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Sample records for ap-1 endosomal coat

  1. An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex.

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    Rajendra Kumar Singh

    2009-12-01

    Full Text Available The down-regulation of the major histocompatibility complex class I (MHC-I from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1. The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxphi, which mediates binding to the medium (micro subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the mu subunit of AP-1 (micro1 as if it contained a Yxxphimotif.Here, we show that a direct interaction between the MHC-I CD/Nef and micro1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of micro1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and micro1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on micro1 for Yxxphi motifs were required for a robust interaction.These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the mu subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in micro1 for interaction with MHC-I CD/Nef.

  2. HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons.

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    Shen, Qing-Tao; Ren, Xuefeng; Zhang, Rui; Lee, Il-Hyung; Hurley, James H

    2015-10-23

    The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat. Copyright © 2015, American Association for the Advancement of Science.

  3. Decoupling internalization, acidification and phagosomal-endosomal/lysosomal fusion during phagocytosis of InlA coated beads in epithelial cells.

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    Craig D Blanchette

    Full Text Available BACKGROUND: Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK and Caco-2 epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA, a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification. CONCLUSIONS/SIGNIFICANCE: Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply

  4. Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

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    Blanchette, C D; Woo, Y; Thomas, C; Shen, N; Sulchek, T A; Hiddessen, A L

    2008-12-22

    Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established, and in several cases, it was treated as a one-step process. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells, such as epithelial cells. Therefore, in this study, we developed a simple and novel method to decouple and accurately measure particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated internalization. We achieved independent measurements of the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, pH sensitive dyes and endosomal/lysosomal dyes, as follows: the rate of InlA bead internalization was measured via antibody quenching of a pH independent dye (Alexa488) conjugated to InlA-beads, the rate at which phagosomes containing internalized InlA beads became acidified was measured using a pH dependent dye (FITC) conjugated to the beads and the rate of phagosomal-endosomal/lysosomal fusion was measured using a combination of unlabeled InlA-beads and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we also exploited the phagosomal acidification

  5. AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis

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    Cécile Campagne

    2018-02-01

    Full Text Available Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1, from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

  6. AP1S3 Mutations Are Associated with Pustular Psoriasis and Impaired Toll-like Receptor 3 Trafficking

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    Setta-Kaffetzi, Niovi; Simpson, Michael A.; Navarini, Alexander A.; Patel, Varsha M.; Lu, Hui-Chun; Allen, Michael H.; Duckworth, Michael; Bachelez, Hervé; Burden, A. David; Choon, Siew-Eng; Griffiths, Christopher E.M.; Kirby, Brian; Kolios, Antonios; Seyger, Marieke M.B.; Prins, Christa; Smahi, Asma; Trembath, Richard C.; Fraternali, Franca; Smith, Catherine H.; Barker, Jonathan N.; Capon, Francesca

    2014-01-01

    Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis. PMID:24791904

  7. The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

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    Fubito Nakatsu

    2014-11-01

    Full Text Available The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis. Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells.

  8. COBRA1 inhibits AP-1 transcriptional activity in transfected cells

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    Zhong Hongjun; Zhu Jianhua; Zhang Hao; Ding Lihua; Sun Yan; Huang Cuifen; Ye Qinong

    2004-01-01

    Mutations in the breast cancer susceptibility gene (BRCA1) account for a significant proportion of hereditary breast and ovarian cancers. Cofactor of BRCA1 (COBRA1) was isolated as a BRCA1-interacting protein and exhibited a similar chromatin reorganizing activity to that of BRCA1. However, the biological role of COBRA1 remains largely unexplored. Here, we report that ectopic expression of COBRA1 inhibited activator protein 1 (AP-1) transcriptional activity in transfected cells in a dose-dependent manner, whereas reduction of endogenous COBRA1 with a small interfering RNA significantly enhanced AP-1-mediated transcriptional activation. COBRA1 physically interacted with the AP-1 family members, c-Jun and c-Fos, and the middle region of COBRA1 bound to c-Fos. Lack of c-Fos binding site in the COBRA1 completely abolished the COBRA1 inhibition of AP-1 trans-activation. These findings suggest that COBRA1 may directly modulate AP-1 pathway and, therefore, may play important roles in cell proliferation, differentiation, apoptosis, and oncogenesis

  9. Luteolin, a flavonoid, inhibits AP-1 activation by basophils

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    Hirano, Toru; Higa, Shinji; Arimitsu, Junsuke; Naka, Tetsuji; Ogata, Atsushi; Shima, Yoshihito; Fujimoto, Minoru; Yamadori, Tomoki; Ohkawara, Tomoharu; Kuwabara, Yusuke; Kawai, Mari; Matsuda, Hisashi; Yoshikawa, Masayuki; Maezaki, Naoyoshi; Tanaka, Tetsuaki; Kawase, Ichiro; Tanaka, Toshio

    2006-01-01

    Flavonoids including luteolin, apigenin, and fisetin are inhibitors of IL-4 synthesis and CD40 ligand expression by basophils. This study was done to search for compounds with greater inhibitory activity of IL-4 expression and to clarify the molecular mechanisms through which flavonoids inhibit their expression. Of the 37 flavonoids and related compounds examined, ayanin, luteolin, and apigenin were the strongest inhibitors of IL-4 production by purified basophils in response to anti-IgE antibody plus IL-3. Luteolin did not suppress Syk or Lyn phosphorylation in basophils, nor did suppress p54/46 SAPK/JNK, p38 MAPK, and p44/42 MAPK activation by a basophilic cell line, KU812 cells, stimulated with A23187 and PMA. However, luteolin did inhibit phosphorylation of c-Jun and DNA binding activity of AP-1 in nuclear lysates from stimulated KU812 cells. These results provide a fundamental structure of flavonoids for IL-4 inhibition and demonstrate a novel action of flavonoids that suppresses the activation of AP-1

  10. Internal structure of magnetic endosomes

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    Rivière, C.; Wilhelm, C.; Cousin, F.; Dupuis, V.; Gazeau, F.; Perzynski, R.

    2007-01-01

    The internal structure of biological vesicles filled with magnetic nanoparticles is investigated using the following complementary analyses: electronic transmission microscopy, dynamic probing by magneto-optical birefringence and structural probing by Small Angle Neutron Scattering (SANS). These magnetic vesicles are magnetic endosomes obtained via a non-specific interaction between cells and anionic magnetic iron oxide nanoparticles. Thanks to a magnetic purification process, they are probed at two different stages of their formation within HeLa cells: (i) adsorption of nanoparticles onto the cellular membrane and (ii) their subsequent internalisation within endosomes. Differences in the microenvironment of the magnetic nanoparticles at those two different stages are highlighted here. The dynamics of magnetic nanoparticles adsorbed onto cellular membranes and confined within endosomes is respectively 3 and 5 orders of magnitude slower than for isolated magnetic nanoparticles in aqueous media. Interestingly, SANS experiments show that magnetic endosomes have an internal structure close to decorated vesicles, with magnetic nanoparticles locally decorating the endosome membrane, inside their inner-sphere. These results, important for future biomedical applications, suggest that multiple fusions of decorated vesicles are the biological processes underlying the endocytosis of that kind of nanometric materials.

  11. Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

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    Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa; San Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2010-11-05

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

  12. Membrane Tethering Complexes in the Endosomal System

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    Spang, Anne

    2016-01-01

    Vesicles that are generated by endocytic events at the plasma membrane are destined to early endosomes. A prerequisite for proper fusion is the tethering of two membrane entities. Tethering of vesicles to early endosomes is mediated by the class C core vacuole/endosome tethering (CORVET) complex, while fusion of late endosomes with lysosomes depends on the homotypic fusion and vacuole protein sorting (HOPS) complex. Recycling through the trans-Golgi network (TGN) and to the plasma membrane is...

  13. Endocytosis and Endosomal Trafficking in Plants.

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    Paez Valencia, Julio; Goodman, Kaija; Otegui, Marisa S

    2016-04-29

    Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants.

  14. The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

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    Diccianni, M B; Imagawa, M; Muramatsu, M

    1992-10-11

    Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis.

  15. The immunobiology and clinical features of type 1 autoimmune polyglandular syndrome (APS-1).

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    Guo, Can-Jie; Leung, Patrick S C; Zhang, Weici; Ma, Xiong; Gershwin, M Eric

    2018-01-01

    Autoimmune Polyglandular Syndrome type 1 (APS-1) is a subtype of the autoimmune polyendocrine syndrome characterized by the simultaneous or sequential dysfunction of multiple endocrine or non-endocrine glands. A clinical diagnosis of APS-1 is typically based on the presence of at least two of three following criteria: chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal insufficiency. The first identified causative mutated gene for APS-1 is autoimmune regulator (AIRE) encoding a critical transcription factor, which is primarily expressed in the medullary thymic epithelial cells (mTECs) for generating central immune tolerance. A wide range of chronic, debilitating complications, with no obvious correlation with genetics, makes a diagnosis of APS-1 challenging early in the disease course. Managing APS-1 is difficult due to its complexity, especially the intricate relationships within manifestations and genetic mutations. The past decades have witnessed dramatic progress in elucidating the function of AIRE and conducting large-scale cohort studies in APS-1. However, no clear evidence-based guidelines have been established in APS-1. In this review, we provide a detailed critical overview of the study history, epidemiology, clinical features, and related mechanisms of autoimmunity in APS-1, as well as currently available therapies for this autoimmune disorder. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice.

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    Hwang, S-K; Jin, H; Kwon, J T; Chang, S-H; Kim, T H; Cho, C-S; Lee, K H; Young, M R; Colburn, N H; Beck, G R; Yang, H-S; Cho, M-H

    2007-09-01

    The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.

  17. Membrane tethering complexes in the endosomal system

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    Anne eSpang

    2016-05-01

    Full Text Available Vesicles that are generated by endocytic events at the plasma membrane are destined to early endosomes. A prerequisite for proper fusion is the tethering of two membrane entities. Tethering of vesicles to early endosomes is mediated by the CORVET complex, while fusion of late endosomes with lysosomes depends on the HOPS complex. Recycling through the TGN and to the plasma membrane is facilitated by the GARP and EARP complexes, respectively. However, there are other tethering functions in the endosomal system as there are multiple pathways through which proteins can be delivered from endosomes to either the TGN or the plasma membrane. Furthermore, complexes that may be part of novel tethering complexes have been recently identified. Thus it is likely that more tethering factors exist. In this review, I will provide an overview of different tethering complexes of the endosomal system and discuss how they may provide specificity in membrane traffic.

  18. Knockdown of CDK2AP1 in human embryonic stem cells reduces the threshold of differentiation.

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    Khaled N Alsayegh

    Full Text Available Recent studies have suggested a role for the Cyclin Dependent Kinase-2 Associated Protein 1 (CDK2AP1 in stem cell differentiation and self-renewal. In studies with mouse embryonic stem cells (mESCs derived from generated mice embryos with targeted deletion of the Cdk2ap1 gene, CDK2AP1 was shown to be required for epigenetic silencing of Oct4 during differentiation, with deletion resulting in persistent self-renewal and reduced differentiation potential. Differentiation capacity was restored in these cells following the introduction of a non-phosphorylatible form of the retinoblastoma protein (pRb or exogenous Cdk2ap1. In this study, we investigated the role of CDK2AP1 in human embryonic stem cells (hESCs. Using a shRNA to reduce its expression in hESCs, we found that CDK2AP1 knockdown resulted in a significant reduction in the expression of the pluripotency genes, OCT4 and NANOG. We also found that CDK2AP1 knockdown increased the number of embryoid bodies (EBs formed when differentiation was induced. In addition, the generated EBs had significantly higher expression of markers of all three germ layers, indicating that CDK2AP1 knockdown enhanced differentiation. CDK2AP1 knockdown also resulted in reduced proliferation and reduced the percentage of cells in the S phase and increased cells in the G2/M phase of the cell cycle. Further investigation revealed that a higher level of p53 protein was present in the CDK2AP1 knockdown hESCs. In hESCs in which p53 and CDK2AP1 were simultaneously downregulated, OCT4 and NANOG expression was not affected and percentage of cells in the S phase of the cell cycle was not reduced. Taken together, our results indicate that the knockdown of CDK2AP1 in hESCs results in increased p53 and enhances differentiation and favors it over a self-renewal fate.

  19. The structure and function of presynaptic endosomes

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    Jähne, Sebastian, E-mail: sebastian.jaehne1@stud.uni-goettingen.de [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); International Max Planck Research School for Neurosciences, 37077 Göttingen (Germany); Rizzoli, Silvio O. [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); Helm, Martin S., E-mail: martin.helm@med.uni-goettingen.de [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); International Max Planck Research School for Molecular Biology, 37077 Göttingen (Germany)

    2015-07-15

    The function of endosomes and of endosome-like structures in the presynaptic compartment is still controversial. This is in part due to the absence of a consensus on definitions and markers for these compartments. Synaptic endosomes are sometimes seen as stable organelles, permanently present in the synapse. Alternatively, they are seen as short-lived intermediates in synaptic vesicle recycling, arising from the endocytosis of large vesicles from the plasma membrane, or from homotypic fusion of small vesicles. In addition, the potential function of the endosome is largely unknown in the synapse. Some groups have proposed that the endosome is involved in the sorting of synaptic vesicle proteins, albeit others have produced data that deny this possibility. In this review, we present the existing evidence for synaptic endosomes, we discuss their potential functions, and we highlight frequent technical pitfalls in the analysis of this elusive compartment. We also sketch a roadmap to definitely determine the role of synaptic endosomes for the synaptic vesicle cycle. Finally, we propose a common definition of synaptic endosome-like structures.

  20. The structure and function of presynaptic endosomes

    International Nuclear Information System (INIS)

    Jähne, Sebastian; Rizzoli, Silvio O.; Helm, Martin S.

    2015-01-01

    The function of endosomes and of endosome-like structures in the presynaptic compartment is still controversial. This is in part due to the absence of a consensus on definitions and markers for these compartments. Synaptic endosomes are sometimes seen as stable organelles, permanently present in the synapse. Alternatively, they are seen as short-lived intermediates in synaptic vesicle recycling, arising from the endocytosis of large vesicles from the plasma membrane, or from homotypic fusion of small vesicles. In addition, the potential function of the endosome is largely unknown in the synapse. Some groups have proposed that the endosome is involved in the sorting of synaptic vesicle proteins, albeit others have produced data that deny this possibility. In this review, we present the existing evidence for synaptic endosomes, we discuss their potential functions, and we highlight frequent technical pitfalls in the analysis of this elusive compartment. We also sketch a roadmap to definitely determine the role of synaptic endosomes for the synaptic vesicle cycle. Finally, we propose a common definition of synaptic endosome-like structures

  1. Enhanced Endosomal Escape by Light-Fueled Liquid-Metal Transformer.

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    Lu, Yue; Lin, Yiliang; Chen, Zhaowei; Hu, Quanyin; Liu, Yang; Yu, Shuangjiang; Gao, Wei; Dickey, Michael D; Gu, Zhen

    2017-04-12

    Effective endosomal escape remains as the "holy grail" for endocytosis-based intracellular drug delivery. To date, most of the endosomal escape strategies rely on small molecules, cationic polymers, or pore-forming proteins, which are often limited by the systemic toxicity and lack of specificity. We describe here a light-fueled liquid-metal transformer for effective endosomal escape-facilitated cargo delivery via a chemical-mechanical process. The nanoscale transformer can be prepared by a simple approach of sonicating a low-toxicity liquid-metal. When coated with graphene quantum dots (GQDs), the resulting nanospheres demonstrate the ability to absorb and convert photoenergy to drive the simultaneous phase separation and morphological transformation of the inner liquid-metal core. The morphological transformation from nanospheres to hollow nanorods with a remarkable change of aspect ratio can physically disrupt the endosomal membrane to promote endosomal escape of payloads. This metal-based nanotransformer equipped with GQDs provides a new strategy for facilitating effective endosomal escape to achieve spatiotemporally controlled drug delivery with enhanced efficacy.

  2. The Role of Activator Protein-1 (AP-1) Family Members in CD30-Positive Lymphomas

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    Garces de los Fayos Alonso, Ines; Lagger, Sabine; Merkel, Olaf; Kenner, Lukas

    2018-01-01

    The Activator Protein-1 (AP-1) transcription factor (TF) family, composed of a variety of members including c-JUN, c-FOS and ATF, is involved in mediating many biological processes such as proliferation, differentiation and cell death. Since their discovery, the role of AP-1 TFs in cancer development has been extensively analysed. Multiple in vitro and in vivo studies have highlighted the complexity of these TFs, mainly due to their cell-type specific homo- or hetero-dimerization resulting in diverse transcriptional response profiles. However, as a result of the increasing knowledge of the role of AP-1 TFs in disease, these TFs are being recognized as promising therapeutic targets for various malignancies. In this review, we focus on the impact of deregulated expression of AP-1 TFs in CD30-positive lymphomas including Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma. PMID:29597249

  3. Synaptic and genomic responses to JNK and AP-1 signaling in Drosophila neurons

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    Bohmann Dirk

    2005-06-01

    Full Text Available Abstract Background The transcription factor AP-1 positively controls synaptic plasticity at the Drosophila neuromuscular junction. Although in motor neurons, JNK has been shown to activate AP-1, a positive regulator of growth and strength at the larval NMJ, the consequences of JNK activation are poorly studied. In addition, the downstream transcriptional targets of JNK and AP-1 signaling in the Drosophila nervous system have yet to be identified. Here, we further investigated the role of JNK signaling at this model synapse employing an activated form of JNK-kinase; and using Serial Analysis of Gene Expression and oligonucleotide microarrays, searched for candidate early targets of JNK or AP-1 dependent transcription in neurons. Results Temporally-controlled JNK induction in postembryonic motor neurons triggers synaptic growth at the NMJ indicating a role in developmental plasticity rather than synaptogenesis. An unexpected observation that JNK activation also causes a reduction in transmitter release is inconsistent with JNK functioning solely through AP-1 and suggests an additional, yet-unidentified pathway for JNK signaling in motor neurons. SAGE profiling of mRNA expression helps define the neural transcriptome in Drosophila. Though many putative AP-1 and JNK target genes arose from the genomic screens, few were confirmed in subsequent validation experiments. One potentially important neuronal AP-1 target discovered, CG6044, was previously implicated in olfactory associative memory. In addition, 5 mRNAs regulated by RU486, a steroid used to trigger conditional gene expression were identified. Conclusion This study demonstrates a novel role for JNK signaling at the larval neuromuscular junction and provides a quantitative profile of gene transcription in Drosophila neurons. While identifying potential JNK/AP-1 targets it reveals the limitations of genome-wide analyses using complex tissues like the whole brain.

  4. Ectopic expression of Jatropha curcas APETALA1 (JcAP1) caused early flowering in Arabidopsis, but not in Jatropha

    Science.gov (United States)

    Tang, Mingyong; Tao, Yan-Bin

    2016-01-01

    Jatropha curcas is a promising feedstock for biofuel production because Jatropha oil is highly suitable for the production of biodiesel and bio-jet fuels. However, Jatropha exhibits a low seed yield as a result of unreliable and poor flowering. APETALA1 (AP1) is a floral meristem and organ identity gene in higher plants. The flower meristem identity genes of Jatropha have not yet been identified or characterized. To better understand the genetic control of flowering in Jatropha, an AP1 homolog (JcAP1) was isolated from Jatropha. An amino acid sequence analysis of JcAP1 revealed a high similarity to the AP1 proteins of other perennial plants. JcAP1 was expressed in inflorescence buds, flower buds, sepals and petals. The highest expression level was observed during the early developmental stage of the flower buds. The overexpression of JcAP1 using the cauliflower mosaic virus (CaMV) 35S promoter resulted in extremely early flowering and abnormal flowers in transgenic Arabidopsis plants. Several flowering genes downstream of AP1 were up-regulated in the JcAP1-overexpressing transgenic plant lines. Furthermore, JcAP1 overexpression rescued the phenotype caused by the Arabidopsis AP1 loss-of-function mutant ap1-11. Therefore, JcAP1 is an ortholog of AtAP1, which plays a similar role in the regulation of flowering in Arabidopsis. However, the overexpression of JcAP1 in Jatropha using the same promoter resulted in little variation in the flowering time and floral organs, indicating that JcAP1 may be insufficient to regulate flowering by itself in Jatropha. This study helps to elucidate the function of JcAP1 and contributes to the understanding of the molecular mechanisms of flower development in Jatropha. PMID:27168978

  5. Ectopic expression of Jatropha curcas APETALA1 (JcAP1 caused early flowering in Arabidopsis, but not in Jatropha

    Directory of Open Access Journals (Sweden)

    Mingyong Tang

    2016-04-01

    Full Text Available Jatropha curcas is a promising feedstock for biofuel production because Jatropha oil is highly suitable for the production of biodiesel and bio-jet fuels. However, Jatropha exhibits a low seed yield as a result of unreliable and poor flowering. APETALA1 (AP1 is a floral meristem and organ identity gene in higher plants. The flower meristem identity genes of Jatropha have not yet been identified or characterized. To better understand the genetic control of flowering in Jatropha, an AP1 homolog (JcAP1 was isolated from Jatropha. An amino acid sequence analysis of JcAP1 revealed a high similarity to the AP1 proteins of other perennial plants. JcAP1 was expressed in inflorescence buds, flower buds, sepals and petals. The highest expression level was observed during the early developmental stage of the flower buds. The overexpression of JcAP1 using the cauliflower mosaic virus (CaMV 35S promoter resulted in extremely early flowering and abnormal flowers in transgenic Arabidopsis plants. Several flowering genes downstream of AP1 were up-regulated in the JcAP1-overexpressing transgenic plant lines. Furthermore, JcAP1 overexpression rescued the phenotype caused by the Arabidopsis AP1 loss-of-function mutant ap1-11. Therefore, JcAP1 is an ortholog of AtAP1, which plays a similar role in the regulation of flowering in Arabidopsis. However, the overexpression of JcAP1 in Jatropha using the same promoter resulted in little variation in the flowering time and floral organs, indicating that JcAP1 may be insufficient to regulate flowering by itself in Jatropha. This study helps to elucidate the function of JcAP1 and contributes to the understanding of the molecular mechanisms of flower development in Jatropha.

  6. Expression of activator protein-1 (AP-1) family members in breast cancer

    International Nuclear Information System (INIS)

    Kharman-Biz, Amirhossein; Gao, Hui; Ghiasvand, Reza; Zhao, Chunyan; Zendehdel, Kazem; Dahlman-Wright, Karin

    2013-01-01

    The activator protein-1 (AP-1) transcription factor is believed to be important in tumorigenesis and altered AP-1 activity was associated with cell transformation. We aimed to assess the potential role of AP-1 family members as novel biomarkers in breast cancer. We studied the expression of AP-1 members at the mRNA level in 72 primary breast tumors and 37 adjacent non-tumor tissues and evaluated its correlation with clinicopathological parameters including estrogen receptor (ER), progesterone receptor (PR) and HER2/neu status. Expression levels of Ubiquitin C (UBC) were used for normalization. Protein expression of AP-1 members was assessed using Western blot analysis in a subset of tumors. We used student’s t-test, one-way ANOVA, logistic regression and Pearson’s correlation coefficient for statistical analyses. We found significant differences in the expression of AP-1 family members between tumor and adjacent non-tumor tissues for all AP-1 family members except Fos B. Fra-1, Fra-2, Jun-B and Jun-D mRNA levels were significantly higher in tumors compared to adjacent non-tumor tissues (p < 0.001), whilst c-Fos and c-Jun mRNA levels were significantly lower in tumors compared with adjacent non-tumor tissues (p < 0.001). In addition, Jun-B overexpression had outstanding discrimination ability to differentiate tumor tissues from adjacent non-tumor tissues as determined by ROC curve analysis. Moreover, Fra-1 was significantly overexpressed in the tumors biochemically classified as ERα negative (p = 0.012) and PR negative (p = 0.037). Interestingly, Fra-1 expression was significantly higher in triple-negative tumors compared with luminal carcinomas (p = 0.01). Expression levels of Fra-1 and Jun-B might be possible biomarkers for prognosis of breast cancer

  7. AP-1 Oligodeoxynucleotides Reduce Aortic Elastolysis in a Murine Model of Marfan Syndrome.

    Science.gov (United States)

    Arif, Rawa; Zaradzki, Marcin; Remes, Anca; Seppelt, Philipp; Kunze, Reiner; Schröder, Hannes; Schwill, Simon; Ensminger, Stephan M; Robinson, Peter N; Karck, Matthias; Müller, Oliver J; Hecker, Markus; Wagner, Andreas H; Kallenbach, Klaus

    2017-12-15

    Marfan syndrome is characterized by high expression of matrix metalloproteinases (MMPs) in aortic smooth muscle cells (AoSMCs) associated with medial elastolysis and aortic root aneurysm. We aimed to reduce aortic elastolysis through decrease of MMP expression with decoy oligodeoxynucleotides (dODNs) neutralizing the transcription factor activating factor-1 (AP-1). AP-1 abundance in nuclear extracts as well as MMP-2 and MMP-9 expression were significantly increased in isolated mAoSMC of mgR/mgR Marfan mice compared to wild-type cells. Exposure to AP-1 neutralizing dODNs resulted in a significant reduction of basal and interleukin-1β-stimulated MMP expression and activity in mAoSMCs. Moreover, increased migration and formation of superoxide radical anions was substantially decreased in mAoSMCs by AP-1 dODN treatment. Aortic grafts from donor Marfan mice were treated with AP-1- dODN ex vivo and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Pretreatment of aortic grafts with AP-1 dODN led to reduced elastolysis, macrophage infiltration, and MMP activity. Permeability of the endothelial monolayer was increased for dODN in mgR/mgR aortae with observed loss of tight junction proteins ZO-1 and occludin, enabling dODN to reach the tunica media. Targeting AP-1 activity offers a new potential strategy to treat the vascular phenotype associated with Marfan syndrome. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Effects of environmental estrogenic chemicals on AP1 mediated transcription with estrogen receptors alpha and beta.

    Science.gov (United States)

    Fujimoto, Nariaki; Honda, Hiroaki; Kitamura, Shigeyuki

    2004-01-01

    There has been much discussion concerning endocrine disrupting chemicals suspected of exerting adverse effects in both wildlife and humans. Since the majority of these compounds are estrogenic, a large number of in vitro tests for estrogenic characteristics have been developed for screening purpose. One reliable and widely used method is the reporter gene assay employing estrogen receptors (ERs) and a reporter gene with a cis-acting estrogen responsive element (ERE). Other elements such as AP1 also mediate estrogenic signals and the manner of response could be quite different from that of ERE. Since this has yet to be explored, the ER mediated AP1 activity in response to a series of environmental estrogens was investigated in comparison with ERE findings. All the compounds exhibited estrogenic properties with ERE-luc and their AP1 responses were quite similar. These was one exception, however, p,p'-DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane) did not exert any AP1-luc activity, while it appeared to be estrogenic at 10(-7) to 10(-5)M with the ERE action. None of the compounds demonstrated ER beta:AP1 activity. These data suggest that significant differences can occur in responses through the two estrogen pathways depending on environmental chemicals.

  9. The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

    OpenAIRE

    Diccianni, M B; Imagawa, M; Muramatsu, M

    1992-01-01

    Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lac...

  10. Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking.

    Directory of Open Access Journals (Sweden)

    Komla Sobo

    Full Text Available BACKGROUND: Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2-3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. CONCLUSIONS/SIGNIFICANCE: These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.

  11. Energy expenditure and bone formation share a common sensitivity to AP-1 transcription in the hypothalamus

    DEFF Research Database (Denmark)

    Rowe, Glenn C; Vialou, Vincent; Sato, Kazusa

    2012-01-01

    ) whether these effects were due to antagonism to AP1. Our results show that stereotactic injection of an adeno-associated virus vector to restrict overexpression of ¿FosB to the ventral hypothalamus of wildtype mice induced a profound increase in both energy expenditure and bone formation and bone mass...

  12. Prevention of Breast Cancer Cell Transformation by Blockade of the AP-1 Transcription Factor

    Science.gov (United States)

    2001-10-01

    to transfection. pLPCX-TAM67 and control plasmids were transfected with Fugene 6 Regent (Roche) according to manufacture’s protocol. Chloroquine was...breast tu- mor growth with nonisomerizable analogues of similar to those in other reports (27,28). whether increasing or inhibiting AP-1 ac- tamoxifen

  13. AP-1-Targeting Anti-Inflammatory Activity of the Methanolic Extract of Persicaria chinensis

    Directory of Open Access Journals (Sweden)

    Muhammad Jahangir Hossen

    2015-01-01

    Full Text Available In traditional Chinese medicine, Persicaria chinensis L. has been prescribed to cure numerous inflammatory disorders. We previously analyzed the bioactivity of the methanol extract of this plant (Pc-ME against LPS-induced NO and PGE2 in RAW264.7 macrophages and found that it prevented HCl/EtOH-induced gastric ulcers in mice. The purpose of the current study was to explore the molecular mechanism by which Pc-ME inhibits activator protein- (AP- 1 activation pathway and mediates its hepatoprotective activity. To investigate the putative therapeutic properties of Pc-ME against AP-1-mediated inflammation and hepatotoxicity, lipopolysaccharide- (LPS- stimulated RAW264.7 and U937 cells, a monocyte-like human cell line, and an LPS/D-galactosamine- (D-GalN- induced acute hepatitis mouse model were employed. The expression of LPS-induced proinflammatory cytokines including interleukin- (IL- 1β, IL-6, and tumor necrosis factor-α (TNF-α was significantly diminished by Pc-ME. Moreover, Pc-ME reduced AP-1 activation and mitogen-activated protein kinase (MAPK phosphorylation in both LPS-stimulated RAW264.7 cells and differentiated U937 cells. Additionally, we highlighted the hepatoprotective and curative effects of Pc-ME pretreated orally in a mouse model of LPS/D-GalN-intoxicated acute liver injury by demonstrating the significant reduction in elevated serum AST and ALT levels and histological damage. Therefore, these results strongly suggest that Pc-ME could function as an antihepatitis remedy suppressing MAPK/AP-1-mediated inflammatory events.

  14. Hepatic stellate cells lack AP-1 responsiveness to electrophiles and phorbol 12-myristate-13-acetate

    International Nuclear Information System (INIS)

    Reichard, John F.; Petersen, Dennis R.

    2004-01-01

    Stellate cell profibrotic gene induction and transdifferentiation are central events in liver fibrosis. Oxidative stress has been implicated as an activator of the transcription factors Nrf2 and AP-1 through shared kinase signaling pathways that also purportedly contribute to stellate cell activation. The present study examined the role of oxidative stress in ARE- and TRE-regulated gene induction in isolated hepatic stellate cells. Using a portion of the human Nqo1 promoter consisting of an ARE imbedded TRE, it was demonstrated that while the ARE was responsible for mediating inducible gene expression in response to the electrophiles 4-HNE and tBHQ, the TRE was refractory to induction by either electrophiles or PMA. It was demonstrated that stellate cells possess nuclear TRE-binding proteins that were identified as JunB, JunD, Fra1, and Fra2, which were unaffected by either electrophiles or PMA treatment. This report demonstrates that, in contrast to the ARE, the TRE and its binding cognate AP-1 did not mediate independent gene induction in hepatic stellate cells. This observation is significant given the presumed importance attributed to AP-1 in mediating profibrogenic gene expression

  15. Co-expression analysis identifies CRC and AP1 the regulator of Arabidopsis fatty acid biosynthesis.

    Science.gov (United States)

    Han, Xinxin; Yin, Linlin; Xue, Hongwei

    2012-07-01

    Fatty acids (FAs) play crucial rules in signal transduction and plant development, however, the regulation of FA metabolism is still poorly understood. To study the relevant regulatory network, fifty-eight FA biosynthesis genes including de novo synthases, desaturases and elongases were selected as "guide genes" to construct the co-expression network. Calculation of the correlation between all Arabidopsis thaliana (L.) genes with each guide gene by Arabidopsis co-expression dating mining tools (ACT) identifies 797 candidate FA-correlated genes. Gene ontology (GO) analysis of these co-expressed genes showed they are tightly correlated to photosynthesis and carbohydrate metabolism, and function in many processes. Interestingly, 63 transcription factors (TFs) were identified as candidate FA biosynthesis regulators and 8 TF families are enriched. Two TF genes, CRC and AP1, both correlating with 8 FA guide genes, were further characterized. Analyses of the ap1 and crc mutant showed the altered total FA composition of mature seeds. The contents of palmitoleic acid, stearic acid, arachidic acid and eicosadienoic acid are decreased, whereas that of oleic acid is increased in ap1 and crc seeds, which is consistent with the qRT-PCR analysis revealing the suppressed expression of the corresponding guide genes. In addition, yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) revealed that CRC can bind to the promoter regions of KCS7 and KCS15, indicating that CRC may directly regulate FA biosynthesis. © 2012 Institute of Botany, Chinese Academy of Sciences.

  16. AP-1 proteins in the adult brain: facts and fiction about effectors of neuroprotection and neurodegeneration.

    Science.gov (United States)

    Herdegen, T; Waetzig, V

    2001-04-30

    Jun and Fos proteins are induced and activated following most physiological and pathophysiological stimuli in the brain. Only few data allow conclusions about distinct functions of AP-1 proteins in neurodegeneration and neuroregeneration, and these functions mainly refer to c-Jun and its activation by JNKs. Apoptotic functions of activated c-Jun affect hippocampal, nigral and primary cultured neurons following excitotoxic stimulation and destruction of the neuron-target-axis including withdrawal of trophic molecules. The inhibition of JNKs might exert neuroprotection by subsequent omission of c-Jun activation. Besides endogenous neuronal functions, the c-Jun/AP-1 proteins can damage the nervous system by upregulation of harmful programs in non-neuronal cells (e.g. microglia) with release of neurodegenerative molecules. In contrast, the differentiation with neurite extension and maturation of neural cells in vitro indicate physiological and potentially neuroprotective functions of c-Jun and JNKs including sensoring for alterations in the cytoskeleton. This review summarizes the multiple molecular interfunctions which are involved in the shift from the physiological role to degenerative effects of the Jun/JNK-axis such as cell type-specific expression and intracellular localization of scaffold proteins and upstream activators, antagonistic phosphatases, interaction with other kinase systems, or the activation of transcription factors competing for binding to JNK proteins and AP-1 DNA elements.

  17. Mutations in ap1b1 cause mistargeting of the Na(+/K(+-ATPase pump in sensory hair cells.

    Directory of Open Access Journals (Sweden)

    Rachel Clemens Grisham

    Full Text Available The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1 gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na(+/K(+-ATPase pump (NKA was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na(+ levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells.

  18. Transcription factor AP-1 in esophageal squamous cell carcinoma: Alterations in activity and expression during Human Papillomavirus infection

    International Nuclear Information System (INIS)

    Hussain, Showket; Bharti, Alok C; Salam, Irfana; Bhat, Mohammad Akbar; Mir, Mohammad Muzaffar; Hedau, Suresh; Siddiqi, Mushtaq A; Basir, Seemi Farhat; Das, Bhudev C

    2009-01-01

    Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related deaths in Jammu and Kashmir (J&K) region of India. A substantial proportion of esophageal carcinoma is associated with infection of high-risk HPV type 16 and HPV18, the oncogenic expression of which is controlled by host cell transcription factor Activator Protein-1 (AP-1). We, therefore, have investigated the role of DNA binding and expression pattern of AP-1 in esophageal cancer with or without HPV infection. Seventy five histopathologically-confirmed esophageal cancer and an equal number of corresponding adjacent normal tissue biopsies from Kashmir were analyzed for HPV infection, DNA binding activity and expression of AP-1 family of proteins by PCR, gel shift assay and immunoblotting respectively. A high DNA binding activity and elevated expression of AP-1 proteins were observed in esophageal cancer, which differed between HPV positive (19%) and HPV negative (81%) carcinomas. While JunB, c-Fos and Fra-1 were the major contributors to AP-1 binding activity in HPV negative cases, Fra-1 was completely absent in HPV16 positive cancers. Comparison of AP-1 family proteins demonstrated high expression of JunD and c-Fos in HPV positive tumors, but interestingly, Fra-1 expression was extremely low or nil in these tumor tissues. Differential AP-1 binding activity and expression of its specific proteins between HPV - positive and HPV - negative cases indicate that AP-1 may play an important role during HPV-induced esophageal carcinogenesis

  19. Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network.

    Science.gov (United States)

    Raub, T J; Koroly, M J; Roberts, R M

    1990-04-01

    By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the

  20. Isolation and characterization of the Jatropha curcas APETALA1 (JcAP1) promoter conferring preferential expression in inflorescence buds.

    Science.gov (United States)

    Tao, Yan-Bin; He, Liang-Liang; Niu, Longjian; Xu, Zeng-Fu

    2016-08-01

    The 1.5 kb JcAP1 promoter from the biofuel plant Jatropha curcas is predominantly active in the inflorescence buds of transgenic plants, in which the -1313/-1057 region is essential for maintaining the activity. Arabidopsis thaliana APETALA1 (AP1) is a MADS-domain transcription factor gene that functions primarily in flower development. We isolated a homolog of AP1 from Jatropha curcas (designated JcAP1), which was shown to exhibit flower-specific expression in Jatropha. JcAP1 is first expressed in inflorescence buds and continues to be primarily expressed in the sepals. We isolated a 1.5 kb JcAP1 promoter and evaluated its activity in transgenic Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. In transgenic Arabidopsis and Jatropha, the inflorescence buds exhibited notable GUS activity, whereas the sepals did not. Against expectations, the JcAP1 promoter was active in the anthers of Arabidopsis and Jatropha and was highly expressed in Jatropha seeds. An analysis of promoter deletions in transgenic Arabidopsis revealed that deletion of the -1313/-1057 region resulted in loss of JcAP1 promoter activity in the inflorescence buds and increased activity in the anthers. These results suggested that some regulatory sequences in the -1313/-1057 region are essential for maintaining promoter activity in inflorescence buds and can partly suppress activity in the anthers. Based on these findings, we hypothesized that other elements located upstream of the 1.5 kb JcAP1 promoter may be required for flower-specific activation. The JcAP1 promoter characterized in this study can be used to drive transgene expression in both the inflorescence buds and seeds of Jatropha.

  1. Temporal pattern of AP-1 DNA-binding activity in the rat hippocampus following a kindled seizure

    Energy Technology Data Exchange (ETDEWEB)

    Shomori, T. [Department of Neurology, Okayama University Medical School, 2-5-1, Shikata-cho Okayama (Japan); Hayabara, T. [Clinical Research Institute, National Sanatorium Minamiokayama Hospital, 4066 Hayashima-cho (Japan); Ishihara, T. [Department of Neuropsychiatry, Okayama University Medical School, 2-5-1 Shikata-cho Okayama (Japan); Okada, S. [Department of Neurology, Okayama University Medical School, 2-5-1, Shikata-cho Okayama (Japan); Akiyama, K. [Department of Neuropsychiatry, Okayama University Medical School, 2-5-1 Shikata-cho Okayama (Japan); Sato, K. [Clinical Research Institute, National Sanatorium Minamiokayama Hospital, 4066 Hayashima-cho (Japan); Kashihara, K. [Department of Neurology, Okayama University Medical School, 2-5-1, Shikata-cho Okayama (Japan)

    1997-07-28

    DNA binding by transcripton factor AP-1 was enhanced remarkably following kindling stimulation in rat amygdala. Maximum increase occurred 2 h after stimulation with return to baseline within 24 h. Supershift and western analyses revealed that 38,000 mol. wt Fos-related antigen and JunD were the main components of the evoked AP-1 complexes at the time their induction reached maximum. AP-1 induction 2 h after the last kindling stimulation was more prominent in samples from previously kindled rats than in those from non-kindled rats. This study sought to establish the role of AP-1 in plastic changes of the hippocampus associated with kindling. Male Sprague-Dawley rats were kindled from the left amygdala until they exhibited Racine [15] class 5 generalized seizures. Nuclear proteins were extracted from dorsal hippocampi obtained from 0 to 24 h after final stimulations. From these, we evaluated the temporal pattern of DNA binding by AP-1 using a gel mobility-shift assay with a {sup 32}P-labelled AP-1 probe. Supershift and western analyses were added to investigate components of the seizure-evoked AP-1 complexes. Our results suggest that the basal level of AP-1 complexes is not associated with the seizure susceptibility in kindling. However, development of kindling appears to facilitate stimulus-evoked AP-1 induction, probably via plastic changes in the central nervous system. AP-1 may mediate such changes by regulating expression of certain genes. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  2. Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1

    International Nuclear Information System (INIS)

    Kwon, Ho-Keun; Lee, Sung Haeng; Park, Zee Yong; Im, Sin-Hyeog; Hwang, Ji-Sun; So, Jae-Seon; Lee, Choong-Gu; Sahoo, Anupama; Ryu, Jae-Ha; Jeon, Won Kyung; Ko, Byoung Seob; Im, Chang-Rok

    2010-01-01

    Cinnamomum cassia bark is the outer skin of an evergreen tall tree belonging to the family Lauraceae containing several active components such as essential oils (cinnamic aldehyde and cinnamyl aldehyde), tannin, mucus and carbohydrate. They have various biological functions including anti-oxidant, anti-microbial, anti-inflammation, anti-diabetic and anti-tumor activity. Previously, we have reported that anti-cancer effect of cinnamon extracts is associated with modulation of angiogenesis and effector function of CD8 + T cells. In this study, we further identified that anti-tumor effect of cinnamon extracts is also link with enhanced pro-apoptotic activity by inhibiting the activities NFκB and AP1 in mouse melanoma model. Water soluble cinnamon extract was obtained and quality of cinnamon extract was evaluated by HPLC (High Performance Liquid Chromatography) analysis. In this study, we tested anti-tumor activity and elucidated action mechanism of cinnamon extract using various types of tumor cell lines including lymphoma, melanoma, cervix cancer and colorectal cancer in vitro and in vivo mouse melanoma model. Cinnamon extract strongly inhibited tumor cell proliferation in vitro and induced active cell death of tumor cells by up-regulating pro-apoptotic molecules while inhibiting NFκB and AP1 activity and their target genes such as Bcl-2, BcL-xL and survivin. Oral administration of cinnamon extract in melanoma transplantation model significantly inhibited tumor growth with the same mechanism of action observed in vitro. Our study suggests that anti-tumor effect of cinnamon extracts is directly linked with enhanced pro-apoptotic activity and inhibition of NFκB and AP1 activities and their target genes in vitro and in vivo mouse melanoma model. Hence, further elucidation of active components of cinnamon extract could lead to development of potent anti-tumor agent or complementary and alternative medicine for the treatment of diverse cancers

  3. Depletion of the AP-1 repressor JDP2 induces cell death similar to apoptosis

    DEFF Research Database (Denmark)

    Lerdrup, Mads; Holmberg, Christian Henrik; Dietrich, Nikolaj

    2005-01-01

    JDP2 is a ubiquitously expressed nuclear protein that efficiently represses the activity of the transcription factor AP-1. Thus far, all studies of JDP2 function have relied on the ectopic expression of the protein. In this study, we use a different approach: depletion of JDP2 from cells. Specific...... depletion of JDP2 resulted in p53-independent cell death that resembles apoptosis and was evident at 72 h. The death mechanism was caspase dependent as the cells could be rescued by treatment with caspase inhibitor zVAD. Our studies suggest that JDP2 functions as a general survival protein, not only...

