-erythroid 2-related factor-2 (NRF2), which is a master switch for antioxidant signaling. Notably, there is fine-tuned crosstalk between AHR and NRF2, which mutually increase or decrease their activation states. Many NRF2-mediated antioxidant phytochemicals are capable of up- and downmodulating AHR signaling. The precise mechanisms by which these phytochemicals differentially affect the AHR and NRF2 system remain largely unknown and warrant future investigation.

  16. Antioxidants for Healthy Skin: The Emerging Role of Aryl Hydrocarbon Receptors and Nuclear Factor-Erythroid 2-Related Factor-2

    Science.gov (United States)

    Furue, Masutaka; Uchi, Hiroshi; Mitoma, Chikage; Hashimoto-Hachiya, Akiko; Chiba, Takahito; Ito, Takamichi; Nakahara, Takeshi; Tsuji, Gaku

    2017-01-01

    Skin is the outermost part of the body and is, thus, inevitably exposed to UV rays and environmental pollutants. Oxidative stress by these hazardous factors accelerates skin aging and induces skin inflammation and carcinogenesis. Aryl hydrocarbon receptors (AHRs) are chemical sensors that are abundantly expressed in epidermal keratinocytes and mediate the production of reactive oxygen species. To neutralize or minimize oxidative stress, the keratinocytes also express nuclear factor-erythroid 2-related factor-2 (NRF2), which is a master switch for antioxidant signaling. Notably, there is fine-tuned crosstalk between AHR and NRF2, which mutually increase or decrease their activation states. Many NRF2-mediated antioxidant phytochemicals are capable of up- and downmodulating AHR signaling. The precise mechanisms by which these phytochemicals differentially affect the AHR and NRF2 system remain largely unknown and warrant future investigation. PMID:28273792

  17. A Structural Investigation into Oct4 Regulation by Orphan Nuclear Receptors, Germ Cell Nuclear Factor (GCNF) and Liver Receptor Homolog-1 (LRH-1).

    Science.gov (United States)

    Weikum, Emily R; Tuntland, Micheal L; Murphy, Michael N; Ortlund, Eric A

    2016-10-27

    Oct4 is a transcription factor required for maintaining pluripotency and self-renewal in stem cells. Prior to differentiation, Oct4 must be silenced to allow for the development of the three germ layers in the developing embryo. This fine-tuning is controlled by the nuclear receptors, liver receptor homolog-1 and germ cell nuclear factor. Liver receptor homolog-1 is responsible for driving the expression of Oct4 where germ cell nuclear factor represses its expression upon differentiation. Both receptors bind to a DR0 motif located within the Oct4 promoter. Here, we present the first structure of mouse germ cell nuclear factor DNA binding domain in complex with the Oct4 DR0. The overall structure revealed two molecules bound in a head-to-tail fashion on opposite sides of the DNA. Additionally, we solved the structure of the human liver receptor homolog-1 DNA binding domain bound to the same element. We explore the structural elements that govern Oct4 recognition by these two nuclear receptors.

  18. Mutations in fibroblast growth factor receptors: Phenotypic consequences during eukaryotic development

    Energy Technology Data Exchange (ETDEWEB)

    Park, W.J.; Bellus, G.A.; Jabs, E.W. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)

    1995-10-01

    Recently, a tremendous amount of excitement and interest has been generated by the rapid succession of discoveries in the human fibroblast growth factor receptor (FGFR) field. In less than a year, mutations in three FGFRs (FGFR1-FGFR3) have been associated with three skeletal dysplasias and four craniosynostotic syndromes. FGFRs are members of the receptor tyrosine kinase family that bind fibroblast growth factors (FGFs). The FGF family consists of structurally related polypeptides that play a key role in numerous aspects of embryogenesis, growth, and homeostasis. FGFs have a potent growth stimulatory and/or differentiation-inducing effect on cells such as those derived from the early-embryonic mesoderm or ectoderm. In addition to mitogenesis and differentiation, FGFs also stimulate chemotaxis, cell survival, and angiogenesis. FGFs mediate cellular responses on binding to and activation of FGFRs. 45 refs., 2 figs., 1 tab.

  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. Nonsmall Cell Lung Cancer Therapy: Insight into Multitargeted Small-Molecule Growth Factor Receptor Inhibitors

    Directory of Open Access Journals (Sweden)

    Mridul Roy

    2013-01-01

    Full Text Available To date, lung cancer is the leading cause of cancer-related death worldwide, among which nonsmall cell lung cancer (NSCLC comprises about 85%. Taking into account the side effects of surgery, radiation, platinum-based doublet chemotherapy, and the growth self-sufficiency characteristic of cancer cells, drugs have been discovered toward growth factor receptor (GFR to treat NSCLC. As expected, these drugs provide a greater benefit. To increase the efficacy of such growth factor receptor tyrosine kinase inhibitors (RTKIs, coinhibition of GFR signaling pathways and combination of inhibitors along with radiation or chemotherapy have drew intense insight. Although clinical trials about single-agent RTKIs or their combination strategies suggest their increase potency against cancer, they are not beyond adverse effects, and sometimes the effects are more deadly than chemotherapy. Nevertheless the hope for RTKIs may be proved true by further researches and digging deep into cancer therapeutics.

  1. 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...... extent this effect involves changes in upstream signaling events is unresolved. We show here that glial overgrowth in egh is strongly linked to increased activation of Insulin and Fibroblast Growth Factor receptors (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants...... 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....

  2. Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor

    DEFF Research Database (Denmark)

    Brakebusch, C; Nophar, Y; Kemper, O;

    1992-01-01

    The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu-p55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor,...

  3. 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

  4. Rationale for targeting fibroblast growth factor receptor signaling in breast cancer

    OpenAIRE

    André, Fabrice; Cortés, Javier

    2015-01-01

    Fibroblast growth factor receptor (FGFR) signaling is involved in multiple biological processes, including cell proliferation, survival, differentiation, migration, and apoptosis during embryonic development and adult tissue homeostasis. Given its role in the activation of critical signaling pathways, aberrant FGFR signaling has been implicated in multiple cancer types. A comprehensive search of PubMed and congress abstracts was conducted to identify reports on FGFR pathway components in brea...

  5. 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.

  6. Cutaneous adverse events of epidermal growth factor receptor inhibitors: A retrospective review of 99 cases

    Directory of Open Access Journals (Sweden)

    Kumutnart Chanprapaph

    2015-01-01

    Full Text Available Background: Previous reports regarding the cutaneous adverse events of epidermal growth factor receptor inhibitors are mostly limited to small case reports and case series, mainly involving Caucasian patients. Aims: We describe the trends in the clinical presentation of Asian patients who had cutaneous adverse events induced by epidermal growth factor receptor inhibitors and to explore the relationship between skin adverse events and tumor response. Methods: From 2006 to 2010, medical records of Thai patients with non-small cell lung cancer receiving epidermal growth factor receptor inhibitors were retrieved and analyzed. Results: In all, 99 patients were reviewed and analyzed. Erlotinib and gefitinib were commenced in 75 (75.8% and 24 (24.2% patients, respectively. Cutaneous adverse events occurred in 43 (57.3% patients receiving erlotinib and in 15 (62.5% patients receiving gefitinib. The most common adverse event was xerosis (52.5%. Less common adverse events included papulo-pustular eruption (27.3%, erythematous maculopapular rash (11.1%, mucositis (6.7%, paronychia (5.1%, and trichomegaly (2%. Elderly patients had a higher occurrence of xerosis. The presence of cutaneous adverse events was significantly higher in subjects who had a tumor response. Limitations: The limitations of study include its retrospective nature, and the initial screening of cutaneous adverse events was done by non-dermatologists. Conclusions: Cutaneous adverse events due to epidermal growth factor receptor inhibitors are not uncommon in the Asian population. We found a positive correlation between the occurrences of cutaneou adverse events and tumor response supporting the view that they are surrogate markers for therapeutic response.

  7. Novel Mutations and Deletions of the KIT (Steel Factor Receptor) Gene in Human Piebaldism

    OpenAIRE

    Ezoe, Kazuhiko; Holmes, Stuart A.; Ho, Lingling; Bennett, Christopher P.; Bolognia, Jean L.; Brueton, Louise; Burn, John; Falabella, Rafael; Gatto, Emilia M.; Ishii, Norihisa; Moss, Celia; Pittelkow, Mark R.; Thompson, Elizabeth; Ward, K. Anne; Spritz, Richard A.

    1995-01-01

    Piebaldism is an autosomal dominant genetic disorder of pigmentation characterized by white patches of skin and hair. Melanocytes are lacking in these hypopigmented regions, the result of mutations of the KIT gene, which encodes the cell surface receptor for steel factor (SLF). We describe the analysis of 26 unrelated patients with piebaldism-like hypopigmentation—17 typical patients, 5 with atypical clinical features or family histories, and 4 with other disorders that involve white spotting...

  8. Glucocorticoid-induced tumour necrosis factor receptor (GITR) and its ligand (GITRL) in atopic dermatitis

    DEFF Research Database (Denmark)

    Baumgartner-Nielsen, Jane; Vestergaard, Christian; Thestrup-Pedersen, K.