  4. Arsenic may be involved in fluoride-induced bone toxicity through PTH/PKA/AP1 signaling pathway.

    Science.gov (United States)

    Zeng, Qi-bing; Xu, Yu-yan; Yu, Xian; Yang, Jun; Hong, Feng; Zhang, Ai-hua

    2014-01-01

    Chronic exposure to combined fluoride and arsenic continues to be a major public health problem worldwide, affecting thousands of people. In recent years, more and more researchers began to focus on the interaction between the fluorine and the arsenic. In this study, the selected investigation site was located in China. The study group was selected from people living in fluoride-arsenic polluted areas due to burning coal. The total number of participants was 196; including the fluoride-arsenic anomaly group (130) and the fluoride-arsenic normal group (63). By observing the changes in gene and protein expression of PTH/PKA/AP1 signaling pathway, the results show that fluoride can increase the expression levels of PTH, PKA, and AP1, but arsenic can only affect the expression of AP1; fluoride and arsenic have an interaction on the expression of AP1. Further study found that fluoride and arsenic can affect the mRNA expression level of c-fos gene (AP1 family members), and have an interaction on the expression of c-fos, but not c-jun. The results indicate that PTH/PKA/AP1 signaling pathway may play an important role in bone toxicity of fluoride. Arsenic can affect the expression of c-fos, thereby affecting the expression of transcription factor AP1, indirectly involved in fluoride-induced bone toxicity. Copyright © 2013. Published by Elsevier B.V.

  5. Neurotrophin signaling endosomes; biogenesis, regulation, and functions

    Science.gov (United States)

    Yamashita, Naoya; Kuruvilla, Rejji

    2016-01-01

    In the nervous system, communication between neurons and their post-synaptic target cells is critical for the formation, refinement and maintenance of functional neuronal connections. Diffusible signals secreted by target tissues, exemplified by the family of neurotrophins, impinge on nerve terminals to influence diverse developmental events including neuronal survival and axonal growth. Key mechanisms of action of target-derived neurotrophins include the cell biological processes of endocytosis and retrograde trafficking of their Trk receptors from growth cones to cell bodies. In this review, we summarize the molecular mechanisms underlying this endosome-mediated signaling, focusing on the instructive role of neurotrophin signaling itself in directing its own trafficking. Recent studies have linked impaired neurotrophin trafficking to neurodevelopmental disorders, highlighting the relevance of neurotrophin endosomes in human health. PMID:27327126

  6. Conformational biosensors reveal GPCR signalling from endosomes

    DEFF Research Database (Denmark)

    Irannejad, R; Tomshine, Jin C; Tomshine, Jon R

    2013-01-01

    A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited...... or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute...... in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane...

  7. c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.

    Science.gov (United States)

    Grassilli, E; Bellesia, E; Salomoni, P; Croce, M A; Sikora, E; Radziszewska, E; Tesco, G; Vergelli, M; Latorraca, S; Barbieri, D; Fagiolo, U; Santacaterina, S; Amaducci, L; Tiozzo, R; Sorbi, S; Franceschi, C

    1996-09-13

    In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully.

  8. The V-ATPase a2-subunit as a putative endosomal pH-sensor.

    Science.gov (United States)

    Marshansky, V

    2007-11-01

    V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

  9. Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

    Directory of Open Access Journals (Sweden)

    Andreea Popa

    2015-02-01

    Full Text Available Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

  10. The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

    Science.gov (United States)

    Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

    2016-10-01

    USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

  11. Andrographolide suppresses high glucose-induced fibronectin expression in mesangial cells via inhibiting the AP-1 pathway.

    Science.gov (United States)

    Lan, Tian; Wu, Teng; Gou, Hongju; Zhang, Qianqian; Li, Jiangchao; Qi, Cuiling; He, Xiaodong; Wu, Pingxiang; Wang, Lijing

    2013-11-01

    Mesangial cells (MCs) proliferation and accumulation of glomerular matrix proteins such as fibronectin (FN) are the early features of diabetic nephropathy, with MCs known to upregulate matrix protein synthesis in response to high glucose. Recently, it has been found that andrographolide has renoprotective effects on diabetic nephropathy. However, the molecular mechanism underlying these effects remains unclear. Cell viability and proliferation was evaluated by MTT. FN expression was examined by immunofluorescence and immunoblotting. Activator protein-1 (AP-1) activation was assessed by immunoblotting, luciferase reporter and electrophoretic mobility shift assays. Andrographolide significantly decreased high glucose-induced cell proliferation and FN expression in MCs. Exposure of MCs to high glucose markedly stimulated the expression of phosphorylated c-jun, whereas the stimulation was inhibited by andrographolide. Plasmid pAP-1-Luc luciferase reporter assay showed that andrographolide blocked high glucose-induced AP-1 transcriptional activity. EMSA assay demonstrated that increased AP-1 binding to an AP-1 binding site at -1,029 in the FN gene promoter upon high glucose stimulation, and the binding were disrupted by andrographolide treatment. These data indicate that andrographolide suppresses high glucose-induced FN expression by inhibiting AP-1-mediated pathway. © 2013 Wiley Periodicals, Inc.

  12. Promotion of Homologous Recombination and Genomic Stability byRAD51AP1 via RAD51 Recombinase Enhancement

    Energy Technology Data Exchange (ETDEWEB)

    Wiese, Claudia; Dray, Eloise; Groesser, Torsten; San Filippo,Joseph; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams,Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-04-11

    Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

  13. Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7.

    Directory of Open Access Journals (Sweden)

    Yu-Wen Chu

    Full Text Available ZAC, an encoding gene mapped at chromosome 6q24-q25 within PSORS1, was previously found over-expressed in the lower compartment of the hyperplastic epidermis in psoriatic lesions. Cytokines produced in the inflammatory dermatoses may drive AP-1 transcription factor to induce responsive gene expressions. We demonstrated that mZac1 can enhance AP-1-responsive S100A7 expression of which the encoding gene was located in PSORS4 with HaCaT keratinocytes. However, the mZac1-enhanced AP-1 transcriptional activity was suppressed by curcumin, indicating the anti-inflammatory property of this botanical agent and is exhibited by blocking the AP-1-mediated cross-talk between PSORS1 and PSORS4. Two putative AP-1-binding sites were found and demonstrated to be functionally important in the regulation of S100A7 promoter activity. Moreover, we found curcumin reduced the DNA-binding activity of AP-1 to the recognition element located in the S100A7 promoter. The S100A7 expression was found to be upregulated in the lesioned epidermis of atopic dermatitis and psoriasis, which is where this keratinocyte-derived chemoattractant engaged in the pro-inflammatory feedback loop. Understanding the regulatory mechanism of S100A7 expression will be helpful to develop therapeutic strategies for chronic inflammatory dermatoses via blocking the reciprocal stimuli between the inflammatory cells and keratinocytes.

  14. MAPK/AP-1-Targeted Anti-Inflammatory Activities of Xanthium strumarium.

    Science.gov (United States)

    Hossen, Muhammad Jahangir; Kim, Mi-Yeon; Cho, Jae Youl

    2016-01-01

    Xanthium strumarium L. (Asteraceae), a traditional Chinese medicine, is prescribed to treat arthritis, bronchitis, and rhinitis. Although the plant has been used for many years, the mechanism by which it ameliorates various inflammatory diseases is not yet fully understood. To explore the anti-inflammatory mechanism of methanol extracts of X. strumarium (Xs-ME) and its therapeutic potential, we used lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells and human monocyte-like U937 cells as well as a LPS/D-galactosamine (GalN)-induced acute hepatitis mouse model. To find the target inflammatory pathway, we used holistic immunoblotting analysis, reporter gene assays, and mRNA analysis. Xs-ME significantly suppressed the up-regulation of both the activator protein (AP)-1-mediated luciferase activity and the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1[Formula: see text], IL-6, and tumor necrosis factor (TNF)-[Formula: see text]. Moreover, Xs-ME strongly inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW264.7 and U937 cells. Additionally, these results highlighted the hepatoprotective and curative effects of Xs-ME in a mouse model of LPS/D-GalN-induced acute liver injury, as assessed by elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and histological damage. Therefore, our results strongly suggest that the ethnopharmacological roles of Xs-ME in hepatitis and other inflammatory diseases might result from its inhibitory activities on the inflammatory signaling of MAPK and AP-1.

  15. AP1 transcription factors are required to maintain the peripheral taste system.

    Science.gov (United States)

    Shandilya, Jayasha; Gao, Yankun; Nayak, Tapan K; Roberts, Stefan G E; Medler, Kathryn F

    2016-10-27

    The sense of taste is used by organisms to achieve the optimal nutritional requirement and avoid potentially toxic compounds. In the oral cavity, taste receptor cells are grouped together in taste buds that are present in specialized taste papillae in the tongue. Taste receptor cells are the cells that detect chemicals in potential food items and transmit that information to gustatory nerves that convey the taste information to the brain. As taste cells are in contact with the external environment, they can be damaged and are routinely replaced throughout an organism's lifetime to maintain functionality. However, this taste cell turnover loses efficiency over time resulting in a reduction in taste ability. Currently, very little is known about the mechanisms that regulate the renewal and maintenance of taste cells. We therefore performed RNA-sequencing analysis on isolated taste cells from 2 and 6-month-old mice to determine how alterations in the taste cell-transcriptome regulate taste cell maintenance and function in adults. We found that the activator protein-1 (AP1) transcription factors (c-Fos, Fosb and c-Jun) and genes associated with this pathway were significantly downregulated in taste cells by 6 months and further declined at 12 months. We generated conditional c-Fos-knockout mice to target K14-expressing cells, including differentiating taste cells. c-Fos deletion caused a severe perturbation in taste bud structure and resulted in a significant reduction in the taste bud size. c-Fos deletion also affected taste cell turnover as evident by a decrease in proliferative marker, and upregulation of the apoptotic marker cleaved-PARP. Thus, AP1 factors are important regulators of adult taste cell renewal and their downregulation negatively impacts taste maintenance.

  16. Role of the Small GTPase Rho3 in Golgi/Endosome trafficking through functional interaction with adaptin in Fission Yeast.

    Directory of Open Access Journals (Sweden)

    Ayako Kita

    Full Text Available BACKGROUND: We had previously identified the mutant allele of apm1(+ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1 complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we isolated rho3(+, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl(- sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl(-, and valproic acid. Green fluorescent protein (GFP-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner. CONCLUSIONS/SIGNIFICANCE: Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence

  17. TLR4-NOX4-AP-1 signaling mediates lipopolysaccharide-induced CXCR6 expression in human aortic smooth muscle cells

    International Nuclear Information System (INIS)

    Patel, Devang N.; Bailey, Steven R.; Gresham, John K.; Schuchman, David B.; Shelhamer, James H.; Goldstein, Barry J.; Foxwell, Brian M.; Stemerman, Michael B.; Maranchie, Jodi K.; Valente, Anthony J.; Mummidi, Srinivas; Chandrasekar, Bysani

    2006-01-01

    CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-κB (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis

  18. Molecular assemblies and membrane domains in multivesicular endosome dynamics

    International Nuclear Information System (INIS)

    Falguieres, Thomas; Luyet, Pierre-Philippe; Gruenberg, Jean

    2009-01-01

    Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo 'back-fusion' with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.

  19. Human TMEM174 that is highly expressed in kidney tissue activates AP-1 and promotes cell proliferation

    International Nuclear Information System (INIS)

    Wang, Pingzhang; Sun, Bo; Hao, Dongxia; Zhang, Xiujun; Shi, Taiping; Ma, Dalong

    2010-01-01

    Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174ΔTM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.

  20. Human TMEM174 that is highly expressed in kidney tissue activates AP-1 and promotes cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Pingzhang [Chinese National Human Genome Center, 3-707 North YongChang Road BDA, Beijing 100191 (China); Laboratory of Medical Immunology, School of Basic Medical Science, Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191 (China); Peking University Center for Human Disease Genomics, No. 38 Xueyuan Road, Beijing 100191 (China); Sun, Bo; Hao, Dongxia [Department of Biology, Northchina Coal Medical College, No. 57 JianShe South Road, Tangshan 063000 (China); Zhang, Xiujun, E-mail: zhangxiujun66@yahoo.com.cn [Department of Biology, Northchina Coal Medical College, No. 57 JianShe South Road, Tangshan 063000 (China); Shi, Taiping, E-mail: taiping_shi@yahoo.com.cn [Chinese National Human Genome Center, 3-707 North YongChang Road BDA, Beijing 100191 (China); Laboratory of Medical Immunology, School of Basic Medical Science, Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191 (China); Peking University Center for Human Disease Genomics, No. 38 Xueyuan Road, Beijing 100191 (China); Ma, Dalong [Chinese National Human Genome Center, 3-707 North YongChang Road BDA, Beijing 100191 (China); Laboratory of Medical Immunology, School of Basic Medical Science, Peking University Health Science Center, No. 38 Xueyuan Road, Beijing 100191 (China); Peking University Center for Human Disease Genomics, No. 38 Xueyuan Road, Beijing 100191 (China)

    2010-04-16

    Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174{Delta}TM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.

  1. AP-1/IRF-3 Targeted Anti-Inflammatory Activity of Andrographolide Isolated from Andrographis paniculata

    Directory of Open Access Journals (Sweden)

    Ting Shen

    2013-01-01

    Full Text Available Andrographolide (AG is an abundant component of plants of the genus Andrographis and has a number of beneficial properties including neuroprotective, anticancer, anti-inflammatory, and antidiabetic effects. Despite numerous pharmacological studies, the precise mechanism of AG is still ambiguous. Thus, in the present study, we investigated the molecular mechanisms of AG and its target proteins as they pertain to anti-inflammatory responses. AG suppressed the production of nitric oxide (NO and prostaglandin E2 (PGE2, as well as the mRNA abundance of inducible NO synthase (iNOS, tumor necrosis factor-alpha (TNF-α, cyclooxygenase (COX-2, and interferon-beta (IFN-β in a dose-dependent manner in both lipopolysaccharide- (LPS- activated RAW264.7 cells and peritoneal macrophages. AG also substantially ameliorated the symptoms of LPS-induced hepatitis and EtOH/HCl-induced gastritis in mice. Based on the results of luciferase reporter gene assays, kinase assays, and measurement of nuclear levels of transcription factors, the anti-inflammatory effects of AG were found to be clearly mediated by inhibition of both (1 extracellular signal-regulated kinase (ERK/activator protein (AP-1 and (2 IκB kinase ε (IKKε/interferon regulatory factor (IRF-3 pathways. In conclusion, we detected a novel molecular signaling pathway by which AG can suppress inflammatory responses. Thus, AG is a promising anti-inflammatory drug with two pharmacological targets.

  2. Interplay between the HTLV-2 Tax and APH-2 proteins in the regulation of the AP-1 pathway

    Directory of Open Access Journals (Sweden)

    Marban Céline

    2012-12-01

    Full Text Available Abstract Background In contrast with human T-cell leukemia virus type 1 (HTLV-1 that causes ATL (adult T-cell leukemia, HTLV-2 has not been causally linked to malignant disease. The minus strand of the HTLV genomes encode the regulatory proteins HTLV-1 bZIP factor (HBZ for HTLV-1 and antisense protein of HTLV-2 (APH-2 for HTLV-2. Unlike the viral proteins Tax1 and Tax2, both HBZ and APH-2 are constitutively expressed in infected cells suggesting that they may play important roles in the pathogenesis of these viruses. To date, very little is known about the function of APH-2 except that it inhibits Tax2-mediated transcription of HTLV-2 genes. In the present study, we investigated the role of APH-2 in basal and Tax2B-mediated activation of the AP-1 pathway. Results We demonstrate that, unlike HBZ, APH-2 stimulates basal AP-1 transcription by interacting with c-Jun and JunB through its non-conventional bZIP domain. In addition, when Tax2 and APH-2 are co-expressed, they physically interact in vivo and in vitro and APH-2 acts as an inhibitor of Tax2-mediated activation of AP-1 transcription. Conclusions This report is the first to document that HTLV-2 can modulate the AP-1 pathway. Altogether our results reveal that, in contrast with HBZ, APH-2 regulates AP-1 activity in a Tax2-dependant manner. As the AP-1 pathway is involved in numerous cellular functions susceptible to affect the life cycle of the virus, these distinct biological properties between HBZ and APH-2 may contribute to the differential pathogenic potential of HTLV-1 and HTLV-2.

  3. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    International Nuclear Information System (INIS)

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E.

    2011-01-01

    Highlights: ► Baculovirus p35 is regulated by both viral and host factors. ► Baculovirus p35 is negatively regulated by SfP53-like factor. ► Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at −1401 while P53 motif is at −1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  4. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    Energy Technology Data Exchange (ETDEWEB)

    Mohareer, Krishnaveni [Laboratory of Molecular and Cell Biology, Center for DNA Fingerprinting and Diagnostics, Hyderabad 500001 (India); Institute of Life Sciences, University of Hyderabad Campus, Prof. C.R. Rao Road, Gachibowli, Hyderabad 500046 (India); Sahdev, Sudhir [Laboratory of Molecular and Cell Biology, Center for DNA Fingerprinting and Diagnostics, Hyderabad 500001 (India); Ranbaxy Pharmaceuticals, Gurgaon, New Delhi (India); Hasnain, Seyed E., E-mail: seh@bioschool.iitd.ac.in [Institute of Life Sciences, University of Hyderabad Campus, Prof. C.R. Rao Road, Gachibowli, Hyderabad 500046 (India); Kusuma School of Biological Sciences, IIT Delhi, New Delhi 110016 (India); ILBS, Vasant Kunj, New Delhi (India); King Saud University, Riyadh, KSA (Saudi Arabia)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  5. Positioning of AMPA Receptor-Containing Endosomes Regulates Synapse Architecture

    Directory of Open Access Journals (Sweden)

    Marta Esteves da Silva

    2015-11-01

    Full Text Available Lateral diffusion in the membrane and endosomal trafficking both contribute to the addition and removal of AMPA receptors (AMPARs at postsynaptic sites. However, the spatial coordination between these mechanisms has remained unclear, because little is known about the dynamics of AMPAR-containing endosomes. In addition, how the positioning of AMPAR-containing endosomes affects synapse organization and functioning has never been directly explored. Here, we used live-cell imaging in hippocampal neuron cultures to show that intracellular AMPARs are transported in Rab11-positive recycling endosomes, which frequently enter dendritic spines and depend on the microtubule and actin cytoskeleton. By using chemically induced dimerization systems to recruit kinesin (KIF1C or myosin (MyosinV/VI motors to Rab11-positive recycling endosomes, we controlled their trafficking and found that induced removal of recycling endosomes from spines decreases surface AMPAR expression and PSD-95 clusters at synapses. Our data suggest a mechanistic link between endosome positioning and postsynaptic structure and composition.

  6. TXRF versus conventional EDXRF using 150 nm AP1 sample support films

    International Nuclear Information System (INIS)

    Wobrauschek, P.; Buzanich, G.; Marosi, N.

    2000-01-01

    Total reflection x-ray fluorescence analysis (TXRF ) is a powerful analytical tool for trace element analysis in the ng/g concentration or pg absolute region for medium Z elements using commercial or own designed TXRF equipment. One of the goals of TXRF is the extreme small penetration depth of the primary radiation into the reflector - substrate of 3-4 run if an incidence angle of about 1 mrad is adjusted. Among others, this effect results in a drastically reduced background in the measured spectra. This is caused by the reduction of elastic and inelastic scattered primary radiation from the substrate. Since there are now thin film supports commercially available having a thickness of 150 nm it is interesting to compare experimental results using TXRF and sample support films in 45 o incident beam and 45 o fluorescence emission geometry. As this film is only about 50 times more in thickness than the penetration depth in a quartz reflector, tests were performed to compare the analytical results and detection limits achievable from the same samples analyzed in total reflection geometry on a quartz reflector and conventional EDRXS geometry on the AP1 film with a dimple in the center for easy placing of the droplet. A volume of 3-5 μl of a liquid sample were used in both cases, pipetted and vacuum dried to prepare the sample. A high power 3 kW Mo-anode X-ray tube was used, for TXRF the line focus and for the conventional geometry the point focus was chosen. As additional results measurements from the TRACOR TN 5000 spectrometer will be presented using an air cooled low power tube with Rh - anode in a compact commercially available unit. A comparison of the results will be given in terms of sensitivity and detection limits, and future aspects for improvements and applications will be discussed. (author)

  7. Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

    Science.gov (United States)

    McKenzie, Jenna E.; Raisley, Brent; Zhou, Xin; Naslavsky, Naava; Taguchi, Tomohiko; Caplan, Steve; Sheff, David

    2012-01-01

    Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and ER. To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes, recycling endosomes, late endosomes and lysosomes. All cargos pass through early endosomes, but may take different routes to the Golgi. Retromer dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-Mannose-6-Phosphate Receptor, which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CD8-Mannose-6-Phosphate Receptor was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the early endosomes, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB. PMID:22540229

  8. Heparin (GAG-hed) inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

    International Nuclear Information System (INIS)

    Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Ángel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

    2006-01-01

    High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G 2 /M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data suggest that GAG-hed could have

  9. Catecholamine up-regulates MMP-7 expression by activating AP-1 and STAT3 in gastric cancer

    Directory of Open Access Journals (Sweden)

    Yu Ming

    2010-10-01

    Full Text Available Abstract Background Stress, anxiety and depression can cause complex physiological and neuroendocrine changes, resulting in increased level of stress related hormone catecholamine, which may constitute a primary mechanism by which physiological factors impact gene expression in tumors. In the present study, we investigated the effects of catecholamine stimulation on MMP-7 expression in gastric cancer cells and elucidated the molecular mechanisms of the up-regulation of MMP-7 level by catecholamine through an adrenergic signaling pathway. Results Increased MMP-7 expression was identified at both mRNA and protein levels in the gastric cancer cells in response to isoproterenol stimulation. β2-AR antigonist effectively abrogated isoproterenol-induced MMP-7 expression. The activation of STAT3 and AP-1 was prominently induced by isoproterenol stimulation and AP-1 displayed a greater efficacy than STAT3 in isoproterenol-induced MMP-7 expression. Mutagenesis of three STAT3 binding sites in MMP-7 promoter failed to repress the transactivation of MMP-7 promoter and silencing STAT3 expression was not effective in preventing isoproterenol-induced MMP-7 expression. However, isoproterenol-induced MMP-7 promoter activities were completely disappeared when the AP-1 site was mutated. STAT3 and c-Jun could physically interact and bind to the AP-1 site, implicating that the interplay of both transcriptional factors on the AP-1 site is responsible for isoproterenol-stimulated MMP-7 expression in gastric cancer cells. The expression of MMP-7 in gastric cancer tissues was found to be at the site where β2-AR was overexpressed and the levels of MMP-7 and β2-AR were the highest in the metastatic locus of gastric cancer. Conclusions Up-regulation of MMP-7 expression through β2-AR-mediated signaling pathway is involved in invasion and metastasis of gastric cancer.

  10. bZIP transcription factor CgAP1 is essential for oxidative stress tolerance and full virulence of the poplar anthracnose fungus Colletotrichum gloeosporioides.

    Science.gov (United States)

    Sun, Yingjiao; Wang, Yonglin; Tian, Chengming

    2016-10-01

    Yeast AP1 transcription factor is a regulator of oxidative stress response. Here, we report the identification and characterization of CgAP1, an ortholog of YAP1 in poplar anthracnose fungus Colletotrichum gloeosporioides. The expression of CgAP1 was highly induced by reactive oxygen species. CgAP1 deletion mutants displayed enhanced sensitivity to oxidative stress compared with the wild-type strain, and their poplar leaf virulence was obviously reduced. However, the mutants exhibited no obvious defects in aerial hyphal growth, conidia production, and appressoria formation. CgAP1::eGFP fusion protein localized to the nucleus after TBH (tert-Butyl hydroperoxide) treatment, suggesting that CgAP1 functions as a redox sensor in C. gloeosporioides. In addition, CgAP1 prevented the accumulation of ROS during early stages of biotrophic growth. CgAP1 also acted as a positive regulator of several ROS-related genes (i.e., Glr1, Hyr1, and Cyt1) involved in the antioxidative response. These results highlight the key regulatory role of CgAP1 transcription factor in oxidative stress response and provide insights into the function of ROS detoxification in virulence of C. gloeosporioides. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Heterotrimeric G protein subunits are located on rat liver endosomes

    Directory of Open Access Journals (Sweden)

    Van Dyke Rebecca W

    2004-01-01

    Full Text Available Abstract Background Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors. Results By Western blotting Gsα, Giα1,2, Giα3 and Gβ were enriched in both canalicular (CM and basolateral (BLM membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC > compartment for uncoupling of receptor and ligand (CURL > multivesicular bodies (MVB >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsα, Giα3 and Gβ corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers. Conclusion We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s of internalized insulin receptors.

  12. Cholesterol transfer at endosomal-organelle membrane contact sites.

    Science.gov (United States)

    Ridgway, Neale D; Zhao, Kexin

    2018-06-01

    Cholesterol is delivered to the limiting membrane of late endosomes by Niemann-Pick Type C1 and C2 proteins. This review summarizes recent evidence that cholesterol transfer from endosomes to the endoplasmic reticulum and other organelles is mediated by lipid-binding proteins that localize to membrane contact sites (MCS). LDL-cholesterol in the late endosomal/lysosomes is exported to the plasma membrane, where most cholesterol resides, and the endoplasmic reticulum, which harbors the regulatory complexes and enzymes that control the synthesis and esterification of cholesterol. A major advance in dissecting these cholesterol transport pathways was identification of frequent and dynamic MCS between endosomes and the endoplasmic reticulum, peroxisomes and plasma membrane. Positioned at these MCS are members of the oxysterol-binding protein (OSBP) and steroidogenic acute regulatory protein-related lipid-transfer family of lipid transfer proteins that bridge the opposing membranes and directly or indirectly mediate cholesterol transfer. OSBP-related protein 1L (ORP1L), ORP5 and ORP6 mediate cholesterol transfer to the endoplasmic reticulum that regulates cholesterol homeostasis. ORP1L and STARD3 also move cholesterol from the endoplasmic reticulum-to-late endosomal/lysosomes under low-cholesterol conditions to facilitate intraluminal vesicle formation. Cholesterol transport also occurs at MCS with peroxisomes and possibly the plasma membrane. Frequent contacts between organelles and the endo-lysosomal vesicles are sites for bidirectional transfer of cholesterol.

  13. Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells

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    Guillaume van Niel

    2015-10-01

    Full Text Available Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

  14. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    Science.gov (United States)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  15. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    Science.gov (United States)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-01-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier. PMID:26123532

  16. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery.

    Science.gov (United States)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-30

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds' escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds' cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  17. Curcumin modulates cellular AP-1, NF-kB, and HPV16 E6 proteins in oral cancer.

    Science.gov (United States)

    Mishra, Alok; Kumar, Rakesh; Tyagi, Abhishek; Kohaar, Indu; Hedau, Suresh; Bharti, Alok C; Sarker, Subhodeep; Dey, Dipankar; Saluja, Daman; Das, Bhudev

    2015-01-01

    In this study, we investigated the effects of the natural antioxidant curcumin on the HPV16-positive oral carcinoma cell line 93VU147T and demonstrated that curcumin is not only a potent inhibitor for the activity of host nuclear transcription factors AP-1 and NF-kB but it also selectively suppresses transcription of the HPV16/E6 oncogene during the carcinogenic process in oral cancer cells. This study suggests a therapeutic potential of curcumin for high-risk human papilloma virus (HPV)-infected oral cancers.

  18. Sugary Kefir Strain Lactobacillus mali APS1 Ameliorated Hepatic Steatosis by Regulation of SIRT-1/Nrf-2 and Gut Microbiota in Rats.

    Science.gov (United States)

    Chen, Yung-Tsung; Lin, Yu-Chun; Lin, Jin-Seng; Yang, Ning-Sun; Chen, Ming-Ju

    2018-04-01

    Non-alcoholic fatty liver disease (NAFLD) is a common disease that is concomitant with obesity, resulting in increased mortality. To date, the efficiency of NAFLD treatment still needs to be improved. Therefore, we aimed to evaluate the effect of Lactobacillus mali APS1, which was isolated from sugary kefir, on hepatic steatosis in rats fed a high-fat diet (HFD). Sprague Dawley rats were fed a control diet, a HFD with saline, and a HFD with APS1 intervention by gavage daily for 12 weeks. The results showed that APS1 significantly reduced body weight and body weight gain in HFD-fed rats. APS1 reduced hepatic lipid accumulation by regulating SIRT-1/PGC-1α/SREBP-1 expression. Moreover, APS1 increased hepatic antioxidant activity by modulating Nrf-2/HO-1 expression. Notably, APS1 manipulated the gut microbiota, resulting in increasing proportions of the phylum Bacteroidetes/Firmicutes and reducing the abundance of specific NAFLD-associated bacteria. These results suggested that APS1 ameliorated hepatic steatosis by modulating lipid metabolism and antioxidant activity via manipulating specific NAFLD-associated gut microbiota in vivo. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape

    Science.gov (United States)

    2015-01-01

    To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In

  20. Ebi/AP-1 suppresses pro-apoptotic genes expression and permits long-term survival of Drosophila sensory neurons.

    Directory of Open Access Journals (Sweden)

    Young-Mi Lim

    Full Text Available Sensory organs are constantly exposed to physical and chemical stresses that collectively threaten the survival of sensory neurons. Failure to protect stressed neurons leads to age-related loss of neurons and sensory dysfunction in organs in which the supply of new sensory neurons is limited, such as the human auditory system. Transducin β-like protein 1 (TBL1 is a candidate gene for ocular albinism with late-onset sensorineural deafness, a form of X-linked age-related hearing loss. TBL1 encodes an evolutionarily conserved F-box-like and WD40 repeats-containing subunit of the nuclear receptor co-repressor/silencing mediator for retinoid and thyroid hormone receptor and other transcriptional co-repressor complexes. Here we report that a Drosophila homologue of TBL1, Ebi, is required for maintenance of photoreceptor neurons. Loss of ebi function caused late-onset neuronal apoptosis in the retina and increased sensitivity to oxidative stress. Ebi formed a complex with activator protein 1 (AP-1 and was required for repression of Drosophila pro-apoptotic and anti-apoptotic genes expression. These results suggest that Ebi/AP-1 suppresses basal transcription levels of apoptotic genes and thereby protects sensory neurons from degeneration.

  1. Activation of transcriptional activities of AP-1 and SRE by a new zinc-finger protein ZNF641

    International Nuclear Information System (INIS)

    Qi Xingzhu; Li Yongqing; Xiao Jing; Yuan Wuzhou; Yan Yan; Wang Yuequn; Liang Shuyuan; Zhu Chuanbing; Chen Yingduan; Liu Mingyao; Wu Xiushan

    2006-01-01

    Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that zinc-finger proteins are involved in the regulation of the MAPK signaling pathways. Here, we report the identification and characterization of a novel human zinc-finger protein, ZNF641. The cDNA of ZNF641 is 4.9 kb, encoding 438 amino acids in the nucleus. The protein is highly conserved in evolution across different vertebrate species from mouse to human. Northern blot analysis indicates that ZNF641 is expressed in most of the examined human tissues, with a high level in skeletal muscle. Overexpression of pCMV-Tag2B-ZNF641 in the COS-7 cells activates the transcriptional activities of AP-1 and SRE. Deletion analysis indicates that the linker between KRAB box and C 2 H 2 -type zinc-fingers represents the basal activation domain. These results suggest that ZNF641 may be a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE

  2. The AP-1 Transcription Factor c-Jun Promotes Arthritis by Regulating Cyclooxygenase-2 and Arginase-1 Expression in Macrophages.

    Science.gov (United States)

    Hannemann, Nicole; Jordan, Jutta; Paul, Sushmita; Reid, Stephen; Baenkler, Hanns-Wolf; Sonnewald, Sophia; Bäuerle, Tobias; Vera, Julio; Schett, Georg; Bozec, Aline

    2017-05-01

    Activation of proinflammatory macrophages is associated with the inflammatory state of rheumatoid arthritis. Their polarization and activation are controlled by transcription factors such as NF-κB and the AP-1 transcription factor member c-Fos. Surprisingly, little is known about the role of the AP-1 transcription factor c-Jun in macrophage activation. In this study, we show that mRNA and protein levels of c-Jun are increased in macrophages following pro- or anti-inflammatory stimulations. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment cluster analyses of microarray data using wild-type and c-Jun-deleted macrophages highlight the central function of c-Jun in macrophages, in particular for immune responses, IL production, and hypoxia pathways. Mice deficient for c-Jun in macrophages show an amelioration of inflammation and bone destruction in the serum-induced arthritis model. In vivo and in vitro gene profiling, together with chromatin immunoprecipitation analysis of macrophages, revealed direct activation of the proinflammatory factor cyclooxygenase-2 and indirect inhibition of the anti-inflammatory factor arginase-1 by c-Jun. Thus, c-Jun regulates the activation state of macrophages and promotes arthritis via differentially regulating cyclooxygenase-2 and arginase-1 levels. Copyright © 2017 by The American Association of Immunologists, Inc.

  3. ZNF328, a novel human zinc-finger protein, suppresses transcriptional activities of SRE and AP-1

    International Nuclear Information System (INIS)

    Ou Ying; Wang Shenqiu; Cai Zhenyu; Wang Yuequn; Wang Canding; Li Yongqing; Li Fang; Yuan Wuzhou; Liu Bisheng; Wu Xiushan; Liu Mingyao

    2005-01-01

    The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C 2 H 2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions

  4. The p25 Subunit of the Dynactin Complex is Required for Dynein-Early Endosome Interaction

    Science.gov (United States)

    2011-01-01

    early endosome movement. In filamentous hyphae , dynein powers the minus end–directed movement of early endosomes (Steinberg and Schuster 2011...observed in time-lapse sequences (Video 1; Abenza et al., 2009). In still images, early endosomes were seen to distribute along the hyphae (Fig. 2 A...nuclear distribution along elongated hyphae and also for the microtubule minus end–directed movement of early endosomes away from the tip (Morris

  5. Structural Basis for Endosomal Targeting by the Bro1 Domain

    Science.gov (United States)

    Kim, Jaewon; Sitaraman, Sujatha; Hierro, Aitor; Beach, Bridgette M.; Odorizzi, Greg; Hurley, James H.

    2010-01-01

    Summary Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs. PMID:15935782

  6. Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

    NARCIS (Netherlands)

    van der Kant, Rik; Jonker, Caspar T. H.; Wijdeven, Ruud H.; Bakker, Jeroen; Janssen, Lennert; Klumperman, Judith; Neefjes, Jacques

    2015-01-01

    Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail

  7. Unconventional secretion of tissue transglutaminase involves phospholipid-dependent delivery into recycling endosomes.

    Directory of Open Access Journals (Sweden)

    Evgeny A Zemskov

    2011-04-01

    Full Text Available Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG. The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.

  8. Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

    Science.gov (United States)

    Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

    2018-01-01

    Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

  9. Arf6, Rab11 and transferrin receptor define distinct populations of recycling endosomes.

    Science.gov (United States)

    Kobayashi, Hotaka; Fukuda, Mitsunori

    2013-09-01

    Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations. Here we report finding that three well-known recycling endosome markers, i.e., Arf6, Rab11 and transferrin receptor (TfR), have different intracellular localizations in PC12 cells. The results of immunofluorescence analyses showed that the signals of endogenous Arf6, Rab11 and TfR in nerve growth factor-stimulated PC12 cells generally differed, although there was some overlapping. Our findings provide new information about recycling endosome markers, and they highlight the heterogeneity of recycling endosomes.

  10. Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

    Science.gov (United States)

    Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

    2009-05-01

    Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

  11. Promyelocytic leukemia bodies tether to early endosomes during mitosis.

    Science.gov (United States)

    Palibrk, Vuk; Lång, Emma; Lång, Anna; Schink, Kay Oliver; Rowe, Alexander D; Bøe, Stig Ove

    2014-01-01

    During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

  12. Enhancing endosomal escape of transduced proteins by photochemical internalisation.

    Directory of Open Access Journals (Sweden)

    Kevin Mellert

    Full Text Available Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.

  13. Enhancing endosomal escape of transduced proteins by photochemical internalisation.

    Science.gov (United States)

    Mellert, Kevin; Lamla, Markus; Scheffzek, Klaus; Wittig, Rainer; Kaufmann, Dieter

    2012-01-01

    Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.

  14. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

    International Nuclear Information System (INIS)

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

    2010-01-01

    Research highlights: → Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. → Adaptor-related protein complex 1 μ1A (AP-1 mu1A) was firstly reported to interact with kAE1. → The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. → AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. → AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl - ) and bicarbonate (HCO 3 - ) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl - /HCO 3 - exchange at the basolateral membrane and failure of proton (H + ) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.

  15. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    Energy Technology Data Exchange (ETDEWEB)

    Sawasdee, Nunghathai; Junking, Mutita [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Ngaojanlar, Piengpaga [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Immunology and Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Sukomon, Nattakan; Ungsupravate, Duangporn [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Akkarapatumwong, Varaporn [Institute of Molecular Biosciences, Mahidol University at Salaya Campus, Nakorn Pathom 73170 (Thailand); Noisakran, Sansanee [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  16. Protein complexes and cholesterol in the control of late endosomal dynamicsCholesterol and multi-protein complexes in the control of late endosomal dynamics

    NARCIS (Netherlands)

    Kant, Rik Henricus Nicolaas van der

    2013-01-01

    Late endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer’s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the

  17. Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jing Lu

    Full Text Available Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced.

  18. Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

    Science.gov (United States)

    Siddiqa, Abida; Massimi, Paola; Pim, David; Broniarczyk, Justyna; Banks, Lawrence

    2018-03-15

    Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans -Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans -Golgi network. Indeed, the loss of

  19. IL12A, MPHOSPH9/CDK2AP1 and RGS1 are novel multiple sclerosis susceptibility loci

    DEFF Research Database (Denmark)

    Sørensen, Per Soelberg

    2010-01-01

    and the same direction of effect observed in the discovery phase. Three loci exceeded genome-wide significance in the joint analysis: RGS1 (P value=3.55 x 10(-9)), IL12A (P=3.08 x 10(-8)) and MPHOSPH9/CDK2AP1 (P=3.96 x 10(-8)). The RGS1 risk allele is shared with celiac disease (CD), and the IL12A risk allele......A recent meta-analysis identified seven single-nucleotide polymorphisms (SNPs) with suggestive evidence of association with multiple sclerosis (MS). We report an analysis of these polymorphisms in a replication study that includes 8,085 cases and 7,777 controls. A meta-analysis across...... the replication collections and a joint analysis with the discovery data set were performed. The possible functional consequences of the validated susceptibility loci were explored using RNA expression data. For all of the tested SNPs, the effect observed in the replication phase involved the same allele...

  20. TRIM45, a novel human RBCC/TRIM protein, inhibits transcriptional activities of ElK-1 and AP-1

    International Nuclear Information System (INIS)

    Wang Yuequn; Li Yongqing; Qi Xinzhu; Yuan Wuzhou; Ai Jianping; Zhu Chuanbing; Cao Lei; Yang Hong; Liu Fang; Wu Xiushan; Liu Mingyao

    2004-01-01

    The tripartite motif (TRIM) proteins play important roles in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, and apoptosis. In this study, we report the identification and characterization of the human tripartite motif-containing protein 45 (TRIM45), a novel member of the TRIM family, from a human embryonic heart cDNA library. TRIM45 has a predicted 580 amino acid open reading frame, encoding a putative 64-kDa protein. The N-terminal region harbors a RING finger, two B-boxes, and a predicted α-helical coiled-coil domain, which together form the RBCC/TRIM motif found in a large family of proteins, whereas the C-terminal region contains a filamin-type immunoglobulin (IG-FLMN) domain. Northern blot analysis indicates that TRIM45 is expressed in a variety of human adult and embryonic tissues. In the cell, TRIM45 protein is expressed both in cytoplasm and in cell nucleus. Overexpression of TRIM45 in COS-7 cells inhibits the transcriptional activities of ElK-1 and AP-1. These results suggest that TRIM45 may act as a new transcriptional repressor in mitogen-activated protein kinase signaling pathway

  1. The Inflammation-Related Gene S100A12 Is Positively Regulated by C/EBPβ and AP-1 in Pigs

    Directory of Open Access Journals (Sweden)

    Xinyun Li

    2014-08-01

    Full Text Available S100A12 is involved in the inflammatory response and is considered an important marker for many inflammatory diseases in humans. Our previous studies indicated that the S100A12 gene was abundant in the immune tissues of pigs and was significantly upregulated during infection with Haemophilus parasuis (HPS or porcine circovirus type 2 (PCV2. In this study, the mechanism of transcriptional regulation of S100A12 was investigated in pigs. Our results showed that S100A12, CCAAT/enhancer-binding protein beta (C/EBPβ and activator protein-1 (AP-1 genes were up-regulated in PK-15 (ATCC, CCL-33 cells when treated with LPS or Poly I: C. Additionally, the promoter activity and expression level of the S100A12 gene were significantly upregulated when C/EBPβ or AP-1 were overexpressed. We utilized electromobility shift assays (EMSA to confirm that C/EBPβ and AP-1 could directly bind the S100A12 gene promoter. We also found that the transcriptional activity and expression levels of C/EBPβ and AP-1 could positively regulate each other. Furthermore, the promoter activity of the S100A12 gene was higher when C/EBPβ and AP-1 were cotransfected than when they were transfected individually. We concluded that the S100A12 gene was cooperatively and positively regulated by C/EBPβ and AP-1 in pigs. Our study offers new insight into the transcriptional regulation of the S100A12 gene.

  2. Penta-acetyl geniposide-induced apoptosis involving transcription of NGF/p75 via MAPK-mediated AP-1 activation in C6 glioma cells

    International Nuclear Information System (INIS)

    Peng, C.-H.; Huang, C.-N.; Hsu, S.-P.; Wang, C.-J.

    2007-01-01

    We have demonstrated the herbal derivative penta-acetyl geniposide ((Ac) 5 GP) induces C6 glioma cell apoptosis through the critical sphingomyelinase (SMase)/nerve growth factor (NGF)/p75 and its downstream signals. It has been reported mitogen-activated protein kinase (MAPK) mediates NGF synthesis induced by SMase activation. In this study, ERK, p38 and JNK are shown to mediate (Ac) 5 GP-induced glioma cell apoptosis and elevation of NGF and p75. Treatment of PD98059 (ERK-specific inhibitor), SB203580 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) decreases the elevation of NGF and p75 mRNA induced by (Ac) 5 GP, indicating possible transcription regulation via MAPKs. The results of nuclear extract blotting and EMSA further confirm (Ac) 5 GP maximally increases AP-1 and NF-κB DNA binding at 6 h. Inhibition of ERK, p38 and JNK block the activation of AP-1 and NF-κB, suggesting these MAPKs are involved in (Ac) 5 GP-induced transcription regulation. We thereby used RT-PCR to analyze cells treated with (Ac) 5 GP, with or without AP-1 or NF-κB inhibitors. AP-1 inhibitor NDGA decreases NGF/p75 and expression of FasL and caspase 3 induced by (Ac) 5 GP, suggesting the importance of AP-1 in mediating NGF/p75 and their downstream apoptotic signals. However, FasL and caspase 3 do not change with the NF-κB inhibitor PDTC; NF-κB might be linked to other cellular events. Overall, we demonstrate that MAPK mediates (Ac) 5 GP-induced activation of AP-1, promoting the transcription of NGF/p75 and downstream apoptotic signals. These results further highlight the potential therapeutic effects of (Ac) 5 GP in chemoprevention or as an anti-tumor agent

  3. Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin

    Science.gov (United States)

    Chia, Pei Zhi Cheryl; Gasnereau, Isabelle; Lieu, Zi Zhao; Gleeson, Paul A.

    2011-01-01

    The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin–TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail. PMID:21693586

  4. Gallic Acid Inhibited Matrix Invasion and AP-1/ETS-1-Mediated MMP-1 Transcription in Human Nasopharyngeal Carcinoma Cells.

    Science.gov (United States)

    Pang, Jong-Hwei S; Yen, Jia-Hau; Wu, Hsiao-Ting; Huang, Sheng-Teng

    2017-06-24

    Gallic acid is a trihydroxybenzoic acid found in natural herbal plants. Gallic acid has been reported to inhibit the migration and invasive capability of various cancers. Little is known about the underlying mechanisms of invasion responsible for cancer metastasis via gallic acid. The present study was intended to investigate the anti-invasive effect of gallic acid on human nasopharyngeal carcinoma cells (NPC-BM1) and its related mechanism. Gallic acid inhibited the invasion of NPC-BM1 cells dose- and time-dependently without significant cytotoxic effect. Affymetrix oligonucleotide microarray analysis revealed matrix metalloproteinase-1 (MMP-1) as the most down-regulated gene in NPC-BM1 cells by gallic acid. The cytosolic and secreted MMP-1 levels were both found to be inhibited by gallic acid as demonstrated by western blot analysis and ELISA respectively. The mRNA expression and transcription of MMP-1 gene was also down-regulated as determined by RT/real-time PCR and promoter activity assay. The expression of two major transcription binding factors in the MMP-1 promoter, AP-1 and ETS-1, were demonstrated to be reduced by gallic acid in NPC-BM1 cells. The effect of gallic acid was associated with the inhibition of p38 MAPK signaling pathway. In addition, gallic acid enhanced the gene expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) which further suppressed the MMP-1 activity. These findings may be useful to develop a novel chemotherapeutic agent to inhibit the metastasis of nasopharyngeal cancer.

  5. The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity

    International Nuclear Information System (INIS)

    Prasad, C. Krishna; Meyers, Craig; Zhan Dejin; You Hong; Chiriva-Internati, Maurizio; Mehta, Jawahar L.; Liu Yong; Hermonat, Paul L.

    2003-01-01

    Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process

  6. Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells.

    Science.gov (United States)

    Dey, Swatee; Bakthavatchalu, Vasudevan; Tseng, Michael T; Wu, Peng; Florence, Rebecca L; Grulke, Eric A; Yokel, Robert A; Dhar, Sanjit Kumar; Yang, Hsin-Sheng; Chen, Yumin; St Clair, Daret K

    2008-10-01

    The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

  7. Interactome analysis of transcriptional coactivator multiprotein bridging factor 1 unveils a yeast AP-1-like transcription factor involved in oxidation tolerance of mycopathogen Beauveria bassiana.

    Science.gov (United States)

    Chu, Xin-Ling; Dong, Wei-Xia; Ding, Jin-Li; Feng, Ming-Guang; Ying, Sheng-Hua

    2018-02-01

    Oxidation tolerance is an important determinant to predict the virulence and biocontrol potential of Beauveria bassiana, a well-known entomopathogenic fungus. As a transcriptional coactivator, multiprotein bridging factor 1 mediates the activity of transcription factor in diverse physiological processes, and its homolog in B. bassiana (BbMBF1) contributes to fungal oxidation tolerance. In this study, the BbMBF1-interactomes under oxidative stress and normal growth condition were deciphered by mass spectrometry integrated with the immunoprecipitation. BbMBF1p factor has a broad interaction with proteins that are involved in various cellular processes, and this interaction is dynamically regulated by oxidative stress. Importantly, a B. bassiana homolog of yeast AP-1-like transcription factor (BbAP-1) was specifically associated with the BbMBF1-interactome under oxidation and significantly contributed to fungal oxidation tolerance. In addition, qPCR analysis revealed that several antioxidant genes are jointly controlled by BbAP-1 and BbMBF1. Conclusively, it is proposed that BbMBF1p protein mediates BbAP-1p factor to transcribe the downstream antioxidant genes in B. bassiana under oxidative stress. This study demonstrates for the first time a proteomic view of the MBF1-interactome in fungi, and presents an initial framework to probe the transcriptional mechanism involved in fungal response to oxidation, which will provide a new strategy to improve the biocontrol efficacy of B. bassiana.

  8. Identification of GATA2 and AP-1 activator elements within the enhancer VNTR occurring in intron 5 of the human SIRT3 gene

    Science.gov (United States)

    Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study ...

  9. Inhibitory effects of curcumin and capsaicin on phorbol ester-induced activation of eukaryotic transcription factors, NF-kappaB and AP-1.

    Science.gov (United States)

    Surh, Y J; Han, S S; Keum, Y S; Seo, H J; Lee, S S

    2000-01-01

    Recently, considerable attention has been focused on identifying dietary and medicinal phytochemicals that can inhibit, retard or reverse the multi-stage carcinogenesis. Spices and herbs contain phenolic substances with potent antioxidative and chemopreventive properties. Curcumin, a yellow colouring agent from turmeric and capsaicin, a pungent principle of red pepper exhibit profound anticarcinogenic and antimutagenic activities. Two well-defined eukaryotic transcription factors, nuclear factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) have been implicated in pathogenesis of many human diseases including cancer. These transcription factors are known to be activated by a wide array of external stimuli, such as tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor, reactive oxygen species, bacterial lipopolysaccharide, and ultraviolet. In the present study, we found that topical application of TPA onto dorsal skin of female ICR mice resulted in marked activation of epidermal NF-kappaB and AP-1. Curcumin and capsaicin, when topically applied prior to TPA, significantly attenuated TPA-induced activation of each transcription factor in mouse skin. Likewise, both compounds inhibited NF-kappaB and AP-1 activation in cultured human promyelocytic leukemia (HL-60) cells stimulated with TPA. Based on these findings, it is likely that curcumin and capsaicin exert anti-tumor promotional effects through suppression of the tumor promoter-induced activation of transcription factors, NF-kappaB and AP-1.

  10. AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial–mesenchymal transition in triple-negative breast cancer

    Science.gov (United States)

    Qiao, Yichun; Shiue, Chiou-Nan; Zhu, Jian; Zhuang, Ting; Jonsson, Philip; Wright, Anthony P.H.; Zhao, Chunyan; Dahlman-Wright, Karin

    2015-01-01

    The molecular determinants of malignant cell behaviour in triple-negative breast cancer (TNBC) are poorly understood. Recent studies have shown that regulators of epithelial-mesenchymal transition (EMT) are potential therapeutic targets for TNBC. In this study, we demonstrate that the inflammatory cytokine TNFα induces EMT in TNBC cells via activation of AP-1 signaling and subsequently induces expression of the EMT regulator ZEB2. We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1. We further investigated in detail AP-1 regulation of ZEB2 expression. We show that two ZEB2 transcripts derived from distinct promoters are both expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression. PMID:25762639

  11. Ethanol extract of Lycoris radiata induces cell death in B16F10 melanoma via p38-mediated AP-1 activation.

    Science.gov (United States)

    Son, Minsik; Kim, Aeyung; Lee, Jaewoo; Park, Chul-Hong; Heo, Jin-Chul; Lee, Hyun-Jin; Lee, Sang-Han

    2010-08-01

    Some active alkaloids isolated from Lycoris, a bulbous perennial herb, was shown to possess various anti-tumor and anti-inflammatory activities. In this study, we evaluated the in vitro apoptotic effect of ethanol extract from Lycoris radiata (LRE) and further probed the underlying molecular mechanisms of LRE effects. The survival rate of B16F10 melanoma cells exposed to LRE was decreased in a dose-dependent manner, cell growth was retarded by arresting cell cycle at G1 phase and apoptotic appearance such as caspase-3 activation as well as DNA fragmentation was observed by LRE treatment. In addition, LRE induced p38 and c-Jun phosphorylation, followed by activation of transcription factor AP-1. Pretreatment with the p38 inhibitor (SB203580) blocked LRE-induced AP-1 transcriptional activity, and curcumin, AP-1 inhibitor, dramatically inhibited LRE-induced apoptosis in B16F10 melanoma cells. Our results collectively indicate that LRE-mediated apoptosis occurs through the activation of p38 and AP-1 pathway and potentially LRE exhibits anti-cancer activity against B16F10 melanoma cells.

  12. Effect of diphtheria toxin T-domain on endosomal pH

    Directory of Open Access Journals (Sweden)

    A. J. Labyntsev

    2015-08-01

    Full Text Available A key step in the mode of cytotoxic action of diphtheria toxin (DT is the transfer of its catalytic domain (Cd from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td, but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT. The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT, which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demons­trated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

  13. A CRE/AP-1-like motif is essential for induced syncytin-2 expression and fusion in human trophoblast-like model.

    Directory of Open Access Journals (Sweden)

    Chirine Toufaily

    Full Text Available Syncytin-2 is encoded by the envelope gene of Endogenous Retrovirus-FRD (ERVFRD-1 and plays a critical role in fusion of placental trophoblasts leading to the formation of the multinucleated syncytiotrophoblast. Its expression is consequently regulated in a strict manner. In the present study, we have identified a forskolin-responsive region located between positions -300 to -150 in the Syncytin-2 promoter region. This 150 bp region in the context of a minimal promoter mediated an 80-fold induction of promoter activity following forskolin stimulation. EMSA analyses with competition experiments with nuclear extracts from forskolin-stimulated BeWo cells demonstrated that the -211 to -177 region specifically bound two forskolin-induced complexes, one of them containing a CRE/AP-1-like motif. Site-directed mutagenesis of the CRE/AP-1 binding site in the context of the Syncytin-2 promoter or a heterologous promoter showed that this motif was mostly essential for forskolin-induced promoter activity. Transfection experiments with dominant negative mutants and constitutively activated CREB expression vectors in addition to Chromatin Immunoprecipitation suggested that a CREB family member, CREB2 was binding and acting through the CRE/AP-1 motif. We further demonstrated the binding of JunD to this same motif. Similar to forskolin and soluble cAMP, CREB2 and JunD overexpression induced Syncytin-2 promoter activity in a CRE/AP-1-dependent manner and Syncytin-2 expression. In addition, BeWo cell fusion was induced by both CREB2 and JunD overexpression, while being repressed following silencing of either gene. These results thereby demonstrate that induced expression of Syncytin-2 is highly dependent on the interaction of bZIP-containing transcription factors to a CRE/AP-1 motif and that this element is important for the regulation of Syncytin-2 expression, which results in the formation of the peripheral syncytiotrophoblast layer.

  14. Induction of activator protein (AP)-1 and nuclear factor-kappaB by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals.

    Science.gov (United States)

    Edmead, C E; Patel, Y I; Wilson, A; Boulougouris, G; Hall, N D; Ward, S G; Sansom, D M

    1996-10-15

    A major obstacle in understanding the signaling events that follow CD28 receptor ligation arises from the fact that CD28 acts as a costimulus to TCR engagement, making it difficult to assess the relative contribution of CD28 signals as distinct from those of the TCR. To overcome this problem, we have exploited the observation that activated human T cell blasts can be stimulated via the CD28 surface molecule in the absence of antigenic challenge; thus, we have been able to observe the response of normal T cells to CD28 activation in isolation. Using this system, we observed that CD28 stimulation by B7-transfected CHO cells induced a proliferative response in T cells that was not accompanied by measurable IL-2 production. However, subsequent analysis of transcription factor generation revealed that B7 stimulation induced both activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) complexes, but not NF-AT. In contrast, engagement of the TCR by class II MHC/superantigen, either with or without CD28 ligation, resulted in the induction of NF-AT, AP-1, and NF-kappaB as well as IL-2 production. Using selective inhibitors, we investigated the signaling pathways involved in the CD28-mediated induction of AP-1 and NF-kappaB. This revealed that NF-kappaB generation was sensitive to chloroquine, an inhibitor of acidic sphingomyelinase, but not to the phosphatidylinositol 3-kinase inhibitor, wortmannin. In contrast, AP-1 generation was inhibited by wortmannin and was also variably sensitive to chloroquine. These data suggest that in activated normal T cells, CD28-derived signals can stimulate proliferation at least in part via NF-kappaB and AP-1 generation, and that this response uses both acidic sphingomyelinase and phosphatidylinositol 3-kinase-linked pathways.

  15. Up-regulation of interleukin-4 production via NF-AT/AP-1 activation in T cells by biochanin A, a phytoestrogen and its metabolites

    International Nuclear Information System (INIS)

    Park, Jin; Chung, Su Wol; Kim, Seung Hyun; Kim, Tae Sung

    2006-01-01

    Phytoestrogens are naturally occurring compounds derived from plants. Although phytoestrogens exhibit many biological functions including estrogen agonist/antagonist properties, the effect on allergic responses remains unclear. In this study, we investigated whether biochanin A, a phytoestrogen and its metabolites, genistein, p-ethylphenol and phenolic acid, affect production of IL-4, a pro-inflammatory cytokine closely associated with allergic immune responses, in primary CD4 + T cells and EL4 T lymphoma cells. Biochanin A, genistein and p-ethylphenol significantly enhanced IL-4 production from both CD4 + T cells and EL4 cells in a dose-dependent manner, while phenolic acid did not. Biochanin A, genistein and p-ethylphenol also enhanced IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 promoter constructs, but this effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of a P4 site carrying NF-AT and AP-1 binding sites. In addition, biochanin A, genistein and p-ethylphenol increased both NF-AT and AP-1 DNA binding activities, indicating that they might enhance IL-4 production via NF-AT/AP-1 activation. Furthermore, biochanin A, genistein and p-ethylphenol increased p38 MAPK phosphorylation and PKC activity, while they did not affect ERK phosphorylation. The enhanced NF-AT DNA binding activities were suppressed by inhibitors for PI3-K and PKC, but not by p38 MAPK inhibitors. In contrast, the enhanced AP-1 DNA binding activities and p38 MAPK phosphorylation were significantly suppressed by specific inhibitors for PKC and p38 MAPK, but not by PI3-K inhibitors. These results demonstrate, for the first time, that biochanin A, genistein and p-ethylphenol enhance IL-4 production in activated T cells by two independent pathways, PI3-K/PKC/NF-AT and PKC/p38 MAPK/AP-1

  16. Small Molecules for Early Endosome-Specific Patch Clamping.

    Science.gov (United States)

    Chen, Cheng-Chang; Butz, Elisabeth S; Chao, Yu-Kai; Grishchuk, Yulia; Becker, Lars; Heller, Stefan; Slaugenhaupt, Susan A; Biel, Martin; Wahl-Schott, Christian; Grimm, Christian

    2017-07-20

    To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Proteolytic processing of epidermal growth factor within endosomes

    International Nuclear Information System (INIS)

    Gorman, R.M.; Savage, C.R. Jr.; Poretz, R.D.; Schaudies, R.P.

    1986-01-01

    The authors have reported previously that EGF enters 3 biochemically distinct non-lysosomal intracellular compartments prior to detection within lysosomes. Earlier studies have demonstrated that EGF is processes by sequential removal of 1, 4 and 1 aminoacyl residues at the C-terminus. The final form, which lacks the 6 residues, accumulates in secondary lysosomes. After subcellular fractionation of fibroblasts exposed to 125 I-EGF, ligand is detected with 3 non-lysosomal endocytic compartments and is fully processed prior to entrance into secondary lysosome. Following internalization, EGF enters an early endosomal compartment (E 1 ). The composition of the ligand (60%, -1 form; 40%, native form) represents an enhancement of the -1 form relative to that on the plasma membrane following the 90 min, 0 0 binding period. The proportion of different EGF forms in E 1 remains constant through the 2 min pulse and chase periods up to 30 min. However, in the ultimate endosomal compartment, E 4 , the proportion of the -6 form increases from 25% at 15 min to greater than 75% in 30 min, with a concomitant decrease of the -1 and -5 forms. Secondary lysosomes contain an EGF composition similar to that found in E 4 at 30 min. Accordingly, it appears that EGF is processed to the -6 form following passage through E 1 and during its tenure in E 4

  18. Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

    Science.gov (United States)

    Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

    2016-03-01

    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.

  19. Mouse polyomavirus enters early endosomes, requires their acidic pH for productive infection, and meets transferrin cargo in rab11-positive endosomes

    Czech Academy of Sciences Publication Activity Database

    Liebl, D.; Difato, F.; Horníková, L.; Mannová, P.; Štokrová, Jitka; Forstová, J.

    2006-01-01

    Roč. 80, č. 9 (2006), s. 4610-4622 ISSN 0022-538X R&D Projects: GA ČR(CZ) GA204/03/0593; GA MŠk(CZ) LC545 Institutional research plan: CEZ:AV0Z50520514 Keywords : Polyomavirus internalization and trafficking * Early endosomes * Dependence of infection on endosomal pH Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.341, year: 2006

  20. Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

    Science.gov (United States)

    Jurgeit, Andreas; McDowell, Robert; Moese, Stefan; Meldrum, Eric; Schwendener, Reto; Greber, Urs F

    2012-01-01

    Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV) and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+)-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

  1. Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

    Directory of Open Access Journals (Sweden)

    Andreas Jurgeit

    Full Text Available Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

  2. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

    Science.gov (United States)

    Sriram, V; Krishnan, K S; Mayor, Satyajit

    2003-05-12

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

  3. An Essential Role of Hrs/Vps27 in Endosomal Cholesterol Trafficking

    Directory of Open Access Journals (Sweden)

    Ximing Du

    2012-01-01

    Full Text Available The endosomal sorting complex required for transport (ESCRT plays a crucial role in the degradation of ubiquitinated endosomal membrane proteins. Here, we report that Hrs, a key protein of the ESCRT-0 complex, is required for the transport of low-density lipoprotein-derived cholesterol from endosomes to the endoplasmic reticulum. This function of Hrs in cholesterol transport is distinct from its previously defined role in lysosomal sorting and downregulation of membrane receptors via the ESCRT pathway. In line with this, knocking down other ESCRT proteins does not cause prominent endosomal cholesterol accumulation. Importantly, the localization and biochemical properties of key cholesterol-sorting proteins, NPC1 and NPC2, appear to be unchanged upon Hrs knockdown. Our data identify Hrs as a regulator of endosomal cholesterol trafficking and provide additional insights into the budding of intralumenal vesicles.

  4. Retrogradely Transported TrkA Endosomes Signal Locally within Dendrites to Maintain Sympathetic Neuron Synapses

    Directory of Open Access Journals (Sweden)

    Kathryn M. Lehigh

    2017-04-01

    Full Text Available Sympathetic neurons require NGF from their target fields for survival, axonal target innervation, dendritic growth and formation, and maintenance of synaptic inputs from preganglionic neurons. Target-derived NGF signals are propagated retrogradely, from distal axons to somata of sympathetic neurons via TrkA signaling endosomes. We report that a subset of TrkA endosomes that are transported from distal axons to cell bodies translocate into dendrites, where they are signaling competent and move bidirectionally, in close proximity to synaptic protein clusters. Using a strategy for spatially confined inhibition of TrkA kinase activity, we found that distal-axon-derived TrkA signaling endosomes are necessary within sympathetic neuron dendrites for maintenance of synapses. Thus, TrkA signaling endosomes have unique functions in different cellular compartments. Moreover, target-derived NGF mediates circuit formation and synapse maintenance through TrkA endosome signaling within dendrites to promote aggregation of postsynaptic protein complexes.

  5. Charcot-Marie-Tooth disease-linked protein SIMPLE functions with the ESCRT machinery in endosomal trafficking

    OpenAIRE

    Lee, Samuel M.; Chin, Lih-Shen; Li, Lian

    2012-01-01

    Mutations in small integral membrane protein of lysosome/late endosome (SIMPLE) cause autosomal dominant, Charcot-Marie-Tooth disease (CMT) type 1C. The cellular function of SIMPLE is unknown and the pathogenic mechanism of SIMPLE mutations remains elusive. Here, we report that SIMPLE interacted and colocalized with endosomal sorting complex required for transport (ESCRT) components STAM1, Hrs, and TSG101 on early endosomes and functioned with the ESCRT machinery in the control of endosome-to...

  6. PTP1B targets the endosomal sorting machinery

    DEFF Research Database (Denmark)

    Stuible, Matthew; Abella, Jasmine V; Feldhammer, Matthew

    2010-01-01

    Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown...... mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined...... phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated...

  7. Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells

    International Nuclear Information System (INIS)

    Looby, Eileen; Abdel-Latif, Mohamed MM; Athié-Morales, Veronica; Duggan, Shane; Long, Aideen; Kelleher, Dermot

    2009-01-01

    The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells. Following exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay. DCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure. DCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate

  8. Chrysin inhibits tumor promoter-induced MMP-9 expression by blocking AP-1 via suppression of ERK and JNK pathways in gastric cancer cells.

    Directory of Open Access Journals (Sweden)

    Yong Xia

    Full Text Available Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9 is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients' survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.

  9. Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells

    Directory of Open Access Journals (Sweden)

    Long Aideen

    2009-06-01

    Full Text Available Abstract Background The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells. Methods Following exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay. Results DCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure. Conclusion DCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate.

  10. Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells.

    LENUS (Irish Health Repository)

    Looby, Eileen

    2009-01-01

    BACKGROUND: The progression from Barrett\\'s metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells. METHODS: Following exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay. RESULTS: DCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1\\/2- and p38 MAPK while Erk1\\/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK\\/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure. CONCLUSION: DCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate.

  11. ERalpha and AP-1 interact in vivo with a specific sequence of the F promoter of the human ERalpha gene in osteoblasts.

    Science.gov (United States)

    Lambertini, Elisabetta; Tavanti, Elisa; Torreggiani, Elena; Penolazzi, Letizia; Gambari, Roberto; Piva, Roberta

    2008-07-01

    Estrogen-responsive genes often have an estrogen response element (ERE) positioned next to activator protein-1 (AP-1) binding sites. Considering that the interaction between ERE and AP-1 elements has been described for the modulation of bone-specific genes, we investigated the 17-beta-estradiol responsiveness and the role of these cis-elements present in the F promoter of the human estrogen receptor alpha (ERalpha) gene. The F promoter, containing the sequence analyzed here, is one of the multiple promoters of the human ERalpha gene and is the only active promoter in bone tissue. Through electrophoretic mobility shift (EMSA), chromatin immunoprecipitation (ChIP), and re-ChIP assays, we investigated the binding of ERalpha and four members of the AP-1 family (c-Jun, c-fos, Fra-2, and ATF2) to a region located approximately 800 bp upstream of the transcriptional start site of exon F of the human ERalpha gene in SaOS-2 osteoblast-like cells. Reporter gene assay experiments in combination with DNA binding assays demonstrated that F promoter activity is under the control of upstream cis-acting elements which are recognized by specific combinations of ERalpha, c-Jun, c-fos, and ATF2 homo- and heterodimers. Moreover, ChIP and re-ChIP experiments showed that these nuclear factors bind the F promoter in vivo with a simultaneous occupancy stimulated by 17-beta-estradiol. Taken together, our findings support a model in which ERalpha/AP-1 complexes modulate F promoter activity under conditions of 17-beta-estradiol stimulation. (c) 2008 Wiley-Liss, Inc.

  12. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation

    Energy Technology Data Exchange (ETDEWEB)

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol [Natural Medicine Center, KIST Gangneung Institute, Gangneung 210-340 (Korea, Republic of); Kang, Ki Sung [College of Korean Medicine, Gachon University, Seongnam 461-701 (Korea, Republic of); Kim, Yong Kee, E-mail: yksnbk@sm.ac.kr [College of Pharmacy, Sookmyung Women’s University, Seoul 140-742 (Korea, Republic of); Kim, Su-Nam, E-mail: snkim@kist.re.kr [Natural Medicine Center, KIST Gangneung Institute, Gangneung 210-340 (Korea, Republic of)

    2014-08-08

    Highlights: • SHQA increases PPARα/γ transactivation and inhibits MMP-2/-9 expression. • SHQA inhibits TNFα-induced AP-1 and MAPK signaling. • SHQA inhibits TNFα-induced p65 translocation and IκBα phosphorylation. • SHQA inhibits TNFα-induced AP-1 and NF-κB signaling via PPARα. - Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ’s role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα.

  13. EGF-R is Expressed and AP-1 and NF-κ:B Are Activated in Stromal Myofibroblasts Surrounding Colon Adenocarcinomas Paralleling Expression of COX-2 and VEGF

    Directory of Open Access Journals (Sweden)

    Panagiotis A. Konstantinopoulos

    2007-01-01

    Full Text Available Background: COX-2 and VEGF are important triggers of colon cancer growth, metastasis and angiogenesis. Cox-2 promoter contains transcriptional regulatory elements for AP-1 and NF-κ:B transcription factors whilst vegf is a known AP-1 downstream target gene. We investigated whether stromal myofibroblasts surrounding colon adenocarcinomas express COX-2 and VEGF and whether activation of AP-1 and NF-κ:B, as well as expression of EGF-R parallel expression of COX-2 and VEGF in these cells. Methods: Immunohistochemical methodology was performed on archival sections from 40 patients with colon adenocarcinomas. We evaluated c-FOS, p-c-JUN (phosphorylated c-JUN, p-Iκ:B-α (phosphorylated Iκ:B-α, EGF-R, COX-2, NF-κ:B and VEGF expression in stromal myofibroblasts surrounding colon adenocarcinomas. Double immunostaining with a-smooth muscle actin and each antibody was done to verify the expression of these molecules in stromal myofibroblasts. Results: VEGF, p-Iκ:B-α, NF-κ:B, c-FOS, p-c-JUN, EGF-R and COX-2 were expressed in stromal myofibroblasts surrounding colon adenocarcinomas in the majority of cases. EGF-R, p-Iκ:B-α, NF-κ:B, c-FOS and p-c-JUN correlated positively with COX-2 and VEGF expression. Conclusion: Stromal myofibroblasts surrounding colon adenocarcinomas are an important source of VEGF and COX-2 production, while AP-1 and NF-κ:B transcription factors are activated and EGF-R is expressed in these cells and associated with COX-2 and VEGF production.

  14. Rhizoma Coptidis Inhibits LPS-Induced MCP-1/CCL2 Production in Murine Macrophages via an AP-1 and NF?B-Dependent Pathway

    OpenAIRE

    Remppis, Andrew; Bea, Florian; Greten, Henry Johannes; Buttler, Annette; Wang, Hongjie; Zhou, Qianxing; Preusch, Michael R.; Enk, Ronny; Ehehalt, Robert; Katus, Hugo; Blessing, Erwin

    2010-01-01

    Introduction. The Chinese extract Rhizoma coptidis is well known for its anti-inflammatory, antioxidative, antiviral, and antimicrobial activity. The exact mechanisms of action are not fully understood. Methods. We examined the effect of the extract and its main compound, berberine, on LPS-induced inflammatory activity in a murine macrophage cell line. RAW 264.7 cells were stimulated with LPS and incubated with either Rhizoma coptidis extract or berberine. Activation of AP-1 and NFB was anal...

  15. Stimulation of Pol III-dependent 5S rRNA and U6 snRNA gene expression by AP-1 transcription factors.

    Science.gov (United States)

    Ahuja, Richa; Kumar, Vijay

    2017-07-01

    RNA polymerase III transcribes structurally diverse group of essential noncoding RNAs including 5S ribosomal RNA (5SrRNA) and U6 snRNA. These noncoding RNAs are involved in RNA processing and ribosome biogenesis, thus, coupling Pol III activity to the rate of protein synthesis, cell growth, and proliferation. Even though a few Pol II-associated transcription factors have been reported to participate in Pol III-dependent transcription, its activation by activator protein 1 (AP-1) factors, c-Fos and c-Jun, has remained unexplored. Here, we show that c-Fos and c-Jun bind to specific sites in the regulatory regions of 5S rRNA (type I) and U6 snRNA (type III) gene promoters and stimulate their transcription. Our chromatin immunoprecipitation studies suggested that endogenous AP-1 factors bind to their cognate promoter elements during the G1/S transition of cell cycle apparently synchronous with Pol III transcriptional activity. Furthermore, the interaction of c-Jun with histone acetyltransferase p300 promoted the recruitment of p300/CBP complex on the promoters and facilitated the occupancy of Pol III transcriptional machinery via histone acetylation and chromatin remodeling. The findings of our study, together, suggest that AP-1 factors are novel regulators of Pol III-driven 5S rRNA and U6 snRNA expression with a potential role in cell proliferation. © 2017 Federation of European Biochemical Societies.

  16. c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

    International Nuclear Information System (INIS)

    Zhang Dongyun; Li Jingxia; Gao Jimin; Huang Chuanshu

    2009-01-01

    Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure

  17. CXCL12 gene silencing down-regulates metastatic potential via blockage of MAPK/PI3K/AP-1 signaling pathway in colon cancer.

    Science.gov (United States)

    Ma, J; Su, H; Yu, B; Guo, T; Gong, Z; Qi, J; Zhao, X; Du, J

    2018-01-05

    To investigate the effect of CXCL12 gene silencing on proliferation,invasion, angiogenesis and the relationship of MAPK/PI3K/AP-1 signaling pathway in colon cancer cells. RT-PCR and Western-blot were used to detect the expression of CXCL12 mRNA and protein in four colon cancer cell lines. Human colon cancer cells were transfected with CXCL12 siRNA carrying by Lipofectamine 2000. The expression of CXCL12 protein was confirmed by immunoblotting. WST-1, invasion and angiogenesis assay were used to examine the effect on proliferation, invasion and angiogenesis in colon cancer cells after CXCL12 siRNA silence, respectively. The phosphorylation of MAPK/PI3K/AP-1 protein levels was detected by Western blotting in CXCL12 siRNA suppression DLD-1 cell. CXCL12 mRNA and proteins were only expressed in DLD-1 colon cancer cell lines. CXCL12 siRNA were transfected into DLD-1 cells, the expression CXCL12 proteins was significantly inhibited (P colon cancer cell. The silencing CXCL12 gene significantly inhibits the proliferation, invasion and angiogenesis ability of some types colon carcinoma cells through down-regulation of MAPK/PI3K/AP-1 signaling pathway.

  18. Opposing roles of C/EBPbeta and AP-1 in the control of fibroblast proliferation and growth arrest-specific gene expression

    DEFF Research Database (Denmark)

    Gagliardi, Mark; Maynard, Scott; Miyake, Tetsuaki

    2003-01-01

    in the levels of AP-1 proteins. Therefore, C/EBPbeta is a negative regulator of AP-1 expression and activity in CEF. The expression of cyclin D1 and cell proliferation were stimulated by the dominant negative mutant of C/EBPbeta but not in the presence of TAM67, a dominant negative mutant of c-Jun and AP-1. CEF......Chicken embryo fibroblasts (CEF) express several growth arrest-specific (GAS) gene products in G0. In contact-inhibited cells, the expression of the most abundant of these proteins, the p20K lipocalin, is activated at the transcriptional level by C/EBPbeta. In this report, we describe the role of C....../EBPbeta in CEF proliferation. We show that the expression of a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) completely inhibited p20K expression at confluence and stimulated the proliferation of CEF without inducing transformation. Mouse embryo fibroblasts nullizygous for C/EBPbeta had...

  19. Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-κB and AP-1.

    Science.gov (United States)

    Yang, Xinyu; Li, Lin; Liu, Jin; Lv, Ben; Chen, Fangping

    2016-01-01

    Extracellular histones have been recognized recently as proinflammatory mediators; they are released from dying cells in response to inflammatory challenge, contributing to endothelial cell dysfunction, thrombin formation, organ failure, and death during sepsis. Clinical studies suggest that the plasma concentration of the histone-DNA complex is correlated with the severity of DIC and is a poor independent prognostic marker in sepsis. In addition, platelet activation stimulates thrombus formation. Whether histones contribute to procoagulant activity in other ways remains elusive. In this study, we confirmed that histones induce tissue factor (TF) expression in a concentration- and time-dependent manner in vascular endothelial cells (ECs) and macrophages. However, histones did not affect TF pathway inhibitor expression. Moreover, blocking the cell surface receptors TLR4 and TLR2 with specific neutralizing antibodies significantly reduced histone-induced TF expression. Furthermore, histones enhanced the nuclear translocation of NF-κB (c-Rel/p65) and AP-1 expression in a time-dependent manner in ECs. Mutating NF-κB and AP-1 significantly reduced histone-induced TF expression. Altogether, our experiments suggest that histone induces TF expression in ECs via cell surface receptors TLR4 and TLR2, simultaneously depending on the activation of the transcription factors NF-κB and AP-1. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Rhizoma coptidis Inhibits LPS-Induced MCP-1/CCL2 Production in Murine Macrophages via an AP-1 and NFB-Dependent Pathway

    Directory of Open Access Journals (Sweden)

    Andrew Remppis

    2010-01-01

    Full Text Available Introduction. The Chinese extract Rhizoma coptidis is well known for its anti-inflammatory, antioxidative, antiviral, and antimicrobial activity. The exact mechanisms of action are not fully understood. Methods. We examined the effect of the extract and its main compound, berberine, on LPS-induced inflammatory activity in a murine macrophage cell line. RAW 264.7 cells were stimulated with LPS and incubated with either Rhizoma coptidis extract or berberine. Activation of AP-1 and NFB was analyzed in nuclear extracts, secretion of MCP-1/CCL2 was measured in supernatants. Results. Incubation with Rhizoma coptidis and berberine strongly inhibited LPS-induced monocyte chemoattractant protein (MCP-1 production in RAW cells. Activation of the transcription factors AP-1 and NFB was inhibited by Rhizoma coptidis in a dose- and time-dependent fashion. Conclusions. Rhizoma coptidis extract inhibits LPS-induced MCP-1/CCL2 production in vitro via an AP-1 and NFB-dependent pathway. Anti-inflammatory action of the extract is mediated mainly by its alkaloid compound berberine.

  1. Rhizoma Coptidis Inhibits LPS-Induced MCP-1/CCL2 Production in Murine Macrophages via an AP-1 and NFκB-Dependent Pathway

    Science.gov (United States)

    Remppis, Andrew; Bea, Florian; Greten, Henry Johannes; Buttler, Annette; Wang, Hongjie; Zhou, Qianxing; Preusch, Michael R.; Enk, Ronny; Ehehalt, Robert; Katus, Hugo; Blessing, Erwin

    2010-01-01

    Introduction. The Chinese extract Rhizoma coptidis is well known for its anti-inflammatory, antioxidative, antiviral, and antimicrobial activity. The exact mechanisms of action are not fully understood. Methods. We examined the effect of the extract and its main compound, berberine, on LPS-induced inflammatory activity in a murine macrophage cell line. RAW 264.7 cells were stimulated with LPS and incubated with either Rhizoma coptidis extract or berberine. Activation of AP-1 and NFκB was analyzed in nuclear extracts, secretion of MCP-1/CCL2 was measured in supernatants. Results. Incubation with Rhizoma coptidis and berberine strongly inhibited LPS-induced monocyte chemoattractant protein (MCP)-1 production in RAW cells. Activation of the transcription factors AP-1 and NFκB was inhibited by Rhizoma coptidis in a dose- and time-dependent fashion. Conclusions. Rhizoma coptidis extract inhibits LPS-induced MCP-1/CCL2 production in vitro via an AP-1 and NFκB-dependent pathway. Anti-inflammatory action of the extract is mediated mainly by its alkaloid compound berberine. PMID:20652055

  2. Stochastic acidification, activation of hemagglutinin and escape of influenza viruses from an endosome

    Science.gov (United States)

    Lagache, Thibault; Sieben, Christian; Meyer, Tim; Herrmann, Andreas; Holcman, David

    2017-06-01

    Influenza viruses enter the cell inside an endosome. During the endosomal journey, acidification triggers a conformational change of the virus spike protein hemagglutinin (HA) that results in escape of the viral genome from the endosome into the cytoplasm. It is still unclear how the interplay between acidification and HA conformation changes affects the kinetics of the viral endosomal escape. We develop here a stochastic model to estimate the change of conformation of HAs inside the endosome nanodomain. Using a Markov process, we model the arrival of protons to HA binding sites and compute the kinetics of their accumulation. We compute the Mean First Passage Time (MFPT) of the number of HA bound sites to a threshold, which is used to estimate the HA activation rate for a given pH concentration. The present analysis reveals that HA proton binding sites possess a high chemical barrier, ensuring a stability of the spike protein at sub-acidic pH. We predict that activating more than 3 adjacent HAs is necessary to trigger endosomal fusion and this configuration prevents premature release of viruses from early endosomes

  3. ER network homeostasis is critical for plant endosome streaming and endocytosis

    Science.gov (United States)

    Stefano, Giovanni; Renna, Luciana; Lai, YaShiuan; Slabaugh, Erin; Mannino, Nicole; Buono, Rafael A; Otegui, Marisa S; Brandizzi, Federica

    2015-01-01

    Eukaryotic cells internalize cargo at the plasma membrane via endocytosis, a vital process that is accomplished through a complex network of endosomal organelles. In mammalian cells, the ER is in close association with endosomes and regulates their fission. Nonetheless, the physiological role of such interaction on endocytosis is yet unexplored. Here, we probed the existence of ER–endosome association in plant cells and assayed its physiological role in endocytosis. Through live-cell imaging and electron microscopy studies, we established that endosomes are extensively associated with the plant ER, supporting conservation of interaction between heterotypic organelles in evolutionarily distant kingdoms. Furthermore, by analyzing ER–endosome dynamics in genetic backgrounds with defects in ER structure and movement, we also established that the ER network integrity is necessary for homeostasis of the distribution and streaming of various endosome populations as well as for efficient endocytosis. These results support a novel model that endocytosis homeostasis depends on a spatiotemporal control of the endosome dynamics dictated by the ER membrane network. PMID:27462431

  4. Cadmium induces matrix metalloproteinase-9 expression via ROS-dependent EGFR, NF-kB, and AP-1 pathways in human endothelial cells

    International Nuclear Information System (INIS)

    Lian, Sen; Xia, Yong; Khoi, Pham Ngoc; Ung, Trong Thuan; Yoon, Hyun Joong; Kim, Nam Ho; Kim, Kyung Keun; Jung, Young Do

    2015-01-01

    Highlights: • Cadmium induces MMP-9 expression through NADPH oxidase-derived ROS. • Cadmium induces MMP-9 through EGFR-mediated Akt, Erk1/2 and JNK1/2 signaling pathways. • Akt, MAPKs (Erk1/2 and JNK1/2) functioned as upstream signals of NF-kB and AP-1 respectively, in cadmium-induced MMP-9 in endothelial cells. • ROS production by NADPH oxidase is the furthest upstream signal in MMP-9 expression in ECV304 cells. - Abstract: Cadmium (Cd), a widespread cumulative pollutant, is a known human carcinogen, associated with inflammation and tumors. Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in tumor metastasis; however, the mechanisms underlying the MMP-9 expression induced by Cd remain obscure in human endothelial cells. Here, Cd elevated MMP-9 expression in dose- and time-dependent manners in human endothelial cells. Cd increased ROS production and the ROS-producing NADPH oxidase. Cd translocates p47 phox , a key subunit of NADPH oxidase, to the cell membrane. Cd also activated the phosphorylation of EGFR, Akt, Erk1/2, and JNK1/2 in addition to promoting NF-kB and AP-1 binding activities. Specific inhibitor and mutagenesis studies showed that EGFR, Akt, Erk1/2, JNK1/2 and transcription factors NF-κB and AP-1 were related to Cd-induced MMP-9 expression in endothelial cells. Akt, Erk1/2, and JNK1/2 functioned as upstream signals in the activation of NF-κB and AP-1, respectively. In addition, N-acetyl-L-cystein (NAC), diphenyleneiodonium chloride (DPI) and apocynin (APO) inhibited the Cd-induced activation of EGFR, Akt, Erk1/2, JNK1/2, and p38 MAPK, indicating that ROS production by NADPH oxidase is the furthest upstream signal in MMP-9 expression. At present, it states that Cd displayed marked invasiveness in ECV304 cells, which was partially abrogated by MMP-9 neutralizing antibodies. These results demonstrated that Cd induces MMP-9 expression via ROS-dependent EGFR- > Erk1/2, JNK1/2- > AP-1 and EGFR- > Akt- > NF-κB signaling pathways and, in turn

  5. Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.