    2006-01-01

    The glucocorticoid-induced tumour necrosis factor receptor-related gene (GITR) is expressed on regulatory T-cells (Treg), which are CD4+CD25+ lymphocytes. Binding of the GITR-ligand (GITRL) leads to downregulation of the regulatory function of Tregs. Patients suffering from a defect in their Treg......-cells are localized in the vicinity of GITRL-expressing cells in atopic dermatitis skin, the GITR/GITRL interaction may serve to perpetuate the inflammation locally....

  9. 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

  10. Impact of epidermal growth factor receptor and transforming growth factor-α on hepatitis C virus-induced hepatocarcinogenesis.

    Science.gov (United States)

    Badawy, Afkar Abdel-Ghany; El-Hindawi, Ali; Hammam, Olfat; Moussa, Mona; Gabal, Samia; Said, Noha

    2015-10-01

    Epidermal growth factor receptor system plays a central hepato-protective and pro-regenerative role in liver. Transforming growth factor-α (TGF-α) is an important autocrine growth regulator of hepatocytes that plays a role in development of hepatocellular carcinoma (HCC) among patients with chronic hepatitis C (CHC). This study was done on 40 core liver biopsies from patients with CHC, 20 liver specimens from HCC cases on top of CHC as well as five normal controls. All were immunohistochemically stained with epidermal growth factor receptor (EGFR) and TGF-α antibodies. Some selected HCC cases were submitted for FISH technique to detect EGFR gene alteration. By immunohistochemistry EGFR and TGF-α were overexpressed in HCC and cirrhotic cases compared to CHC cases without cirrhosis. Also, their expression was stronger in CHC cases with higher grades of activity and stages of fibrosis compared to lower ones. FISH positive results for EGFR were detected in 33.3% of the examined HCC cases. EGFR and TGF-α can be used as predictive markers for activity, fibrosis, and carcinogenesis in CHC patients. Overexpression of EGFR in HCC patients can be promising in selecting those who can get benefit from anti-EGFR target therapy.

  11. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†

    Science.gov (United States)

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-01-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

  12. The role of tumour necrosis factor alpha and soluble tumour necrosis factor alpha receptors in the symptomatology of schizophrenia.

    Science.gov (United States)

    Turhan, Levent; Batmaz, Sedat; Kocbiyik, Sibel; Soygur, Arif Haldun

    2016-07-01

    Background Immunological mechanisms may be responsible for the development and maintenance of schizophrenia symptoms. Aim The aim of this study is to measure tumour necrosis factor-alpha (TNF-α), soluble tumour necrosis factor-alpha receptor I (sTNF-αRI), and soluble tumour necrosis factor-alpha receptor II (sTNF-αRII) levels in patients with schizophrenia and healthy individuals, and to determine their relationship with the symptoms of schizophrenia. Methods Serum TNF-α, sTNF-αRI and sTNF-αRII levels were measured. The Positive and Negative Syndrome Scale (PANSS) was administered for patients with schizophrenia (n = 35), and the results were compared with healthy controls (n = 30). Hierarchical regression analyses were undertaken to predict the levels of TNF-α, sTNF-αRI and sTNF-αRII. Results No significant difference was observed in TNF-α levels, but sTNF-αRI and sTNF-αRII levels were lower in patients with schizophrenia. Serum sTNF-αRI and sTNF-αRII levels were found to be negatively correlated with the negative subscale score of the PANSS, and sTNF-αRI levels were also negatively correlated with the total score of the PANSS. Smoking, gender, body mass index were not correlated with TNF-α and sTNF-α receptor levels. Conclusions These results suggest that there may be a change in anti-inflammatory response in patients with schizophrenia due to sTNF-αRI and sTNF-αRII levels. The study also supports low levels of TNF activity in schizophrenia patients with negative symptoms.

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

    Directory of Open Access Journals (Sweden)

    Jeivad F

    2012-01-01

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

  14. Therapy for acute pancreatitis with platelet-activating factor receptor antagonists

    Institute of Scientific and Technical Information of China (English)

    Chong Chen; Shi-Hai Xia; Hong Chen; Xiao-Hong Li

    2008-01-01

    Acute pancreatitis (AP) causes release of plateletactivating factor (PAF),which induces systemic effects that contribute to circulatory disturbances and multiple organ failure.PAF is a cell surface secretion of bioactive lipid,which could produce physiological and pathological effects by binding to its cell surface receptor called platelet-activating factor receptor (PAF-R).Studies showed that PAF participates in the occurrence and development of AP and administration of platelet-activating factor receptor antagonists (PAF-RAs) could significantly reduce local and systemic events after AP.PAF has also been implicated as a key mediator in the progression of severe AP,which can lead to complications and unacceptably high mortality rates.Several classes of PAF-RA show PAFRAs significant local and systemic effects on reducing inflammatory changes.As a preventive treatment,PAF-RA could block a series of PAF-mediatedinflammatory injury and thus improve the prognosis of AR This review introduces the important role of PAF-RA in the treatment of AP.

  15. Dopamine receptor activation increases glial cell line-derived neurotrophic factor in experimental stroke.

    Science.gov (United States)

    Kuric, Enida; Wieloch, Tadeusz; Ruscher, Karsten

    2013-09-01

    Treatment with levodopa enhances functional recovery after experimental stroke but its mechanisms of action are elusive. Reactive astrocytes in the ischemic hemisphere are involved in mechanisms promoting recovery and also express dopamine 1 (D1) and dopamine 2 (D2) receptors. Here we investigated if the activation of astrocytic dopamine receptors (D1 and D2) regulates the expression of glial cell line-derived neurotrophic factor (GDNF) after combined in vitro hypoxia/aglycemia (H/A) and studied the expression of GDNF in the ischemic brain after treatment with levodopa/benserazide following transient occlusion of the middle cerebral artery (tMCAO) in the rat. Twenty-four hours after H/A, GDNF levels were upregulated in exposed astrocytes compared to normoxic control cultures and further elevated by the addition of the selective D1 receptor agonist (R)-(+)-SKF-38393 hydrochloride while D1 receptor antagonism by R(+)-SCH-23390 hydrochloride significantly reduced GDNF. No effect on GDNF levels was observed by the application of the D2 receptor agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide hydrate or S-(-)-eticlopride hydrochloride (D2 receptor antagonist). After tMCAO, GDNF was upregulated in D1 expressing reactive astrocytes in the peri-infarct area. In addition, treatment with levodopa/benserazide significantly increased GDNF levels in the infarct core and peri-infarct area after tMCAO without affecting the expression of glial fibrillar acidic protein (GFAP), an intermediate filament and marker of reactive gliosis. After stroke, GDNF levels increase in the ischemic hemisphere in rats treated with levodopa, implicating GDNF in the mechanisms of tissue reorganization and plasticity and in l-DOPA enhanced recovery of lost brain function. Our results support levodopa treatment as a potential recovery enhancing therapy in stroke patients.

  16. 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.

  17. 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...

  18. Dynamic changes in the expression of growth factor receptors in the myocardium microvascular endothelium after murine myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    WANG Xin-hong; ZHANG Guo-ping; JIN Hui-ming; CHEN Si-feng

    2007-01-01

    Background After myocardial infarction, specific growth factors promote cardiac angiogenisis, leading to a therapeutic effect. Although this effect is mediated by specific receptors in the endothelium of the cardiac microvasculature, few studies have investigated dynamic changes in their expression. We explored this phenomenon in a murine model.Methods We observed the mRNA expression of receptors by specific angiogenesis gene microarray at day 3 and day 7after infarction. The vascular endothelial growth factor (VEGF) receptor Fik-1 was observed at the protein level at day 3and day 7 by immunohistochemistry. The dynamic expression of fibroblast growth factor receptor-1 (FGFR-1) mRNA in the border zone and the noninfarcted zone at day 3, day 7, day 14, and day 42 was investigated by real-time PCR.Statistical significance was analyzed with SPSS 10.0 software using one-way analysis of variance (ANOVA).Results Three days after infarction, 9 receptors in the border zone and 7 receptors in the noninfarcted zone were down-regulated. Two receptors in the infarct edge and 5 receptors in the distant myocardium were up-regulated. However,at day 7, 11 receptors in the border zone were up-regulated, and only one was down-regulated. In the border zone, Fik-1levels decreased at day 3 but increased significantly at day 7. Real-time PCR showed that FGFR-1 mRNA decreased markedly in the border zone at day 3 but increased afterward for at least 6 weeks. In the early stage (3 days) after infarction, the expression of receptors had decreased to some extent. However, at day 7, receptor expression was active and had moved from the distant noninfarcted zone to the border zone as a part of the acute repair process.Conclusion Selecting the proper growth factors to target receptors with protective activity, and determining appropriate therapeutic timing may be important to the success of therapeutic angiogenesis.