    Directory of Open Access Journals (Sweden)

    Claudia A Bertuccio

    Full Text Available The intermediate conductance, Ca2+-activated K+ channel (KCa3.1 targets to the basolateral (BL membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3

  6. Functional and evolutionary analysis of the AP1/SEP/AGL6 superclade of MADS-box genes in the basal eudicot Epimedium sagittatum.

    Science.gov (United States)

    Sun, Wei; Huang, Wenjun; Li, Zhineng; Song, Chi; Liu, Di; Liu, Yongliang; Hayward, Alice; Liu, Yifei; Huang, Hongwen; Wang, Ying

    2014-03-01

    MADS-box transcriptional regulators play important roles during plant development. Based on phylogenetic reconstruction, the AP1/SEP/AGL6 superclade of floral MADS-box genes underwent one or two duplication events in the common ancestor of the core eudicots. However, the functional evolution of the AP1/SEP/AGL6 superclade in basal eudicots remains uncharacterized. Epimedium sagittatum is a basal eudicot species valued for its medicinal properties and showing unique floral morphology. In this study, structural and functional variation of FUL-like (AP1 subfamily), SEP-like and AGL6-like genes in this species was investigated to further our understanding of flower evolution in angiosperms. Detailed investigations into the microsynteny and evolutionary history of the floral A and E class MADS-box genes in eudicots were undertaken and used to trace their genomic rearrangements. One AP1-like gene, two SEP-like genes and one AGL6-like gene were cloned from E. sagittatum. Their expression patterns were examined using quantitative RT-PCR in different vegetative and reproductive organs at two developmental stages. Yeast two-hybrid assays were carried out among AP1/SEP/AGL6 superclade, AP3/PI and AGAMOUS subfamily members for elucidation of dimerization patterns. In addition, possible formation of a ternary complex involving B class proteins with the A class protein EsFUL-like, the E class SEP-like protein EsAGL2-1 or the AGL6-class protein EsAGL6 were detected using yeast three-hybrid assays. Transgenic Arabidopsis or tobacco plants expressing EsFUL-like, EsAGL2-1 and EsAGL6-like under the cauliflower mosaic virus (CaMV) 35S promoter were generated and analysed. Genomic studies of AP1 syntenic regions in arabidopsis, columbine, strawberry, papaya, peach, grapevine and tomato were conducted for microsyntenic analyses. Sequence and phylogenetic analyses showed that EsFUL-like is a member of the AP1 (A class) subfamily, EsAGL2-1 and EsAGL2-2 belong to the SEP-like (E class

  7. Recycling endosomes in apical plasma membrane domain formation and epithelial cell polarity

    NARCIS (Netherlands)

    Golachowska, Magdalena R.; Hoekstra, Dick; van IJzendoorn, Sven C. D.

    2010-01-01

    Recycling endosomes have taken central stage in the intracellular sorting and polarized trafficking of apical and basolateral plasma membrane components. Molecular players in the underlying mechanisms are now emerging, including small GTPases, class V myosins and adaptor proteins. In particular,

  8. An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport

    NARCIS (Netherlands)

    Jongsma, Marlieke L. M.; Berlin, Ilana; Wijdeven, Ruud H. M.; Janssen, Lennert; Janssen, George M. C.; Garstka, Malgorzata A.; Janssen, Hans; Mensink, Mark; van Veelen, Peter A.; Spaapen, Robbert M.; Neefjes, Jacques

    2016-01-01

    Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent.

  9. Erythroid cell mitochondria receive endosomal iron by a "kiss-and-run" mechanism.

    Science.gov (United States)

    Hamdi, Amel; Roshan, Tariq M; Kahawita, Tanya M; Mason, Anne B; Sheftel, Alex D; Ponka, Prem

    2016-12-01

    In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe 2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the "kiss-and-run" hypothesis). Using a novel method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different fluorophores. We have identified a double-labeled population definitively representing endosomes interacting with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome-mitochondrion association is reversible, since a "chase" with unlabeled holotransferrin causes a time-dependent decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of 59 Fe by reticulocytes and diminishes 59 Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a "traffic jam" leading to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster

    OpenAIRE

    Sriram, V.; Krishnan, K.S.; Mayor, Satyajit

    2003-01-01

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defec...

  11. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    Energy Technology Data Exchange (ETDEWEB)

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  12. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

    International Nuclear Information System (INIS)

    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi; Rhim, Hyangshuk; Bae, Yong Soo; Choi, Soo Young; Park, Jinseu

    2014-01-01

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction

  13. Fluocinolone acetonide partially restores the mineralization of LPS-stimulated dental pulp cells through inhibition of NF-κB pathway and activation of AP-1 pathway

    Science.gov (United States)

    Liu, Zhongning; Jiang, Ting; Wang, Xinzhi; Wang, Yixiang

    2013-01-01

    BACKGROUND AND PURPOSE Fluocinolone acetonide (FA) is commonly used as a steroidal anti-inflammatory drug. We recently found that in dental pulp cells (DPCs) FA has osteo-/odonto-inductive as well as anti-inflammatory effects. However, the mechanism by which FA induces these effects in DPCs is poorly understood. EXPERIMENTAL APPROACH The effect of FA on the mineralization of DPCs during inflammatory conditions and the underlying mechanism were investigated by real-time PCR, Western blot, EMSA, histochemical staining, immunostaining and pathway blockade assays. KEY RESULTS FA significantly inhibited the inflammatory response in LPS-treated DPCs not only by down-regulating the expression of pro–inflammation-related genes, but also by up-regulating the expression of the anti-inflammatory gene PPAR-γ and mineralization-related genes. Moreover, histochemical staining and immunostaining showed that FA could partially restore the expressions of alkaline phosphatase, osteocalcin and dentin sialophosphoprotein (DSPP) and mineralization in LPS-stimulated DPCs. Real-time PCR and Western blot analysis revealed that FA up-regulated DSPP and runt-related transcription factor 2 expression by inhibiting the expression of phosphorylated-NF-κB P65 and activating activator protein-1 (AP-1) (p-c-Jun and Fra-1). These results were further confirmed through EMSA, by detection of NF-κB DNA-binding activity and pathway blockade assays using a NF-κB pathway inhibitor, AP-1 pathway inhibitor and glucocorticoid receptor antagonist. CONCLUSIONS AND IMPLICATIONS Inflammation induced by LPS suppresses the mineralization process in DPCs. FA partially restored this osteo-/odonto-genesis process in LPS-treated DPCs and had an anti-inflammatory effect through inhibition of the NF-κB pathway and activation of the AP-1 pathway. Hence, FA is a potential new treatment for inflammation-associated bone/teeth diseases. PMID:24024985

  14. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

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    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi [Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Rhim, Hyangshuk [Department of Biomedical Sciences, Department of Medical Life Sciences, College of Medicine, the Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Bae, Yong Soo [Department of Biological Science, College of Natural Sciences, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Choi, Soo Young [Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of); Park, Jinseu, E-mail: jinpark@hallym.ac.kr [Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702 (Korea, Republic of)

    2014-10-01

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction.

  15. Activation of Nrf2 Reduces UVA-Mediated MMP-1 Upregulation via MAPK/AP-1 Signaling Cascades: The Photoprotective Effects of Sulforaphane and Hispidulin

    Science.gov (United States)

    Chaiprasongsuk, Anyamanee; Lohakul, Jinaphat; Soontrapa, Kitipong; Sampattavanich, Somponnat; Akarasereenont, Pravit

    2017-01-01

    UVA irradiation plays a role in premature aging of the skin through triggering oxidative stress-associated stimulation of matrix metalloproteinase-1 (MMP-1) responsible for collagen degradation, a hallmark of photoaged skin. Compounds that can activate nuclear factor E2-related factor 2 (Nrf2), a transcription factor regulating antioxidant gene expression, should therefore serve as effective antiphotoaging agents. We investigated whether genetic silencing of Nrf2 could relieve UVA-mediated MMP-1 upregulation via activation of mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) signaling using human keratinocyte cell line (HaCaT). Antiphotoaging effects of hispidulin (HPD) and sulforaphane (SFN) were assessed on their abilities to activate Nrf2 in controlling MMP-1 and collagen expressions in association with phosphorylation of MAPKs (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38), c-Jun, and c-Fos, using the skin of BALB/c mice subjected to repetitive UVA irradiation. Our findings suggested that depletion of Nrf2 promoted both mRNA expression and activity of MMP-1 in the UVA-irradiated HaCaT cells. Treatment of Nrf2 knocked-down HaCaT cells with MAPK inhibitors significantly suppressed UVA-induced MMP-1 and AP-1 activities. Moreover, pretreatment of the mouse skin with HPD and SFN, which could activate Nrf2, provided protective effects against UVA-mediated MMP-1 induction and collagen depletion in correlation with the decreased levels of phosphorylated MAPKs, c-Jun, and c-Fos in the mouse skin. In conclusion, Nrf2 could influence UVA-mediated MMP-1 upregulation through the MAPK/AP-1 signaling cascades. HPD and SFN may therefore represent promising antiphotoaging candidates. PMID:28011874

  16. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    Science.gov (United States)

    Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

    2012-01-01

    Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

  17. A Role for EHD4 in the Regulation of Early Endosomal Transport

    Science.gov (United States)

    Sharma, Mahak; Naslavsky, Naava; Caplan, Steve

    2009-01-01

    All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway. PMID:18331452

  18. Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

    International Nuclear Information System (INIS)

    Mesa, Rosana; Magadan, Javier; Barbieri, Alejandro; Lopez, Cecilia; Stahl, Philip D.; Mayorga, Luis S.

    2005-01-01

    The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN)

  19. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

    Science.gov (United States)

    Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

    2015-01-01

    The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

  20. PIKfyve mediates the motility of late endosomes and lysosomes in neuronal dendrites.

    Science.gov (United States)

    Tsuruta, Fuminori; Dolmetsch, Ricardo E

    2015-09-25

    The endosome/lysosome system in the nervous system is critically important for a variety of neuronal functions such as neurite outgrowth, retrograde transport, and synaptic plasticity. In neurons, the endosome/lysosome system is crucial for the activity-dependent internalization of membrane proteins and contributes to the regulation of lipid level on the plasma membrane. Although homeostasis of membrane dynamics plays important roles in the properties of central nervous systems, it has not been elucidated how endosome/lysosome system is regulated. Here, we report that phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) mediates the motility of late endosomes and lysosomes in neuronal dendrites. Endosomes and lysosomes are highly motile in resting neurons, however knockdown of PIKfyve led to a significant reduction in late endosomes and lysosomes motility. We also found that vesicle acidification is crucial for their motility and PIKfyve is associated with this process indirectly. These data suggest that PIKfyve mediates vesicle motility through the regulation of vesicle integrity in neurons. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. The ESCRT regulator Did2 maintains the balance between long-distance endosomal transport and endocytic trafficking.

    Directory of Open Access Journals (Sweden)

    Carl Haag

    2017-04-01

    Full Text Available In highly polarised cells, like fungal hyphae, early endosomes function in both endocytosis as well as long-distance transport of various cargo including mRNA and protein complexes. However, knowledge on the crosstalk between these seemingly different trafficking processes is scarce. Here, we demonstrate that the ESCRT regulator Did2 coordinates endosomal transport in fungal hyphae of Ustilago maydis. Loss of Did2 results in defective vacuolar targeting, less processive long-distance transport and abnormal shuttling of early endosomes. Importantly, the late endosomal protein Rab7 and vacuolar protease Prc1 exhibit increased shuttling on these aberrant endosomes suggesting defects in endosomal maturation and identity. Consistently, molecular motors fail to attach efficiently explaining the disturbed processive movement. Furthermore, the endosomal mRNP linker protein Upa1 is hardly present on endosomes resulting in defects in long-distance mRNA transport. In conclusion, the ESCRT regulator Did2 coordinates precise maturation of endosomes and thus provides the correct membrane identity for efficient endosomal long-distance transport.

  2. Aggregation of endosomal-vacuolar compartments in the Aovps24-deleted strain in the filamentous fungus Aspergillus oryzae

    International Nuclear Information System (INIS)

    Tatsumi, Akinori; Shoji, Jun-ya; Kikuma, Takashi; Arioka, Manabu; Kitamoto, Katsuhiko

    2007-01-01

    Previously, we found that deletion of Aovps24, an ortholog of Saccharomyces cerevisiae VPS24, that encodes an ESCRT (endosomal sorting complex required for transport)-III component required for late endosomal function results in fragmented and aggregated vacuoles. Although defective late endosomal function is likely responsible for this phenotype, critical lack of our knowledge on late endosomes in filamentous fungi prevented us from further characterization. In this study, we identified late endosomes of Aspergillus oryzae, by expressing a series of fusion proteins of fluorescent proteins with orthologs of late endosomal proteins. Using these fusion proteins as markers, we observed late endosomes in the wild type strain and the Aovps24 disruptant and demonstrated that late endosomes are aberrantly aggregated in the Aovps24 disruptant. Moreover, we revealed that the aggregated late endosomes have features of vacuoles as well. As deletion of another ESCRT-III component-encoding gene, Aovps2, resulted in similar phenotypes to that in the Aovps24 disruptant, phenotypes of the Aovps24 disruptant are probably due to defective late endosomal function

  3. Terminalia catappa Exerts Antimetastatic Effects on Hepatocellular Carcinoma through Transcriptional Inhibition of Matrix Metalloproteinase-9 by Modulating NF-κB and AP-1 Activity

    Directory of Open Access Journals (Sweden)

    Chao-Bin Yeh

    2012-01-01

    Full Text Available High mortality and morbidity rates for hepatocellular carcinoma (HCC in Taiwan primarily result from uncontrolled tumor metastasis. Previous studies have identified that Terminalia catappa leaf extracts (TCE exert hepatoprotective, antioxidative, antiinflammatory, anticancer, and antimetastatic activities. However, the effects of TCE on HCC and the underlying molecular mechanisms of its activities have yet to be fully elucidated. The present study's findings demonstrate that TCE concentration dependently inhibits human HCC migration/invasion. Zymographic and western blot analyses revealed that TCE inhibited the activities and expression of matrix metalloproteinase-9 (MMP-9. Assessment of mRNA levels, using reverse transcriptase polymerase chain reaction (PCR and real-time PCR, and promoter assays confirmed the inhibitory effects of TCE on MMP-9 expression in HCC cells. The inhibitory effects of TCE on MMP-9 proceeded by upregulating tissue inhibitor of metalloproteinase-1 (TIMP-1, as well as suppressing nuclear translocation and DNA binding activity of nuclear factor-kappa B (NF-κB and activating protein-1 (AP-1 on the MMP-9 promoter in Huh7 cells. In conclusion, TCE inhibits MMP-9 expression and HCC cell metastasis and, thus, has potential use as a chemopreventive agent. Its inhibitory effects are associated with downregulation of the binding activities of the transcription factors NF-κB and AP-1.

  4. [6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling.

    Directory of Open Access Journals (Sweden)

    E K Radhakrishnan

    Full Text Available We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale, in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer.

  5. Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis

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    Akshay Bhinge

    2017-04-01

    Full Text Available Summary: Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS, it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential. : In this article, Bhinge, Stanton, and colleagues use genome editing of patient-derived iPSCs to model ALS phenotypic defects in vitro. Transcriptomic analysis of disease MNs reveals activation of MAPK, AP1, WNT, cell-cycle, and p53 signaling in ALS MNs. Pharmacological screening uncovers activated ERK and JNK signaling as therapeutic targets in ALS. Keywords: ALS, SOD1, FUS, CRISPR-Cas9, p38, ERK, JNK, WNT, TP53, JUN

  6. Cinnamoyloxy-mammeisin Isolated from Geopropolis Attenuates Inflammatory Process by Inhibiting Cytokine Production: Involvement of MAPK, AP-1, and NF-κB.

    Science.gov (United States)

    Franchin, Marcelo; Rosalen, Pedro Luiz; da Cunha, Marcos Guilherme; Silva, Rangel Leal; Colón, David F; Bassi, Gabriel Shimizu; de Alencar, Severino Matias; Ikegaki, Masaharu; Alves-Filho, José C; Cunha, Fernando Q; Beutler, John A; Cunha, Thiago Mattar

    2016-07-22

    Chemical compounds belonging to the class of coumarins have promising anti-inflammatory potential. Cinnamoyloxy-mammeisin (CNM) is a 4-phenylcoumarin that can be isolated from Brazilian geopropolis. To our knowledge, its anti-inflammatory activity has never been studied. Therefore, the present study investigated the anti-inflammatory activity of CNM and elucidated its mechanism of action on isolated macrophages. Pretreatment with CNM reduced neutrophil migration into the peritoneal and joint cavity of mice. Likewise, CNM reduced the in vitro and in vivo release of TNF-α and CXCL2/MIP-2. Regarding the possible molecular mechanism of action, CNM reduced the phosphorylation of proteins ERK 1/2, JNK, p38 MAPK, and AP-1 (subunit c-jun) in PG-stimulated macrophages. Pretreatment with CNM also reduced NF-κB activation in RAW 264.7 macrophages stably expressing the NF-κB-luciferase reporter gene. On the other hand, it did not alter IκBα degradation or nuclear translocation of p65. Thus, the results of this study demonstrate promising anti-inflammatory activity of CNM and provide an explanation of its mechanism of action in macrophages via inhibition of MAPK signaling, AP-1, and NF-κB.

  7. Bee Venom Inhibits Porphyromonas gingivalis Lipopolysaccharides-Induced Pro-Inflammatory Cytokines through Suppression of NF-κB and AP-1 Signaling Pathways.

    Science.gov (United States)

    Kim, Woon-Hae; An, Hyun-Jin; Kim, Jung-Yeon; Gwon, Mi-Gyeong; Gu, Hyemin; Park, Jae-Bok; Sung, Woo Jung; Kwon, Yong-Chul; Park, Kyung-Duck; Han, Sang Mi; Park, Kwan-Kyu

    2016-11-10

    Periodontitis is a chronic inflammatory disease that leads to destruction of tooth supporting tissues. Porphyromonas gingivalis ( P. gingivalis ), especially its lipopolysaccharides (LPS), is one of major pathogens that cause periodontitis. Bee venom (BV) has been widely used as a traditional medicine for various diseases. Previous studies have demonstrated the anti-inflammatory, anti-bacterial effects of BV. However, a direct role and cellular mechanism of BV on periodontitis-like human keratinocytes have not been explored. Therefore, we investigated the anti-inflammatory mechanism of BV against P. gingivalis LPS (PgLPS)-induced HaCaT human keratinocyte cell line. The anti-inflammatory effect of BV was demonstrated by various molecular biological methods. The results showed that PgLPS increased the expression of Toll-like receptor (TLR)-4 and pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, and interferon (IFN)-γ. In addition, PgLPS induced activation of the signaling pathways of inflammatory cytokines-related transcription factors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1). BV effectively inhibited those pro-inflammatory cytokines through suppression of NF-κB and AP-1 signaling pathways. These results suggest that administration of BV attenuates PgLPS-induced inflammatory responses. Furthermore, BV may be a useful treatment to anti-inflammatory therapy for periodontitis.

  8. Salvia miltiorrhiza extract inhibits TPA-induced MMP-9 expression and invasion through the MAPK/AP-1 signaling pathway in human breast cancer MCF-7 cells.

    Science.gov (United States)

    Kim, Jeong-Mi; Noh, Eun-Mi; Song, Hyun-Kyung; Lee, Minok; Lee, Soo Ho; Park, Sueng Hyuk; Ahn, Chan-Keun; Lee, Guem-San; Byun, Eui-Baek; Jang, Beom-Su; Kwon, Kang-Beom; Lee, Young-Rae

    2017-09-01

    Cancer cell invasion is crucial for metastasis. A major factor in the capacity of cancer cell invasion is the activation of matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix. Salvia miltiorrhiza has been used as a promotion for blood circulation to remove blood stasis. Numerous previous studies have demonstrated that S. miltiorrhiza extracts (SME) decrease lipid levels and inhibit inflammation. However, the mechanism behind the effect of SME on breast cancer invasion has not been identified. The inhibitory effects of SME on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression were assessed using western blotting, reverse transcription-quantitative polymerase chain reaction and zymography assays. MMP-9 upstream signal proteins, including mitogen-activated protein kinases and activator protein 1 (AP-1) were also investigated. Cell invasion was assessed using a matrigel invasion assay. The present study demonstrated the inhibitory effects of the SME ethanol solution on MMP-9 expression and cell invasion in TPA-treated MCF-7 breast cancer cells. SME suppressed TPA-induced MMP-9 expression and MCF-7 cell invasion by blocking the transcriptional activation of AP-1. SME may possess therapeutic potential for inhibiting breast cancer cell invasiveness.

  9. Benzyl alcohol derivatives from the mushroom Hericium erinaceum attenuate LPS-stimulated inflammatory response through the regulation of NF-κB and AP-1 activity.

    Science.gov (United States)

    Noh, Hyung Jun; Yoon, Ju Young; Kim, Geum Sook; Lee, Seung Eun; Lee, Dae Young; Choi, Je Hun; Kim, Seung Yu; Kang, Ki Sung; Cho, Jae Youl; Kim, Ki Hyun

    2014-10-01

    On the search for anti-inflammatory compounds from natural Korean medicinal sources, a bioassay-guided fractionation and chemical investigation of the MeOH extract from the fruiting bodies of Hericium erinaceum resulted in the isolation and identification of five benzyl alcohol derivatives (1-5). In this study, their anti-inflammatory effects on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW 264.7 macrophage cells. The structures of isolates were identified by comparing their spectroscopic data with previously reported values. The analysis of their inhibitory activities on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells showed that erinacerin B (2) and hericenone E (4) decreased the levels of NO and PGE2 production in a concentration-dependent manner. Next, this study was performed to examine their mechanism of action on the regulation of NO and PGE2 production. Compounds 2 and 4 were found to block the LPS-induced phosphorylation of two major inflammatory transcription factors, NF-κB (p65/p50) and AP-1 (c-Jun and c-Fos). Taken together, these results suggest that down-regulation of LPS-induced NO and PGE2 production by compounds 2 and 4 is mediated through the modulation of NF-κB and AP-1 activation in macrophage cells. These results impact the development of potential health products for preventing and treating inflammatory diseases.

  10. Radical Scavenging Activity-Based and AP-1-Targeted Anti-Inflammatory Effects of Lutein in Macrophage-Like and Skin Keratinocytic Cells

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    Jueun Oh

    2013-01-01

    Full Text Available Lutein is a naturally occurring carotenoid with antioxidative, antitumorigenic, antiangiogenic, photoprotective, hepatoprotective, and neuroprotective properties. Although the anti-inflammatory effects of lutein have previously been described, the mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, in the present study, we aimed to investigate the regulatory activity of lutein in the inflammatory responses of skin-derived keratinocytes or macrophages and to elucidate the mechanism of its inhibitory action. Lutein significantly reduced several skin inflammatory responses, including increased expression of interleukin-(IL- 6 from LPS-treated macrophages, upregulation of cyclooxygenase-(COX- 2 from interferon-γ/tumor necrosis-factor-(TNF- α-treated HaCaT cells, and the enhancement of matrix-metallopeptidase-(MMP- 9 level in UV-irradiated keratinocytes. By evaluating the intracellular signaling pathway and the nuclear transcription factor levels, we determined that lutein inhibited the activation of redox-sensitive AP-1 pathway by suppressing the activation of p38 and c-Jun-N-terminal kinase (JNK. Evaluation of the radical and ROS scavenging activities further revealed that lutein was able to act as a strong anti-oxidant. Taken together, our findings strongly suggest that lutein-mediated AP-1 suppression and anti-inflammatory activity are the result of its strong antioxidative and p38/JNK inhibitory activities. These findings can be applied for the preparation of anti-inflammatory and cosmetic remedies for inflammatory diseases of the skin.

  11. Syndapin/SDPN-1 is required for endocytic recycling and endosomal actin association in the Caenorhabditis elegans intestine

    Science.gov (United States)

    Gleason, Adenrele M.; Nguyen, Ken C. Q.; Hall, David H.; Grant, Barth D.

    2016-01-01

    Syndapin/pascin-family F-BAR domain proteins bind directly to membrane lipids and are associated with actin dynamics at the plasma membrane. Previous reports also implicated mammalian syndapin 2 in endosome function during receptor recycling, but precise analysis of a putative recycling function for syndapin in mammalian systems is difficult because of its effects on the earlier step of endocytic uptake and potential redundancy among the three separate genes that encode mammalian syndapin isoforms. Here we analyze the endocytic transport function of the only Caenorhabditis elegans syndapin, SDPN-1. We find that SDPN-1 is a resident protein of the early and basolateral recycling endosomes in the C. elegans intestinal epithelium, and sdpn-1 deletion mutants display phenotypes indicating a block in basolateral recycling transport. sdpn-1 mutants accumulate abnormal endosomes positive for early endosome and recycling endosome markers that are normally separate, and such endosomes accumulate high levels of basolateral recycling cargo. Furthermore, we observed strong colocalization of endosomal SDPN-1 with the F-actin biosensor Lifeact and found that loss of SDPN-1 greatly reduced Lifeact accumulation on early endosomes. Taken together, our results provide strong evidence for an in vivo function of syndapin in endocytic recycling and suggest that syndapin promotes transport via endosomal fission. PMID:27630264

  12. Establishment and characterization of cetuximab resistant head and neck squamous cell carcinoma cell lines: focus on the contribution of the AP-1 transcription factor

    Science.gov (United States)

    Boeckx, Carolien; Blockx, Lina; de Beeck, Ken Op; Limame, Ridha; Camp, Guy Van; Peeters, Marc; Vermorken, Jan B; Specenier, Pol; Wouters, An; Baay, Marc; Lardon, Filip

    2015-01-01

    Background: After an initial response to EGFR targeted therapy, secondary resistance almost invariably ensues, thereby limiting the clinical benefit of the drug. Hence, it has been recognized that the successful implementation of targeted therapy in the treatment of HNSCC cancer is very much dependent on predictive biomarkers for patient selection. Methods: We generated an in vitro model of acquired cetuximab resistance by chronically exposing three HNSCC cell lines to increasing cetuximab doses. Gene expression profiles of sensitive parental cells and resistant daughter cells were compared using microarray analysis. Growth inhibitory experiments were performed with an HB-EGF antibody and the MMP inhibitor, both in combination with cetuximab. Characteristics of EMT were analyzed using migration and invasion assays, immunofluorescent vimentin staining and qRT-PCR for several genes involved in this process. The function of the transcription factor AP-1 was investigated using qRT-PCR for several genes upregulated or downregulated in cetuximab resistant cells. Furthermore, anchorage-independent growth was investigated using the soft agar assay. Results: Gene expression profiling shows that cetuximab resistant cells upregulate several genes, including interleukin 8, the EGFR ligand HB-EGF and the metalloproteinase ADAM19. Cytotoxicity experiments with neutralizing HB-EGF antibody could not induce any growth inhibition, whereas an MMP inhibitor inhibited cell growth in cetuximab resistant cells. However, no synergetic effects combined with cetuximab could be observed. Cetuximab resistant cells showed traits of EMT, as witnessed by increased migratory potential, increased invasive potential, increased vimentine expression and increased expression of several genes involved in EMT. Furthermore, expression of upregulated genes could be repressed by the treatment with apigenin. The cetuximab resistant LICR-HN2 R10.3 cells tend to behave differently in cell culture, forming

  13. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    Directory of Open Access Journals (Sweden)

    Ewoud Bernardus Compeer

    2012-03-01

    Full Text Available The cross-presentation of endocytosed antigen as peptide/class I MHC complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells (APC capable of antigen cross-presentation, description of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC, there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlight DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, recycling and maturation including the sorting of membrane proteins, dynamic remodeling of endosomal structures and cell-surface directed endosomal trafficking. We will conclude with description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

  14. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-01-01

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

  15. Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes.

    Science.gov (United States)

    Tornieri, Karine; Zlatic, Stephanie A; Mullin, Ariana P; Werner, Erica; Harrison, Robert; L'hernault, Steven W; Faundez, Victor

    2013-12-20

    Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome-lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion.

  16. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-05-18

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

  17. Dietary turmeric modulates DMBA-induced p21ras, MAP kinases and AP-1/NF-κB pathway to alter cellular responses during hamster buccal pouch carcinogenesis

    International Nuclear Information System (INIS)

    Garg, Rachana; Ingle, Arvind; Maru, Girish

    2008-01-01

    The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12 weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished the DMBA-induced mRNA expression of proto-oncogenes (c-jun, c-fos) and NF-κB, leading to decreased protein levels and in further attenuation of DMBA-induced AP-1/NF-κB DNA-binding in the buccal pouch nuclear extracts. Besides, buccal pouch of hamsters receiving turmeric diet showed significant alterations in DMBA-induced effects: (a) decrease in cell proliferation (diminished PCNA and Bcl2 expression), (b) enhanced apoptosis (increased expression of Bax, caspase-3 and apoptotic index), (c) decrease in inflammation (levels of Cox-2, the downstream target of AP-1/NF-κB, and PGE2) and (d) aberrant expression of differentiation markers, the cytokeratins (1, 5, 8, and 18). Together, the protective effects of dietary turmeric converge on augmenting apoptosis of the initiated cells and decreasing cell proliferation in DMBA-treated animals, which in turn, is reflected in decreased tumor burden, multiplicity and enhanced latency period. Some of these biomarkers are likely to be helpful in monitoring clinical trials and evaluating drug effect measurements

  18. 5-Methoxyl Aesculetin Abrogates Lipopolysaccharide-Induced Inflammation by Suppressing MAPK and AP-1 Pathways in RAW 264.7 Cells

    Directory of Open Access Journals (Sweden)

    Lei Wu

    2016-03-01

    Full Text Available For the first time, a pale amorphous coumarin derivative, 5-methoxyl aesculetin (MOA, was isolated from the dried bark of Fraxinus rhynchophylla Hance (Oleaceae. MOA modulates cytokine expression in lipopolysaccharide (LPS-treated RAW 264.7 macrophages, but the precise mechanisms are still not fully understood. We determined the effects of MOA on the production of inflammatory mediators and pro-inflammatory cytokines in the LPS-induced inflammatory responses of RAW 264.7 macrophages. MOA significantly inhibited the LPS-induced production of nitric oxide (NO, prostaglandin E2 (PGE2, tumor necrosis factor-α (TNF-α, interleukin-6, and interleukin-1β. It also effectively attenuated inducible nitric oxide (NO synthase, cyclooxygenase-2, and TNF-α mRNA expression and significantly decreased the levels of intracellular reactive oxygen species. It inhibited phosphorylation of the extracellular signal-regulated kinase (ERK1/2, thus blocking nuclear translocation of activation protein (AP-1. In a molecular docking study, MOA was shown to target the binding site of ERK via the formation of three hydrogen bonds with two residues of the kinase, which is sufficient for the inhibition of ERK. These results suggest that the anti-inflammatory effects of MOA in RAW 264.7 macrophages derive from its ability to block both the activation of mitogen-activated protein kinases (MAPKs and one of their downstream transcription factors, activator protein-1 (AP-1. Our observations support the need for further research into MOA as a promising therapeutic agent in inflammatory diseases.

  19. NF-κB/AP-1-Targeted Inhibition of Macrophage-Mediated Inflammatory Responses by Depigmenting Compound AP736 Derived from Natural 1,3-Diphenylpropane Skeleton

    Directory of Open Access Journals (Sweden)

    Van Thai Ha

    2014-01-01

    Full Text Available AP736 was identified as an antimelanogenic drug that can be used for the prevention of melasma, freckles, and dark spots in skin by acting as a suppressor of melanin synthesis and tyrosinase expression. Since macrophage-mediated inflammatory responses are critical for skin health, here we investigated the potential anti-inflammatory activity of AP736. The effects of AP736 on various inflammatory events such as nitric oxide (NO/prostaglandin (PG E2 production, inflammatory gene expression, phagocytic uptake, and morphological changes were examined in RAW264.7 cells. AP736 was found to strongly inhibit the production of both NO and PGE2 in lipopolysaccharide- (LPS- treated RAW264.7 cells. In addition, AP736 strongly inhibited both LPS-induced morphological changes and FITC-dextran-induced phagocytic uptake. Furthermore, AP736 also downregulated the expression of multiple inflammatory genes, such as inducible NO synthase (iNOS, cyclooxygenase- (COX- 2, and interleukin- (IL- 1β in LPS-treated RAW264.7 cells. Transcription factor analysis, including upstream signalling events, revealed that both NF-κB and AP-1 were targeted by AP736 via inhibition of the IKK/IκBα and IRAK1/TAK1 pathways. Therefore, our results strongly suggest that AP736 is a potential anti-inflammatory drug due to its suppression of NF-κB-IKK/IκBα and AP-1-IRAK1/TAK1 signalling, which may make AP736 useful for the treatment of macrophage-mediated skin inflammation.

  20. 5-Methoxyl Aesculetin Abrogates Lipopolysaccharide-Induced Inflammation by Suppressing MAPK and AP-1 Pathways in RAW 264.7 Cells

    Science.gov (United States)

    Wu, Lei; Li, Xueqin; Wu, Haifeng; Long, Wei; Jiang, Xiaojian; Shen, Ting; Qiang, Qian; Si, Chuanling; Wang, Xinfeng; Jiang, Yunyao; Hu, Weicheng

    2016-01-01

    For the first time, a pale amorphous coumarin derivative, 5-methoxyl aesculetin (MOA), was isolated from the dried bark of Fraxinus rhynchophylla Hance (Oleaceae). MOA modulates cytokine expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, but the precise mechanisms are still not fully understood. We determined the effects of MOA on the production of inflammatory mediators and pro-inflammatory cytokines in the LPS-induced inflammatory responses of RAW 264.7 macrophages. MOA significantly inhibited the LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-6, and interleukin-1β. It also effectively attenuated inducible nitric oxide (NO) synthase, cyclooxygenase-2, and TNF-α mRNA expression and significantly decreased the levels of intracellular reactive oxygen species. It inhibited phosphorylation of the extracellular signal-regulated kinase (ERK1/2), thus blocking nuclear translocation of activation protein (AP)-1. In a molecular docking study, MOA was shown to target the binding site of ERK via the formation of three hydrogen bonds with two residues of the kinase, which is sufficient for the inhibition of ERK. These results suggest that the anti-inflammatory effects of MOA in RAW 264.7 macrophages derive from its ability to block both the activation of mitogen-activated protein kinases (MAPKs) and one of their downstream transcription factors, activator protein-1 (AP-1). Our observations support the need for further research into MOA as a promising therapeutic agent in inflammatory diseases. PMID:26938526

  1. Anticancer effects of monocarbonyl analogs of curcumin: oxidative stress, nuclear translocation and modulation of AP-1 and NF-κB

    Directory of Open Access Journals (Sweden)

    Brian Adams

    2015-01-01

    Full Text Available Purpose: In order to elucidate anticancer effects of monocarbonyl analogs of curcumin (MACs, we have undertaken the present study to obtain information regarding drug targets by using a microarray approach, and to study the cellular localization of EF24 and the activity of two key transcription factors, AP-1 and NF-κB, involved in complex cellular responses of cell survival and death. Methods: Cytotoxic activity of various drugs was evaluated using a Neutral Red Dye assay. Cellular localization of biotinylated EF24 (active and reduced EF24 (inactive was determined using light and confocal microscopy. Measurement of transcription factor binding was carried out using Transfactor ELISA kits (BD Clontech, Palo Alto, CA. Gene microarray processing was performed at Expression Analysis, Inc (Durham, NC using Affymetrix Human U133A Gene Chips.Results: In this study, we demonstrated that EF24 and UBS109 exhibit much more potent cytotoxic activity against pancreatic cancer than the current standard chemotherapeutic agent gemcitabine. EF24, rapidly localizes to the cell nucleus. The compound modulates the DNA binding activity of NF-κB and AP-1 in MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells. Immunohistochemical studies utilizing biotinylated-EF24 and chemically-reduced EF24 show that the unsaturated compound and biotinylated EF24, but not reduced EF24, translocates to the nucleus within 30 minutes after the addition of drug. Through a gene microarray study, EF24 is shown to affect genes directly involved in cytoprotection, tumor growth, angiogenesis, metastasis and apoptosis. Conclusion: EF24 and UBS109 warrant further investigation for development of pancreatic cancer therapy. The dualistic modulations of gene expression may be a manifestation of the cell responses for survival against oxidative stress by EF24. However, the cytotoxic action of EF24 ultimately prevails to kill the cells.

  2. Thermoresponsive pegylated bubble liposome nanovectors for efficient siRNA delivery via endosomal escape

    KAUST Repository

    Alamoudi, Kholod; Martins, Patricia; Croissant, Jonas G.; Patil, Sachin; Omar, Haneen; Khashab, Niveen M.

    2017-01-01

    Improving the delivery of siRNA into cancer cells via bubble liposomes. Designing a thermoresponsive pegylated liposome through the introduction of ammonium bicarbonate salt into liposomes so as to control their endosomal escape for gene therapy.A sub-200 nm nanovector was fully characterized and examined for cellular uptake, cytotoxicity, endosomal escape and gene silencing.The siRNA-liposomes were internalized into cancer cells within 5 min and then released siRNAs in the cytosol prior to lysosomal degradation upon external temperature elevation. This was confirmed by confocal bioimaging and gene silencing reaching up to 90% and further demonstrated by the protein inhibition of both target genes.The thermoresponsiveness of ammonium bicarbonate containing liposomes enabled the rapid endosomal escape of the particles and resulted in an efficient gene silencing.

  3. Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Hirokazu Tanaka

    2013-05-01

    Full Text Available PIN-FORMED (PIN proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.

  4. Thermoresponsive pegylated bubble liposome nanovectors for efficient siRNA delivery via endosomal escape

    KAUST Repository

    Alamoudi, Kholod

    2017-05-19

    Improving the delivery of siRNA into cancer cells via bubble liposomes. Designing a thermoresponsive pegylated liposome through the introduction of ammonium bicarbonate salt into liposomes so as to control their endosomal escape for gene therapy.A sub-200 nm nanovector was fully characterized and examined for cellular uptake, cytotoxicity, endosomal escape and gene silencing.The siRNA-liposomes were internalized into cancer cells within 5 min and then released siRNAs in the cytosol prior to lysosomal degradation upon external temperature elevation. This was confirmed by confocal bioimaging and gene silencing reaching up to 90% and further demonstrated by the protein inhibition of both target genes.The thermoresponsiveness of ammonium bicarbonate containing liposomes enabled the rapid endosomal escape of the particles and resulted in an efficient gene silencing.

  5. G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

    Science.gov (United States)

    Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

    2015-03-13

    Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Thermal fatigue behavior of thermal barrier coatings by air plasma spray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Han Sang; Kim, Eui Hyun [Korea Electric Power Research Institute, Daejeon (Korea, Republic of); Lee, Jung Hyuk [Korea Plant Service and Engineering Co. Ltd., Incheon (Korea, Republic of)

    2008-06-15

    Effects of top coat morphology and thickness on thermal fatigue behavior of Thermal Barrier Coatings (TBC) were investigated in this study. Thermal fatigue tests were conducted on three coating specimens with different top coat morphology and thickness, and then the test data were compared via microstructures, cycles to failure, and fracture surfaces. In the air plasma spray specimens (APS1, APS2), top coat were 200 and 300 {mu}m respectively. The thickness of top coat was about 700 {mu}m in the Perpendicular Cracked Specimen (PCS). Under thermal fatigue condition at 1,100 .deg. C, the cycles to top coat failure of APS1, APS2, and PCS were 350, 560 and 480 cycles, respectively. The cracks were initiated at the interface of top coat and Thermally Grown Oxide (TGO) and propagated into TGO or top coat as the number of thermal fatigue cycles increased. For the PCS specimen, additive cracks were initiated and propagated at the starting points of perpendicular cracks in the top coat. Also, the thickness of TGO and the decrease of aluminium concentration in bond coat do not affect the cycles to failure.

  7. GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

    Science.gov (United States)

    Toh, Wei Hong; Chia, Pei Zhi Cheryl; Hossain, Mohammed Iqbal; Gleeson, Paul A.

    2018-01-01

    The diversion of the membrane-bound β-site amyloid precursor protein–(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aβ production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aβ production. PMID:29142073

  8. Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment

    Directory of Open Access Journals (Sweden)

    Shravan Babu Girada

    2017-10-01

    Conclusions: The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex.

  9. Neuron specific Rab4 effector GRASP-1 coordinates membrane specialization and maturation of recycling endosomes

    NARCIS (Netherlands)

    C.C. Hoogenraad (Casper); I. Popa (Ioana); K. Futai (Kensuke); E. Sanchez-Martinez (Emma); P. Wulf (Phebe); T. van Vlijmen (Thijs); B.R. Dortland (Bjorn); V. Oorschot (Viola); R. Govers (Robert); M. Monti (Maria); A.J.R. Heck (Albert); M. Sheng (Morgan); J. Klumperman (Judith); H. Rehmann (Holger); D. Jaarsma (Dick); L.C. Kapitein (Lukas); P. van der Sluijs

    2010-01-01

    textabstractThe endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1

  10. Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation

    Directory of Open Access Journals (Sweden)

    Chao-Yang Lai

    2017-01-01

    Full Text Available Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs, particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.

  11. TLR2 ligands induce NF-κB activation from endosomal compartments of human monocytes.

    Directory of Open Access Journals (Sweden)

    Karim J Brandt

    Full Text Available Localization of Toll-like receptors (TLR in subcellular organelles is a major strategy to regulate innate immune responses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomal compartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we show that the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-κB-dependent signaling from endosomal compartments in human monocytes and in a NF-κB sensitive reporter cell line, despite the expression of TLR2 at the cell surface. Further analyses indicate that TLR2-induced NF-κB activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findings establish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-κB activation. These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependent signaling pathways.