  19. The structure and function of vertebrate fibroblast growth factor receptor 1.

    Science.gov (United States)

    Groth, Casper; Lardelli, Michael

    2002-01-01

    The vertebrate fibroblast growth factor receptor 1 (FGFR1) is alternatively spliced generating multiple splice variants that are differentially expressed during embryo development and in the adult body. The restricted expression patterns of FGFR1 isoforms, together with differential expression and binding of specific ligands, leads to activation of common FGFR1 signal transduction pathways, but may result in distinctively different biological responses as a result of differences in cellular context. FGFR1 isoforms are also present in the nucleus in complex with various fibroblast growth factors where they function to regulate transcription of target genes.

  20. Proteinuria, a modifiable risk factor: angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs).

    Science.gov (United States)

    Dykeman-Sharpe, Jennifer

    2003-01-01

    Microalbuminuria and proteinuria have been determined to be modifiable risk factors for the progression of chronic kidney disease as well as risk factors for cardiovascular events. Angiotensin converting enzyme inhibitors and angiotensin II receptor blockers have been demonstrated to decrease proteinuria at all stages and slow the progression of renal disease. Proteinuria can be used as a marker of successful treatment in patients with chronic kidney disease in combination with other established targets. This article discusses the various diagnostic tests used for the detection of microalbuminuria and proteinuria and appropriate pharmaceutical treatment.

  1. Therapeutic targeting of epidermal growth factor receptor in human cancer: successes and limitations%Therapeutic targeting of epidermal growth factor receptor in humancancer: successes and limitations

    Institute of Scientific and Technical Information of China (English)

    Jill Wykosky; Tim Fenton; Frank Furnari; Webster K. Cavenee

    2011-01-01

    Epidermal growth factor receptor (EGFR) is one of the most commonly altered genes in human cancer by way of over-expression, amplification, and mutation. Targeted inhibition of EGFR activity suppresses signal transduction pathways which control tumor cell growth, proliferation, and resistance to apoptosis. Small molecule tyrosine kinase inhibitors and monoclonal antibodies are among the most common EGFR-targeting agents and have been used clinically for treating various malignancies. This review discusses the successes and challenges of targeting EGFR in human cancer. The genetic alterations of EGFR tend to occur more often in some solid tumors than others, as do the mechanisms of resistance to targeted inhibition. The clinical and basic science experiences with these agents thus far have important implications for the future of therapeutic targeting of EGFR.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-06-10

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

  3. 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.

  4. 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.

  5. Tissue-specific pioneer factors associate with androgen receptor cistromes and transcription programs

    Science.gov (United States)

    Pihlajamaa, Päivi; Sahu, Biswajyoti; Lyly, Lauri; Aittomäki, Viljami; Hautaniemi, Sampsa; Jänne, Olli A

    2014-01-01

    Androgen receptor (AR) binds male sex steroids and mediates physiological androgen actions in target tissues. ChIP-seq analyses of AR-binding events in murine prostate, kidney and epididymis show that in vivo AR cistromes and their respective androgen-dependent transcription programs are highly tissue specific mediating distinct biological pathways. This high order of tissue specificity is achieved by the use of exclusive collaborating factors in the three androgen-responsive tissues. We find two novel collaborating factors for AR signaling in vivo—Hnf4α (hepatocyte nuclear factor 4α) in mouse kidney and AP-2α (activating enhancer binding protein 2α) in mouse epididymis—that define tissue-specific AR recruitment. In mouse prostate, FoxA1 serves for the same purpose. FoxA1, Hnf4α and AP-2α motifs are over-represented within unique AR-binding loci, and the cistromes of these factors show substantial overlap with AR-binding events distinct to each tissue type. These licensing or pioneering factors are constitutively bound to chromatin and guide AR to specific genomic loci upon hormone exposure. Collectively, liganded receptor and its DNA-response elements are required but not sufficient for establishment of tissue-specific transcription programs. PMID:24451200

  6. Platelet-derived growth factors and their receptors in normal and malignant hematopoiesis

    Science.gov (United States)

    Demoulin, Jean-Baptiste; Montano-Almendras, Carmen P.

    2012-01-01

    Platelet-derived growth factors (PDGF) bind to two closely related receptor tyrosine kinases, PDGF receptor α and β, which are encoded by the PDGFRA and PDGFRB genes. Aberrant activation of PDGF receptors occurs in myeloid malignancies associated with hypereosinophilia, due to chromosomal alterations that produce fusion genes, such as ETV6-PDGFRB or FIP1L1-PDGFRA. Most patients are males and respond to low dose imatinib, which is particularly effective against PDGF receptor kinase activity. Recently, activating point mutations in PDGFRA were also described in hypereosinophilia. In addition, autocrine loops have been identified in large granular lymphocyte leukemia and HTLV-transformed lymphocytes, suggesting new possible indications for tyrosine kinase inhibitor therapy. Although PDGF was initially purified from platelets more than 30 years ago, its physiological role in the hematopoietic system remains unclear. Hematopoietic defects in PDGF-deficient mice have been reported but appear to be secondary to cardiovascular and placental abnormalities. Nevertheless, PDGF acts directly on several hematopoietic cell types in vitro, such as megakaryocytes, platelets, activated macrophages and, possibly, certain lymphocyte subsets and eosinophils. The relevance of these observations for normal human hematopoiesis remains to be established. PMID:22432087

  7. Receptor binding sites for atrial natriuretic factor are expressed by brown adipose tissue

    Energy Technology Data Exchange (ETDEWEB)

    Bacay, A.C.; Mantyh, C.R.; Vigna, S.R.; Mantyh, P.W. (Wadsworth VA Medical Center, Los Angeles, CA (USA))

    1988-09-01

    To explore the possibility that atrial natriuretic factor (ANF) is involved in thermoregulation we used quantitative receptor autoradiography and homogenate receptor binding assays to identify ANF bindings sites in neonatal rat and sheep brown adipose tissue, respectively. Using quantitative receptor autoradiography were were able to localize high levels of specific binding sites for {sup 125}I-rat ANF in neonatal rat brown adipose tissue. Homogenate binding assays on sheep brown fat demonstrated that the radioligand was binding to the membrane fraction and that the specific binding was not due to a lipophilic interaction between {sup 125}I-rat ANF and brown fat. Specific binding of {sup 125}I-rat ANF to the membranes of brown fat cells was inhibited by unlabeled rat ANF with a Ki of 8.0 x 10(-9) M, but not by unrelated peptides. These studies demonstrate that brown fat cells express high levels of ANF receptor binding sites in neonatal rat and sheep and suggest that ANF may play a role in thermoregulation.

  8. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  9. Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation.

    Science.gov (United States)

    Zhao, Haotian; Yang, Tianyu; Madakashira, Bhavani P; Thiels, Cornelius A; Bechtle, Chad A; Garcia, Claudia M; Zhang, Huiming; Yu, Kai; Ornitz, David M; Beebe, David C; Robinson, Michael L

    2008-06-15

    The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27(kip1) and p57(kip2), increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and alpha-, beta- and gamma-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.

  10. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hanazono, Y.; Hosoi, T.; Kuwaki, T.; Matsuki, S.; Miyazono, K.; Miyagawa, K.; Takaku, F. (Univ. of Tokyo (Japan))

    1990-11-01

    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. Identification and characterization of tumor necrosis factor receptor (TNFR)-associated factor 3 from humphead snapper, Lutjanus sanguineus.

    Science.gov (United States)

    Cai, Jia; Xia, Hongli; Huang, Yucong; Tang, Jufen; Jian, Jichang; Wu, Zaohe; Lu, Yishan

    2015-10-01

    Tumor necrosis factor receptor (TNFR)-associated factor 3(TRAF3) is a key regulator in TNFR and Toll-like receptor (TLRs)/RIG-I-like receptors (RLRs) signal pathway. Here, a TRAF3 gene (Ls-TRAF3, GenBank Accession No: KJ789921) is cloned from humphead snapper (Lutjanus sanguineus). The Ls-TRAF3 cDNA contains an open reading frame of 1788 bp, which encodes a polypeptide of 595 amino acids. The deduced amino acid of Ls-TRAF3 possesses a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Ls-TRAF3 protein shares high identities with other known TRAF3 proteins. In healthy fish, Ls-TRAF3 transcripts were broadly expressed in all examined tissues with highest expression levels in spleen, liver and head kidney. Quantitative real-time PCR (qRT-PCR) analysis revealed that Ls-TRAF3 could be induced by bacteria or viral PAMP poly I:C stimulation in vivo. Here, we also showed Ls-TRAF3 that, positively regulated IRF3 and Mx upon poly I:C stimuli, whereas prevented production of proinflammatory cytokine IL-6 after LPS injection. Moreover, over-expression of wide type (WT) Ls-TRAF3 and truncated forms, including ΔZinc finger 1, ΔZinc finger 2 and Δcoiled-coil suppressed NF-κB activity significantly, whereas the inhibitory effect of NF-κB was partially impaired when the RING finger or MATH domain deletion, suggesting the latter was more important for downstream signal transduction. Taken together, these results implicated that Ls-TRAF3 might play regulatory roles in immune response to pathogen invasion.

  13. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...

  14. Molecular characterization and functional analysis of tumor necrosis factor receptor-associated factor 2 in the Pacific oyster.

    Science.gov (United States)

    Huang, Baoyu; Zhang, Linlin; Du, Yishuai; Li, Li; Tang, Xueying; Zhang, Guofan

    2016-01-01

    Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are a family of crucial adaptors, playing vital roles in mediating signal transduction in immune signaling pathways, including RIG-I-like receptor (RLR) signaling pathway. In the present study, a new TRAF family member (CgTRAF2) was identified in the Pacific oyster, Crassostrea gigas. Comparison and phylogenetic analysis revealed that CgTRAF2 could be a new member of the invertebrate TRAF2 family. Quantitative real-time PCR revealed that CgTRAF2 mRNA was highly expressed in the digestive gland, gills, and hemocytes, and it was significantly up-regulated after Vibrio alginolyticus and ostreid herpesvirus 1 (OsHV-1) challenge. The CgTRAF2 mRNA expression profile in different developmental stages of oyster larvae suggested that CgTRAF2 could function in early larval development. CgTRAF2 mRNA expression pattern, after the silence of CgMAVS (Mitochondrial Antiviral Signaling) -like, indicated that CgTRAF2 might function downstream of CgMAVS-like. Moreover, the subcellular localization analysis revealed that CgTRAF2 was localized in cytoplasm, and it may play predominately important roles in signal transduction. Collectively, these results demonstrated that CgTRAF2 might play important roles in the innate immunity and larval development of the Pacific oyster.

  15. Germ cell nuclear factor directly represses the transcription of peroxisome proliferator-activated receptor delta gene

    Institute of Scientific and Technical Information of China (English)

    Chengqiang He; Naizheng Ding; Jie Kang

    2008-01-01

    Germ cell nuclear factor (GCNF) is a transcription factor that can repress gene transcription and plays an important role during spermatogenesis. Peroxisome proliferator-activated receptor delta (PPARδ) is a nuclear hormone receptor belonging to the steroid receptor superfamily.It can activate the expression of many genes,including those involved in lipid metabolism.In this report,we showed that GCNF specifically interacts with PPARδ promoter.Overexpression of GCNF in African green monkey SV40 transformed kidney fibroblast COS7 cells and mouse embryo fibroblast NIH 3T3 cells represses the activity of PPARδ promoter.The mutation of GCNF response element in PPARδ promoter relieves the repression in NIH 3T3 cells and mouse testis.Moreover,we showed that GCNF in nuclear extracts of mouse testis is able to bind to PPARδ promoter directly.We also found that GCNF and PPARδ mRNA were expressed with different patterns in mouse testis by in situ hybridization.These results suggested that GCNF might be a negative regulator of PPARδ gene expression through its direct interaction with PPARδ promoter in mouse testis.

  16. EXPRESSION OF EPIDERMAL GROWTH FACTOR RECEPTOR IN DIFFERENT SALIVARY ADENOID CYSTIC CARCINOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    MA Jie; ZONG Zhi-hong; WANG Zhao-yuan

    2005-01-01

    Objective: To investigate the expression of epidermal growth factor receptor, a receptor tyrosine protein kinase, in the subcellular fractions of human salivary adenoid cystic carcinoma cell lines SACC-83 and SACC-LM. Methods: Low metastatic and high metastatic cells of the adenoid cystic carcinoma, SACC-83 and SACC-LM, were cultured. Their subcellular fractions were extracted. The expression of epidermal growth factor receptor was detected with Western blot method, and the results of protein expression were quantitatively analyzed by FluorChem V2.0 software. Results: The results of Western blot analysis indicated that, EGFR expression on the membrane of SACC-83 cells was significantly higher than that of SACC-LM cells, but its expression in cytoplasm was significantly less in the former than the later (P<0.01). In SACC-83 cell line, EGFR was over-expressed in membrane (P<0.01), but in SACC-LM cell line, EGFR was over-expressed in cytoplasm (P<0.01). Conclusion: The results suggest that the obtaining of metastasis ability is related to the high expression of EGFR protein in cytoplasm, so the molecular targeting therapy to EGFR may be an ideal treatment for the invasion and metastasis of salivary adenoid cystic carcinoma.

  17. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment*

    OpenAIRE

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-01-01

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by th...

  18. Surface proteome analysis identifies platelet derived growth factor receptor-alpha as a critical mediator of transforming growth factor-beta-induced collagen secretion.

    Science.gov (United States)

    Heinzelmann, Katharina; Noskovičová, Nina; Merl-Pham, Juliane; Preissler, Gerhard; Winter, Hauke; Lindner, Michael; Hatz, Rudolf; Hauck, Stefanie M; Behr, Jürgen; Eickelberg, Oliver

    2016-05-01

    Fibroblasts are extracellular matrix-producing cells in the lung. Fibroblast activation by transforming growth factor-beta leads to myofibroblast-differentiation and increased extracellular matrix deposition, a hallmark of pulmonary fibrosis. While fibroblast function with respect to migration, invasion, and extracellular matrix deposition has been well-explored, little is known about the surface proteome of lung fibroblasts in general and its specific response to fibrogenic growth factors, in particular transforming growth factor-beta. We thus performed a cell-surface proteome analysis of primary human lung fibroblasts in presence/absence of transforming growth factor-beta, followed by characterization of our findings using FACS analysis, Western blot, and siRNA-mediated knockdown experiments. We identified 213 surface proteins significantly regulated by transforming growth factor-beta, platelet derived growth factor receptor-alpha being one of the top down-regulated proteins. Transforming growth factor beta-induced downregulation of platelet derived growth factor receptor-alpha induced upregulation of platelet derived growth factor receptor-beta expression and phosphorylation of Akt, a downstream target of platelet derived growth factor signaling. Importantly, collagen type V expression and secretion was strongly increased after forced knockdown of platelet derived growth factor receptor-alpha, an effect that was potentiated by transforming growth factor-beta. We therefore show previously underappreciated cross-talk of transforming growth factor-beta and platelet derived growth factor signaling in human lung fibroblasts, resulting in increased extracellular matrix deposition in a platelet derived growth factor receptor-alpha dependent manner. These findings are of particular importance for the treatment of lung fibrosis patients with high pulmonary transforming growth factor-beta activity.

  19. Effect of liver regeneration after partial hepatectomy and ischemia-reperfusion on expression of growth factor receptors

    Institute of Scientific and Technical Information of China (English)

    P Baier; G Wolf-Vorbeck; S Hempel; UT Hopt; E von Dobschuetz

    2006-01-01

    AIM: To investigate the effects of experimental partial hepatectomy and normothermic ischemia-reperfusion damage on the time course of the expression of four different growth factor receptors in liver regeneration.This is relevant due to the potential therapeutic use of growth factors in stimulating liver regeneration.METHODS: For partial hepatectomy (PH) 80% of the liver mass was resected in Sprague Dawley rats.Ischemia and reperfusion (I/R) were induced by occlusion of the portal vein and the hepatic artery for 15 min. The epidermal growth factor receptor, hepatic growth factor receptor, fibroblast growth factor receptor and tumour necrosis factor receptor-1 were analysed by immunohistochemistry up to 72 h after injury.Quantitative RT-PCR was performed at the time point of minimal receptor expression (24 h).RESULTS: In immunohistochemistry, EGFR, HGFR,FGFR and TNFR1 showed biphasic kinetics after partial hepatectomy with a peak up to 12 h, a nadir after 24 h and another weak increase up to 72 h. During liver regeneration, after ischemia and reperfusion, the receptor expression was lower; the nadir at 24 h after reperfusion was the same. To evaluate whether this nadir was caused by a lack of mRNA transcription, or due to a posttranslational regulation, RT-PCR was performed at 24 h and compared to resting liver. In every probe there was specific mRNA for the receptors. EGFR, FGFR and TNFR1 mRNA expression was equal or lower than in resting liver, HGFR expression after I/R was stronger than in the control.CONCLUSION: At least partially due to a post-transcriptional process, there is a nadir in the expression of the analysed receptors 24 h after liver injury. Therefore,a therapeutic use of growth factors to stimulate liver regeneration 24 h after the damage might be not successful.

  20. 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.

  1. Receptor Signaling Directs Global Recruitment of Pre-existing Transcription Factors to Inducible Elements

    Science.gov (United States)

    Cockerill, Peter N.