  12. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

    Science.gov (United States)

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M.; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-01-01

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion. PMID:24743596

  13. Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules

    DEFF Research Database (Denmark)

    van Weering, Jan R.T.; Sessions, Richard B.; Traer, Colin J.

    2012-01-01

    that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip...... and organizes the tubular endosomal network....

  14. Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons.

    Science.gov (United States)

    Cai, Qian; Lu, Li; Tian, Jin-Hua; Zhu, Yi-Bing; Qiao, Haifa; Sheng, Zu-Hang

    2010-10-06

    Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Endosomal gene expression: a new indicator for prostate cancer patient prognosis?

    LENUS (Irish Health Repository)

    Johnson, Ian R D

    2015-11-10

    Prostate cancer continues to be a major cause of morbidity and mortality in men, but a method for accurate prognosis in these patients is yet to be developed. The recent discovery of altered endosomal biogenesis in prostate cancer has identified a fundamental change in the cell biology of this cancer, which holds great promise for the identification of novel biomarkers that can predict disease outcomes. Here we have identified significantly altered expression of endosomal genes in prostate cancer compared to non-malignant tissue in mRNA microarrays and confirmed these findings by qRT-PCR on fresh-frozen tissue. Importantly, we identified endosomal gene expression patterns that were predictive of patient outcomes. Two endosomal tri-gene signatures were identified from a previously published microarray cohort and had a significant capacity to stratify patient outcomes. The expression of APPL1, RAB5A, EEA1, PDCD6IP, NOX4 and SORT1 were altered in malignant patient tissue, when compared to indolent and normal prostate tissue. These findings support the initiation of a case-control study using larger cohorts of prostate tissue, with documented patient outcomes, to determine if different combinations of these new biomarkers can accurately predict disease status and clinical progression in prostate cancer patients.

  16. Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

    Science.gov (United States)

    Ho, Ernest; Ivanova, Iordanka A; Dagnino, Lina

    2016-12-01

    The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions. Although the mechanisms that mediate E-cadherin delivery to the plasma membrane have been widely studied in simple epithelia, this process is less well understood in the stratified epidermis. In this study, we have investigated the role of Engulfment and Cell Motility 2 (ELMO2) and integrin-linked kinase (ILK) in the positioning of E-cadherin-containing recycling endosomes during establishment of cell-cell contacts in differentiating keratinocytes. We now show that induction of keratinocyte differentiation by Ca 2+ is accompanied by localization of ELMO2 and ILK to Rab4- and Rab11a-containing recycling endosomes. The positioning of long-loop Rab11a-positive endosomes at areas adjacent to cell-cell contacts is disrupted in ELMO2- or ILK-deficient keratinocytes, and is associated with impaired localization of E-cadherin to cell borders. Our studies show a previously unrecognized role for ELMO2 and ILK in modulation of endosomal positioning, which may play key roles in epidermal sheet maintenance and permeability barrier function. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

    Science.gov (United States)

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2017-04-01

    The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

  18. Eucalyptus globulus extract protects against UVB-induced photoaging by enhancing collagen synthesis via regulation of TGF-β/Smad signals and attenuation of AP-1.

    Science.gov (United States)

    Park, Bom; Hwang, Eunson; Seo, Seul A; Cho, Jin-Gyeong; Yang, Jung-Eun; Yi, Tae-Hoo

    2018-01-01

    UV irradiation triggers the overproduction of matrix metalloproteinases and collagen degradation, which in turn causes increased pigmentation, dryness, and deep wrinkling of the skin. These chronic symptoms are collectively referred to as photoaging. Eucalyptus globulus is an evergreen tree that is widely used in cosmetics because of its antimicrobial activity. In this study, we investigated the protective effect of 50% ethanol extracts of Eucalyptus globulus on UV-induced photoaging in vitro and in vivo. Normal human dermal fibroblasts were treated with Eucalyptus globulus at concentrations ranging from 1 to 100 μg/mL after UVB or non-UVB irradiation. We found that Eucalyptus globulus suppressed the expression of MMPs and IL-6, but increased the expression of TGF-β1 and procollagen type 1. In addition, Eucalyptus globulus inhibited activation of the AP-1 transcription factor, an inducer of MMPs. Eucalyptus globulus was also found to regulate TGF-β/Smad signaling by reversing the activity of negative Smad regulators. Lastly, in vivo studies showed that topical application of Eucalyptus globulus on UVB-irradiated hairless mice reduced wrinkle formation and dryness by down-regulating MMP-1 and up-regulating expression of elastin, TGF-β1, and procollagen type 1. Taken together, these data suggest that Eucalyptus globulus may be a useful agent in cosmetic products. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway.

    Directory of Open Access Journals (Sweden)

    Juin-Hua Huang

    2015-07-01

    Full Text Available Collaboration between heterogeneous pattern recognition receptors (PRRs leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3 and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA, genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection.

  20. Prostaglandin E1 and Its Analog Misoprostol Inhibit Human CML Stem Cell Self-Renewal via EP4 Receptor Activation and Repression of AP-1.

    Science.gov (United States)

    Li, Fengyin; He, Bing; Ma, Xiaoke; Yu, Shuyang; Bhave, Rupali R; Lentz, Steven R; Tan, Kai; Guzman, Monica L; Zhao, Chen; Xue, Hai-Hui

    2017-09-07

    Effective treatment of chronic myelogenous leukemia (CML) largely depends on the eradication of CML leukemic stem cells (LSCs). We recently showed that CML LSCs depend on Tcf1 and Lef1 factors for self-renewal. Using a connectivity map, we identified prostaglandin E1 (PGE1) as a small molecule that partly elicited the gene expression changes in LSCs caused by Tcf1/Lef1 deficiency. Although it has little impact on normal hematopoiesis, we found that PGE1 treatment impaired the persistence and activity of LSCs in a pre-clinical murine CML model and a xenograft model of transplanted CML patient CD34 + stem/progenitor cells. Mechanistically, PGE1 acted on the EP4 receptor and repressed Fosb and Fos AP-1 factors in a β-catenin-independent manner. Misoprostol, an FDA-approved EP4 agonist, conferred similar protection against CML. These findings suggest that activation of this PGE1-EP4 pathway specifically targets CML LSCs and that the combination of PGE1/misoprostol with conventional tyrosine-kinase inhibitors could provide effective therapy for CML. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Masato, E-mail: okadam@biken.osaka-u.ac.jp [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  2. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

    Directory of Open Access Journals (Sweden)

    Sofie Ignoul

    Full Text Available BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432 have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y, endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4 and not with late endosomal/lysosomal markers (LAMP-1, Rab7. Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6

  3. Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-κB and AP-1 signaling

    Directory of Open Access Journals (Sweden)

    Simon James

    2011-05-01

    Full Text Available Abstract Background Mushrooms are well recognized for their culinary properties as well as for their potency to enhance immune response. In the present study, we evaluated anti-inflammatory properties of an edible oyster mushroom (Pleurotus ostreatus in vitro and in vivo. Methods RAW264.7 murine macrophage cell line and murine splenocytes were incubated with the oyster mushroom concentrate (OMC, 0-100 μg/ml in the absence or presence of lipopolysacharide (LPS or concanavalin A (ConA, respectively. Cell proliferation was determined by MTT assay. Expression of cytokines and proteins was measured by ELISA assay and Western blot analysis, respectively. DNA-binding activity was assayed by the gel-shift analysis. Inflammation in mice was induced by intraperitoneal injection of LPS. Results OMC suppressed LPS-induced secretion of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6, and IL-12p40 from RAW264.7 macrophages. OMC inhibited LPS-induced production of prostaglandin E2 (PGE2 and nitric oxide (NO through the down-regulation of expression of COX-2 and iNOS, respectively. OMC also inhibited LPS-dependent DNA-binding activity of AP-1 and NF-κB in RAW264.7 cells. Oral administration of OMC markedly suppressed secretion of TNF-α and IL-6 in mice challenged with LPS in vivo. Anti-inflammatory activity of OMC was confirmed by the inhibition of proliferation and secretion of interferon-γ (IFN-γ, IL-2, and IL-6 from concanavalin A (ConA-stimulated mouse splenocytes. Conclusions Our study suggests that oyster mushroom possesses anti-inflammatory activities and could be considered a dietary agent against inflammation. The health benefits of the oyster mushroom warrant further clinical studies.

  4. Cyr61/CCN1 induces CCL20 production by keratinocyte via activating p38 and JNK/AP-1 pathway in psoriasis.

    Science.gov (United States)

    Li, Huidan; Li, Haichuan; Huo, Rongfen; Wu, Pinru; Shen, Zhengyu; Xu, Hui; Shen, Baihua; Li, Ningli

    2017-10-01

    Psoriasis is a common chronic skin disease characterized by epidermal hyperplasia and inflammation. Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) has recently been implicated in psoriasis pathogenesis by promoting keratinocyte activation. However, the mechanisms by which CCN1 enhances cutaneous inflammation are not fully understood. In this study, we investigated the role of CCN1 on the expression of CCL20 in human keratinocyte. By double-label immunofluorescence staining, we first identified that the expression of CCN1 colocalized well with CCL20 production in the epidermis of psoriasis skin lesion. Furthermore, in vivo, blocking or knockdown CCN1 expression ameliorated skin inflammation and reduced the expression of CCL20 in both imiquimod and IL-23-induced psoriasis-like mouse models, which indicated that CCN1 might be involved in the regulation of CCL20 production in psoriasis. Next, in vitro, we stimulated primary normal human epidermal keratinocyte (NHEK) with exogenous protein CCN1 and found that CCN1 directly upregulated CCL20 production independent of TNF-α, IL-22 and IL-17 pathway. Lastly, the signaling pathway study showed that CCN1 enhanced the binding of AP-1 to the CCL20 promoter via crosstalk with p38 and JNK. Our study demonstrates that CCN1 stimulates CCL20 production in vitro and in vivo, and thus supports the notion that overexpressed CCN1 in hyperproliferating keratinocyte is functionally involved in the recruitment of inflammatory cells to skin lesions affected by psoriasis. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  5. Amitriptyline up-regulates connexin43-gap junction in rat cultured cortical astrocytes via activation of the p38 and c-Fos/AP-1 signalling pathway.

    Science.gov (United States)

    Morioka, N; Suekama, K; Zhang, F F; Kajitani, N; Hisaoka-Nakashima, K; Takebayashi, M; Nakata, Y

    2014-06-01

    Intercellular communication via gap junctions, comprised of connexin (Cx) proteins, allow for communication between astrocytes, which in turn is crucial for maintaining CNS homeostasis. The expression of Cx43 is decreased in post-mortem brains from patients with major depression. A potentially novel mechanism of tricyclic antidepressants is to increase the expression and functioning of gap junctions in astrocytes. The effect of amitriptyline on the expression of Cx43 and gap junction intercellular communication (GJIC) in rat primary cultured cortical astrocytes was investigated. We also investigated the role of p38 MAPK intracellular signalling pathway in the amitriptyline-induced expression of Cx43 and GJIC. Treatment with amitriptyline for 48 h significantly up-regulated Cx43 mRNA, protein and GJIC. The up-regulation of Cx43 was not monoamine-related since noradrenaline, 5-HT and dopamine did not induce Cx43 expression and pretreatment with α- and β-adrenoceptor antagonists had no effect. Intracellular signalling involved p38 MAPK, as amitriptyline significantly increased p38 MAPK phosphorylation and Cx43 expression and GJIC were significantly blocked by the p38 inhibitor SB 202190. Furthermore, amitriptyline-induced Cx43 expression and GJIC were markedly reduced by transcription factor AP-1 inhibitors (curcumin and tanshinone IIA). The translocation of c-Fos from the cytosol and the nucleus of cortical astrocytes was increased by amitriptyline, and this response was dependent on p38 activity. These findings indicate a novel mechanism of action of amitriptyline through cortical astrocytes, and further suggest that targeting this mechanism could lead to the development of a new class of antidepressants. © 2014 The British Pharmacological Society.

  6. Transcriptional Inhibition of Matrix Metal loproteinase 9 (MMP-9 Activity by a c-fos/Estrogen Receptor Fusion Protein is Mediated by the Proximal AP-1 Site of the MMP-9 Promoter and Correlates with Reduced Tumor Cell Invasion

    Directory of Open Access Journals (Sweden)

    David L. Crowe

    1999-10-01

    Full Text Available Tumor cell invasion of basement membranes is one of the hallmarks of malignant transformation. Tumor cells secrete proteolytic enzymes known as matrix metalloproteinases (MMPs which degrade extracellular matrix molecules. Increased expression of MMP-9 has been associated with acquisition of invasive phenotype in many tumors. However, multiple mechanisms for regulation of MMP-9 gene expression by tumor cell lines have been proposed. A number of transcription factor binding sites have been characterized in the upstream regulatory region of the MMP-9 gene, including those for AP-1. To determine how a specific AP-1 family member, c-fos, regulates MMP-9 promoter activity through these sites, we used an expression vector containing the c-fos coding region fused to the estrogen receptor (ER ligand binding domain. This construct is activated upon binding estradiol. Stable expression of this construct in ER negative squamous cell carcinoma (SCC lines produced an estradiol dependent decrease in the number of cells that migrated through a reconstituted basement membrane. This decreased invasiveness was accompanied by estradiol dependent downregulation of MMP-9 activity as determined by gelatin zymography. Estradiol also produced transcriptional downregulation of an MMP-9 promoter construct in cells transiently transfected with the c-fosER expression vector. This downregulation was mediated by the AP-1 site at —79 by in the MMP-9 promoter. We concluded that the proximal AP-1 site mediated the transcriptional downregulation of the MMP-9 promoter by a conditionally activated c-fos fusion protein.

  7. Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway.

    Science.gov (United States)

    Ko, Su Hyuk; Jeon, Jong Ik; Myung, Hyun Soo; Kim, Young-Jeon; Kim, Jung Mogg

    2017-10-01

    Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. Copyright © 2017 American Society for Microbiology.

  8. LMTK1 regulates dendritic formation by regulating movement of Rab11A-positive endosomes.

    Science.gov (United States)

    Takano, Tetsuya; Urushibara, Tomoki; Yoshioka, Nozomu; Saito, Taro; Fukuda, Mitsunori; Tomomura, Mineko; Hisanaga, Shin-Ichi

    2014-06-01

    Neurons extend two types of neurites-axons and dendrites-that differ in structure and function. Although it is well understood that the cytoskeleton plays a pivotal role in neurite differentiation and extension, the mechanisms by which membrane components are supplied to growing axons or dendrites is largely unknown. We previously reported that the membrane supply to axons is regulated by lemur kinase 1 (LMTK1) through Rab11A-positive endosomes. Here we investigate the role of LMTK1 in dendrite formation. Down-regulation of LMTK1 increases dendrite growth and branching of cerebral cortical neurons in vitro and in vivo. LMTK1 knockout significantly enhances the prevalence, velocity, and run length of anterograde movement of Rab11A-positive endosomes to levels similar to those expressing constitutively active Rab11A-Q70L. Rab11A-positive endosome dynamics also increases in the cell body and growth cone of LMTK1-deficient neurons. Moreover, a nonphosphorylatable LMTK1 mutant (Ser34Ala, a Cdk5 phosphorylation site) dramatically promotes dendrite growth. Thus LMTK1 negatively controls dendritic formation by regulating Rab11A-positive endosomal trafficking in a Cdk5-dependent manner, indicating the Cdk5-LMTK1-Rab11A pathway as a regulatory mechanism of dendrite development as well as axon outgrowth. © 2014 Takano et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  9. Endosomal protein sorting and autophagy genes contribute to the regulation of yeast life span.

    Science.gov (United States)

    Longo, Valter D; Nislow, Corey; Fabrizio, Paola

    2010-11-01

    Accumulating evidence from various organisms points to a role for autophagy in the regulation of life span. By performing a genome-wide screen to identify novel life span determinants in Saccharomyces cerevisiae, we have obtained further insights into the autophagy-related and -unrelated degradation processes that may be important for preventing cellular senescence. The generation of multivesicular bodies and their fusion with the vacuole in the endosomal pathway emerged as novel cell functions involved in yeast chronological survival and longevity extension.

  10. Endosome-based protein trafficking and Ca2+ homeostasis in the heart

    Directory of Open Access Journals (Sweden)

    Jerry eCurran

    2015-02-01

    Full Text Available The ability to dynamically regulate, traffic, retain, and recycle proteins within the cell membrane is fundamental to life and central to the normal function of the heart and cardiovascular system. In the heart, these systems are essential for the regulation of cardiac calcium, both at the level of the plasma membrane, but also at local domains of the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, nucleus, and nuclear envelope. One intracellular pathway often overlooked in relation to cardiovascular calcium regulation and signaling is the endosome-based trafficking pathway. Highlighting its importance, this system and its molecular components are evolutionarily conserved across all metazoans. However, remarkably little is known of how endosome-based protein trafficking and recycling functions within mammalian cells systems, especially in the heart. The vast majority of what is known has been derived from heterologous cell systems. However, recently, more appropriate cell and animal models been developed that have allowed researchers to begin to understand how this system functions within the intact physiological environment. All excitable cells, including cardiomyocytes, depend on the proper expression and organization of multiple ion channels, pumps, exchangers, and transporters within the plasma membrane. As the endosomal system acts to regulate the expression and localization of membrane proteins, understanding the in vivo function of this system in the heart is important. This review will focus on endosome-based protein trafficking in the heart in both health and disease. Special emphasis will be given to the role played by the family of endocytic regulatory proteins, C-terminal Eps15 homology domain -containing proteins (EHDs, as recent data demonstrates that this family of proteins is essential for the proper trafficking and localization and of key proteins involved in excitation-contraction coupling.

  11. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    Science.gov (United States)

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

    Science.gov (United States)

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-01-01

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

  13. Involvement of the endosomal-lysosomal system correlates with regional pathology in Creutzfeldt-Jakob disease

    DEFF Research Database (Denmark)

    Kovács, Gábor G; Gelpi, Ellen; Ströbel, Thomas

    2007-01-01

    The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes...... correlate with regional pathology. Overloading of this system might impair the function of lysosomal enzymes and thus may mimic some features of lysosomal storage disorders. Udgivelsesdato: 2007-Jul...

  14. Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

    Science.gov (United States)

    Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

    2016-02-01

    Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    International Nuclear Information System (INIS)

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose; Okada, Masato

    2012-01-01

    Highlights: ► p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. ► We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. ► The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. ► Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. ► The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome–lysosome fusion, which is required for processing of various macromolecules.

  16. Molecular determinants of the interaction between Doa1 and Hse1 involved in endosomal sorting.

    Science.gov (United States)

    Han, Seungsu; Shin, Donghyuk; Choi, Hoon; Lee, Sangho

    2014-03-28

    Yeast Doa1/Ufd3 is an adaptor protein for Cdc48 (p97 in mammal), an AAA type ATPase associated with endoplasmic reticulum-associated protein degradation pathway and endosomal sorting into multivesicular bodies. Doa1 functions in the endosomal sorting by its association with Hse1, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hse1 was previously reported to be mediated between PFU domain of Doa1 and SH3 of Hse1. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doa1/PFU interacts with Hse1/SH3 with a moderate affinity of 5 μM. Asn-438 of Doa1/PFU and Trp-254 of Hse1/SH3 are found to be critical in the interaction while Phe-434, implicated in ubiquitin binding via a hydrophobic interaction, is not. Small-angle X-ray scattering measurements combined with molecular docking and biochemical analysis yield the solution structure of the Doa1/PFU:Hse1/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hse1/SH3. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. dOCRL maintains immune cell quiescence by regulating endosomal traffic.

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    Steven J Del Signore

    2017-10-01

    Full Text Available Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.

  18. ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

    Science.gov (United States)

    Wu, Yu; Pons, Valérie; Goudet, Amélie; Panigai, Laetitia; Fischer, Annette; Herweg, Jo-Ana; Kali, Sabrina; Davey, Robert A; Laporte, Jérôme; Bouclier, Céline; Yousfi, Rahima; Aubenque, Céline; Merer, Goulven; Gobbo, Emilie; Lopez, Roman; Gillet, Cynthia; Cojean, Sandrine; Popoff, Michel R; Clayette, Pascal; Le Grand, Roger; Boulogne, Claire; Tordo, Noël; Lemichez, Emmanuel; Loiseau, Philippe M; Rudel, Thomas; Sauvaire, Didier; Cintrat, Jean-Christophe; Gillet, Daniel; Barbier, Julien

    2017-11-14

    Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

  19. Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval.

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    Jennifer Hirst

    2018-01-01

    Full Text Available The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR, GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

  20. Cholesterol Regulates Syntaxin 6 Trafficking at trans-Golgi Network Endosomal Boundaries

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    Meritxell Reverter

    2014-05-01

    Full Text Available Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN. Here, using Chinese hamster ovary (CHO Niemann-Pick type C1 (NPC1 mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6 accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs. This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

  1. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

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    Hendrik Fuchs

    2016-07-01

    Full Text Available The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

  2. Methods of analysis of the membrane trafficking pathway from recycling endosomes to lysosomes.

    Science.gov (United States)

    Matsui, Takahide; Fukuda, Mitsunori

    2014-01-01

    The transferrin receptor (TfR) is responsible for iron uptake through its trafficking between the plasma membrane and recycling endosomes, and as a result it has become a well-known marker for recycling endosomes. Although the molecular basis of the TfR recycling pathway has been thoroughly investigated, the TfR degradation mechanism has been poorly understood. Exposure of cultured cells to two drugs, the protein synthesis inhibitor cycloheximide and the V-ATPase inhibitor bafilomycin A1, recently showed that TfR is not only recycled back to the plasma membrane after endocytosis but is constitutively transported to lysosomes for degradation. The results of genome-wide screening of mouse Rab small GTPases (common regulators of membrane trafficking in all eukaryotes) have indicated that Rab12 regulates TfR trafficking to lysosomes independently of the known membrane trafficking pathways, for example, the conventional endocytic pathway and recycling pathway. This chapter summarizes the methods that the authors used to analyze the membrane trafficking pathway from recycling endosomes to lysosomes that is specifically regulated by Rab12. © 2014 Elsevier Inc. All rights reserved.

  3. Integration of two RAB5 groups during endosomal transport in plants

    Science.gov (United States)

    Ebine, Kazuo; Choi, Seung-won; Ichinose, Sakura; Uemura, Tomohiro; Nakano, Akihiko

    2018-01-01

    RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells. PMID:29749929

  4. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

    Science.gov (United States)

    Canfrán-Duque, Alberto; Barrio, Luis C; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A; Busto, Rebeca

    2016-03-18

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality.

  5. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    Directory of Open Access Journals (Sweden)

    Alberto Canfrán-Duque

    2016-03-01

    Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2 and LBPA (lysobisphosphatidic acid, which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1 and coatomer subunit β (β-COP were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality.

  6. Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

    Science.gov (United States)

    Liu, Ou; Grant, Barth D.

    2015-01-01

    The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions. PMID:26393361

  7. The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin

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    Miranda Arnold

    2016-09-01

    Full Text Available AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3 and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1. Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD and schizophrenia (SZ; yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines, and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse orthologue of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function.

  8. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    Science.gov (United States)

    Canfrán-Duque, Alberto; Barrio, Luis C.; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A.; Busto, Rebeca

    2016-01-01

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality. PMID:26999125

  9. Lysosomal and endosomal heterogeneity in the liver: A comparison of the intracellular pathways of endocytosis in rat liver cells

    International Nuclear Information System (INIS)

    Kindberg, G.M.; Tolleshaug, H.; Gjoen, T.; Berg, T.

    1991-01-01

    Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients

  10. Functional conservation and divergence of four ginger AP1/AGL9 MADS-box genes revealed by analysis of their expression and protein-protein interaction, and ectopic expression of AhFUL gene in Arabidopsis.

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    Xiumei Li

    Full Text Available Alpinia genus are known generally as ginger-lilies for showy flowers in the ginger family, Zingiberaceae, and their floral morphology diverges from typical monocotyledon flowers. However, little is known about the functions of ginger MADS-box genes in floral identity. In this study, four AP1/AGL9 MADS-box genes were cloned from Alpinia hainanensis, and protein-protein interactions (PPIs and roles of the four genes in floral homeotic conversion and in floral evolution are surveyed for the first time. AhFUL is clustered to the AP1 lineage, AhSEP4 and AhSEP3b to the SEP lineage, and AhAGL6-like to the AGL6 lineage. The four genes showed conserved and divergent expression patterns, and their encoded proteins were localized in the nucleus. Seven combinations of PPI (AhFUL-AhSEP4, AhFUL-AhAGL6-like, AhFUL-AhSEP3b, AhSEP4-AhAGL6-like, AhSEP4-AhSEP3b, AhAGL6-like-AhSEP3b, and AhSEP3b-AhSEP3b were detected, and the PPI patterns in the AP1/AGL9 lineage revealed that five of the 10 possible combinations are conserved and three are variable, while conclusions cannot yet be made regarding the other two. Ectopic expression of AhFUL in Arabidopsis thaliana led to early flowering and floral organ homeotic conversion to sepal-like or leaf-like. Therefore, we conclude that the four A. hainanensis AP1/AGL9 genes show functional conservation and divergence in the floral identity from other MADS-box genes.

  11. Transforming growth factor-beta 1 (TGF-beta1) promotes IL-2 mRNA expression through the up-regulation of NF-kappaB, AP-1 and NF-AT in EL4 cells.

    Science.gov (United States)

    Han, S H; Yea, S S; Jeon, Y J; Yang, K H; Kaminski, N E

    1998-12-01

    Transforming growth factor beta1 (TGF-beta1) has been previously shown to modulate interleukin 2 (IL-2) secretion by activated T-cells. In the present studies, we determined that TGF-beta1 induced IL-2 mRNA expression in the murine T-cell line EL4, in the absence of other stimuli. IL-2 mRNA expression was significantly induced by TGF-beta1 (0.1-1 ng/ml) over a relatively narrow concentration range, which led to the induction of IL-2 secretion. Under identical condition, we examined the effect of TGF-beta1 on the activity of nuclear factor AT (NF-AT), nuclear factor kappaB (NF-kappaB), activator protein-1 (AP-1) and octamer, all of which contribute to the regulation of IL-2 gene expression. Electrophoretic mobility shift assays showed that TGF-beta1 markedly increased NF-AT, NF-kappaB and AP-1 binding to their respective cognate DNA binding sites, whereas octamer binding remained constant, as compared with untreated cells. Employing a reporter gene expression system with p(NF-kappaB)3-CAT, p(NF-AT)3-CAT and p(AP-1)3-CAT, TGF-beta1 treatment of transfected EL4 cells induced a dose-related increase in chloramphenicol acetyltransferase activity that correlated well with the DNA binding profile found in the electrophoretic mobility shift assay studies. These results show that TGF-beta1, in the absence of any additional stimuli, up-regulates the activity of key transcription factors involved in IL-2 gene expression, including NF-AT, NF-kappaB and AP-1, to help promote IL-2 mRNA expression by EL4 cells.

  12. Wnt-11 signaling leads to down-regulation of the Wnt/β-catenin, JNK/AP-1 and NF-κB pathways and promotes viability in the CHO-K1 cells

    International Nuclear Information System (INIS)

    Railo, Antti; Nagy, Irina I.; Kilpelaeinen, Pekka; Vainio, Seppo

    2008-01-01

    The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical β-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical β-catenin mediated Wnt signaling but also JNK/AP-1 and NF-κB signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-κB pathway. Consistent with the central role of Akt, JNK and NF-κB in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways

  13. Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation.

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    Bin Fan

    Full Text Available Over activation of microglia results in the production of proinflammatory agents that have been implicated in various brain diseases. Pycnogenol is a patented extract from French maritime pine bark (Pinus pinaster Aiton with strong antioxidant and anti-inflammatory potency. The present study investigated whether pycnogenol may be associated with the production of proinflammatory mediators in lipopolysaccharide-stimulated BV2 (mouse-derived microglia. It was found that pycnogenol treatment was dose-dependently associated with significantly less release of nitricoxide (NO, TNF-α, IL-6 and IL-1β, and lower levels of intercellular adhesion molecule1 (ICAM-1 and perilipin 2 (PLIN2. Furthermore, this effect was replicated in primary brain microglia. Levels of inducible NO synthase mRNA and protein were attenuated, whereas there was no change in the production of the anti-inflammatory cytokine IL-10. Further evidence indicated that pycnogenol treatment led to the suppression of NF-κB activation through inhibition of p65 translocation into the nucleus and inhibited DNA binding of AP-1, suggesting that these proinflammatory factors are associated with NF-κB and AP-1. We conclude that pycnogenol exerts anti-inflammatory effects through inhibition of the NF-κB and AP-1pathway, and may be useful as a therapeutic agent in the prevention of diseases caused by over activation of microglia.

  14. Adiponectin release and insulin receptor targeting share trans-Golgi-dependent endosomal trafficking routes

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    Maria Rödiger

    2018-02-01

    Full Text Available Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The assembly of cargo-laden vesicles at the trans-Golgi network is initiated by the ARF family of small GTPases. Here, we demonstrate the role of the trans-Golgi localized monomeric GTPase ARFRP1 in endosomal-mediated vesicle trafficking of mature adipocytes. Methods: Control (Arfrp1flox/flox and inducible fat-specific Arfrp1 knockout (Arfrp1iAT−/− mice were metabolically characterized. In vitro experiments on mature 3T3-L1 cells and primary mouse adipocytes were conducted to validate the impact of ARFRP1 on localization of adiponectin and the insulin receptor. Finally, secretion and transferrin-based uptake and recycling assays were performed with HeLa and HeLa M-C1 cells. Results: We identified the ARFRP1-based sorting machinery to be involved in vesicle trafficking relying on the endosomal compartment for cell surface delivery. Secretion of adiponectin from fat depots was selectively reduced in Arfrp1iAT−/− mice, and Arfrp1-depleted 3T3-L1 adipocytes revealed an accumulation of adiponectin in Rab11-positive endosomes. Plasma adiponectin deficiency of Arfrp1iAT−/− mice resulted in deteriorated hepatic insulin sensitivity, increased gluconeogenesis and elevated fasting blood glucose levels. Additionally, the insulin receptor, undergoing endocytic recycling after ligand binding, was less abundant at the plasma membrane of adipocytes lacking Arfrp1. This had detrimental effects on adipose insulin signaling, followed by insufficient suppression of basal lipolytic activity and impaired adipose tissue expansion. Conclusions: Our findings suggest that adiponectin secretion and insulin receptor surface targeting utilize the same post-Golgi trafficking pathways that are essential for an appropriate systemic insulin sensitivity and glucose homeostasis. Keywords: Adiponectin, ARFRP1, Exocytosis, Insulin receptor, trans-Golgi

  15. Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Heenan, Erin J.; Vanhooke, Janeen L.; Temple, Brenda R.; Betts, Laurie; Sondek, John E.; Dohlman, Henrik G.; (UNC)

    2009-09-11

    G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light, and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein {alpha} subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein {beta} subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical G{beta} subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential G{gamma} protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 G{beta}-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.

  16. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  17. A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events.

    Science.gov (United States)

    Kaufmann, A M; Goldman, S D B; Krise, J P

    2009-03-01

    Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders-Niemann-Pick type C, mucolipidosis type IV, and Sandhoff's disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.

  18. A novel chimeric cell-penetrating peptide with membrane-disruptive properties for efficient endosomal escape.

    Science.gov (United States)

    Salomone, Fabrizio; Cardarelli, Francesco; Di Luca, Mariagrazia; Boccardi, Claudia; Nifosì, Riccardo; Bardi, Giuseppe; Di Bari, Lorenzo; Serresi, Michela; Beltram, Fabio

    2012-11-10

    Efficient endocytosis into a wide range of target cells and low toxicity make the arginine-rich Tat peptide (Tat(11): YGRKKRRQRRR, residues 47-57 of HIV-1 Tat protein) an excellent transporter for delivery purposes. Unfortunately, molecules taken up by endocytosis undergo endosomal entrapment and possible metabolic degradation. Escape from the endosome is therefore actively researched. In this context, antimicrobial peptides (AMPs) provide viable templates for the design of new membrane-disruptive motifs. In particular the Cecropin-A and Melittin hybrids (CMs) are among the smallest and most effective peptides with membrane-perturbing abilities. Here we present a novel chimeric peptide in which the Tat(11) motif is fused to the CM(18) hybrid (KWKLFKKIGAVLKVLTTG, residues 1-7 of Cecropin-A and 2-12 of Melittin). When administered to cells, CM(18)-Tat(11) combines the two desired functionalities: efficient uptake and destabilization of endocytotic-vesicle membranes. We show that this chimeric peptide effectively increases cargo-molecule cytoplasm availability and allows the subsequent intracellular localization of diverse membrane-impermeable molecules (i.e. Tat(11)-EGFP fusion protein, calcein, dextrans, and plasmidic DNA) with no detectable cytotoxicity. The present results open the way to the rational engineering of "modular" cell-penetrating peptides (CPPs) that combine (i) efficient translocation from the extracellular milieu into vesicles and (ii) efficient release of molecules from vesicles into the cytoplasm. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. An Inside Job: How Endosomal Na+/H+ Exchangers Link to Autism and Neurological Disease

    Directory of Open Access Journals (Sweden)

    Kalyan C. Kondapalli

    2014-06-01

    Full Text Available Autism imposes a major impediment to childhood development and a huge emotional and financial burden on society. In recent years, there has been rapidly accumulating genetic evidence that links the eNHE, a subset of Na+/H+ exchangers that localize to intracellular vesicles, to a variety of neurological conditions including autism, attention deficit hyperactivity disorder, intellectual disability and epilepsy. By providing a leak pathway for protons pumped by the V-ATPase, eNHE determine luminal pH and regulate cation (Na+, K+ content in early and recycling endosomal compartments. Loss-of-function mutations in eNHE cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways. Two isoforms, NHE6 and NHE9 are highly expressed in brain, including hippocampus and cortex. Here, we summarize evidence for the importance of luminal cation content and pH on processing, delivery and fate of cargo and on the surface expression and function of membrane receptors and neurotransmitter transporters, drawing upon insights from model organisms and mammalian cells. These studies lead to cellular models of eNHE activity in pre- and post-synaptic neurons and astrocytes, where they could impact synapse development and plasticity. The study of eNHE has provided new insight on the mechanism of autism and other debilitating neurological disorders and opened up new possibilities for therapeutic intervention.

  20. The Annexin A1 Receptor FPR2 Regulates the Endosomal Export of Influenza Virus

    Directory of Open Access Journals (Sweden)

    Fryad Rahman

    2018-05-01

    Full Text Available The Formyl Peptide Receptor 2 (FPR2 is a novel promising target for the treatment of influenza. During viral infection, FPR2 is activated by annexinA1, which is present in the envelope of influenza viruses; this activation promotes virus replication. Here, we investigated whether blockage of FPR2 would affect the genome trafficking of influenza virus. We found that, upon infection and cell treatment with the specific FPR2 antagonist WRW4 or the anti-FPR2 monoclonal antibody, FN-1D6-AI, influenza viruses were blocked into endosomes. This effect was independent on the strain and was observed for H1N1 and H3N2 viruses. In addition, blocking FPR2signaling in alveolar lung A549 epithelial cells with the monoclonal anti-FPR2 antibody significantly inhibited virus replication. Altogether, these results show that FPR2signaling interferes with the endosomal trafficking of influenza viruses and provides, for the first time, the proof of concept that monoclonal antibodies directed against FPR2 inhibit virus replication. Antibodies-based therapeutics have emerged as attractive reagents in infectious diseases. Thus, this study suggests that the use of anti-FPR2 antibodies against influenza hold great promise for the future.

  1. Altered neurological function in mice immunized with early endosome antigen 1

    Directory of Open Access Journals (Sweden)

    Fritzler Marvin J

    2004-01-01

    Full Text Available Abstract Background Autoantibodies directed against the 160 kDa endosome protein early endosome antigen 1 (EEA1 are seen in patients with neurological diseases. To determine if antibodies to EEA1 have a neuropathological effect, mice from three major histocompatability haplotype backgrounds (H2q, H2b and H2d were immunized with EEA1 (amino acids 82–1411 that was previously shown to contain the target EEA1 epitopes. The mice were then subjected to five neuro-behavioural tests: grid walking, forelimb strength, open field, reaching and rotarod. Results The immunized SWR/J mice with sustained anti-EEA1 antibodies had significantly reduced forelimb strength than the control non-immune mice of the same strain, and BALB/CJ immune mice demonstrated significantly more forelimb errors on the grid walk test than the control group. Conclusions Antibodies to recombinant EEA1 in mice may mediate neurological deficits that are consistent with clinical features of some humans that spontaneously develop anti-EEA1 autoantibodies.

  2. Ebola Viral Glycoprotein Bound to Its Endosomal Receptor Niemann-Pick C1.

    Science.gov (United States)

    Wang, Han; Shi, Yi; Song, Jian; Qi, Jianxun; Lu, Guangwen; Yan, Jinghua; Gao, George F

    2016-01-14

    Filoviruses, including Ebola and Marburg, cause fatal hemorrhagic fever in humans and primates. Understanding how these viruses enter host cells could help to develop effective therapeutics. An endosomal protein, Niemann-Pick C1 (NPC1), has been identified as a necessary entry receptor for this process, and priming of the viral glycoprotein (GP) to a fusion-competent state is a prerequisite for NPC1 binding. Here, we have determined the crystal structure of the primed GP (GPcl) of Ebola virus bound to domain C of NPC1 (NPC1-C) at a resolution of 2.3 Å. NPC1-C utilizes two protruding loops to engage a hydrophobic cavity on head of GPcl. Upon enzymatic cleavage and NPC1-C binding, conformational change in the GPcl further affects the state of the internal fusion loop, triggering membrane fusion. Our data therefore provide structural insights into filovirus entry in the late endosome and the molecular basis for design of therapeutic inhibitors of viral entry. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

    Science.gov (United States)

    Caviston, Juliane P.; Zajac, Allison L.; Tokito, Mariko; Holzbaur, Erika L.F.

    2011-01-01

    Huntingtin (Htt) is a membrane-associated scaffolding protein that interacts with microtubule motors as well as actin-associated adaptor molecules. We examined a role for Htt in the dynein-mediated intracellular trafficking of endosomes and lysosomes. In HeLa cells depleted of either Htt or dynein, early, recycling, and late endosomes (LE)/lysosomes all become dispersed. Despite altered organelle localization, kinetic assays indicate only minor defects in intracellular trafficking. Expression of full-length Htt is required to restore organelle localization in Htt-depleted cells, supporting a role for Htt as a scaffold that promotes functional interactions along its length. In dynein-depleted cells, LE/lysosomes accumulate in tight patches near the cortex, apparently enmeshed by cortactin-positive actin filaments; Latrunculin B-treatment disperses these patches. Peripheral LE/lysosomes in dynein-depleted cells no longer colocalize with microtubules. Htt may be required for this off-loading, as the loss of microtubule association is not seen in Htt-depleted cells or in cells depleted of both dynein and Htt. Inhibition of kinesin-1 relocalizes peripheral LE/lysosomes induced by Htt depletion but not by dynein depletion, consistent with their detachment from microtubules upon dynein knockdown. Together, these data support a model of Htt as a facilitator of dynein-mediated trafficking that may regulate the cytoskeletal association of dynamic organelles. PMID:21169558

  4. NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes*

    Science.gov (United States)

    Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

    2016-01-01

    NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. PMID:26987901

  5. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

    Science.gov (United States)

    Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

    2015-11-01

    Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

  6. The NKG2D ligand ULBP2 is specifically regulated through an invariant chain-dependent endosomal pathway

    DEFF Research Database (Denmark)

    Uhlenbrock, Franziska Katharina; Hagemann-Jensen, Michael Henrik; Kehlet, Stephanie

    2014-01-01

    by affecting endosomal/lysosomal integrity and protein kinase C activity. The invariant chain was further essential for endosomal transport of ULBP2. This novel pathway was identified through screening experiments by which methylselenic acid was found to possess notable NKG2D ligand regulatory properties....... The protein kinase C inhibitor methylselenic acid induced MICA/B surface expression but dominantly blocked ULBP2 surface transport. Remarkably, by targeting this novel pathway we could specifically block the production of soluble ULBP2 from different, primary melanomas. Our findings strongly suggest...

  7. The N-terminal domains of Vps3 and Vps8 are critical for localization and function of the CORVET tethering complex on endosomes.

    Directory of Open Access Journals (Sweden)

    Nadine Epp

    Full Text Available Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.

  8. Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47{sup phox} pathway

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Ming-Horng [Department of Pediatrics, Division of Neonatology and Pediatric Hematology/Oncology, Chang Gung Memorial Hospital, Yunlin, Taiwan (China); Lin, Zih-Chan [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Liang, Chan-Jung [Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Yen, Feng-Lin [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Institute of Biomedical Sciences, Sun Yat-Sen University, 70 Lienhai Rd., Kaohsiung, Taiwan (China); Chiang, Yao-Chang [Center for Drug Abuse and Addiction, China Medical University Hospital, Taichung, Taiwan (China); China Medical University, Taichung, Taiwan (China); Lee, Chiang-Wen, E-mail: cwlee@gw.cgust.edu.tw [Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi, Taiwan (China); Chronic Diseases and Health Promotion Research Center, Chang Gung University of Science and Technology, Chia-Yi, Taiwan (China); Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan (China)

    2014-09-01

    Eupafolin, a major active component found in the methanol extracts of Phyla nodiflora, has been used to treat inflammation of skin. We examined its effects on cyclooxygenase-2 (COX-2) expression in LPS-treated human dermal fibroblasts. Lipopolysaccharide (LPS) significantly increased prostaglandin-E2 (PGE2) production associated with increased COX-2 expression in Hs68 cells. This effect was blocked by eupafolin, TLR-4 antibody, antioxidants (APO and NAC), as well as inhibitors, including U0126 (ERK1/2), SB202190 (p38), SP600125 (JNK1/2), and Tanshinone IIA (AP-1). In gene regulation level, qPCR and promoter assays revealed that COX-2 expression was attenuated by eupafolin. In addition, eupafolin also ameliorated LPS-induced p47 phox activation and decreased reactive oxygen species (ROS) generation and NADPH oxidase (Nox) activity. Moreover, pretreatment with eupafolin and APO led to reduced LPS-induced phosphorylation of ERK1/2, JNK, and p38. Further, eupafolin attenuated LPS-induced increase in AP-1 transcription factor binding activity as well as the increase in the phosphorylation of c-Jun and c-Fos. In vivo studies have shown that in dermal fibroblasts of LPS treated mice, eupafolin exerted anti-inflammation effects by decreasing COX-2 protein levels. Our results reveal a novel mechanism for anti-inflammatory and anti-oxidative effects of eupafolin that involved inhibition of LPS-induced ROS generation, suppression of MAPK phosphorylation, diminished DNA binding activity of AP-1 and attenuated COX-2 expression leading to reduced production of prostaglandin E2 (PGE2). Our results demonstrate that eupafolin may be used to treat inflammatory responses associated with dermatologic diseases. - Highlights: • LPS activates the Nox2/p47{sup phox}/JNK/AP-1 and induces COX2 expression in Hs68 cells. • Eupafolin inhibits LPS-induced COX-2 expression via Nox2/p47{sup phox} inhibition. • Eupafolin may be used in the treatment of skin diseases involving inflammation.