    2016-01-01

    Gene expression programs are largely regulated by the tissue-specific expression of lineage-defining transcription factors or by the inducible expression of transcription factors in response to specific stimuli. Here I will review our own work over the last 20 years to show how specific activation signals also lead to the wide-spread re-distribution of pre-existing constitutive transcription factors to sites undergoing chromatin reorganization. I will summarize studies showing that activation of kinase signaling pathways creates open chromatin regions that recruit pre-existing factors which were previously unable to bind to closed chromatin. As models I will draw upon genes activated or primed by receptor signaling in memory T cells, and genes activated by cytokine receptor mutations in acute myeloid leukemia. I also summarize a hit-and-run model of stable epigenetic reprograming in memory T cells, mediated by transient Activator Protein 1 (AP-1) binding, which enables the accelerated activation of inducible enhancers. PMID:28018147

  2. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

    Science.gov (United States)

    Smiley, P L; Stremler, K E; Prescott, S M; Zimmerman, G A; McIntyre, T M

    1991-06-15

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows

  3. Epidermal growth factor receptor in the prawn Macrobrachium rosenbergii: function and putative signaling cascade.

    Science.gov (United States)

    Sharabi, Omri; Ventura, Tomer; Manor, Rivka; Aflalo, Eliahu D; Sagi, Amir

    2013-09-01

    Epidermal growth factor receptors (EGFRs) are highly conserved members of the tyrosine kinase receptor superfamily found in metazoans and plants. In arthropods, EGFRs are vital for the proper development of embryos and of adult limbs, gonads, and eyes as well as affecting body size. In searching for genes involved in the growth and development of our model organism, the decapod crustacean (Macrobrachium rosenbergii), a comprehensive transcript library was established using next-generation sequencing. Using this library, the expression of several genes assigned to the signal transduction pathways mediated by EGFRs was observed, including a transcript encoding M. rosenbergii EGFR (Mr-EGFR), several potential ligands upstream to the receptor, and most of the putative downstream signal transducer genes. The deduced protein encoded by Mr-EGFR, representing the first such receptor reported thus far in crustaceans, shows sequence similarity to other arthropod EGFRs. The M. rosenbergii gene is expressed in most tested tissues. The role of Mr-EGFR was revealed by temporarily silencing the transcript through weekly injections of double-stranded Mr-EGFR RNA. Such treatment resulted in a significant reduction in growth and a delay in the appearance of a male secondary sexual characteristic, namely the appendix masculina. An additional function of Mr-EGFR was revealed with respect to eye development. Although the optic ganglion appeared to have retained its normal morphology, Mr-EGFR-silenced individuals developed abnormal eyes that presented irregular organization of the ommatidia, reflected by unorganized receptor cells occupying large areas of the dioptric portion and by a shortened crystalline tract layer.

  4. Molecular assembly of the ternary granulocyte-macrophage colony-stimulating factor receptor complex.

    Science.gov (United States)

    McClure, Barbara J; Hercus, Timothy R; Cambareri, Bronwyn A; Woodcock, Joanna M; Bagley, Christopher J; Howlett, Geoff J; Lopez, Angel F

    2003-02-15

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the production and functional activity of granulocytes and macrophages, properties that have encouraged its clinical use in bone marrow transplantation and in certain infectious diseases. Despite the importance of GM-CSF in regulating myeloid cell numbers and function, little is known about the exact composition and mechanism of assembly of the GM-CSF receptor complex. We have now produced soluble forms of the GM-CSF receptor alpha chain (sGMRalpha) and beta chain (sbetac) and utilized GM-CSF, the GM-CSF antagonist E21R (Glu21Arg), and the betac-blocking monoclonal antibody BION-1 to define the molecular assembly of the GM-CSF receptor complex. We found that GM-CSF and E21R were able to form low-affinity, binary complexes with sGMRalpha, each having a stoichiometry of 1:1. Importantly, GM-CSF but not E21R formed a ternary complex with sGMRalpha and sbetac, and this complex could be disrupted by E21R. Significantly, size-exclusion chromatography, analytical ultracentrifugation, and radioactive tracer experiments indicated that the ternary complex is composed of one sbetac dimer with a single molecule each of sGMRalpha and of GM-CSF. In addition, a hitherto unrecognized direct interaction between betac and GM-CSF was detected that was absent with E21R and was abolished by BION-1. These results demonstrate a novel mechanism of cytokine receptor assembly likely to apply also to interleukin-3 (IL-3) and IL-5 and have implications for our molecular understanding and potential manipulation of GM-CSF activation of its receptor.

  5. Expression of fibroblast growth factor (FGF)-8 isoforms and FGF receptors in human ovarian tumors.

    Science.gov (United States)

    Valve, E; Martikainen, P; Seppänen, J; Oksjoki, S; Hinkka, S; Anttila, L; Grenman, S; Klemi, P; Härkönen, P

    2000-12-01

    FGF-8 is a mitogenic growth factor, which is widely expressed during embryonic development but only at a very low level in adult tissues. Alternative splicing of the human FGF-8 gene potentially allows coding for 4 protein isoforms (a, b, e, f), which differ in their transforming capacity. The FGF-8 isoforms preferentially activate the receptors FGFR1IIIc, FGFR2IIIc, FGFR3IIIc and FGFR4. FGF-8 is over-expressed in human breast and prostate cancers. Expression has also been found in RT-PCR studies of human ovarian and testicular cancers. The present study was undertaken to examine which FGF-8 isoforms are expressed in ovarian cancer and whether FGF-8 receptors are also expressed. Specimens from 5 normal human ovaries and 51 ovarian tumors (1 benign tumor, 8 borderline malignancies, 42 malignant tumors of different histopathological types) were studied by RT-PCR and immunohistochemistry. FGF-8 isoform b was expressed in all ovarian tumors and in all 7 ovarian-cancer cell lines studied. Isoform a was co-expressed in 9 malignant ovarian tumors. FGF-8 mRNA was not detected by RT-PCR of 3 normal ovary samples. Immunohistochemical staining localized FGF-8 protein to cancer cells. In general, the increased intensity of FGF-8 staining was associated with loss of differentiation within the tumors (Bowker's test, p = 0.37). FGF-8 staining of surface epithelium observed on 2 normal ovaries was very faint. RT-PCR showed that FGFR1IIIc, FGFR2IIIc and FGFR4 were the FGF-8 receptors expressed in normal ovaries and in ovarian tumors. FGF-8 receptor immunoreactivity was preferentially found in normal ovary surface epithelium and tumor cells but also in some stromal cells. Collectively, our results show that ovarian cancers of a wide variety of histological types expressing receptors for FGF-8 have acquired the capacity of expressing FGF-8. This suggests that FGF-8 has an important role in ovarian tumorigenesis.

  6. Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.

    Directory of Open Access Journals (Sweden)

    Marie-Laure Plissonnier

    2016-03-01

    Full Text Available Hepatitis C virus (HCV is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC. The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+ and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR, a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

  7. Transient downregulation of glomerular atrial natriuretic factor receptors in high output heart failure in the rat.

    Science.gov (United States)

    Gauquelin, G; Bonhomme, M C; Garcia, R

    1995-03-01

    The renal response to exogenous atrial natriuretic factor (ANF) is blunted in chronic heart failure. The aim of the present studies was to investigate whether renal ANF receptor regulation in chronic heart failure is a time related event. Glomerular ANF receptors were analysed in radioligand binding experiments at 0, 1, 2, 6, 12, 24, and 48 h, as well as at 1, 2, 4, and 8 weeks after the induction of an aortocaval shunt. Rats with aortocaval shunts had lower packed cell volume and body weight and higher relative heart weight than sham operated controls. Plasma ANF C and N terminal levels were increased in shunt rats as early as 5 min after establishment of the shunt. Right and left atrial ANF concentrations were decreased and ventricular ANF concentration was increased in shunt rats at 6 and 12 h respectively. Competitive inhibition of 125I-ANF binding showed that at 6 h the density (Bmax) of glomerular ANF receptors was significantly lower than in the controls [518(SEM 10) v 759(12) fmol.mg-1 protein] without differences in their affinity (Kd). The low Bmax in shunt animals persisted at 12, 24, and 48 h, even at 1 week [Bmax: 400(29) and 713(28) fmol.mg-1 protein; Kd: 80(2) and 70(4) pM, for AC rats and controls, respectively]. Bmax values were not significantly different at 2, 4, and 8 weeks. In 24 h animals, C-ANF displaced 65% of total binding, with both total and C ANF binding sites being 38% lower in shunt animals. Downregulation of glomerular ANF receptors is a transient event during the development of high output heart failure in the rat. Thus the blunted renal response to ANF during chronic heart failure is not likely to be due to a decrease in renal ANF receptor density or affinity.

  8. Potentiation of nerve growth factor-induced neurite outgrowth by fluvoxamine: role of sigma-1 receptors, IP3 receptors and cellular signaling pathways.

    Directory of Open Access Journals (Sweden)

    Tomoko Nishimura

    Full Text Available BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs have been widely used and are a major therapeutic advance in psychopharmacology. However, their pharmacology is quite heterogeneous. The SSRI fluvoxamine, with sigma-1 receptor agonism, is shown to potentiate nerve-growth factor (NGF-induced neurite outgrowth in PC 12 cells. However, the precise cellular and molecular mechanisms underlying potentiation by fluvoxamine are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and sigma-1 receptor agonists. METHODS AND FINDINGS: The effects of three SSRIs (fluvoxamine, sertraline, paroxetine and three sigma-1 receptor agonists (SA4503, 4-phenyl-1-(4-phenylbutyl piperidine (PPBP, and dehydroepiandrosterone (DHEA-sulfate on NGF-induced neurite outgrowth in PC12 cells were examined. Also examined were the effects of the sigma-1 receptor antagonist NE-100, inositol 1,4,5-triphosphate (IP(3 receptor antagonist, and specific inhibitors of signaling pathways in the potentiation of NGF-induced neurite outgrowth by selective sigma-1 receptor agonist SA4503. Fluvoxamine (but not sertraline or paroxetine and the sigma-1 receptor agonists SA4503, PPBP, and DHEA-sulfate significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by fluvoxamine and the three sigma-1 receptor agonists was blocked by co-administration of the selective sigma-1 receptor antagonist NE-100, suggesting that sigma-1 receptors play a role in blocking the enhancement of NGF-induced neurite outgrowth. Moreover, the potentiation by SA4503 was blocked by co-administration of the IP(3 receptor antagonist xestospongin C. In addition, the specific inhibitors of phospholipase C (PLC-gamma, phosphatidylinositol 3-kinase (PI3K, p38MAPK, c-Jun N-terminal kinase (JNK, and the Ras/Raf/mitogen-activated protein kinase (MAPK

  9. Immunomodulator CD200 promotes neurotrophic activity by interacting with and activating the fibroblast growth factor receptor

    DEFF Research Database (Denmark)

    Pankratova, Stanislava; Bjornsdottir, Halla; Christensen, Claus;

    2016-01-01

    in the suppression of microglia activation. We for the first time demonstrated that CD200 can interact with and transduce signaling through activation of the fibroblast growth factor receptor (FGFR), thereby inducing neuritogenesis and promoting neuronal survival in primary neurons. CD200-induced FGFR...... phosphorylation was abrogated by CD200R, whereas FGF2-induced FGFR activation was inhibited by CD200. We also identified a sequence motif located in the first Ig-like module of CD200, likely representing the minimal CD200 binding site for FGFR. The FGFR binding motif overlaps with the CD200R binding site......, suggesting that they can compete for CD200 binding in cells that express both receptors. We propose that CD200 in neurons functions as a ligand of FGFR....

  10. Implication of platelet-derived growth factor receptor alpha in prostate cancer skeletal metastasis

    Institute of Scientific and Technical Information of China (English)

    Qingxin Liu; Danielle Jernigan; Yun Zhang; Alessandro Fatatis

    2011-01-01

    Metastasis represents by far the most feared complication of prostate carcinoma and is the main cause of death for patients.The skeleton is frequently targeted by disseminated cancer cells andrepresents the sole site of spread in more than 80% of prostate cancer cases.Compatibility between select malignant phenotypes and the microenvironment of colonized tissues is broadly recognized as the culprit for the organ-tropism of cancer cells.Here,we review our recent studies showing that the expression of platelet-derived growth factor receptor alpha (PDGFRα) supports the survival and growth of prostate cancer cells in the skeleton and that the soluble fraction of bone marrow activates PDGFRα in a ligand-independent fashion.Finally,we offer pre-clinical evidence that this receptor is a viable target for therapy.

  11. Ramucirumab (IMC-1121B): Monoclonal antibody inhibition of vascular endothelial growth factor receptor-2.

    Science.gov (United States)

    Spratlin, Jennifer

    2011-04-01

    Angiogenesis, a well-recognized characteristic of malignancy, has been exploited more than any other pathway targeted by biologic anti-neoplastic therapies. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the critical receptor involved in malignant angiogenesis with its activation inducing a number of other cellular modifications resulting in tumor growth and metastases. Ramucirumab (IMC-1121B; ImClone Systems Corporation, Branchburg, NJ) is a fully human monoclonal antibody developed to specifically inhibit VEGFR-2. Ramucirumab is currently being investigated in multiple clinical trials across a variety of tumor types. Herein, angiogenesis inhibition in cancer is reviewed and up-to-date information on the clinical development of ramucirumab is presented.

  12. Treatment related changes of the serum epidermal growth factor receptor in advanced colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, K G; Aalund Olsen, Dorte; Brandslund, I

    2009-01-01

    .001), and a correlation between the magnitude of increase and a higher degree of skin toxicity, a well known indicator of clinical benefit to EGFR inhibitors. sEGFR in rectal cancer patients displayed a decreasing tendency during CRT (p...e22096 Background: The epidermal growth factor receptor (EGFR) is an established target for therapy in colorectal cancer. The extracellular domain of the receptor is shed into circulation and detectable by ELISA. We investigated the changes in sEGFR levels during preoperative chemoradiation (CRT......) in rectal cancer patients and third-line treatment with cetuximab and irinotecan (CETIRI) in advanced disease, to elucidate the predictive or prognostic value in these settings. METHODS: We included 126 healthy controls and 118 patients with chemorefractory mCRC treated with cetuximab (initial 400/m(2) mg...

  13. Fibroblast growth factor receptor 2 plays an essential role in telencephalic progenitors.

    Science.gov (United States)

    Ever, Leah; Zhao, Rui-Jing; Eswarakumar, Veraragavan P; Gaiano, Nicholas

    2008-01-01

    We used loss-of-function analysis to determine the role of fibroblast growth factor receptor 2 (FGFR2) in telencephalic progenitors, and also to examine interactions between FGFR and Notch signaling. While the telencephalon of FGFR2 mutants appears grossly normal, mutant telencephalic progenitors exhibit altered proliferative behavior in vivo and in vitro. Based upon our prior finding that Notch1 activation increased neurosphere frequency in FGF2, we tested whether this effect is mediated by FGFR1 or FGFR2. We found that Notch1 activation increased neurosphere frequency in cells mutant for either FGFR1 or FGFR2, but had no effect on the reduced size of neurospheres mutant for those receptors. Additional analyses revealed biochemical changes in the adult neocortex mutant for the IIIc isoform of FGFR2, and essential roles for FGFR2 in nasopharynx, eyelid, and cornea development.

  14. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-27

    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  15. 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.

  16. Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo.

    Science.gov (United States)

    Watson, A J; Hogan, A; Hahnel, A; Wiemer, K E; Schultz, G A

    1992-02-01

    The sensitive technique of mRNA phenotyping with the reverse transcription-polymerase chain reaction was employed to determine the patterns of gene expression for several growth factor ligand and receptor genes during bovine preimplantation development. Several thousand bovine embryos encompassing a developmental series from one-cell zygotes to hatched blastocysts were produced by the application of in vitro maturation, fertilization, and oviductal epithelial cell embryo coculture methods. Transcripts for transforming growth factor (TGF-alpha) and platelet-derived growth factor (PDGF-A) are detectable in all preimplantation bovine stages as observed in the mouse. Transcripts for TGF-beta 2 and insulin-like growth factor (IGF-II) and the receptors for PDGF-alpha, insulin, IGF-I, and IGF-II are also detectable throughout bovine preimplantation development, suggesting that these mRNAs are products of both the maternal and the embryonic genomes in the cow, whereas in the mouse they are present only following the activation of the embryonic genome at the two-cell stage. In contrast to the mouse embryo, IGF-I mRNA was detected within preimplantation bovine embryos. Basic fibroblast growth factor (bFGF) is a maternal message in the bovine embryo, since it is only detectable up until the eight-cell embryo stage. Bovine trophoblast protein (bTP) mRNA was detectable within day 8 bovine blastocysts. As was observed in the mouse, the transcripts for insulin, epidermal growth factor (EGF), or nerve growth factor (NGF) were not detectable in any bovine embryo stage. Analyses of this type should aid the development of a completely defined culture medium for the more efficient production of preimplantation bovine embryos.

  17. Featured Article: Nuclear export of opioid growth factor receptor is CRM1 dependent.

    Science.gov (United States)

    Kren, Nancy P; Zagon, Ian S; McLaughlin, Patricia J

    2016-02-01

    Opioid growth factor receptor (OGFr) facilitates growth inhibition in the presence of its specific ligand opioid growth factor (OGF), chemically termed [Met(5)]-enkephalin. The function of the OGF-OGFr axis requires the receptor to translocate to the nucleus. However, the mechanism of nuclear export of OGFr is unknown. In this study, endogenous OGFr, as well as exogenously expressed OGFr-EGFP, demonstrated significant nuclear accumulation in response to leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export, suggesting that OGFr is exported in a CRM1-dependent manner. One consensus sequence for a nuclear export signal (NES) was identified. Mutation of the associated leucines, L217 L220 L223 and L225, to alanine resulted in decreased nuclear accumulation. NES-EGFP responded to LMB, indicating that this sequence is capable of functioning as an export signal in isolation. To determine why the sequence functions differently in isolation than as a full length protein, the localization of subNES was evaluated in the presence and absence of MG132, a potent inhibitor of proteosomal degradation. MG132 had no effect of subNES localization. The role of tandem repeats located at the C-terminus of OGFr was examined for their role in nuclear trafficking. Six of seven tandem repeats were removed to form deltaTR. DeltaTR localized exclusively to the nucleus indicating that the tandem repeats may contribute to the localization of the receptor. Similar to the loss of cellular proliferation activity (i.e. inhibition) recorded with subNES, deltaTR also demonstrated a significant loss of inhibitory activity indicating that the repeats may be integral to receptor function. These experiments reveal that OGFr contains one functional NES, L217 L220 L223 and L225 and can be exported from the nucleus in a CRM1-dependent manner.