  9. Doubly truncated FosB isoform (Delta2DeltaFosB) induces osteosclerosis in transgenic mice and modulates expression and phosphorylation of Smads in osteoblasts independent of intrinsic AP-1 activity

    DEFF Research Database (Denmark)

    Sabatakos, George; Rowe, Glenn C; Kveiborg, Marie

    2008-01-01

    DeltaFosB and a further truncated isoform (Delta2DeltaFosB) that lacks known transactivation domains but, like DeltaFosB, induces increased expression of osteoblast marker genes. MATERIALS AND METHODS: To test Delta2DeltaFosB's ability to induce bone formation in vivo, we generated transgenic mice......6 expression. CONCLUSIONS: DeltaFosB's AP-1 transactivating function is not needed to induce increased bone formation, and Delta2DeltaFosB may act, at least in part, by increasing Smad1 expression, phosphorylation, and translocation to the nucleus....

  10. Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47phox pathway

    International Nuclear Information System (INIS)

    Tsai, Ming-Horng; Lin, Zih-Chan; Liang, Chan-Jung; Yen, Feng-Lin; Chiang, Yao-Chang; Lee, Chiang-Wen

    2014-01-01

    Eupafolin, a major active component found in the methanol extracts of Phyla nodiflora, has been used to treat inflammation of skin. We examined its effects on cyclooxygenase-2 (COX-2) expression in LPS-treated human dermal fibroblasts. Lipopolysaccharide (LPS) significantly increased prostaglandin-E2 (PGE2) production associated with increased COX-2 expression in Hs68 cells. This effect was blocked by eupafolin, TLR-4 antibody, antioxidants (APO and NAC), as well as inhibitors, including U0126 (ERK1/2), SB202190 (p38), SP600125 (JNK1/2), and Tanshinone IIA (AP-1). In gene regulation level, qPCR and promoter assays revealed that COX-2 expression was attenuated by eupafolin. In addition, eupafolin also ameliorated LPS-induced p47 phox activation and decreased reactive oxygen species (ROS) generation and NADPH oxidase (Nox) activity. Moreover, pretreatment with eupafolin and APO led to reduced LPS-induced phosphorylation of ERK1/2, JNK, and p38. Further, eupafolin attenuated LPS-induced increase in AP-1 transcription factor binding activity as well as the increase in the phosphorylation of c-Jun and c-Fos. In vivo studies have shown that in dermal fibroblasts of LPS treated mice, eupafolin exerted anti-inflammation effects by decreasing COX-2 protein levels. Our results reveal a novel mechanism for anti-inflammatory and anti-oxidative effects of eupafolin that involved inhibition of LPS-induced ROS generation, suppression of MAPK phosphorylation, diminished DNA binding activity of AP-1 and attenuated COX-2 expression leading to reduced production of prostaglandin E2 (PGE2). Our results demonstrate that eupafolin may be used to treat inflammatory responses associated with dermatologic diseases. - Highlights: • LPS activates the Nox2/p47 phox /JNK/AP-1 and induces COX2 expression in Hs68 cells. • Eupafolin inhibits LPS-induced COX-2 expression via Nox2/p47 phox inhibition. • Eupafolin may be used in the treatment of skin diseases involving inflammation

  11. HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

    Science.gov (United States)

    Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

    2018-03-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes

  12. Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

    NARCIS (Netherlands)

    Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

    2011-01-01

    The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

  13. Characterization of PEBBLEs as a Tool for Real-Time Measurement of Dictyostelium discoideum Endosomal pH

    Directory of Open Access Journals (Sweden)

    Everett Moding

    2009-01-01

    Full Text Available The measurement of intracellular ion concentration change is important for understanding the cellular mechanisms for communication. Recently developed nanosensors, (Photonic Explorers for Biomedical use with Biologically Localized Embedding PEBBLEs, have a number of advantages for measuring ions in cells over established methods using microelectrodes, unbound fluorescent dyes, or NMR. PEBBLE sensors have been shown to work in principle for measuring dynamic ion changes, but few in vivo applications have been demonstrated. We modified the protocol for the fabrication of pH sensing PEBBLEs and developed a protocol for the utilization of these sensors for the monitoring of dynamic pH changes in the endosomes of slime mold Dictyostelium discoideum (D. discoideum. Oregon Green 514-CdSe Quantum Dot PEBBLEs were used to measure real-time pH inside D. discoideum endosomes during cAMP stimulation. Endosomal pH was shown to decrease during cAMP signaling, demonstrating a movement of protons into the endosomes of D. discoideum amoebae.

  14. COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

    NARCIS (Netherlands)

    Phillips-Krawczak, Christine A.; Singla, Amika; Starokadomskyy, Petro; Deng, Zhihui; Osborne, Douglas G.; Li, Haiying; Dick, Christopher J.; Gomez, Timothy S.; Koenecke, Megan; Zhang, Jin-San; Dai, Haiming; Sifuentes-Dominguez, Luis F.; Geng, Linda N.; Kaufmann, Scott H.; Hein, Marco Y.; Wallis, Mathew; McGaughran, Julie; Gecz, Jozef; De Sluis, Bart van; Billadeau, Daniel D.; Burstein, Ezra

    2015-01-01

    COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex

  15. Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

    NARCIS (Netherlands)

    Peden, A.A.; Oorschot, V.; Hesser, B.A.; Austin, C.D.; Scheller, R.H.; Klumperman, J.

    2004-01-01

    The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal

  16. CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

    NARCIS (Netherlands)

    Bartuzi, Paulina; Billadeau, Daniel D.; Favier, Robert; Rong, Shunxing; Dekker, Daphne; Fedoseienko, Alina; Fieten, Hille; Wijers, Melinde; Levels, Johannes H.; Huijkman, Nicolette; Kloosterhuis, Niels; Van der Molen, Henk; Brufau, Gemma; Groen, Albert K.; Elliott, Alison M.; Kuivenhoven, Jan Albert; Plecko, Barbara; Grangl, Gernot; McGaughran, Julie; Horton, Jay D.; Burstein, Ezra; Hofker, Marten H.; van de Sluis, Bart

    2016-01-01

    The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal

  17. CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

    NARCIS (Netherlands)

    Bartuzi, Paulina; Billadeau, Daniel D.; Favier, Robert; Rong, Shunxing; Dekker, Daphne; Fedoseienko, Alina; Fieten, Hille; Wijers, Melinde; Levels, Johannes H.; Huijkman, Nicolette; Kloosterhuis, Niels; van der Molen, Henk; Brufau, Gemma; Groen, Albert K.; Elliott, Alison M.; Kuivenhoven, Jan Albert; Plecko, Barbara; Grangl, Gernot; McGaughran, Julie; Horton, Jay D.; Burstein, Ezra; Hofker, Marten H.; van de Sluis, Bart

    The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal

  18. Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction.

    Science.gov (United States)

    Liao, Edward H; Gray, Lindsay; Tsurudome, Kazuya; El-Mounzer, Wassim; Elazzouzi, Fatima; Baim, Christopher; Farzin, Sarah; Calderon, Mario R; Kauwe, Grant; Haghighi, A Pejmun

    2018-01-01

    Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons.

  19. IL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

    International Nuclear Information System (INIS)

    Wainszelbaum, Marisa J.; Proctor, Brandon M.; Pontow, Suzanne E.; Stahl, Philip D.; Barbieri, M. Alejandro

    2006-01-01

    The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E 2 (PGE 2 ) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells

  20. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

    Science.gov (United States)

    Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P.

    2015-01-01

    Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. PMID:26071406

  1. Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant.

    Science.gov (United States)

    Cunningham, Margaret R; Nisar, Shaista P; Cooke, Alexandra E; Emery, Elizabeth D; Mundell, Stuart J

    2013-05-01

    P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient. © 2013 John Wiley & Sons A/S.

  2. Oleoylethanolamide exerts anti-inflammatory effects on LPS-induced THP-1 cells by enhancing PPARα signaling and inhibiting the NF-κB and ERK1/2/AP-1/STAT3 pathways.

    Science.gov (United States)

    Yang, Lichao; Guo, Han; Li, Ying; Meng, Xianglan; Yan, Lu; Dan Zhang; Wu, Sangang; Zhou, Hao; Peng, Lu; Xie, Qiang; Jin, Xin

    2016-10-10

    The present study aimed to examine the anti-inflammatory actions of oleoylethanolamide (OEA) in lipopolysaccharide (LPS)-induced THP-1 cells. The cells were stimulated with LPS (1 μg/ml) in the presence or absence of OEA (10, 20 and 40 μM). The pro-inflammatory cytokines were evaluated by qRT-PCR and ELISA. The THP-1 cells were transiently transfected with PPARα small-interfering RNA, and TLR4 activity was determined with a blocking test using anti-TLR4 antibody. Additionally, a special inhibitor was used to analyse the intracellular signaling pathway. OEA exerted a potent anti-inflammatory effect by reducing the production of pro-inflammatory cytokines and TLR4 expression, and by enhancing PPARα expression. The modulatory effects of OEA on LPS-induced inflammation depended on PPARα and TLR4. Importantly, OEA inhibited LPS-induced NF-κB activation, IκBα degradation, expression of AP-1, and the phosphorylation of ERK1/2 and STAT3. In summary, our results demonstrated that OEA exerts anti-inflammatory effects by enhancing PPARα signaling, inhibiting the TLR4-mediated NF-κB signaling pathway, and interfering with the ERK1/2-dependent signaling cascade (TLR4/ERK1/2/AP-1/STAT3), which suggests that OEA may be a therapeutic agent for inflammatory diseases.

  3. Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells.

    Directory of Open Access Journals (Sweden)

    Emilia Galperin

    Full Text Available Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2. To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF receptor (EGFR, we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

  4. Hard coatings

    International Nuclear Information System (INIS)

    Dan, J.P.; Boving, H.J.; Hintermann, H.E.

    1993-01-01

    Hard, wear resistant and low friction coatings are presently produced on a world-wide basis, by different processes such as electrochemical or electroless methods, spray technologies, thermochemical, CVD and PVD. Some of the most advanced processes, especially those dedicated to thin film depositions, basically belong to CVD or PVD technologies, and will be looked at in more detail. The hard coatings mainly consist of oxides, nitrides, carbides, borides or carbon. Over the years, many processes have been developed which are variations and/or combinations of the basic CVD and PVD methods. The main difference between these two families of deposition techniques is that the CVD is an elevated temperature process (≥ 700 C), while the PVD on the contrary, is rather a low temperature process (≤ 500 C); this of course influences the choice of substrates and properties of the coating/substrate systems. Fundamental aspects of the vapor phase deposition techniques and some of their influences on coating properties will be discussed, as well as the very important interactions between deposit and substrate: diffusions, internal stress, etc. Advantages and limitations of CVD and PVD respectively will briefly be reviewed and examples of applications of the layers will be given. Parallel to the development and permanent updating of surface modification technologies, an effort was made to create novel characterisation methods. A close look will be given to the coating adherence control by means of the scratch test, at the coating hardness measurement by means of nanoindentation, at the coating wear resistance by means of a pin-on-disc tribometer, and at the surface quality evaluation by Atomic Force Microscopy (AFM). Finally, main important trends will be highlighted. (orig.)

  5. Radiation-induced apoptosis in developing rats and kainic acid-induced excitotoxicity in adult rats are associated with distinctive morphological and biochemical c-Jun/AP-1 (N) expression

    Energy Technology Data Exchange (ETDEWEB)

    Pozas, E. [Unitat de Neuropatologia, Servei d' Anatomia Patologica, Hospital Princeps d' Espanya, Universitat de Barcelona, 08907 Hospitalet de Llobregat (Spain); Planas, A.M. [Departament de Farmacologia i Toxicologia, IIBB, CSIC Barcelona (Spain); Ferrer, I. [Unitat de Neuropatologia, Servei d' Anatomia Patologica, Hospital Princeps d' Espanya, Universitat de Barcelona, 08907 Hospitalet de Llobregat (Spain)

    1997-07-14

    Ionizing radiation produces apoptosis in the developing rat brain. Strong c-Jun immunoreactivity, as revealed with the antibody c-Jun/AP-1 (N) which is raised against the amino acids 91-105 mapping with the amino terminal domain of mouse c-Jun p39, is simultaneously observed in the nucleus and cytoplasm of apoptotic cells. Western blotting of total brain homogenates, using the same antibody, shows a p39 band in control rats which is accompanied by a strong, phosphorylated p62 double-band in irradiated animals. In addition, increased c-Jun N-terminal kinase 1 expression, as found on western blots, is found in irradiated rats when compared with controls. Intraperitoneal injection of kainic acid at convulsant doses to the adult rat produces cell death with morphological features of necrosis, together with the appearance of cells with fine granular chromatin degeneration and small numbers of apoptotic-like cells, in the entorhinal and piriform cortices, basal amygdala, certain thalamic nuclei, and CA1 region of the hippocampus. c-Jun expression in kainic acid-treated rats, as revealed with the c-Jun/AP-1 (N) antibody, is found in the nuclei of a minority of cells in the same areas. The vast majority of c-Jun-immunoreactive cells have normal nuclear morphology, whereas necrotic cells are negative and only a few cells with fine granular chromatin condensation and apoptotic cells following kainic acid injection are stained with c-Jun antibodies. Western blotting, using the same antibody, shows a p39 band in control rats, which is accompanied by a band at about p26 from 6 h onwards following kainic acid injection. Decreased c-Jun N-terminal kinase 1 expression, as revealed on western blots, is observed in kainic acid-treated rats.These results show that the antibody c-Jun/AP-1 (N) recognizes three different forms of c-Jun-related immunoreactivity in normal and pathological states, which are associated with the different outcome of cells. These results stress the necessity

  6. A putative SUMO interacting motif in the B30.2/SPRY domain of rhesus macaque TRIM5α important for NF-κB/AP-1 signaling and HIV-1 restriction

    Directory of Open Access Journals (Sweden)

    Marie-Édith Nepveu-Traversy

    2016-01-01

    Full Text Available TRIM5α from the rhesus macaque (TRIM5αRh is a restriction factor that shows strong activity against HIV-1. TRIM5αRh binds specifically to HIV-1 capsid (CA through its B30.2/PRYSPRY domain shortly after entry of the virus into the cytoplasm. Recently, three putative SUMO interacting motifs (SIMs have been identified in the PRYSPRY domain of human and macaque TRIM5α. However, structural modeling of this domain suggested that two of them were buried in the hydrophobic core of the protein, implying that interaction with SUMO was implausible, while the third one was not relevant to restriction. In light of these results, we re-analyzed the TRIM5αRh PRYSPRY sequence and identified an additional putative SIM (435VIIC438 which we named SIM4. This motif is exposed at the surface of the PRYSPRY domain, allowing potential interactions with SUMO or SUMOylated proteins. Introducing a double mutation in SIM4 (V435K, I436K did not alter stability, unlike mutations in SIM1. SIM4-mutated TRIM5αRh failed to bind HIV-1CA and lost the ability to restrict this virus. Accordingly, SIM4 undergoes significant variation among primates and substituting this motif with naturally occurring SIM4 variants affected HIV-1 restriction by TRIM5αRh, suggesting a direct role in capsid recognition. Interestingly, SIM4-mutated TRIM5αRh also failed to activate NF-κB and AP-1-mediated transcription. Although there is no direct evidence that SIM4 is involved in direct interaction with SUMO or a SUMOylated protein, mutating this motif strongly reduced co-localization of TRIM5αRh with SUMO-1 and with PML, a SUMOylated nuclear protein. In conclusion, this new putative SIM is crucial for both direct interaction with incoming capsids and for NF-κB/AP-1 signaling. We speculate that the latter function is mediated by interactions of SIM4 with a SUMOylated protein involved in the NF-κB/AP-1 signaling pathways.

  7. Glycosaminoglycan-resistant and pH-sensitive lipid-coated DNA complexes produced by detergent removal method.

    Science.gov (United States)

    Lehtinen, Julia; Hyvönen, Zanna; Subrizi, Astrid; Bunjes, Heike; Urtti, Arto

    2008-10-21

    Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.

  8. Protective Coatings

    Science.gov (United States)

    1980-01-01

    General Magnaplate Corporation's pharmaceutical machine is used in the industry for high speed pressing of pills and capsules. Machine is automatic system for molding glycerine suppositories. These machines are typical of many types of drug production and packaging equipment whose metal parts are treated with space spinoff coatings that promote general machine efficiency and contribute to compliance with stringent federal sanitation codes for pharmaceutical manufacture. Collectively known as "synergistic" coatings, these dry lubricants are bonded to a variety of metals to form an extremely hard slippery surface with long lasting self lubrication. The coatings offer multiple advantages; they cannot chip, peel or be rubbed off. They protect machine parts from corrosion and wear longer, lowering maintenance cost and reduce undesired heat caused by power-robbing friction.

  9. Stimulation of Alpha7 Nicotinic Acetylcholine Receptor Attenuates Nicotine-Induced Upregulation of MMP, MCP-1, and RANTES through Modulating ERK1/2/AP-1 Signaling Pathway in RAW264.7 and MOVAS Cells

    Directory of Open Access Journals (Sweden)

    Liping Liu

    2017-01-01

    Full Text Available Vagus nerve stimulation through alpha7 nicotine acetylcholine receptors (α7-nAChR signaling had been demonstrated attenuation of inflammation. This study aimed to determine whether PNU-282987, a selective α7-nAChR agonist, affected activities of matrix metalloproteinase (MMP and inflammatory cytokines in nicotine-treatment RAW264.7 and MOVAS cells and to assess the underlying molecular mechanisms. RAW264.7 and MOVAS cells were treated with nicotine at different concentrations (0, 1, 10, and 100 ng/ml for 0–120 min. Nicotine markedly stimulated the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2 and c-Jun in RAW264.7 cells. Pretreatment with U0126 significantly suppressed phosphorylation of ERK1/2 and further attenuated nicotine-induced activation of c-Jun and upregulation of MMP-2, MMP-9, monocyte chemotactic protein- (MCP- 1, and regulated upon activation normal T cell expressed and secreted (RANTES. Similarly, nicotine treatment also increased phosphorylation of c-Jun and expressions of MMP-2, MMP-9, MCP-1, and RANTES in MOVAS cells. When cells were pretreated with PNU-282987, nicotine-induced activations of ERK1/2 and c-Jun in RAW264.7 cells and c-Jun in MOVAS cells were effectively inhibited. Furthermore, nicotine-induced secretions of MMP-2, MMP-9, MCP-1, and RANTES were remarkably downregulated. Treatment with α7-nAChR agonist inhibits nicotine-induced upregulation of MMP and inflammatory cytokines through modulating ERK1/2/AP-1 signaling in RAW264.7 cells and AP-1 in MOVAS cells, providing a new therapeutic for abdominal aortic aneurysm.

  10. Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

    Science.gov (United States)

    Sun, Ying-Jie; Nishikawa, Kaori; Yuda, Hideki; Wang, Yu-Lai; Osaka, Hitoshi; Fukazawa, Nobuna; Naito, Akira; Kudo, Yoshihisa; Wada, Keiji; Aoki, Shunsuke

    2006-09-01

    With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

  11. Augmented internalisation of ferroportin to late endosomes impairs iron uptake by enterocyte-like IEC-6 cells.

    Science.gov (United States)

    Oates, Phillip S; Thomas, Carla

    2005-08-01

    Absorption of iron occurs by duodenal enterocytes, involving uptake by the divalent metal transporter-1 (DMT1) and release by ferroportin. Ferroportin responds to the hepatocyte-produced 25-amino-acid-peptide hepcidin-25 by undergoing internalisation to late endosomes that impair iron release. Ferroportin is also expressed on the apical membrane of polarised Caco-2 cells, rat intestinal cells and in IEC-6 cells (an intestinal epithelial cell line). A blocking antibody to ferroportin also impairs the uptake, but not the release, of iron. In this study IEC-6 cells were used to study the mechanism of impairment or recovery from impairment produced by the blocking antibody and the fate of DMT1 and ferroportin. Uptake of 1 muM Fe(II) was studied by adding the antibody from time 0 and after adding or removing the antibody once a steady state had been reached. Surface binding, maximum iron transport rate V(max) and transporter affinity (K(m)) were measured after impairment of iron uptake. Ferroportin and DMT1 distribution were assessed by immunofluorescence microscopy. Antibody-mediated impairment, or recovery from impairment, of Fe(II) uptake occurred within minutes. Impairment was lost when the antibody was combined with the immunizing peptide. DMT1 and ferroportin undergo internalisation to late endosomes and, in the presence of the antibody, augmented internalisation of DMT1 and ferroportin caused swelling of late endosomes. Surface binding of Fe(II) and iron transport V(max) were reduced by 50%, indicating that the antibody removed membrane-bound DMT1. The ferroportin antibody induced rapid turnover of membrane ferroportin and DMT1 and its internalisation to late endosomes, resulting in impaired Fe(II) uptake.

  12. Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs.

    Science.gov (United States)

    Chen, Mo; Qiu, Tao; Wu, Jiajie; Yang, Yang; Wright, Graham D; Wu, Min; Ge, Ruowen

    2018-03-09

    Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE-Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE-Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE-Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.

  13. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

    Science.gov (United States)

    Nixon, Ralph A

    2017-07-01

    Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and β-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote β-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies β-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-β, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease. © FASEB.

  14. Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity

    DEFF Research Database (Denmark)

    Yarani, Reza; Shiraishi, Takehiko; Nielsen, Peter E.

    2018-01-01

    Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginin...... indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore....

  15. Smart DNA vectors based on cyclodextrin polymers: compaction and endosomal release.

    Science.gov (United States)

    Wintgens, Véronique; Leborgne, Christian; Baconnais, Sonia; Burckbuchler, Virginie; Le Cam, Eric; Scherman, Daniel; Kichler, Antoine; Amiel, Catherine

    2012-02-01

    Neutral β-cyclodextrin polymers (polyβCD) associated with cationic adamantyl derivatives (Ada) can be used to deliver plasmid DNA into cells. In absence of an endosomolytic agent, transfection efficiency remains low because most complexes are trapped in the endosomal compartment. We asked whether addition of an imidazole-modified Ada can increase efficiency of polyβCD/cationic Ada-based delivery system. We synthesized two adamantyl derivatives: Ada5, which has a spacer arm between the Ada moiety and a bi-cationic polar head group, and Ada6, which presents an imidazole group. Strength of association between polyβCD and Ada derivatives was evaluated by fluorimetric titration. Gel mobility shift assay, zeta potential, and dark field transmission electron microscopy experiments demonstrated the system allowed for efficient DNA compaction. In vitro transfection experiments performed on HepG2 and HEK293 cells revealed the quaternary system polyβCD/Ada5/Ada6/DNA has efficiency comparable to cationic lipid DOTAP. We successfully designed fine-tuned DNA vectors based on cyclodextrin polymers combined with two new adamantyl derivatives, leading to significant transfection associated with low toxicity.

  16. Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome.

    Directory of Open Access Journals (Sweden)

    Fumitaka Momose

    Full Text Available Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs. Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM. However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD of Rab11 family interacting proteins (Rab11-FIPs. Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

  17. Endosomal sorting complexes required for ESCRTing cells toward death during neurogenesis, neurodevelopment and neurodegeneration.

    Science.gov (United States)

    Kaul, Zenia; Chakrabarti, Oishee

    2018-03-25

    The endosomal sorting complexes required for transport (ESCRT) proteins help in the recognition, sorting and degradation of ubiquitinated cargoes from the cell surface, long-lived proteins or aggregates, and aged organelles present in the cytosol. These proteins take part in the endo-lysosomal system of degradation. The ESCRT proteins also play an integral role in cytokinesis, viral budding and mRNA transport. Many neurodegenerative diseases are caused by toxic accumulation of cargo in the cell, which causes stress and ultimately leads to neuronal death. This accumulation of cargo occurs because of defects in the endo-lysosomal degradative pathway-loss of function of ESCRTs has been implicated in this mechanism. ESCRTs also take part in many survival processes, lack of which can culminate in neuronal cell death. While the role played by the ESCRT proteins in maintaining healthy neurons is known, their role in neurodegenerative diseases is still poorly understood. In this review, we highlight the importance of ESCRTs in maintaining healthy neurons and then suggest how perturbations in many of the survival mechanisms governed by these proteins could eventually lead to cell death; quite often these correlations are not so obviously laid out. Extensive neuronal death eventually culminates in neurodegeneration. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Aggregation of nanoparticles in endosomes and lysosomes produces surface-enhanced Raman spectroscopy

    Science.gov (United States)

    Lucas, Leanne J.; Chen, Xiaoke K.; Smith, Aaron J.; Korbelik, Mladen; Zeng, Haishan; Lee, Patrick W. K.; Hewitt, Kevin Cecil

    2015-01-01

    The purpose of this study was to explore the use of surface-enhanced Raman spectroscopy (SERS) to image the distribution of epidermal growth factor receptor (EGFR) in cells. To accomplish this task, 30-nm gold nanoparticles (AuNPs) tagged with antibodies to EGFR (1012 per mL) were incubated with cells (106 per mL) of the A431 human epidermoid carcinoma and normal human bronchial epithelial cell lines. Using the 632.8-nm excitation line of a He-Ne laser, Raman spectroscopy measurements were performed using a point mapping scheme. Normal cells show little to no enhancement. SERS signals were observed inside the cytoplasm of A431 cells with an overall enhancement of 4 to 7 orders of magnitude. Raman intensity maps of the 1450 and 1583 cm-1 peaks correlate well with the expected distribution of EGFR and AuNPs, aggregated following uptake by endosomes and lysosomes. Spectral features from tyrosine and tryptophan residues dominate the SERS signals.

  19. Peroxisomes, lipid droplets, and endoplasmic reticulum "hitchhike" on motile early endosomes.

    Science.gov (United States)

    Guimaraes, Sofia C; Schuster, Martin; Bielska, Ewa; Dagdas, Gulay; Kilaru, Sreedhar; Meadows, Ben R A; Schrader, Michael; Steinberg, Gero

    2015-12-07

    Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3- and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell. © 2015 Guimaraes et al.

  20. Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells.

    Science.gov (United States)

    Fearnley, Gareth W; Smith, Gina A; Odell, Adam F; Latham, Antony M; Wheatcroft, Stephen B; Harrison, Michael A; Tomlinson, Darren C; Ponnambalam, Sreenivasan

    2014-01-01

    The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function. © 2014 Elsevier Inc. All rights reserved.

  1. Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression.

    Science.gov (United States)

    Schwartz, Verena; Friedrich, Katharina; Polleichtner, Georg; Gründer, Stefan

    2015-12-15

    Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment.

  2. Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

    Science.gov (United States)

    Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George

    2018-01-01

    Background Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein we aimed to determine how new perforin transits to the synapse if not via lytic granules. Results We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. Conclusions The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response. PMID:28822075

  3. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

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    Bruno Hernáez

    2016-04-01

    Full Text Available African swine fever virus (ASFV is a nucleocytoplasmic large DNA virus (NCLDV that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

  4. Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

    Science.gov (United States)

    Mundy, Dorothy I.; Li, Wei Ping; Luby-Phelps, Katherine; Anderson, Richard G. W.

    2012-01-01

    Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking. PMID:22238363

  5. Modeling the signaling endosome hypothesis: Why a drive to the nucleus is better than a (random walk

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    Howe Charles L

    2005-10-01

    Full Text Available Abstract Background Information transfer from the plasma membrane to the nucleus is a universal cell biological property. Such information is generally encoded in the form of post-translationally modified protein messengers. Textbook signaling models typically depend upon the diffusion of molecular signals from the site of initiation at the plasma membrane to the site of effector function within the nucleus. However, such models fail to consider several critical constraints placed upon diffusion by the cellular milieu, including the likelihood of signal termination by dephosphorylation. In contrast, signaling associated with retrogradely transported membrane-bounded organelles such as endosomes provides a dephosphorylation-resistant mechanism for the vectorial transmission of molecular signals. We explore the relative efficiencies of signal diffusion versus retrograde transport of signaling endosomes. Results Using large-scale Monte Carlo simulations of diffusing STAT-3 molecules coupled with probabilistic modeling of dephosphorylation kinetics we found that predicted theoretical measures of STAT-3 diffusion likely overestimate the effective range of this signal. Compared to the inherently nucleus-directed movement of retrogradely transported signaling endosomes, diffusion of STAT-3 becomes less efficient at information transfer in spatial domains greater than 200 nanometers from the plasma membrane. Conclusion Our model suggests that cells might utilize two distinct information transmission paradigms: 1 fast local signaling via diffusion over spatial domains on the order of less than 200 nanometers; 2 long-distance signaling via information packets associated with the cytoskeletal transport apparatus. Our model supports previous observations suggesting that the signaling endosome hypothesis is a subset of a more general hypothesis that the most efficient mechanism for intracellular signaling-at-a-distance involves the association of signaling

  6. Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.

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    Heather E Miller

    2018-04-01

    Full Text Available Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1 and cationic-independent mannose-6-phosphate receptor (CI-M6PR, respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the

  7. The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation*

    Science.gov (United States)

    Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob; Gether, Ulrik; Jørgensen, Trine Nygaard

    2014-01-01

    The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation. PMID:24973209

  8. Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Mate Suzanne E

    2012-09-01

    Full Text Available Abstract Background Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ. We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs. Methods Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle. Results Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle. Conclusion EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs.

  9. A novel berbamine derivative inhibits cell viability and induces apoptosis in cancer stem-like cells of human glioblastoma, via up-regulation of miRNA-4284 and JNK/AP-1 signaling.

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    Fan Yang

    Full Text Available Glioblastoma (GBM is the most common primary brain tumor, accounting for approximately 40% of all central nervous system malignancies. Despite standard treatment consisting of surgical resection, radiotherapy and/or chemotherapy, the prognosis for GBM is poor; with a median survival of 14.6 months. The cancer stem cell or cancer-initiating cell model has provided a new paradigm for understanding development and recurrence of GBM following treatment. Berbamine (BBM is a natural compound derived from the Berberis amurensis plant, and along with its derivatives, has been shown to exhibit antitumor activity in several cancers. Here, we reported that a novel synthetic Berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of cancer stem-like cells (CSCs in a time- and dose-dependent manner when the CSCs from four GBM patients (PBT003, PBT008, PBT022, and PBT030 were cultured. These CSCs grew in neurospheres and expressed CD133 and nestin as markers. Treatment with BBMD3 destroyed the neurosphere morphology, and led to the induction of apoptosis in the CSCs. Induction of apoptosis in these CSCs is dependent upon activation of caspase-3 and cleavage of poly (ADP-ribose polymerase (PARP. MicroRNA-4284 (miR-4284 was shown to be over-expressed about 4-fold in the CSCs following BBMD3 treatment. Furthermore, transfection of synthetic anti-sense oligonucleotide against human miR-4284 partially blocked the anticancer effects of BBMD3 on the GBM derived CSCs. BBMD3 also increased phosphorylation of the c-Jun N-terminal kinase (JNK/stress-activated protein kinase (SAPK, resulting in an increase expression of phosphorylated c-Jun and total c-Fos; the major components of transcriptional factor AP-1. The JNK-c-Jun/AP-1 signaling pathway plays an important role in the induction of apoptosis in response to UV irradiation and some drug treatments. Targeting glioblastoma stem-like cells with BBMD3 is therefore novel, and may have promise as an

  10. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar CellsSummary

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    Scott W. Messenger

    2015-11-01

    Full Text Available Background & Aims: Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI3P. PI(3,5P2 is necessary for maturation of early endosomes (EE to late endosomes (LE. Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods: Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1, and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8, bile acids, and cigarette toxin was determined. Results: PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions: These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular

  11. Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda.

    Science.gov (United States)

    Yoon, June-Sun; Gurusamy, Dhandapani; Palli, Subba Reddy

    2017-11-01

    RNA interference (RNAi) efficiency varies among insects studied. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. We recently showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into small interference RNAs (siRNAs) because they are trapped in acidic bodies. In the current study, we focused on the identification of acidic bodies in which dsRNAs accumulate in Sf9 cells. Time-lapse imaging studies showed that dsRNAs enter Sf9 cells and accumulate in acidic bodies within 20 min after their addition to the medium. CypHer-5E-labeled dsRNA also accumulated in the midgut and fat body dissected from Spodoptera frugiperda larvae with similar patterns observed in Sf9 cells. Pharmacological inhibitor assays showed that the dsRNAs use clathrin mediated endocytosis pathway for transport into the cells. We investigated the potential dsRNA accumulation sites employing LysoTracker and double labeling experiments using the constructs to express a fusion of green fluorescence protein with early or late endosomal marker proteins and CypHer-5E-labeled dsRNA. Interestingly, CypHer-5E-labeled dsRNA accumulated predominantly in early and late endosomes. These data suggest that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi response in lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

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    Tracy P. M. Chong

    2011-03-01

    Full Text Available The potent mitogenic toxin from Pasteurella multocida (PMT is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH4Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn and cholera toxin (CT, the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity.

  13. The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

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    Ravikrishna Ramanujam

    Full Text Available In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA, Adenylate cyclase and Pth11 (a non-canonical GPCR in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

  14. The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

    Science.gov (United States)

    Shepherd, Dawn; Booth, Sarah; Waithe, Dominic; Reis e Sousa, Caetano

    2015-01-01

    TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. PMID:25917086

  15. The PDZ protein GIPC regulates trafficking of the LPA1 receptor from APPL signaling endosomes and attenuates the cell's response to LPA.

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    Tal Varsano

    Full Text Available Lysophosphatidic acid (LPA mediates diverse cellular responses through the activation of at least six LPA receptors--LPA(1-6, but the interacting proteins and signaling pathways that mediate the specificity of these receptors are largely unknown. We noticed that LPA(1 contains a PDZ binding motif (SVV identical to that present in two other proteins that interact with the PDZ protein GIPC. GIPC is involved in endocytic trafficking of several receptors including TrkA, VEGFR2, lutropin and dopamine D2 receptors. Here we show that GIPC binds directly to the PDZ binding motif of LPA(1 but not that of other LPA receptors. LPA(1 colocalizes and coimmunoprecipitates with GIPC and its binding partner APPL, an activator of Akt signaling found on APPL signaling endosomes. GIPC depletion by siRNA disturbed trafficking of LPA(1 to EEA1 early endosomes and promoted LPA(1 mediated Akt signaling, cell proliferation, and cell motility. We propose that GIPC binds LPA(1 and promotes its trafficking from APPL-containing signaling endosomes to EEA1 early endosomes and thus attenuates LPA-mediated Akt signaling from APPL endosomes.

  16. The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.

    Science.gov (United States)

    Zehner, Matthias; Marschall, Andrea L; Bos, Erik; Schloetel, Jan-Gero; Kreer, Christoph; Fehrenschild, Dagmar; Limmer, Andreas; Ossendorp, Ferry; Lang, Thorsten; Koster, Abraham J; Dübel, Stefan; Burgdorf, Sven

    2015-05-19

    The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

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    Gastaldelli Michele

    2009-10-01

    Full Text Available Abstract Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5 cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein, which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for

  18. Coating materials

    International Nuclear Information System (INIS)

    Ozeki, Takao; Kimura, Tadashi; Kobayashi, Juichi; Maeda, Yutaka; Nakamoto, Hideo.

    1969-01-01

    A non-solvent type coating material composition having properties as good as thermosetting acrylic or amino alkid resins is provided by employing active energy irradiation, particularly electron beams, using a radically polymerizable low molecular compound (A) (hereafter called an oligomer) containing at least two vinyl radicals in one molecule. This oligomer is produced by reacting an epoxy-containing vinyl monomer with alpha-, beta-ethylene unsaturated carboxylic acids or their anhydrides. The composition (I) contains 10% - 100% of this oligomer. In embodiments, an oligomer having a fiberous trivinyl construction is produced by reacting 180 parts by weight of glycidyl methacrylate ester with 130 parts of itaconic acid in the presence of a polymerization-inhibitor and an addition reaction catalyst at 90 0 C for 6 hours. In practice, the coating material compositions (1), consist of the whole oligomer [I-1]; (2), consist of 10-90% of (A) component and 90%-10% of vinyl monomers containing at least 30% (meth) acrylic monomer [I-2]; (3), 10%-90% of component (A) and 90%-10% of other monomers containing at least two vinyl radicals [I-3]; (4), a mixture of (I-2) and (I-3), [I-4]; and (5), consist of 50% or less unsaturated polyester of 500-5,000 molecular weight range or drying oil, or alkyd resin of 500-5,000 molecular weight range modified by drying oil, [I-5]. As a catalyst a tertiary amino vinyl compound is preferred. Five examples are given. (Iwakiri, K.)

  19. Coatings and Corrosion Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — Purpose: The mission of the Coatings and Corrosion Laboratory is to develop and analyze the effectiveness of innovative coatings test procedures while evaluating the...

  20. Graphene Coatings

    DEFF Research Database (Denmark)

    Stoot, Adam Carsten; Camilli, Luca; Bøggild, Peter

    2014-01-01

    Owing to its remarkable electrical and mechanical properties, graphene has been attracting tremendous interest in materials science. In particular, its chemical stability and impermeability make it a promising protective membrane. However, recent investigations reveal that single layer graphene...... cannot be used as a barrier in the long run, due to galvanic corrosion phenomena arising when oxygen or water penetrate through graphene cracks or domain boundaries. Here, we overcome this issue by using a multilayered (ML) graphene coating. Our lab- as well as industrial-scale tests demonstrate that ML...... graphene can effectively protect Ni in harsh environments, even after long term exposure. This is made possible by the presence of a high number of graphene layers, which can efficiently mask the cracks and domain boundaries defects found in individual layers of graphene. Our findings thus show...

  1. Reorganization of the Endosomal System in Salmonella-Infected Cells: The Ultrastructure of Salmonella-Induced Tubular Compartments

    Science.gov (United States)

    Krieger, Viktoria; Liebl, David; Zhang, Yuying; Rajashekar, Roopa; Chlanda, Petr; Giesker, Katrin; Chikkaballi, Deepak; Hensel, Michael

    2014-01-01

    During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella. PMID:25254663

  2. Coating materials

    International Nuclear Information System (INIS)

    Ozeki, Takao; Kimura, Tadashi; Kobayashi, Juichi; Maeda, Yutaka; Nakamoto, Hideo.

    1969-01-01

    A non-solvent type coating material composition is provided which can be hardened by irradiation with active energy, particularly electron beams, using a composition which contains 10%-100% of a radically polymerizable low molecular compound (A), (hereafter called an oligomer), having at least two vinyl radicals in one molecule. These compositions have a high degree of polymerization and characteristics equivalent to thermosetting acrylic or amino alkyd resin. The oligomer (A) is produced by reacting an epoxy-containing vinyl monomer with saturated polycarboxylic acids or anhydrides. In one embodiment, 146 parts by weight of adipic acid and 280 parts of glycidyl methacrylate ester undergo addition reaction in the presence of a polymerization-inhibitor and a catalyst at 90 0 C for 6 hours to produce an oligomer having a fiberous divinyl construction. The coating composition utilizes this oligomer in the forms of (I-1), a whole oligomer; (I-2), 0%-90% of this oligomer and 90%-10% of a vinyl monomer containing at least 30% of (meth) acrylic monomer; (I-3), 10%-90% of such oligomer and 90%-10% of other monomers containing at least two vinyl radicals in one molecule; (I-4), a mixture of (I-2) and (I-3) in proportion of 1/9 to 9/1, and (I-5), above four compositions each containing 50% or less unsaturated polyester or drying oil having 500-5,000 molecules or a drying oil-modified alkyd resin having 500-5,000 molecules. Four examples are given. (Iwakiri, K.)

  3. Electrocurtain coating process for coating solar mirrors

    Science.gov (United States)

    Kabagambe, Benjamin; Boyd, Donald W.; Buchanan, Michael J.; Kelly, Patrick; Kutilek, Luke A.; McCamy, James W.; McPheron, Douglas A.; Orosz, Gary R.; Limbacher, Raymond D.

    2013-10-15

    An electrically conductive protective coating or film is provided over the surface of a reflective coating of a solar mirror by flowing or directing a cation containing liquid and an anion containing liquid onto the conductive surface. The cation and the anion containing liquids are spaced from, and preferably out of contact with one another on the surface of the reflective coating as an electric current is moved through the anion containing liquid, the conductive surface between the liquids and the cation containing liquid to coat the conductive surface with the electrically conductive coating.

  4. Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez-Martinez, Cristina; Grueso, Esther [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Cantoblanco, Madrid (Spain); Carroll, Miles [Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury SP4 OJG, Wilts (United Kingdom); Rommelaere, Jean [Deutsches Krebsforschungszentrum Division F010, Im Neuenheimer Feld 242, D-69120 Heidelberg (Germany); Almendral, Jose M., E-mail: jmalmendral@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Cantoblanco, Madrid (Spain)

    2012-10-10

    The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus. The VEGFR2-binding peptide (V1) inserted at both 2Nt and VP1-Nt efficiently assembled in virions, but V1 disrupted VP1 and VP2 entry functions. The VP2 defect correlated with restricted externalization of V1-2Nt out of the coat. The specific infectivity of MVM and wtVP-pseudotyped mosaic MVM-V1 virions, upon heating and/or partial 2Nt cleavage, demonstrated that some 2Nt domains become intracellularly translocated out of the virus shell and cleaved to initiate entry. The V1 insertion defines a VP2-driven endosomal enlargement of the channel as an essential structural rearrangement performed by the MVM virion to infect.