  18. Decreased expression of fibroblast and keratinocyte growth factor isoforms and receptors during scarless repair.

    Science.gov (United States)

    Dang, Catherine M; Beanes, Steven R; Soo, Chia; Ting, Kang; Benhaim, Prosper; Hedrick, Marc H; Lorenz, H Peter

    2003-05-01

    Fibroblast growth factors (FGFs) are a family of 21 cytokines with a broad spectrum of activities, including regulation of cell proliferation, differentiation, and migration. The various FGFs bind to one or more of four different tyrosine kinase receptor types. FGFs 1, 2, 5, 7, and 10 are up-regulated during adult cutaneous wound healing. However, the expression of FGFs during fetal skin development and scarless wound healing has not been characterized. It was hypothesized that differential expression of FGF isoforms and receptors occurs during fetal skin development and that this differential expression pattern may regulate the transition from scarless repair to healing with scar formation. Excisional wounds (2 mm) were created on fetal rats at gestational days 16.5 (scarless) (one wound per fetus, n = 36 fetuses) and 19.5 (scarring) (one wound per fetus, n = 36 fetuses). Wounds were harvested at 24, 48, and 72 hours. Survival until wound harvest ranged from 66 to 75 percent for the gestational day 16 fetuses, and from 83 to 92 percent for the gestational day 19 fetuses. Nonwounded fetal skin from littermates (n = 12 fetuses per wound harvest time point) was used as the control. Wounds/skins were pooled by harvest time point, and RNA was isolated from pooled wounds/skins. Reduced-cycle, specific-primer reverse transcriptase-polymerase chain reaction was performed to determine the expression of FGF isoforms 2, 5, 7, 9, and 10 and FGF receptors 1, 2, and 4 in wounds relative to unwounded skin.In unwounded fetal skin, FGF isoform 5 expression more than doubled at birth. FGF 10 expression doubled during the transition period. FGF 7 expression increased more than sevenfold at birth. Expression of FGF isoforms 2 and 9 did not change during late fetal skin development. The expression of FGF receptors 1, 2, and 4 increased at birth. After wounding, expression of FGF isoforms 7 and 10 was down-regulated in scarless wounds, whereas FGF receptor 2 expression decreased in

  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. Integration of estrogen and progesterone receptors with pathological and molecular prognostic factors in breast cancer patients.

    Science.gov (United States)

    Gago, F E; Tello, O M; Diblasi, A M; Ciocca, D R

    1998-12-01

    In this study we have examined biopsies from women with localized primary breast cancer to investigate the prognostic performance of estrogen receptors (ER) and progesterone receptors (PR) for estimating the metastatic probability of the patients, and to explore whether discrimination gets better by combining clinicopathological and other molecular parameters into a score. This prospective study involved 205 patients with a median follow-up of 5 y. Among the evaluated clinicopathological data were: patient's age; tumor size; axillary lymph node involvement; and tumor grade. The most representative tumor samples were derived to a single laboratory for immunohistochemical evaluation of the following molecular markers: ER, PR, proliferating cell nuclear antigen (PCNA), p53 protein product, erbB-2 (HER-2/neu) oncoprotein, and P170 glycoprotein (mdrl gen product). Distant metastases (study endpoint) appeared in 19.5% (40/205) of the patients, most of these patients presented a mixture of poor, regular and good prognostic factors. Disease-free survival analysis procedures (Kaplan-Meier method) identified tumor size, axillary lymph node involvement, tumor grade, receptor status, PCNA, p53, erbB-2 and P170 as useful prognostic factors. Proportional hazard regression analysis (Cox) identified in order of importance erbB-2, tumor size, receptors status, tumor grade and PCNA as useful prognostic factors. To facilitate the evaluation of the prognostic factors, a practical and simple score system was derived. A high pathological score identified 65% of the patients that developed distant metastases, while a high molecular score was obtained in 57% of patients with metastatic disease. There was a significant improvement in the diagnosis of probability of being with distant metastases when the pathological score was combined with the molecular score, 82% of the patients with distant metastases showed an elevated combined score. Validation of this scoring system will need further

  1. Nuclear Factor-κB: Activation and Regulation during Toll-like Receptor Signaling

    Institute of Scientific and Technical Information of China (English)

    Ruaidhrí J. Carmody; Youhai H. Chen

    2007-01-01

    Toll-like receptors (TLRs) recognize distinct microbial components to initiate the innate and adaptive immune responses. TLR activation culminates in the expression of appropriate pro-inflammatory and immunomodulatory factors to meet pathogenic challenges. The transcription factor NF-κB is the master regulator of all TLR-induced responses and its activation is the pivotal event in TLR-mediated activation of the innate immune response. Many of the key molecular events required for TLR-induced NF-κB activation have been elucidated. However, much remain to be learned about the ability of TLRs to generate pathogen-specific responses using a limited number of transcription factors. This review will focus on our current understanding of NF-κB activation by TLRs and potential mechanisms for achieving a signal-specific response through NF-κB.

  2. Targeted therapy with bispecific single chain antibodies for acute leukemia%双特异单链抗体靶向治疗急性白血病

    Institute of Scientific and Technical Information of China (English)

    崔鹤; 张王刚

    2015-01-01

    目前除急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)以外的急性白血病( acute leukemia,AL)疗效堪忧,急需研发新的治疗药物。采用基因工程抗体技术制备的双特异性单链抗体能同时结合两个特异性抗原,与其他抗体药物相比有明显的优越性,是一种有很好临床应用前景的免疫治疗药物,本文对双特异性单链抗体靶向治疗急性白血病的最新进展做一综述。%At present,there is a urgent need to develop new drugs for acute leukemia patients because of their poor effect except for acute promyelocytic leukemia. The bispecific single chain antibodies can combine with two specific antigens using gene engineering antibody technology,show more obvious advantage than other antibody drugs,and po-tential prospect in clinical application. We described the current new targeted therapy with bispecific single chain anti-bodies for acute leukemia in this article.

  3. Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library.

    Science.gov (United States)

    Wu, Qian; Wang, Xiaodong; Gu, Yong; Zhang, Xiao; Qin, Yao; Chen, Heng; Xu, Xinyu; Yang, Tao; Zhang, Mei

    2016-07-01

    Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

  4. Treatment of pancreatic cancer with epidermal growth factor receptor-targeted therapy

    Directory of Open Access Journals (Sweden)

    Bryan A Faller

    2009-09-01

    Full Text Available Bryan A Faller, Barbara BurtnessDepartment of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA, USAAbstract: Pancreatic adenocarcinoma is a common malignancy that remains refractory to available therapies. Gemcitabine has long been the standard, first-line agent in advanced disease. The epidermal growth factor receptor (EGFR is a commonly expressed target in pancreatic cancer that is involved in tumor proliferation, metastasis, and induction of angiogenesis. The addition of the EGFR inhibitor erlotinib to gemcitabine has recently been demonstrated to provide a small, yet statistically significant, survival benefit in advanced disease. This has prompted further research into the applications of EGFR-targeted therapy in pancreatic cancer, albeit with disappointing results. Resistance to these therapies seems highly prevalent and has been implicated in their limited efficacy. The development of rash is associated with treatment efficacy and suggests that predictive factors may one day be identified to guide appropriate patient selection for these agents. Preclinical research has shown promise that resistance to EGFR-targeted therapies can be overcome through a variety of approaches. Application of this research in clinical trials may ultimately yield an unquestioned role for EGFR-targeted therapy in the management of this disease.Keywords: cetuximab, drug resistance, epidermal growth factor receptor, erlotinib, gemcitabine, pancreatic cancer

  5. Brain-derived neurotrophic factor and its receptors in Bergmann glia cells.

    Science.gov (United States)

    Poblete-Naredo, Irais; Guillem, Alain M; Juárez, Claudia; Zepeda, Rossana C; Ramírez, Leticia; Caba, Mario; Hernández-Kelly, Luisa C; Aguilera, José; López-Bayghen, Esther; Ortega, Arturo

    2011-12-01

    Brain-derived neurotrophic factor is an abundant and widely distributed neurotrophin expressed in the Central Nervous System. It is critically involved in neuronal differentiation and survival. The expression of brain-derived neurotrophic factor and that of its catalytic active cognate receptor (TrkB) has been extensively studied in neuronal cells but their expression and function in glial cells is still controversial. Despite of this fact, brain-derived neurotrophic factor is released from astrocytes upon glutamate stimulation. A suitable model to study glia/neuronal interactions, in the context of glutamatergic synapses, is the well-characterized culture of chick cerebellar Bergmann glia cells. Using, this system, we show here that BDNF and its functional receptor are present in Bergmann glia and that BDNF stimulation is linked to the activation of the phosphatidyl-inositol 3 kinase/protein kinase C/mitogen-activated protein kinase/Activator Protein-1 signaling pathway. Accordingly, reverse transcription-polymerase chain reaction (RT-PCR) experiments predicted the expression of full-length and truncated TrkB isoforms. Our results suggest that Bergmann glia cells are able to express and respond to BDNF stimulation favoring the notion of their pivotal role in neuroprotection. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. 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.

  7. Neuritogenic and neuroprotective properties of peptide agonists of the fibroblast growth factor receptor

    DEFF Research Database (Denmark)

    Li, Shizhong; Bock, Elisabeth Marianne; Berezin, Vladimir

    2010-01-01

    Fibroblast growth factor receptors (FGFRs) interact with their cognate ligands, FGFs, and with a number of cell adhesion molecules (CAMs), such as the neural cell adhesion molecule (NCAM), mediating a wide range of events during the development and maintenance of the nervous system. Determination...... of protein structure, in silico modeling and biological studies have recently resulted in the identification of FGFR binding peptides derived from various FGFs and NCAM mimicking the effects of these molecules with regard to their neuritogenic and neuroprotective properties. This review focuses on recently...... developed functional peptide agonists of FGFR with possible therapeutic potential....

  8. Neuritogenic and Neuroprotective Properties of Peptide Agonists of the Fibroblast Growth Factor Receptor

    Directory of Open Access Journals (Sweden)

    Shizhong Li

    2010-05-01

    Full Text Available Fibroblast growth factor receptors (FGFRs interact with their cognate ligands, FGFs, and with a number of cell adhesion molecules (CAMs, such as the neural cell adhesion molecule (NCAM, mediating a wide range of events during the development and maintenance of the nervous system. Determination of protein structure, in silico modeling and biological studies have recently resulted in the identification of FGFR binding peptides derived from various FGFs and NCAM mimicking the effects of these molecules with regard to their neuritogenic and neuroprotective properties. This review focuses on recently developed functional peptide agonists of FGFR with possible therapeutic potential.

  9. Expression and functional characterization of platelet-derived growth factor receptor-like gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate the role of platelet-derived growth factor receptor-like gene(PDGFRL)in the anti-cancer therapy for colorectal cancers(CRC).METHODS:PDGFRL mRNA and protein levels were measured by reverse transcription-polymerase chain reaction(RT-PCR)and immunohistochemistry in CRC and colorectal normal tissues.PDGFRL prokaryotic expression vector was carried out in Escherichia coli(E.coli),and purified by immobilized metal affinity chromatography.The effect of PDGFRL protein on CRC HCT-116 cells was det...

  10. Epidermal Growth Factor Receptor Overexpression as a Target for Auger Electron Radiotherapy of Breast Cancer

    Science.gov (United States)

    2002-08-01

    radiotherapeutic agent for breast cancer. © 2001 Elsevier Science Inc . All rights reserved. Keywords: Breast cancer; Indium-111; Epidermal growth factor receptor... Science Inc . All rights reserved. PII: S0969-8051(01)00262-1 896 .1. Wang et al. / Nuclear Medicine and Biology 28 (2001) 895-902 A NW3 rn- NH, + NHC...raymond.reilly@utoronto.ca (R.M. Reilly). (cDTPAA) with an E-amino group on a lysine residue or the 0969-8051/01/$ - see front matter © 2001 Elsevier

  11. Regulation of tumour necrosis factor (TNF) induced apoptosis by soluble TNF receptors in Helicobacter pylori infection

    OpenAIRE

    Shibata, J; Goto, H.; Arisawa, T.; Niwa, Y.; Hayakawa, T.; Nakayama, A.; Mori, N.

    1999-01-01

    BACKGROUND—Tumour necrosis factor (TNF) is a predominant cytokine produced in the gastric mucosa of patients with Helicobacter pylori infection. TNF induces apoptosis in a variety of cells. The soluble TNF receptors (sTNF-Rs) can be divided into sTNF-RI and sTNF-RII, both of which inhibit TNF activity. However, their precise mechanisms remain unclear.
AIM—To investigate the role of sTNF-Rs in H pylori infection.
METHODS—In 40 patients, production of TNF and sTNF-Rs in gastric mucosa was measu...

  12. Fibroblast growth factor (FGF)-21 signals through both FGF receptor-1 and 2

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Fibroblast growth factor (FGF)-21 is a member of the FGF superfamily based on sequence homology. However, unlike most members of this family it does not show any mitogenic activity in all cell types tested. The objective of this study is to identify and characterize receptors for this molecule. Sequencing of the cDNA clones from 3T3-L1 adipocytes indicates that the only isoforms for FGFR-1 and 2 expressed in 3T3-L1 cells are 1IIIc and 2IIIc, respectively, suggesting that FGF-21 regulates glucose metabolism in 3T3-L1 adipocytes through FGFR-1IIIc and FGFR-2IIIc.

  13. Identification of soluble forms of the fibroblast growth factor receptor in blood.

    OpenAIRE

    Hanneken, A; Ying, W; Ling, N; Baird, A.

    1994-01-01

    We have purified three acidic (FGF-1) and basic (FGF-2) fibroblast growth factor binding proteins (FGF-BP1, FGF-BP2, and FGF-BP3) from human plasma and calf serum and demonstrate the presence of these circulating FGF-BPs in blood. Each are truncated forms of the high-affinity FGF receptor (FGFR-1). FGF-BP1 and FGF-BP2 have estimated molecular masses of 70-85 kDa and 55-60 kDa, respectively, and are detected by using 125I-labeled FGF-2 ligand blotting. Immunoblotting with four distinct antibod...

  14. Insulin-like growth factor-I receptor in proliferation and motility of pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Minoru; Tomizawa; Fuminobu; Shinozaki; Takao; Sugiyama; Shigenori; Yamamoto; Makoto; Sueishi; Takanobu; Yoshida

    2010-01-01

    AIM:To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol3 kinase (PI3K), and PD980...

  15. The cytokines cardiotrophin-like cytokine/cytokine-like factor-1 (CLC/CLF) and ciliary neurotrophic factor (CNTF) differ in their receptor specificities.

    Science.gov (United States)

    Tormo, Aurélie Jeanne; Letellier, Marie-Claude; Lissilaa, Rami; Batraville, Laurie-Anne; Sharma, Mukut; Ferlin, Walter; Elson, Greg; Crabé, Sandrine; Gauchat, Jean-François

    2012-12-01

    Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC) are two cytokines with neurotrophic and immunomodulatory activities. CNTF is a cytoplasmic factor believed to be released upon cellular damage, while CLC requires interaction with a soluble cytokine receptor, cytokine-like factor 1 (CLF), to be efficiently secreted. Both cytokines activate a receptor complex comprising the cytokine binding CNTF receptor α (CNTFRα) and two signaling chains namely, leukemia inhibitory factor receptor β (LIFRβ) and gp130. Human CNTF can recruit and activate an alternative receptor in which CNTFRα is substituted by IL-6Rα. As both CNTF and CLC have immune-regulatory activities in mice, we compared their ability to recruit mouse receptors comprising both gp130 and LIFRβ signaling chains and either IL-6Rα or IL-11Rα which, unlike CNTFRα, are expressed by immune cells. Our results indicate that 1) mouse CNTF, like its human homologue, can activate cells expressing gp130/LIFRβ with either CNTFRα or IL-6Rα and, 2) CLC/CLF is more restricted in its specificity in that it activates only the tripartite CNTFR. Several gp130 signaling cytokines influence T helper cell differentiation. We therefore investigated the effect of CNTF on CD4 T cell cytokine production. We observed that CNTF increased the number of IFN-γ producing CD4 T cells. As IFN-γ is considered a mediator of the therapeutic effect of IFN-β in multiple sclerosis, induction of IFN-γ by CNTF may contribute to the beneficial immunomodulatory effect of CNTF in mouse multiple sclerosis models. Together, our results indicate that CNTF activates the same tripartite receptors in mouse and human cells and further validate rodent models for pre-clinical investigation of CNTF and CNTF derivatives. Furthermore, CNTF and CLC/CLF differ in their receptor specificities. The receptor α chain involved in the immunomodulatory effects of CLC/CLF remains to be identified.

  16. 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

  17. Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors

    DEFF Research Database (Denmark)

    Aderka, D; Engelmann, H; Maor, Y;

    1992-01-01

    activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on prolonged treatment with this cytokine are augmented, reflecting an attenuation by the sTNF-Rs of spontaneous......The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF...... TNF activity decay. Evidence that this stabilization of TNF activity by the sTNF-Rs follows from stabilization of TNF structure within the complexes that TNF forms with the sTNF-Rs is presented here, suggesting that the sTNF-Rs can affect TNF activity not only by interfering with its binding to cells...

  18. 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.

  19. Immunotoxin Therapies for the Treatment of Epidermal Growth Factor Receptor-Dependent Cancers

    Directory of Open Access Journals (Sweden)

    Nathan Simon

    2016-05-01

    Full Text Available Many epithelial cancers rely on enhanced expression of the epidermal growth factor receptor (EGFR to drive proliferation and survival pathways. Development of therapeutics to target EGFR signaling has been of high importance, and multiple examples have been approved for human use. However, many of the current small molecule or antibody-based therapeutics are of limited effectiveness due to the inevitable development of resistance and toxicity to normal tissues. Recombinant immunotoxins are therapeutic molecules consisting of an antibody or receptor ligand joined to a protein cytotoxin, combining the specific targeting of a cancer-expressed receptor with the potent cell killing of cytotoxic enzymes. Over the decades, many bacterial- or plant-based immunotoxins have been developed with the goal of targeting the broad range of cancers reliant upon EGFR overexpression. Many examples demonstrate excellent anti-cancer properties in preclinical development, and several EGFR-targeted immunotoxins have progressed to human trials. This review summarizes much of the past and current work in the development of immunotoxins for targeting EGFR-driven cancers.

  20. 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 mechanism