  5. Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry

    International Nuclear Information System (INIS)

    Sánchez-Martínez, Cristina; Grueso, Esther; Carroll, Miles; Rommelaere, Jean; Almendral, José M.

    2012-01-01

    The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus. The VEGFR2-binding peptide (V1) inserted at both 2Nt and VP1-Nt efficiently assembled in virions, but V1 disrupted VP1 and VP2 entry functions. The VP2 defect correlated with restricted externalization of V1-2Nt out of the coat. The specific infectivity of MVM and wtVP-pseudotyped mosaic MVM-V1 virions, upon heating and/or partial 2Nt cleavage, demonstrated that some 2Nt domains become intracellularly translocated out of the virus shell and cleaved to initiate entry. The V1 insertion defines a VP2-driven endosomal enlargement of the channel as an essential structural rearrangement performed by the MVM virion to infect.

  6. Low Temperature Powder Coating

    Science.gov (United States)

    2011-02-09

    of volatile organic compounds (VOCs) and hazardous air pollutants (HAPs) • Legacy primers contain hexavalent chrome • Conventional powder coatings...coatings both in laboratory and field service evaluations • LTCPC allows environmental cost reductions through VOC/HAP elimination and hexavalent ... chrome reduction. • The LTCPC process greatly shortens the coating operation (LTCPC cures much more rapidly then conventional wet coatings) resulting in

  7. Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.

    Science.gov (United States)

    Mallet, W G; Maxfield, F R

    1999-07-26

    Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N.,W.G. Mallet,T.T. Soe,T.E.McGraw, and F.R. Maxfield.1998.J. Cell Biol.142:923-936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independentpathways for endosomal transport of proteins to the TGN from the plasma membrane.

  8. Increased expression of endosomal members of toll-like receptor family abrogates wound healing in patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Singh, Kanhaiya; Agrawal, Neeraj K; Gupta, Sanjeev K; Mohan, Gyanendra; Chaturvedi, Sunanda; Singh, Kiran

    2016-10-01

    The inflammatory phase of wound healing cascade is an important determinant of the fate of the wound. Acute inflammation is necessary to initiate proper wound healing, while chronic inflammation abrogates wound healing. Different endosomal members of toll-like receptor (TLR) family initiate inflammatory signalling via a range of different inflammatory mediators such as interferons, internal tissue damaged-associated molecular patterns (DAMPs) and hyperactive effector T cells. Sustained signalling of TLR9 and TLR7 contributes to chronic inflammation by activating the plasmacytoid dendritic cells. Diabetic wounds are also characterised by sustained inflammatory phase. The objective of this study was to analyse the differential expression of endosomal TLRs in human diabetic wounds compared with control wounds. We analysed the differential expression of TLR7 and TLR9 both at transcriptional and translational levels in wounds of 84 patients with type 2 diabetes mellitus (T2DM) and 6 control subjects without diabetes using quantitative real-time polymerase chain reaction (RT-PCR), western blot and immunohistochemistry. TLR7 and TLR9 were significantly up-regulated in wounds of the patients with T2DM compared with the controls and were dependent on the infection status of the diabetic wounds, and wounds with microbial infection exhibited lower expression levels of endosomal TLRs. Altered endosomal TLR expression in T2DM subjects might be associated with wound healing impairment. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  9. Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger

    NARCIS (Netherlands)

    Oude Blenke, E.; Sleszynska, M.; Evers, M. J W; Storm, G.; Martin, N. I.; Mastrobattista, E.

    2017-01-01

    Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high

  10. Flow coating apparatus and method of coating

    Science.gov (United States)

    Hanumanthu, Ramasubrahmaniam; Neyman, Patrick; MacDonald, Niles; Brophy, Brenor; Kopczynski, Kevin; Nair, Wood

    2014-03-11

    Disclosed is a flow coating apparatus, comprising a slot that can dispense a coating material in an approximately uniform manner along a distribution blade that increases uniformity by means of surface tension and transfers the uniform flow of coating material onto an inclined substrate such as for example glass, solar panels, windows or part of an electronic display. Also disclosed is a method of flow coating a substrate using the apparatus such that the substrate is positioned correctly relative to the distribution blade, a pre-wetting step is completed where both the blade and substrate are completed wetted with a pre-wet solution prior to dispensing of the coating material onto the distribution blade from the slot and hence onto the substrate. Thereafter the substrate is removed from the distribution blade and allowed to dry, thereby forming a coating.

  11. Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework

    KAUST Repository

    Alsaiari, Shahad K.

    2017-12-22

    CRISPR/Cas9 is a combined protein (Cas9) and an engineered single guide RNA (sgRNA) genome editing platform that offers revolutionary solutions to genetic diseases. It has, however, a double delivery problem owning to the large protein size and the highly charged RNA component. In this work, we report the first example of CRISPR/Cas9 encapsulated by nanoscale zeolitic imidazole frameworks (ZIFs) with a loading efficiency of 17% and enhanced endosomal escape promoted by the protonated imidazole moieties. The gene editing potential of CRISPR/Cas9 encapsulated by ZIF-8 (CC-ZIFs) is further verified by knocking down the gene expression of green fluorescent protein by 37% over 4 days employing CRISPR/Cas9 machinery. The nanoscale CC-ZIFs are biocompatible and easily scaled-up offering excellent loading capacity and controlled co-delivery of intact Cas9 protein and sgRNA.

  12. Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework

    KAUST Repository

    Alsaiari, Shahad K.; Patil, Sachin; Alyami, Mram Z.; Alamoudi, Kholod; Aleisa, Fajr A; Merzaban, Jasmeen; Li, Mo; Khashab, Niveen M.

    2017-01-01

    CRISPR/Cas9 is a combined protein (Cas9) and an engineered single guide RNA (sgRNA) genome editing platform that offers revolutionary solutions to genetic diseases. It has, however, a double delivery problem owning to the large protein size and the highly charged RNA component. In this work, we report the first example of CRISPR/Cas9 encapsulated by nanoscale zeolitic imidazole frameworks (ZIFs) with a loading efficiency of 17% and enhanced endosomal escape promoted by the protonated imidazole moieties. The gene editing potential of CRISPR/Cas9 encapsulated by ZIF-8 (CC-ZIFs) is further verified by knocking down the gene expression of green fluorescent protein by 37% over 4 days employing CRISPR/Cas9 machinery. The nanoscale CC-ZIFs are biocompatible and easily scaled-up offering excellent loading capacity and controlled co-delivery of intact Cas9 protein and sgRNA.

  13. Concerted regulation of npc2 binding to endosomal/lysosomal membranes by bis(monoacylglycero)phosphate and sphingomyelin

    DEFF Research Database (Denmark)

    Enkavi, Giray; Mikkolainen, Heikki; Güngör, Burçin

    2017-01-01

    remained elusive. Here, based on an extensive set of atomistic simulations and free energy calculations, we clarify the mechanism and energetics of npc2-membrane binding and characterize the roles of physiologically relevant key lipids associated with the binding process. Our results capture in atomistic......Niemann-Pick Protein C2 (npc2) is a small soluble protein critical for cholesterol transport within and from the lysosome and the late endosome. Intriguingly, npc2-mediated cholesterol transport has been shown to be modulated by lipids, yet the molecular mechanism of npc2-membrane interactions has......). This mode is associated with cholesterol uptake and release. On the other hand, the second mode (Supine) places the cholesterol binding pocket away from the membrane surface, but has overall higher membrane binding affinity. We determined that bis(monoacylglycero)phosphate (bmp) is specifically required...

  14. Constitutively internalized dopamine transporter is targeted to late endosomes and lysosomal degradation in heterologous cell lines and dopaminergic neurons

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Madsen, Kenneth; Vægter, Christian Bjerggaard

    and amphetamine, a substrate of the DAT. In antibody feeding experiments we observed that Tac-DAT was constitutively internalized faster than Tac alone and using an ELISA based assay we could quantify time-dependent intracellular accumulation of the transporter. Incubation with inhibitors of lysosomal degradation...... (leupeptin, chloroquine, or ammonium chloride) increased the amount of transporter accumulated intracellularly over time, suggesting that constitutively endocytosed transporter was targeted to lysosomal degradation. This was further supported by expression of Tac-DAT in the immortalized dopaminergic cell...... dopaminergic neurons and visualized the DAT directly in the neurons using the fluorescent cocaine analog JHC 1-064. These data showed pronounced colocalization upon constitutive internalization with Lysotracker, a late endosomal/lysosomal marker; however only little co-lolization was observed with Alexa488...

  15. The endosomal recycling of FgSnc1 by FgSnx41-FgSnx4 heterodimer is essential for polarized growth and pathogenicity in Fusarium graminearum.

    Science.gov (United States)

    Zheng, Wenhui; Lin, Yahong; Fang, Wenqin; Zhao, Xu; Lou, Yi; Wang, Guanghui; Zheng, Huawei; Liang, Qifu; Abubakar, Yakubu Saddeeq; Olsson, Stefan; Zhou, Jie; Wang, Zonghua

    2018-04-20

    Endosomal sorting machineries regulate the transport of their cargoes among intracellular compartments. However, the molecular nature of such intracellular trafficking processes in pathogenic fungal development and pathogenicity remains unclear. Here, we dissect the roles and molecular mechanisms of two sorting nexin proteins and their cargoes in endosomal recycling in Fusarium graminearum using high-resolution microscopy and high-throughput co-immunoprecipitation strategies. We show that the sorting nexins, FgSnx41 and FgSnx4, interact with each other and assemble into a functionally interdependent heterodimer through their respective BAR domains. Further analyses demonstrate that the dimer localizes to the early endosomal membrane and coordinates endosomal sorting. The small GTPase FgRab5 regulates the correct localization of FgSnx41-FgSnx4 and is consequently required for its trafficking function. The protein FgSnc1 is a cargo of FgSnx41-FgSnx4 and regulates the fusion of secreted vesicles with the fungal growing apex and plasma membrane. In the absence of FgSnx41 or FgSnx4, FgSnc1 is mis-sorted and degraded in the vacuole, and null deletion of either component causes defects in the fungal polarized growth and virulence. Overall, for the first time, our results reveal the mechanism of FgSnc1 endosomal recycling by FgSnx41-FgSnx4 heterodimer which is essential for polarized growth and pathogenicity in F. graminearum. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  16. Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment.

    Science.gov (United States)

    Girada, Shravan Babu; Kuna, Ramya S; Bele, Shilpak; Zhu, Zhimeng; Chakravarthi, N R; DiMarchi, Richard D; Mitra, Prasenjit

    2017-10-01

    Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  17. European coatings conference - Marine coatings. Proceedings

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2008-07-01

    This volume contains 13 lectures (manuscripts or powerpoint foils) with the following topics: 1. Impact of containerization on polyurethane and polyurea in marine and protective coatings (Malte Homann); 2. The application of combinatorial/high-throughput methods to the development of marine coatings (Bret Chisholm); 3. Progress and perspectives in the AMBIO (advanced nanostructured surfaces for the control of biofouling) Project (James Callow); 4. Release behaviour due to shear and pull-off of silicone coatings with a thickness gradient (James G. Kohl); 5. New liquid rheology additives for high build marine coatings (Andreas Freytag); 6. Effective corrosion protection with polyaniline, polpyrrole and polythiophene as anticorrosice additives for marine paints (Carlos Aleman); 7. Potential applications of sol gel technology for marine applications (Robert Akid); 8: Performance of biocide-free Antifouling Coatings for leisure boats (Bernd Daehne); 9. Novel biocidefree nanostructured antifouling coatings - can nano do the job? (Corne Rentrop); 10. One component high solids, VOC compliant high durability finish technology (Adrian Andrews); 11. High solid coatings - the hybrid solution (Luca Prezzi); 12. Unique organofunctional silicone resins for environmentally friendly high-performance coatings (Dieter Heldmann); 13. Silicone-alkyd paints for marine applications: from battleship-grey to green (Thomas Easton).

  18. The synergistic transactivation of the hepatitis B viral (HBV) pregenomic promoter by the E6 protein of human papillomavirus type 16 (HPV-16 E6) with HBV X protein was mediated through the AP1 site of E element in the enhancer I (EnI) in human liver cell.

    Science.gov (United States)

    Lee, D H; Choi, B H; Rho, H M

    1999-11-01

    Infection by HBV of a cell already infected with other viral species or vice versa has been suggested as being involved in hepatocellular carcinoma. Using the CAT assay method, we investigated the interactive roles of HBx and potentially oncogenic and transactivating viral early proteins such as Ad5 E1A, HPV-16 E6, and SV40 T ag. In the presence of HBx, only HPV-16 E6 showed significant synergistic transactivation of EnI. We further investigated the function of the HPV-16 E6 using deletion, heterologous promoter, and mutation analyses on the EnI promoter. The results showed that the synergistic effect was mediated through the AP1 site of the E element in EnI by the direct activation of AP1 and support the idea that the infection by HBV of the cell with other viral species such as HPV-16 could increase the transcription activity of the HBV and other oncogenes containing an AP1 site in the promoter. Copyright 1999 Academic Press.

  19. PIT Coating Requirements Analysis

    International Nuclear Information System (INIS)

    MINTEER, D.J.

    2000-01-01

    This study identifies the applicable requirements for procurement and installation of a coating intended for tank farm valve and pump pit interior surfaces. These requirements are intended to be incorporated into project specification documents and design media. This study also evaluates previously recommended coatings and identifies requirement-compliant coating products

  20. PIT Coating Requirements Analysis

    Energy Technology Data Exchange (ETDEWEB)

    MINTEER, D.J.

    2000-10-20

    This study identifies the applicable requirements for procurement and installation of a coating intended for tank farm valve and pump pit interior surfaces. These requirements are intended to be incorporated into project specification documents and design media. This study also evaluates previously recommended coatings and identifies requirement-compliant coating products.

  1. Coatings for laser fusion

    International Nuclear Information System (INIS)

    Lowdermilk, W.H.

    1981-01-01

    Optical coatings are used in lasers systems for fusion research to control beam propagation and reduce surface reflection losses. The performance of coatings is important in the design, reliability, energy output, and cost of the laser systems. Significant developments in coating technology are required for future lasers for fusion research and eventual power reactors

  2. Fuel particle coating data

    International Nuclear Information System (INIS)

    Hollabaugh, C.M.; Wagner, P.; Wahman, L.A.; White, R.W.

    1977-01-01

    Development of coating on nuclear fuel particles for the High-Temperature Fuels Technology program at the Los Alamos Scientific Laboratory included process studies for low-density porous and high-density isotropic carbon coats, and for ZrC and ''alloy'' C/ZrC coats. This report documents the data generated by these studies

  3. Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

    Science.gov (United States)

    Hurwitz, Stephanie N; Cheerathodi, Mujeeb R; Nkosi, Dingani; York, Sara B; Meckes, David G

    2018-03-01

    The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major

  4. ATHENA optimized coating design

    DEFF Research Database (Denmark)

    Ferreira, Desiree Della Monica; Christensen, Finn Erland; Jakobsen, Anders Clemen

    2012-01-01

    The optimization of coating design for the ATHENA mission si described and the possibility of increasing the telescope effective area in the range between 0.1 and 10 keV is investigated. An independent computation of the on-axis effective area based on the mirror design of ATHENA is performed...... in order to review the current coating baseline. The performance of several material combinations, considering a simple bi-layer, simple multilayer and linear graded multilayer coatings are tested and simulation of the mirror performance considering both the optimized coating design and the coating...

  5. Metallic coating of microspheres

    International Nuclear Information System (INIS)

    Meyer, S.F.

    1980-01-01

    Extremely smooth, uniform metal coatings of micrometer thicknesses on microscopic glass spheres (microspheres) are often needed as targets for inertial confinement fusion (ICF) experiments. The first part of this paper reviews those methods used successfully to provide metal coated microspheres for ICF targets, including magnetron sputtering, electro- and electroless plating, and chemical vapor pyrolysis. The second part of this paper discusses some of the critical aspects of magnetron sputter coating of microspheres, including substrate requirements, the sticking of microspheres during coating (preventing a uniform coating), and the difficulties in growing the desired dense, smooth, uniform microstructure on continuously moving spherical substrates

  6. Antibacterial polymer coatings.

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Mollye C.; Allen, Ashley N.; Barnhart, Meghan; Tucker, Mark David; Hibbs, Michael R.

    2009-09-01

    A series of poly(sulfone)s with quaternary ammonium groups and another series with aldehyde groups are synthesized and tested for biocidal activity against vegetative bacteria and spores, respectively. The polymers are sprayed onto substrates as coatings which are then exposed to aqueous suspensions of organisms. The coatings are inherently biocidal and do not release any agents into the environment. The coatings adhere well to both glass and CARC-coated coupons and they exhibit significant biotoxicity. The most effective quaternary ammonium polymers kills 99.9% of both gram negative and gram positive bacteria and the best aldehyde coating kills 81% of the spores on its surface.

  7. Coated Aerogel Beads

    Science.gov (United States)

    Littman, Howard (Inventor); Plawsky, Joel L. (Inventor); Paccione, John D. (Inventor)

    2014-01-01

    Methods and apparatus for coating particulate material are provided. The apparatus includes a vessel having a top and a bottom, a vertically extending conduit having an inlet in the vessel and an outlet outside of the vessel, a first fluid inlet in the bottom of the vessel for introducing a transfer fluid, a second fluid inlet in the bottom of the vessel for introducing a coating fluid, and a fluid outlet from the vessel. The method includes steps of agitating a material, contacting the material with a coating material, and drying the coating material to produce a coated material. The invention may be adapted to coat aerogel beads, among other materials. A coated aerogel bead and an aerogel-based insulation material are also disclosed.

  8. Anticorrosive coatings: a review

    DEFF Research Database (Denmark)

    Sørensen, Per Aggerholm; Kiil, Søren; Dam-Johansen, Kim

    2009-01-01

    of volatile organic compounds (VOCs) have caused significant changes in the anticorrosive coating industry. The requirement for new VOC-compliant coating technologies means that coating manufacturers can no longer rely on the extensive track record of their time-served products to convince consumers...... of their suitability for use. An important aspect in the development of new VOC-compliant, high-performance anticorrosive coating systems is a thorough knowledge of the components in anticorrosive coatings, their interactions, their advantages and limitations, as well as a detailed knowledge on the failure modes......, and inhibitive coatings are outlined. In the past decades, several alternatives to organic solvent-borne coatings have reached the commercial market. This review also presents some of these technologies and discusses some of their advantages and limitations. Finally, some of the mechanisms leading to degradation...

  9. Pixelated coatings and advanced IR coatings

    Science.gov (United States)

    Pradal, Fabien; Portier, Benjamin; Oussalah, Meihdi; Leplan, Hervé

    2017-09-01

    Reosc developed pixelated infrared coatings on detector. Reosc manufactured thick pixelated multilayer stacks on IR-focal plane arrays for bi-spectral imaging systems, demonstrating high filter performance, low crosstalk, and no deterioration of the device sensitivities. More recently, a 5-pixel filter matrix was designed and fabricated. Recent developments in pixelated coatings, shows that high performance infrared filters can be coated directly on detector for multispectral imaging. Next generation space instrument can benefit from this technology to reduce their weight and consumptions.

  10. Combustion chemical vapor desposited coatings for thermal barrier coating systems

    Energy Technology Data Exchange (ETDEWEB)

    Hampikian, J.M.; Carter, W.B. [Georgia Institute of Technology, Atlanta, GA (United States)

    1995-10-01

    The new deposition process, combustion chemical vapor deposition, shows a great deal of promise in the area of thermal barrier coating systems. This technique produces dense, adherent coatings, and does not require a reaction chamber. Coatings can therefore be applied in the open atmosphere. The process is potentially suitable for producing high quality CVD coatings for use as interlayers between the bond coat and thermal barrier coating, and/or as overlayers, on top of thermal barrier coatings.

  11. Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells.

    Science.gov (United States)

    Ahmad, Waqas; Li, Yingying; Guo, Yidi; Wang, Xinyu; Duan, Ming; Guan, Zhenhong; Liu, Zengshan; Zhang, Maolin

    2017-06-01

    Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID 50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

  12. Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger.

    Science.gov (United States)

    Oude Blenke, E; Sleszynska, M; Evers, M J W; Storm, G; Martin, N I; Mastrobattista, E

    2017-02-15

    Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high membranolytic activity. However, such lytic peptides also impose a significant safety risk when applied in vivo as they often have similar activity against red blood cells and other nontarget cell membranes. Our aim is to control the membrane-disrupting capacity of these peptides in time and space by physically constraining them to a nanocarrier surface in such a way that they only become activated when delivered inside acidic endosomes. To this end, a variety of chemical approaches for the coupling of lytic peptides to liposomes via functionalized PEG-lipids were explored, including maleimide-thiol chemistry, click-chemistry, and aldehyde-hydrazide chemistry. The latter enables reversible conjugation via a hydrazone bond, allowing for release of the peptide under endosomal conditions. By carefully choosing the conjugation site and by using a pH activated analog of the melittin peptide, lytic activity toward a model membrane is completely inhibited at physiological pH. At endosomal pH the activity is restored by hydrolysis of the acid-labile hydrazone bond, releasing the peptide in its most active, free form. Furthermore, using an analogue containing a nonhydrolyzable bond as a control, it was shown that the activity observed can be completely attributed to release of the peptide, validating dynamic covalent conjugation as a suitable strategy to maintain safety during circulation.

  13. Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)

    International Nuclear Information System (INIS)

    Duke, Elizabeth M.H.; Razi, Minoo; Weston, Anne; Guttmann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Tooze, Sharon A.; Collinson, Lucy M.

    2014-01-01

    Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities. - Highlights: • We image whole, unstained mammalian cells using cryo-soft X-ray tomography. • Endosomes are identified using a gold marker for the transferrin receptor. • A new workflow for correlative cryo-fluorescence and cryo-SXT is used to locate early autophagosomes. • Interactions between endosomes, endoplasmic reticulum and forming autophagosomes are mapped in 3D. • Multiple omegasomes are shown to form at ‘hotspots’ on the endoplasmic reticulum

  14. The small G protein Arl5 contributes to endosome-to-Golgi traffic by aiding the recruitment of the GARP complex to the Golgi

    Directory of Open Access Journals (Sweden)

    Cláudia Rosa-Ferreira

    2015-03-01

    Full Text Available The small G proteins of the Arf family play critical roles in membrane trafficking and cytoskeleton organization. However, the function of some members of the family remains poorly understood including Arl5 which is widely conserved in eukaryotes. Humans have two closely related Arl5 paralogues (Arl5a and Arl5b, and both Arl5a and Arl5b localize to the trans-Golgi with Arl5b being involved in retrograde traffic from endosomes to the Golgi apparatus. To investigate the function of Arl5, we have used Drosophila melanogaster as a model system. We find that the single Arl5 orthologue in Drosophila also localizes to the trans-Golgi, but flies lacking the Arl5 gene are viable and fertile. By using both liposome and column based affinity chromatography methods we find that Arl5 interacts with the Golgi-associated retrograde protein (GARP complex that acts in the tethering of vesicles moving from endosomes to the trans-Golgi network (TGN. In Drosophila tissues the GARP complex is partially displaced from the Golgi when Arl5 is absent, and the late endosomal compartment is enlarged. In addition, in HeLa cells GARP also becomes cytosolic upon depletion of Arl5b. These phenotypes are consistent with a role in endosome-to-Golgi traffic, but are less severe than loss of GARP itself. Thus it appears that Arl5 is one of the factors that directs the recruitment of the GARP complex to the trans-Golgi, and this function is conserved in both flies and humans.

  15. L-Type Voltage-Gated Ca2+ Channels Regulate Synaptic-Activity-Triggered Recycling Endosome Fusion in Neuronal Dendrites

    Directory of Open Access Journals (Sweden)

    Brian G. Hiester

    2017-11-01

    Full Text Available The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca2+ from distinct sources. NMDA receptor Ca2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca2+ channels (L-VGCCs regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.

  16. The Human ABCG1 Transporter Mobilizes Plasma Membrane and Late Endosomal Non-Sphingomyelin-Associated-Cholesterol for Efflux and Esterification

    Directory of Open Access Journals (Sweden)

    Edward B. Neufeld

    2014-12-01

    Full Text Available We have previously shown that GFP-tagged human ABCG1 on the plasma membrane (PM and in late endosomes (LE mobilizes sterol on both sides of the membrane lipid bilayer, thereby increasing cellular cholesterol efflux to lipid surfaces. In the present study, we examined ABCG1-induced changes in membrane cholesterol distribution, organization, and mobility. ABCG1-GFP expression increased the amount of mobile, non-sphingomyelin(SM-associated cholesterol at the PM and LE, but not the amount of SM-associated-cholesterol or SM. ABCG1-mobilized non-SM-associated-cholesterol rapidly cycled between the PM and LE and effluxed from the PM to extracellular acceptors, or, relocated to intracellular sites of esterification. ABCG1 increased detergent-soluble pools of PM and LE cholesterol, generated detergent-resistant, non-SM-associated PM cholesterol, and increased resistance to both amphotericin B-induced (cholesterol-mediated and lysenin-induced (SM-mediated cytolysis, consistent with altered organization of both PM cholesterol and SM. ABCG1 itself resided in detergent-soluble membrane domains. We propose that PM and LE ABCG1 residing at the phase boundary between ordered (Lo and disordered (Ld membrane lipid domains alters SM and cholesterol organization thereby increasing cholesterol flux between Lo and Ld, and hence, the amount of cholesterol available for removal by acceptors on either side of the membrane bilayer for either efflux or esterification.

  17. The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

    Science.gov (United States)

    Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

    2014-06-27

    The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    2016-10-01

    Full Text Available Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs. The VRCs built by Tomato bushy stunt virus (TBSV are enriched with phosphatidylethanolamine (PE through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5-positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

  19. The functional interplay of Rab11, FIP3 and Rho proteins on the endosomal recycling pathway controls cell shape and symmetry.

    Science.gov (United States)

    Bouchet, Jérôme; McCaffrey, Mary W; Graziani, Andrea; Alcover, Andrés

    2018-07-04

    Several families of small GTPases regulate a variety of fundamental cellular processes, encompassing growth factor signal transduction, vesicular trafficking and control of the cytoskeleton. Frequently, their action is hierarchical and complementary, but much of the detail of their functional interactions remains to be clarified. It is well established that Rab family members regulate a variety of intracellular vesicle trafficking pathways. Moreover, Rho family GTPases are pivotal for the control of the actin and microtubule cytoskeleton. However, the interplay between these 2 types of GTPases has been rarely reported. We discuss here our recent findings showing that Rab11, a key regulator of endosomal recycling, and Rac1, a central actin cytoskeleton regulator involved in lamellipodium formation and cell migration, interplay on endosomes through the Rab11 effector FIP3. In the context of the rapidly reactive T lymphocytes, Rab11-Rac1 endosomal functional interplay is important to control cell shape changes and cell symmetry during lymphocyte spreading and immunological synapse formation and ultimately modulate T cell activation.

  20. The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

    Science.gov (United States)

    Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

    2016-09-01

    Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Preliminary coating design and coating developments for ATHENA

    DEFF Research Database (Denmark)

    Jakobsen, Anders Clemen; Ferreira, Desiree Della Monica; Christensen, Finn Erland

    2011-01-01

    We present initial novel coating design for ATHENA. We make use of both simple bilayer coatings of Ir and B4C and more complex constant period multilayer coatings to enhance the effective area and cover the energy range from 0.1 to 10 keV. We also present the coating technology used...... for these designs and present test results from coatings....

  2. Plasma sprayed thermoregulating coatings

    International Nuclear Information System (INIS)

    Kudinov, V.V.; Puzanov, A.A.; Zambrzhitskij, A.P.; Soboleva, V.V.

    1979-01-01

    Shown is the possibility of plasma spraying application for thermoregulating coating formation. Given are test results of service properties of BeO, Al 2 O 2 plasma coatings on the substrates of the MA2-1 magnesium alloy. Described is a device for studying durability of coating optical parameters under ultraviolet irradiation in deep vacuum. Dynamics of absorption coefficient, growth caused by an increase in absorption centers amount under such irradiation is investigated

  3. Zinc phosphate conversion coatings

    Science.gov (United States)

    Sugama, Toshifumi

    1997-01-01

    Zinc phosphate conversion coatings for producing metals which exhibit enhanced corrosion prevention characteristics are prepared by the addition of a transition-metal-compound promoter comprising a manganese, iron, cobalt, nickel, or copper compound and an electrolyte such as polyacrylic acid, polymethacrylic acid, polyitaconic acid and poly-L-glutamic acid to a phosphating solution. These coatings are further improved by the incorporation of Fe ions. Thermal treatment of zinc phosphate coatings to generate .alpha.-phase anhydrous zinc phosphate improves the corrosion prevention qualities of the resulting coated metal.

  4. Silica coatings on clarithromycin.

    Science.gov (United States)

    Bele, Marjan; Dmitrasinovic, Dorde; Planinsek, Odon; Salobir, Mateja; Srcic, Stane; Gaberscek, Miran; Jamnik, Janko

    2005-03-03

    Pre-crystallized clarithromycin (6-O-methylerythromycin A) particles were coated with silica from the tetraethyl orthosilicate (TEOS)-ethanol-aqueous ammonia system. The coatings had a typical thickness of 100-150 nm and presented about 15 wt.% of the silica-drug composite material. The properties of the coatings depended on reactant concentration, temperature and mixing rate and, in particular, on the presence of a cationic surfactant (cetylpyridinium chloride). In the presence of cetylpyridinium chloride the silica coatings slightly decreased the rate of pure clarithromycin dissolution.

  5. Superhydrophobic silica coating by dip coating method

    International Nuclear Information System (INIS)

    Mahadik, Satish A.; Parale, Vinayak; Vhatkara, Rajiv S.; Mahadik, Dinesh B.; Kavale, Mahendra S.; Wagh, Pratap B.; Gupta, Satish; Gurav, Jyoti

    2013-01-01

    Herein, we report a simple and low cost method for the fabrication of superhydrophobic coating surface on quartz substrates via sol-gel dip coating method at room temperature. Desired surface chemistry and texture growth for superhydrophobicity developed under double step sol–gel process at room temperature. The resultant superhydrophobic surfaces were characterized by Field-emission scanning electron microscopy (FE-SEM), Atomic force microscopy (AFM), water contact angle (WCA) measurement, differential thermal gravimetric analysis-differential thermal analysis (TGA-DTA) calorimetry and optical spectrometer. Coating shows the ultra high water contact angle about 168 ± 2° and water sliding angle 3 ± 1° and superoleophilic with petroleum oils. This approach allows a simple strategy for the fabrication process of superhydrophilic–superhydrophobic on same surfaces with high thermal stability of superhydrophobicity up to 560 °C. Thus, durability, special wettability and thermal stability of superhydrophobicity expand their application fields.

  6. Multifunctional Cationic Lipid-Based Nanoparticles Facilitate Endosomal Escape and Reduction-Triggered Cytosolic siRNA Release

    Science.gov (United States)

    Gujrati, Maneesh; Malamas, Anthony; Shin, Tesia; Jin, Erlei; Sun, Lulu; Lu, Zheng-Rong

    2015-01-01

    Small interfering RNA (siRNA) has garnered much attention in recent years as a promising avenue for cancer gene therapy due to its ability to silence disease-related genes. Effective gene silencing is contingent upon the delivery of siRNA into the cytosol of target cells and requires the implementation of delivery systems possessing multiple functionalities to overcome delivery barriers. The present work explores the multifunctional properties and biological activity of a recently developed cationic lipid carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)propionamide]) (ECO). The physicochemical properties and biological activity of ECO/siRNA nanoparticles were assessed over a range of N/P ratios to optimize the formulation. Potent and sustained luciferase silencing in a U87 glioblastoma cell line was observed, even in the presence of serum proteins. ECO/siRNA nanoparticles exhibited pH-dependent membrane disruption at pH levels corresponding to various stages of the intracellular trafficking pathway. It was found that disulfide linkages created during nanoparticle formation enhanced the protection of siRNA from degradation and facilitated site-specific siRNA release in the cytosol by glutathione-mediated reduction. Confocal microscopy confirmed that ECO/siRNA nanoparticles readily escaped from late endosomes prior to cytosolic release of the siRNA cargo. These results demonstrate that the rationally designed multifunctionality of ECO/siRNA nanoparticles is critical for intracellular siRNA delivery and the continuing development of safe and effective delivery systems. PMID:25020033

  7. Photochemical internalisation, a minimally invasive strategy for light-controlled endosomal escape of cancer stem cell-targeting therapeutics.

    Science.gov (United States)

    Selbo, Pål Kristian; Bostad, Monica; Olsen, Cathrine Elisabeth; Edwards, Victoria Tudor; Høgset, Anders; Weyergang, Anette; Berg, Kristian

    2015-08-01

    Despite progress in radio-, chemo- and photodynamic-therapy (PDT) of cancer, treatment resistance still remains a major problem for patients with aggressive tumours. Cancer stem cells (CSCs) or tumour-initiating cells are intrinsically and notoriously resistant to conventional cancer therapies and are proposed to be responsible for the recurrence of tumours after therapy. According to the CSC hypothesis, it is imperative to develop novel anticancer agents or therapeutic strategies that take into account the biology and role of CSCs. The present review outlines our recent study on photochemical internalisation (PCI) using the clinically relevant photosensitiser TPCS2a/Amphinex® as a rational, non-invasive strategy for the light-controlled endosomal escape of CSC-targeting drugs. PCI is an intracellular drug delivery method based on light-induced ROS-generation and a subsequent membrane-disruption of endocytic vesicles, leading to cytosolic release of the entrapped drugs of interest. In different proof-of-concept studies we have demonstrated that PCI of CSC-directed immunotoxins targeting CD133, CD44, CSPG4 and EpCAM is a highly specific and effective strategy for killing cancer cells and CSCs. CSCs overexpressing CD133 are PDT-resistant; however, this is circumvented by PCI of CD133-targeting immunotoxins. In view of the fact that TPCS2a is not a substrate of the efflux pumps ABCG2 and P-glycoprotein (ABCB1), the PCI-method is a promising anti-CSC therapeutic strategy. Due to a laser-controlled exposure, PCI of CSC-targeting drugs will be confined exclusively to the tumour tissue, suggesting that this drug delivery method has the potential to spare distant normal stem cells.

  8. Pannexin2 oligomers localize into endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane

    Directory of Open Access Journals (Sweden)

    Daniela eBoassa

    2015-02-01

    Full Text Available Pannexin2 (Panx2 is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS have been documented. Whereas Pannexin1 (Panx1 is fairly ubiquitous and Pannexin3 (Panx3 is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa and HEK293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the

  9. A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

    Directory of Open Access Journals (Sweden)

    Xuejun Tian

    2015-03-01

    Full Text Available Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj, causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.

  10. Coated electroactive materials

    Science.gov (United States)

    Amine, Khalil; Abouimrane, Ali

    2016-08-30

    A process includes suspending an electroactive material in a solvent, suspending or dissolving a carbon precursor in the solvent; and depositing the carbon precursor on the electroactive material to form a carbon-coated electroactive material. Compositions include a graphene-coated electroactive material prepared from a solution phase mixture or suspension of an electroactive material and graphene, graphene oxide, or a mixture thereof.

  11. Rock-hard coatings

    OpenAIRE

    Muller, M.

    2007-01-01

    Aircraft jet engines have to be able to withstand infernal conditions. Extreme heat and bitter cold tax coatings to the limit. Materials expert Dr Ir. Wim Sloof fits atoms together to develop rock-hard coatings. The latest invention in this field is known as ceramic matrix composites. Sloof has signed an agreement with a number of parties to investigate this material further.

  12. Metallurgical coating system

    International Nuclear Information System (INIS)

    Daniels, L.C.; Whittaker, G.S.

    1984-01-01

    The present invention relates to a novel metallurgical coating system which provides corrosion resistance and non-stick properties to metallic components which are subjected to unusually severe operating conditions. The coating system comprises a first layer comprising tantalum which is deposited upon a substrate and a second layer comprising molybdenum disilicide which is deposited upon the first layer

  13. Rock-hard coatings

    NARCIS (Netherlands)

    Muller, M.

    2007-01-01

    Aircraft jet engines have to be able to withstand infernal conditions. Extreme heat and bitter cold tax coatings to the limit. Materials expert Dr Ir. Wim Sloof fits atoms together to develop rock-hard coatings. The latest invention in this field is known as ceramic matrix composites. Sloof has

  14. Unobtrusive graphene coatings

    NARCIS (Netherlands)

    Mugele, Friedrich Gunther

    2012-01-01

    The contact angle of water drops on substrates for which the wettability is dominated by van der Waals forces remains unchanged when the substrates are coated with a monolayer of graphene. Such 'wetting transparency' could lead to superior conducting and hydrophobic graphene-coated surfaces with

  15. Coating thickness measurement

    International Nuclear Information System (INIS)

    1976-12-01

    The standard specifies measurements of the coating thickness, which make use of beta backscattering and/or x-ray fluorescence. For commonly used combinations of coating material and base material the appropriate measuring ranges and radionuclides to be used are given for continuous as well as for discontinuous measurements

  16. Duplex aluminized coatings

    Science.gov (United States)

    Gedwill, M. A.; Grisaffe, S. J. (Inventor)

    1975-01-01

    The surface of a metallic base system is initially coated with a metallic alloy layer that is ductile and oxidation resistant. An aluminide coating is then applied to the metallic alloy layer. The chemistry of the metallic alloy layer is such that the oxidation resistance of the subsequently aluminized outermost layer is not seriously degraded.

  17. Coated ceramic breeder materials

    Science.gov (United States)

    Tam, Shiu-Wing; Johnson, Carl E.

    1987-01-01

    A breeder material for use in a breeder blanket of a nuclear reactor is disclosed. The breeder material comprises a core material of lithium containing ceramic particles which has been coated with a neutron multiplier such as Be or BeO, which coating has a higher thermal conductivity than the core material.

  18. Radiation curable coating compositions

    International Nuclear Information System (INIS)

    Jenkinson, R.D.; Carder, C.H.

    1979-01-01

    The present invention provides a low-toxicity diluent component for radiation curable coating compositions that contain an acrylyl or methacryly oligomer or resin component such as an acrylyl urethane oligomer. The low-toxicity diluent component of this invention is chosen from the group consisting of tetraethlorthosilicate and tetraethoxyethylorthosilicate. When the diluent component is used as described, benefits in addition to viscosity reduction, may be realized. Application characteristics of the uncured coatings composition, such as flowability, leveling, and smoothness are notably improved. Upon curing by exposure to actinic radiation, the coating composition forms a solid, non-tacky surface free of pits, fissures or other irregularities. While there is no readily apparent reactive mechanism by which the orthosilicate becomes chemically bonded to the cured coating, the presence of silicon in the cured coating has been confirmed by scanning electron microscopy. 12 drawing

  19. Charged-particle coating

    International Nuclear Information System (INIS)

    Johnson, W.L.; Crane, J.K.; Hendricks, C.D.

    1980-01-01

    Advanced target designs require thicker (approx. 300 μm) coatings and better surface finishes that can be produced with current coating techniques. An advanced coating technique is proposed to provide maximum control of the coating flux and optimum manipulation of the shell during processing. In this scheme a small beam of ions or particles of known incident energy are collided with a levitated spherical mandrel. Precise control of the incident energy and angle of the deposition flux optimizes the control of the coating morphology while controlled rotation and noncontact support of the shell minimizes the possibility of particulate or damage generated defects. Almost infinite variability of the incident energy and material in this process provides increased flexibility of the target designs which can be physically realized

  20. Coating thickness measuring device

    International Nuclear Information System (INIS)

    Joffe, B.B.; Sawyer, B.E.; Spongr, J.J.

    1984-01-01

    A device especially adapted for measuring the thickness of coatings on small, complexly-shaped parts, such as, for example, electronic connectors, electronic contacts, or the like. The device includes a source of beta radiation and a radiation detector whereby backscatter of the radiation from the coated part can be detected and the thickness of the coating ascertained. The radiation source and detector are positioned in overlying relationship to the coated part and a microscope is provided to accurately position the device with respect to the part. Means are provided to control the rate of descent of the radiation source and radiation detector from its suspended position to its operating position and the resulting impact it makes with the coated part to thereby promote uniformity of readings from operator to operator, and also to avoid excessive impact with the part, thereby improving accuracy of measurement and eliminating damage to the parts

  1. Fluorine Based Superhydrophobic Coatings

    Directory of Open Access Journals (Sweden)

    Jean-Denis Brassard

    2012-05-01

    Full Text Available Superhydrophobic coatings, inspired by nature, are an emerging technology. These water repellent coatings can be used as solutions for corrosion, biofouling and even water and air drag reduction applications. In this work, synthesis of monodispersive silica nanoparticles of ~120 nm diameter has been realized via Stöber process and further functionalized using fluoroalkylsilane (FAS-17 molecules to incorporate the fluorinated groups with the silica nanoparticles in an ethanolic solution. The synthesized fluorinated silica nanoparticles have been spin coated on flat aluminum alloy, silicon and glass substrates. Functionalization of silica nanoparticles with fluorinated groups has been confirmed by Fourier Transform Infrared spectroscopy (FTIR by showing the presence of C-F and Si-O-Si bonds. The water contact angles and surface roughness increase with the number of spin-coated thin films layers. The critical size of ~119 nm renders aluminum surface superhydrophobic with three layers of coating using as-prepared nanoparticle suspended solution. On the other hand, seven layers are required for a 50 vol.% diluted solution to achieve superhydrophobicity. In both the cases, water contact angles were more than 150°, contact angle hysteresis was less than 2° having a critical roughness value of ~0.700 µm. The fluorinated silica nanoparticle coated surfaces are also transparent and can be used as paint additives to obtain transparent coatings.

  2. PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

    Science.gov (United States)

    Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

    2001-09-18

    It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

  3. Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH.

    Science.gov (United States)

    Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

    2015-04-24

    The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Coatings to prevent frost

    DEFF Research Database (Denmark)

    Lusada, Ricardo; Holberg, Stefan; Bennedsen, Jeanette Marianne Dalgaard

    2016-01-01

    The ability of hydrophobic, organic–inorganic hybrid coatings to decelerate frost propagation was investigated. Compared to a bare aluminum surface, the coatings do not significantly reduce the freezing probability of supercooled water drops. On both surfaces, the probability for ice nucleation...... at temperatures just below 0°C, for example at −4°C, is low. Freezing of a single drop on aluminum leads, however, to instant freezing of the complete surface. On hydrophobic coatings, such a freezing drop is isolated; the frozen area grows slowly. At −4°C surface temperature in a +12°C/90% relative humidity...

  5. Thermally joining and/or coating or thermally separating the workpieces having heat-sensitive coating, comprises restoring coating by thermally coating the coating material after thermally joining and/or coating or thermally separating

    OpenAIRE

    Riedel, Frank; Winkelmann, Ralf; Puschmann, Markus

    2011-01-01

    The method for thermally joining and/or coating or thermally separating the workpieces (1), which have a heat-sensitive coating (2), comprises restoring the coating by thermally coating a coating material (3) after thermally joining and/or coating or thermally separating the workpieces. A part of the thermal energy introduced in the workpiece for joining and/or coating or separating or in the workpieces is used for thermally coating the coating material. Two workpieces are welded or soldered ...

  6. POWDER COAT APPLICATIONS

    Science.gov (United States)

    The report discusses an investigation of critical factors that affect the use of powder coatings on the environment, cost, quality, and production. The investigation involved a small business representative working with the National Defense Center for Environmental Excellence (ND...

  7. Inorganic Coatings Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — The inorganic Coatings Lab provides expertise to Navy and Joint Service platforms acquisition IPTs to aid in materials and processing choices which balance up-front...

  8. Robust Fiber Coatings

    National Research Council Canada - National Science Library

    Goettler, Richard

    2002-01-01

    The highly desired ceramic matrix composite is the one in which the high strength and strain-to-failure is achieved through judicious selection of a fiber coating that can survive the high-temperature...

  9. Coating of substrates

    International Nuclear Information System (INIS)

    Cairns, J.A.; Nelson, R.L.; Woodhead, J.L.

    1979-01-01

    The process is concerned with providing substrates with coatings obtainable from sols, for example to protect the substrate (such as in nuclear reactors or hydrocarbon cracking plant) or to provide a carrier for catalytically active material. Hitherto, coatings obtained from sols have had a high porosity and high surface area so that they have not been entirely satisfactory for the above applications. In the process described, dense, low-porosity coatings are provided by contacting the substrate with a sol of refractory material (e.g. CeO 2 or SiO 2 ) convertible to a gel of density at least 40% of the theoretical density of the refractory material, and converting the sol to the gel. Optionally, the gel may be converted to a ceramic coating by firing. (author)

  10. Friction surfaced Stellite6 coatings

    International Nuclear Information System (INIS)

    Rao, K. Prasad; Damodaram, R.; Rafi, H. Khalid; Ram, G.D. Janaki; Reddy, G. Madhusudhan; Nagalakshmi, R.

    2012-01-01

    Solid state Stellite6 coatings were deposited on steel substrate by friction surfacing and compared with Stellite6 cast rod and coatings deposited by gas tungsten arc and plasma transferred arc welding processes. Friction surfaced coatings exhibited finer and uniformly distributed carbides and were characterized by the absence of solidification structure and compositional homogeneity compared to cast rod, gas tungsten arc and plasma transferred coatings. Friction surfaced coating showed relatively higher hardness. X-ray diffraction of samples showed only face centered cubic Co peaks while cold worked coating showed hexagonally close packed Co also. - Highlights: ► Stellite6 used as coating material for friction surfacing. ► Friction surfaced (FS) coatings compared with casting, GTA and PTA processes. ► Finer and uniformly distributed carbides in friction surfaced coatings. ► Absence of melting results compositional homogeneity in FS Stellite6 coatings.

  11. Radiation hardening coating material

    International Nuclear Information System (INIS)

    McDonald, W.H.; Prucnal, P.J.; DeMajistre, Robert.

    1977-01-01

    This invention concerns a radiation hardening coating material. First a resin is prepared by reaction of bisphenol diglycidylic ether with acrylic or methacrylic acids. Then the reactive solvent is prepared by reaction of acrylic or methacrylic acids with epichlorhydrine or epibromhydrine. Then a solution consisting of the resin dissolved in the reactive solvent is prepared. A substrate (wood, paper, polyesters, polyamines etc.) is coated with this composition and exposed to ionizing radiations (electron beams) or ultraviolet radiations [fr

  12. Hydroxyapatite coatings for biomedical applications

    CERN Document Server

    Zhang, Sam

    2013-01-01

    Hydroxyapatite coatings are of great importance in the biological and biomedical coatings fields, especially in the current era of nanotechnology and bioapplications. With a bonelike structure that promotes osseointegration, hydroxyapatite coating can be applied to otherwise bioinactive implants to make their surface bioactive, thus achieving faster healing and recovery. In addition to applications in orthopedic and dental implants, this coating can also be used in drug delivery. Hydroxyapatite Coatings for Biomedical Applications explores developments in the processing and property characteri

  13. Biocompatibility of Niobium Coatings

    Directory of Open Access Journals (Sweden)

    René Olivares-Navarrete

    2011-09-01

    Full Text Available Niobium coatings deposited by magnetron sputtering were evaluated as a possible surface modification for stainless steel (SS substrates in biomedical implants. The Nb coatings were deposited on 15 mm diameter stainless steel substrates having an average surface roughness of 2 mm. To evaluate the biocompatibility of the coatings three different in vitro tests, using human alveolar bone derived cells, were performed: cellular adhesion, proliferation and viability. Stainless steel substrates and tissue culture plastic were also studied, in order to give comparative information. No toxic response was observed for any of the surfaces, indicating that the Nb coatings act as a biocompatible, bioinert material. Cell morphology was also studied by immune-fluorescence and the results confirmed the healthy state of the cells on the Nb surface. X-ray diffraction analysis of the coating shows that the film is polycrystalline with a body centered cubic structure. The surface composition and corrosion resistance of both the substrate and the Nb coating were also studied by X-ray photoelectron spectroscopy and potentiodynamic tests. Water contact angle measurements showed that the Nb surface is more hydrophobic than the SS substrate.

  14. Coated particle waste form development

    International Nuclear Information System (INIS)

    Oma, K.H.; Buckwalter, C.Q.; Chick, L.A.

    1981-12-01

    Coated particle waste forms have been developed as part of the multibarrier concept at Pacific Northwest Laboratory under the Alternative Waste Forms Program for the Department of Energy. Primary efforts were to coat simulated nuclear waste glass marbles and ceramic pellets with low-temperature pyrolytic carbon (LT-PyC) coatings via the process of chemical vapor deposition (CVD). Fluidized bed (FB) coaters, screw agitated coaters (SAC), and rotating tube coaters were used. Coating temperatures were reduced by using catalysts and plasma activation. In general, the LT-PyC coatings did not provide the expected high leach resistance as previously measured for carbon alone. The coatings were friable and often spalled off the substrate. A totally different concept, thermal spray coating, was investigated at PNL as an alternative to CVD coating. Flame spray, wire gun, and plasma gun systems were evaluated using glass, ceramic, and metallic coating materials. Metal plasma spray coatings (Al, Sn, Zn, Pb) provided a two to three orders-of-magnitude increase in chemical durability. Because the aluminum coatings were porous, the superior leach resistance must be due to either a chemical interaction or to a pH buffer effect. Because they are complex, coated waste form processes rank low in process feasibility. Of all the possible coated particle processes, plasma sprayed marbles have the best rating. Carbon coating of pellets by CVD ranked ninth when compared with ten other processes. The plasma-spray-coated marble process ranked sixth out of eleven processes

  15. Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

    Directory of Open Access Journals (Sweden)

    Margaret Veselits

    Full Text Available Casitas B-lineage lymphoma-b (Cbl-b is a ubiquitin ligase (E3 that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9 into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

  16. Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

    Science.gov (United States)

    Verma, Jitender Kumar; Rastogi, Ruchir

    2017-01-01

    Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment. PMID:28650977

  17. Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

    Science.gov (United States)

    Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

    2011-01-01

    Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

  18. Involvement of Gβγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes.

    Science.gov (United States)

    Kajimoto, Taketoshi; Mohamed, Nesma Nabil Ibrahim; Badawy, Shaymaa Mohamed Mohamed; Matovelo, Shubi Ambwene; Hirase, Mitsuhiro; Nakamura, Shunsuke; Yoshida, Daisuke; Okada, Taro; Ijuin, Takeshi; Nakamura, Shun-Ichi

    2018-01-05

    Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gβγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gβγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles

    Directory of Open Access Journals (Sweden)

    Carlos A.M. Carvalho

    2017-04-01

    Full Text Available Mayaro virus (MAYV is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell.

  20. MiR-153 Regulates Amelogenesis by Targeting Endocytotic and Endosomal/lysosomal Pathways-Novel Insight into the Origins of Enamel Pathologies.

    Science.gov (United States)

    Yin, Kaifeng; Lin, Wenting; Guo, Jing; Sugiyama, Toshihiro; Snead, Malcolm L; Hacia, Joseph G; Paine, Michael L

    2017-03-13

    Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The involvement of epigenetic regulation in the pathogenesis of AI is yet to be clarified due to a lack of knowledge about amelogenesis. Our previous genome-wide microRNA and mRNA transcriptome analyses suggest a key role for miR-153 in endosome/lysosome-related pathways during amelogenesis. Here we show that miR-153 is significantly downregulated in maturation ameloblasts compared with secretory ameloblasts. Within ameloblast-like cells, upregulation of miR-153 results in the downregulation of its predicted targets including Cltc, Lamp1, Clcn4 and Slc4a4, and a number of miRNAs implicated in endocytotic pathways. Luciferase reporter assays confirmed the predicted interactions between miR-153 and the 3'-UTRs of Cltc, Lamp1 (in a prior study), Clcn4 and Slc4a4. In an enamel protein intake assay, enamel cells transfected with miR-153 show a decreased ability to endocytose enamel proteins. Finally, microinjection of miR-153 in the region of mouse first mandibular molar at postnatal day 8 (PN8) induced AI-like pathologies when the enamel development reached maturity (PN12). In conclusion, miR-153 regulates maturation-stage amelogenesis by targeting key genes involved in the endocytotic and endosomal/lysosomal pathways, and disruption of miR-153 expression is a potential candidate etiologic factor contributing to the occurrence of AI.

  1. Coatings for directional eutectics

    Science.gov (United States)

    Rairden, J. R.; Jackson, M. R.

    1976-01-01

    Coatings developed to provide oxidation protection for the directionally-solidified eutectic alloy NiTaC-B (4.4 weight percent Cr) were evaluated. Of seven Co-, Fe- and Ni-base coatings that were initially investigated, best resistance to cyclic oxidation was demonstrated by duplex coatings fabricated by depositing a layer of NiCrAl(Y) by vacuum evaporation from an electron beam source followed by deposition of an Al overlayer using the pack cementation process. It was found that addition of carbon to the coating alloy substantially eliminated the problem of fiber denudation in TaC-type eutectic alloys. Burner rig cycled NiTaC-B samples coated with Ni-20Cr-5Al-0.1C-0.1Y+Al and rupture-tested at 1100 deg C performed as well as or better than uncoated, vacuum cycled and air-tested NiTaC-13; however, a slight degradation with respect to uncoated material was noted in air-stress rupture tests at 870 deg C for both cycled and uncycled samples.

  2. METHOD OF PROTECTIVELY COATING URANIUM

    Science.gov (United States)

    Eubank, L.D.; Boller, E.R.

    1959-02-01

    A method is described for protectively coating uranium with zine comprising cleaning the U for coating by pickling in concentrated HNO/sub 3/, dipping the cleaned U into a bath of molten zinc between 430 to 600 C and containing less than 0 01% each of Fe and Pb, and withdrawing and cooling to solidify the coating. The zinccoated uranium may be given a; econd coating with another metal niore resistant to the corrosive influences particularly concerned. A coating of Pb containing small proportions of Ag or Sn, or Al containing small proportions of Si may be applied over the zinc coatings by dipping in molten baths of these metals.

  3. Zirconium nitride hard coatings

    International Nuclear Information System (INIS)

    Roman, Daiane; Amorim, Cintia Lugnani Gomes de; Soares, Gabriel Vieira; Figueroa, Carlos Alejandro; Baumvol, Israel Jacob Rabin; Basso, Rodrigo Leonardo de Oliveira

    2010-01-01

    Zirconium nitride (ZrN) nanometric films were deposited onto different substrates, in order to study the surface crystalline microstructure and also to investigate the electrochemical behavior to obtain a better composition that minimizes corrosion reactions. The coatings were produced by physical vapor deposition (PVD). The influence of the nitrogen partial pressure, deposition time and temperature over the surface properties was studied. Rutherford backscattering spectrometry (RBS), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), scanning electron microscopy (SEM) and corrosion experiments were performed to characterize the ZrN hard coatings. The ZrN films properties and microstructure changes according to the deposition parameters. The corrosion resistance increases with temperature used in the films deposition. Corrosion tests show that ZrN coating deposited by PVD onto titanium substrate can improve the corrosion resistance. (author)

  4. Nanophase hardfaced coatings

    Energy Technology Data Exchange (ETDEWEB)

    Reisgen, U.; Stein, L.; Balashov, B.; Geffers, C. [RWTH Aachen University (Germany). ISF - Welding and Joining Institute

    2009-08-15

    This paper demonstrates the possibility of producing iron or chromium-based nanophase hardfaced coatings by means of common arc welding methods (TIG, PTA). The appropriate composition of the alloys to be deposited allows to control the structural properties and thus also the coating properties of the weld metal. Specific variations of the alloying elements allow also the realisation of a nanostructured solidification of the carbides and borides with cooling rates that are common for arc surfacing processes. The hardfaced coatings, which had been thus produced, showed phase dimensions of approximately 100-300 nm. Based on the results it is established that the influence of the surfacing parameters and of the coating thickness and thus the influence of the heat control on the nanostructuring process is, compared with the influence of the alloy composition, of secondary importance. The generation of nanoscale structures in hardfaced coatings allows the improvement of mechanical properties, wear resistance and corrosion resistance. Potential applications for these types of hardfaced coatings lie, in particular, in the field of cutting tools that are exposed to corrosion and wear. (Abstract Copyright [2009], Wiley Periodicals, Inc.) [German] Diese Arbeit demonstriert die Moeglichkeit zur Herstellung Eisen- und Chrom-basierter nanophasiger Hartauftragschweissschichten mithilfe ueblicher Lichtbogenschweissverfahren (WIG-, Plasma-Pulver-Auftragschweissen - PPA). Eine geeignete Zusammensetzung der aufzutragenden Legierungen ermoeglicht es, die Gefuegeeigenschaften und damit die Schichteigenschaften des Schweissgutes zu kontrollieren. Gezielte Variationen der Legierungselemente erlauben die Realisierung einer nanostrukturierten Erstarrung der Karbide und Boride bei fuer Lichtbogen-Auftragschweissprozessen ueblichen Abkuehlgeschwindigkeiten. In den so erzeugten Hartschichten werden Phasengroessen von ca. 100-300 nm erreicht. Auf Basis der gewonnenen Ergebnisse kann

  5. Tribology and coatings

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-06-01

    The future use of fuel-efficient, low-emission, advanced transportation systems (for example, those using low-heat-rejection diesel engines or advanced gas turbines) presents new challenges to tribologists and materials scientists. High service temperatures, corrosive environments, and extreme contact pressures are among the concerns that make necessary new tribological designs, novel materials, and effective lubrication concepts. Argonne is working on methods to reduce friction, wear and corrosion, such as soft metal coatings on ceramics, layered compounds, diamond coatings, and hard surfaces.

  6. Active Packaging Coatings

    Directory of Open Access Journals (Sweden)

    Luis J. Bastarrachea

    2015-11-01

    Full Text Available Active food packaging involves the packaging of foods with materials that provide an enhanced functionality, such as antimicrobial, antioxidant or biocatalytic functions. This can be achieved through the incorporation of active compounds into the matrix of the commonly used packaging materials, or by the application of coatings with the corresponding functionality through surface modification. The latter option offers the advantage of preserving the packaging materials’ bulk properties nearly intact. Herein, different coating technologies like embedding for controlled release, immobilization, layer-by-layer deposition, and photografting are explained and their potential application for active food packaging is explored and discussed.

  7. Mechanically Invisible Polymer Coatings

    DEFF Research Database (Denmark)

    2014-01-01

    phase comprises particles, said particles comprising a filler material and an encapsulating coating of a second polymeric material, wherein the backbones of the first and second polymeric materials are the same. The composition may be used in electroactive polymers (EAPs) in order to obtain mechanically......The present invention relates to a composition comprising encapsulated particles in a polymeric material. The composition comprises a continuous phase and a discontinuous phase incorporated therein, wherein the continuous phase comprises a first polymeric material and wherein the discontinuous...... invisible polymer coatings....

  8. Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin by photochemical internalization - A minimally invasive cancer stem cell-targeting strategy.

    Science.gov (United States)

    Bostad, Monica; Olsen, Cathrine Elisabeth; Peng, Qian; Berg, Kristian; Høgset, Anders; Selbo, Pål Kristian

    2015-05-28

    The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin (PCIAC133-saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133-saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133-saporin. PCI of picomolar levels of AC133-saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133-saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133-saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133-saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133-saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133-saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133+ cells in localized primary tumors or metastasis. Copyright © 2015

  9. Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects.

    Science.gov (United States)

    Stein, Karina; Brand, Stephanie; Jenckel, André; Sigmund, Anna; Chen, Zhijian James; Kirschning, Carsten J; Kauth, Marion; Heine, Holger

    2017-02-01

    Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4 + T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. L lactis G121-treated murine BMDCs and human moDCs released T H 1-polarizing cytokines and induced T H 1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of T H 1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13 -/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The T H 1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8. Copyright © 2016 American Academy of Allergy, Asthma & Immunology

  10. Methods and means for coating paper by film coating

    NARCIS (Netherlands)

    van der Maarel, Marc; Ter Veer, Arend Berend Cornelis; Vrieling-Smit, Annet; Delnoye, Pierre

    2015-01-01

    This invention relates to the field of paper coating, more in particular to means and methods for providing paper with at least one layer of pigment using film coating to obtain a well printable surface. Provided is a method for preparing coated paper comprising the steps of: a) providing a

  11. AntiReflection Coating D

    International Nuclear Information System (INIS)

    AIKEN, DANIEL J.

    1999-01-01

    Analytical expressions used to optimize AR coatings for single junction solar cells are extended for use in monolithic, series interconnected multi-junction solar cell AR coating design. The result is an analytical expression which relates the solar cell performance (through J(sub sc)) directly to the AR coating design through the device reflectance. It is also illustrated how AR coating design be used to provide an additional degree of freedom for current matching multi-junction devices

  12. UV Coatings, Polarization, and Coronagraphy

    Science.gov (United States)

    Bolcar, Matthew R.; Quijada, Manuel; West, Garrett; Balasubramanian, Bala; Krist, John; Martin, Stefan; Sabatke, Derek

    2016-01-01

    Presenation for the Large UltraViolet Optical Infrared (LUVOIR) and Habitable Exoplanet Imager (HabEx) Science and Technology Definition Teams (STDT) on technical considerations regarding ultraviolet coatings, polarization, and coronagraphy. The presentations review the state-of-the-art in ultraviolet coatings, how those coatings generate polarization aberrations, and recent study results from both the LUVOIR and HabEx teams.

  13. Coatings for transport industry

    Directory of Open Access Journals (Sweden)

    Krzysztof LUKASZKOWICZ

    2014-09-01

    Full Text Available The investigations concerned structural analysis, as well as mechanical properties and wear resistant of MeN/DLC double-layer coating deposited by hybrid PVD/PACVD method. In sliding dry friction conditions, after the break-in time, the friction coefficient for the investigated elements is set in the range between 0.03-0.06.

  14. Ion Deposited Carbon Coatings.

    Science.gov (United States)

    1983-07-01

    PAGE ("’hen Dita t,,I,, efl TABLE OF CONTENTS Section No. Title Page No. 1.0 OBJECTIVE 1 2.0 SCOPE 2 3.0 BACKGROUND 3 4.0 COATINGS DEPOSITION 4 4.1...scientific, ards of measure. The Committee, and Confer- technical, practical, and teaching purposes.ence voting members, are leading professional On the

  15. Polydopamine-coated capsules

    Science.gov (United States)

    White, Scott R.; Sottos, Nancy R.; Kang, Sen; Baginska, Marta B.

    2018-04-17

    One aspect of the invention is a polymer material comprising a capsule coated with PDA. In certain embodiments, the capsule encapsulates a functional agent. The encapsulated functional agent may be an indicating agent, healing agent, protecting agent, pharmaceutical drug, food additive, or a combination thereof.

  16. Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport across Phospholipid Membranes Induced by a Proton Gradient

    Directory of Open Access Journals (Sweden)

    Fatemeh Madani

    2011-01-01

    Full Text Available Detergent-mediated reconstitution of bacteriorhodopsin (BR into large unilamellar vesicles (LUVs was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

  17. An Azole-Tolerant Endosomal Trafficking Mutant of Candida albicans Is Susceptible to Azole Treatment in a Mouse Model of Vaginal Candidiasis.

    Science.gov (United States)

    Peters, Brian M; Luna-Tapia, Arturo; Tournu, Hélène; Rybak, Jeffrey M; Rogers, P David; Palmer, Glen E

    2017-06-01

    We recently reported that a Candida albicans endosomal trafficking mutant continues to grow after treatment with the azole antifungals. Herein, we report that the vps21 Δ/Δ mutant does not have a survival advantage over wild-type isolates after fluconazole treatment in a mouse model of vaginal candidiasis. Furthermore, loss of VPS21 does not synergize with established mechanisms of azole resistance, such as overexpression of efflux pumps or of Erg11p, the target enzyme of the azoles. In summary, although loss of VPS21 function enhances C. albicans survival after azole treatment in vitro , it does not seem to affect azole susceptibility in vivo . Copyright © 2017 American Society for Microbiology.

  18. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

    International Nuclear Information System (INIS)

    Kosheverova, Vera V.; Kamentseva, Rimma S.; Gonchar, Ilya V.; Kharchenko, Marianna V.; Kornilova, Elena S.

    2016-01-01

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

  19. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kosheverova, Vera V., E-mail: kosheverova_vera@incras.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kamentseva, Rimma S., E-mail: rkamentseva@yandex.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Gonchar, Ilya V., E-mail: ample@mail.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kharchenko, Marianna V., E-mail: mariannakharchenko@gmail.com [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kornilova, Elena S., E-mail: lenkor@mail.cytspb.rssi.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, 29, Polytechnicheskaya, St.Petersburg, 195251 (Russian Federation)

    2016-04-22

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

  20. Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

    Science.gov (United States)

    Connor, Michael G.; Pulsifer, Amanda R.; Ceresa, Brian K.

    2018-01-01

    ABSTRACT Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV. PMID:29463656

  1. MiR-153 Regulates Amelogenesis by Targeting Endocytotic and Endosomal/lysosomal Pathways–Novel Insight into the Origins of Enamel Pathologies

    Science.gov (United States)

    Yin, Kaifeng; Lin, Wenting; Guo, Jing; Sugiyama, Toshihiro; Snead, Malcolm L.; Hacia, Joseph G.; Paine, Michael L.

    2017-01-01

    Amelogenesis imperfecta (AI) is group of inherited disorders resulting in enamel pathologies. The involvement of epigenetic regulation in the pathogenesis of AI is yet to be clarified due to a lack of knowledge about amelogenesis. Our previous genome-wide microRNA and mRNA transcriptome analyses suggest a key role for miR-153 in endosome/lysosome-related pathways during amelogenesis. Here we show that miR-153 is significantly downregulated in maturation ameloblasts compared with secretory ameloblasts. Within ameloblast-like cells, upregulation of miR-153 results in the downregulation of its predicted targets including Cltc, Lamp1, Clcn4 and Slc4a4, and a number of miRNAs implicated in endocytotic pathways. Luciferase reporter assays confirmed the predicted interactions between miR-153 and the 3′-UTRs of Cltc, Lamp1 (in a prior study), Clcn4 and Slc4a4. In an enamel protein intake assay, enamel cells transfected with miR-153 show a decreased ability to endocytose enamel proteins. Finally, microinjection of miR-153 in the region of mouse first mandibular molar at postnatal day 8 (PN8) induced AI-like pathologies when the enamel development reached maturity (PN12). In conclusion, miR-153 regulates maturation-stage amelogenesis by targeting key genes involved in the endocytotic and endosomal/lysosomal pathways, and disruption of miR-153 expression is a potential candidate etiologic factor contributing to the occurrence of AI. PMID:28287144

  2. Active coatings technologies for tailorable military coating systems

    Science.gov (United States)

    Zunino, J. L., III

    2007-04-01

    The main objective of the U.S. Army's Active Coatings Technologies Program is to develop technologies that can be used in combination to tailor coatings for utilization on Army Materiel. The Active Coatings Technologies Program, ACT, is divided into several thrusts, including the Smart Coatings Materiel Program, Munitions Coatings Technologies, Active Sensor packages, Systems Health Monitoring, Novel Technology Development, as well as other advanced technologies. The goal of the ACT Program is to conduct research leading to the development of multiple coatings systems for use on various military platforms, incorporating unique properties such as self repair, selective removal, corrosion resistance, sensing, ability to modify coatings' physical properties, colorizing, and alerting logistics staff when tanks or weaponry require more extensive repair. A partnership between the U.S. Army Corrosion Office at Picatinny Arsenal, NJ along with researchers at the New Jersey Institute of Technology, NJ, Clemson University, SC, University of New Hampshire, NH, and University of Massachusetts (Lowell), MA, are developing the next generation of Smart Coatings Materiel via novel technologies such as nanotechnology, Micro-electromechanical Systems (MEMS), meta-materials, flexible electronics, electrochromics, electroluminescence, etc. This paper will provide the reader with an overview of the Active Coatings Technologies Program, including an update of the on-going Smart Coatings Materiel Program, its progress thus far, description of the prototype Smart Coatings Systems and research tasks as well as future nanotechnology concepts, and applications for the Department of Defense.

  3. New temperable solar coatings: Tempsol

    Science.gov (United States)

    Demiryont, Hulya

    2001-11-01

    This paper deals with the large area deposition and coating properties of the thermo-stable (temperable/bendable) solar coating material, CuO, and some new optical coating systems comprising CuO films for architectural and automotive/transportation applications. The CuO solar coating is combined with other coating layers, for example, an anti-reflection film, a reflection film, a coloration coating layer, etc., which are also thermo-stable. The film systems are developed at the research laboratory by D.C. Magnetron reactive sputtering process. The new developed technologies then transferred to the production line. Product performances are compared before and after heat treatment of the coating systems. Performance tables and other physical properties, including optical parameters, mechanical and environmental stability, storage properties, etc., are also presented for this new product series.

  4. Flow accelerated organic coating degradation

    Science.gov (United States)

    Zhou, Qixin

    Applying organic coatings is a common and the most cost effective way to protect metallic objects and structures from corrosion. Water entry into coating-metal interface is usually the main cause for the deterioration of organic coatings, which leads to coating delamination and underfilm corrosion. Recently, flowing fluids over sample surface have received attention due to their capability to accelerate material degradation. A plethora of works has focused on the flow induced metal corrosion, while few studies have investigated the flow accelerated organic coating degradation. Flowing fluids above coating surface affect corrosion by enhancing the water transport and abrading the surface due to fluid shear. Hence, it is of great importance to understand the influence of flowing fluids on the degradation of corrosion protective organic coatings. In this study, a pigmented marine coating and several clear coatings were exposed to the laminar flow and stationary immersion. The laminar flow was pressure driven and confined in a flow channel. A 3.5 wt% sodium chloride solution and pure water was employed as the working fluid with a variety of flow rates. The corrosion protective properties of organic coatings were monitored inline by Electrochemical Impedance Spectroscopy (EIS) measurement. Equivalent circuit models were employed to interpret the EIS spectra. The time evolution of coating resistance and capacitance obtained from the model was studied to demonstrate the coating degradation. Thickness, gloss, and other topography characterizations were conducted to facilitate the assessment of the corrosion. The working fluids were characterized by Fourier Transform Infrared Spectrometer (FTIR) and conductivity measurement. The influence of flow rate, fluid shear, fluid composition, and other effects in the coating degradation were investigated. We conclude that flowing fluid on the coating surface accelerates the transport of water, oxygen, and ions into the coating, as

  5. Coating of metals

    International Nuclear Information System (INIS)

    Smith, F.

    1978-01-01

    A method is described for coating the surface of an article of Ti, Zr or Ta, or an alloy thereof, with a tinning metal or alloy, the article having a shape other than that of a sheet. The method comprises contacting the surface of the article at an elevated temperature with the molten tinning metal and moving an ultrasonically excited probe over the surface to be coated, the probe being in contact with the surface of the article and with the tinning metal. The tinning metal may be Sn or Zn or a binary alloy of Sn with Zn, Cd or Bi at a temperature of 300 0 to 450 0 C. The head of the probe may be shaped to conform with the surface of the article. The method may be used to form composite articles, and may be applied to a pre-tinned steel article. (U.K.)

  6. Coat of Arms.

    Science.gov (United States)

    Smith, Bryan

    1998-01-01

    Describes an activity, the "coat of arms," that can serve as an ice-breaker or warm-up for the first day of an English-as-a-Second/Foreign-Language class, as a motivating start to the week, or act as an innovative segue between skill lessons. The technique can be adapted for students ranging from elementary school to adult language learners of all…

  7. Scientific coats of arms.

    Science.gov (United States)

    Fara, Patricia

    2005-09-01

    With their mythical creatures and arcane symbolism, coats of arms seem to have little connection with modern science. Yet despite its chivalric origins, the ancient language of heraldry has long fascinated famous scientists. Although this idiosyncratic tradition was parodied by Victorian geologists, who laughingly replaced unicorns and griffins with images of dinosaurs that they had recently discovered, it has been perpetuated since by Ernest Rutherford, who liked to present himself as a new alchemist.

  8. Anti-Corrosion Coating

    Science.gov (United States)

    1986-01-01

    SuperSpan RM 8000 is an anti-corrosion coating which effectively counteracts acid degradation, abrasive wear, and cracking in power industry facilities. It was developed by RM Industrial Products Company, Inc. with NERAC assistance. It had previously been necessary to shut down plants to repair or replace corroded duct-work in coal burning utilities. NASA-developed technology was especially useful in areas relating to thermoconductivity of carbon steel and the bonding characteristics of polymers. The product has sold well.

  9. for zeolite coating

    Directory of Open Access Journals (Sweden)

    Carlos Renato Rambo

    2006-01-01

    Full Text Available Biotemplating is the processing of microcellular ceramics by reproduction of natural morphologies, where the microstructural features of the biotemplate are maintained in the biomorphic ceramic. Different biotemplates with distinct pore anatomies were used to produce biomorphic supports for the zeolite coating: wood, cardboard, sea-sponge and sisal. The biomorphic ceramics were produced by distinguished processing routes: Al-gas infiltration-reaction, liquid-metal infiltration, dip-coating and sol-gel synthesis, in order to produce nitrides, carbides and oxides, depending on the processing conditions. The zeolite coating was performed by hydrothermal growth of MFI-type (Silicalite-1 and ZSM-5 zeolite crystals onto the internal pore walls of the biomorphic templates. The final biomorphic ceramic-zeolite composites were physically characterized, evaluated in terms of their gas adsorption capabilities and correlated to their microstructure and specific pore anatomy. The combination of the properties of the biomorphic ceramics with the adsorption properties of zeolites results in materials with distinct properties as potential candidates for adsorption and catalytic applications due to their characteristic porosity, molecular sieving capabilities and high thermo-mechanical strength.

  10. Coating and curing apparatus and methods

    Science.gov (United States)

    Brophy, Brenor L; Maghsoodi, Sina; Neyman, Patrick J; Gonsalves, Peter R; Hirsch, Jeffrey G; Yang, Yu S

    2015-02-24

    Disclosed are coating apparatus including flow coating and roll-coating that may be used for uniform sol-gel coating of substrates such as glass, solar panels, windows or part of an electronic display. Also disclosed are methods for substrate preparation, flow coating and roll coating. Lastly systems and methods for skin curing sol-gel coatings deposited onto the surface of glass substrates using a high temperature air-knife are disclosed.

  11. Lotus Dust Mitigation Coating and Molecular Adsorber Coating

    Science.gov (United States)

    O'Connor, Kenneth M.; Abraham, Nithin S.

    2015-01-01

    NASA Goddard Space Flight Center has developed two unique coating formulations that will keep surfaces clean and sanitary and contain contaminants.The Lotus Dust Mitigation Coating, modeled after the self-cleaning, water-repellant lotus leaf, disallows buildup of dust, dirt, water, and more on surfaces. This coating, has been successfully tested on painted, aluminum, glass, silica, and some composite surfaces, could aid in keeping medical assets clean.The Molecular Adsorber Coating is a zeolite-based, sprayable molecular adsorber coating, designed to prevent outgassing in materials in vacuums. The coating works well to adsorb volatiles and contaminates in manufacturing and processing, such as in pharmaceutical production. The addition of a biocide would also aid in controlling bacteria levels.

  12. Composition superconductive plumbous coatings

    International Nuclear Information System (INIS)

    Volodin, V.N.; Tuleushev, A.Zh.; Tuleushev, Yu.Zh.; Lisizin, V.N.

    2002-01-01

    Independent dispersion of two or more targets, precipitation of pulverized material on substrate and possibility of composition change in wide range of component concentrations made possible ion-plasma forming of film composition materials from materials with different chemical and physical qualities, particularly in lead-aluminum, lead-beryllium and lead-graphite systems. Named systems are characterized in wide sphere of immiscibility in solid and liquid state and absence of intermediate compounds. It is impossible to receive materials from them in traditional method in conditions of gravitational field. In lead-aluminum system there was received a number of film coatings with aluminum content up to 95 at. % at coating thickness up to 2 μm. Owing to X-ray investigations it is fixed that lead and aluminum have been performed by separate phases. Lead in sprayed layer represents well-crystallized phase with grain size more than 100 nm; texturing is not found. Study of physical qualities has shown that materials with lead base 21.6 at. % Al) have enough high crystalline current in comparison with compact lead, which reaches (2.5-3.0)·10 5 A)·cm 2 , while materials with aluminum base (21.6 at. % Al) loose this effect and critical temperature of transition is reduced from 7.1 to 5.8 K. It was impossible to carry out X-rayed analysis for lead-beryllium film because of weak intensity of beryllium lines against a background of lead owing to a quite large difference of atomic balance. Cryogen tests have shown the increase of critical current strength up to (3.1-3.6)·10 4 A)·cm 2 or composition coating of lead-beryllium (56.99 at. % or 5,45 mas. % Be), at that the critical temperature of transition does not differ from lead temperature. Samples of lead edge of state diagram have been received in the lead-graphite system. X-ray investigation subjected coating contained 6.81 at. % (55.82 mas. %) of lead. Choice of the composition is conditioned on possibilities of

  13. Nanoparticle/Polymer Nanocomposite Bond Coat or Coating

    Science.gov (United States)

    Miller, Sandi G.

    2011-01-01

    This innovation addresses the problem of coatings (meant to reduce gas permeation) applied to polymer matrix composites spalling off in service due to incompatibility with the polymer matrix. A bond coat/coating has been created that uses chemically functionalized nanoparticles (either clay or graphene) to create a barrier film that bonds well to the matrix resin, and provides an outstanding barrier to gas permeation. There is interest in applying clay nanoparticles as a coating/bond coat to a polymer matrix composite. Often, nanoclays are chemically functionalized with an organic compound intended to facilitate dispersion of the clay in a matrix. That organic modifier generally degrades at the processing temperature of many high-temperature polymers, rendering the clay useless as a nano-additive to high-temperature polymers. However, this innovation includes the use of organic compounds compatible with hightemperature polymer matrix, and is suitable for nanoclay functionalization, the preparation of that clay into a coating/bondcoat for high-temperature polymers, the use of the clay as a coating for composites that do not have a hightemperature requirement, and a comparable approach to the preparation of graphene coatings/bond coats for polymer matrix composites.

  14. Modeling of Thermal Barrier Coatings

    Science.gov (United States)

    Ferguson, B. L.; Petrus, G. J.; Krauss, T. M.

    1992-01-01

    The project examined the effectiveness of studying the creep behavior of thermal barrier coating system through the use of a general purpose, large strain finite element program, NIKE2D. Constitutive models implemented in this code were applied to simulate thermal-elastic and creep behavior. Four separate ceramic-bond coat interface geometries were examined in combination with a variety of constitutive models and material properties. The reason for focusing attention on the ceramic-bond coat interface is that prior studies have shown that cracking occurs in the ceramic near interface features which act as stress concentration points. The model conditions examined include: (1) two bond coat coefficient of thermal expansion curves; (2) the creep coefficient and creep exponent of the bond coat for steady state creep; (3) the interface geometry; and (4) the material model employed to represent the bond coat, ceramic, and superalloy base.

  15. Tablet coating by injection molding technology - Optimization of coating formulation attributes and coating process parameters.

    Science.gov (United States)

    Desai, Parind M; Puri, Vibha; Brancazio, David; Halkude, Bhakti S; Hartman, Jeremy E; Wahane, Aniket V; Martinez, Alexander R; Jensen, Keith D; Harinath, Eranda; Braatz, Richard D; Chun, Jung-Hoon; Trout, Bernhardt L

    2018-01-01

    We developed and evaluated a solvent-free injection molding (IM) coating technology that could be suitable for continuous manufacturing via incorporation with IM tableting. Coating formulations (coating polymers and plasticizers) were prepared using hot-melt extrusion and screened via stress-strain analysis employing a universal testing machine. Selected coating formulations were studied for their melt flow characteristics. Tablets were coated using a vertical injection molding unit. Process parameters like softening temperature, injection pressure, and cooling temperature played a very important role in IM coating processing. IM coating employing polyethylene oxide (PEO) based formulations required sufficient room humidity (>30% RH) to avoid immediate cracks, whereas other formulations were insensitive to the room humidity. Tested formulations based on Eudrajit E PO and Kollicoat IR had unsuitable mechanical properties. Three coating formulations based on hydroxypropyl pea starch, PEO 1,000,000 and Opadry had favorable mechanical (35% elongation, >95×10 4 J/m 3 toughness) and melt flow (>0.4g/min) characteristics, that rendered acceptable IM coats. These three formulations increased the dissolution time by 10, 15 and 35min, respectively (75% drug release), compared to the uncoated tablets (15min). Coated tablets stored in several environmental conditions remained stable to cracking for the evaluated 8-week time period. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Article Including Environmental Barrier Coating System

    Science.gov (United States)

    Lee, Kang N. (Inventor)

    2015-01-01

    An enhanced environmental barrier coating for a silicon containing substrate. The enhanced barrier coating may include a bond coat doped with at least one of an alkali metal oxide and an alkali earth metal oxide. The enhanced barrier coating may include a composite mullite bond coat including BSAS and another distinct second phase oxide applied over said surface.

  17. Coatings for fusion reactor environments

    International Nuclear Information System (INIS)

    Mattox, D.M.

    1979-01-01

    The internal surfaces of a tokamak fusion reactor control the impurity injection and gas recycling into the fusion plasma. Coating of internal surfaces may provide a desirable and possibly necessary design flexibility for achieving the temperatures, ion densities and containment times necessary for net energy production from fusion reactions to take place. In this paper the reactor environments seen by various componentare reviewed along with possible materials responses. Characteristics of coating-substrate systems, important to fusion applications, are delineated and the present status of coating development for fusion applications is reviewed. Coating development for fusion applications is just beginning and poses a unique and important challenge for materials development

  18. Properties of radiation cured coatings

    International Nuclear Information System (INIS)

    Larson, E.G.; Spencer, D.S.; Boettcher, T.E.; Melbauer, M.A.; Skarjune, R.P.

    1987-01-01

    Coatings were prepared from acrylate or methacrylate functionalized resins to study the effect of end group functionality on the physical properties of u.v. and electron beam cured coatings. Cure response was measured by solid state NMR and gel extraction, as expected, methacrylate resins cured much slower. Thermal Gravimetric Analysis (TGA) revealed acrylate coatings have greater thermal stability. Properties such as tensile strength and hardness showed little effect of end group functionality or curing method. The O 2 and H 2 O permeabilities of the coating were correlated with the processing conditions. (author)

  19. Decontamination and coating of lead

    International Nuclear Information System (INIS)

    Rankin, W.N.; Bush, S.P.; Lyon, C.E.; Walker, V.

    1988-01-01

    Technology is being developed to decontaminate lead used in shielding applications in contaminated environments for recycle as shieldings. Technology is also being developed to coat either decontaminated lead or new lead before it is used in contaminated environments. The surface of the coating is expected to be much easier to decontaminate than the original lead surface. If contamination becomes severely embedded in the coating and cannot be removed, it can be easily cut with a knife and removed from the lead. The used coating can be disposed of as radioactive (hot hazardous) waste. The lead can then be recoated for further use as a shielding material

  20. Coatings Technology Integration Office (CTIO)

    Data.gov (United States)

    Federal Laboratory Consortium — CTIO serves as the Air Force's central resource for aircraft coating systems and their applications. CTIO's primary objectives are pollution prevention and improved...

  1. Coating material composition

    International Nuclear Information System (INIS)

    Kimura, Tadashi; Ozeki, Takao; Kobayashi, Juichi; Nakamoto, Hideo; Maeda, Yutaka.

    1969-01-01

    A coating material composition is provided which can easily be cross-linked by irradiation with active energy, particularly electron beams and ultraviolet light, using a mixture of a prepolymer (a) with an addition reaction product (b). Such compositions have coating properties as good as thermosetting acrylic or amino alkyd resins. The prepolymer (a) is produced by primarily reacting at least 0.1 mol of saturated cyclocarboxylic acid anhydrides and/or alpha-, beta-ethylene unsaturated carboxylic acid anhydrides by addition reaction with one mol of hydroxyl radicals of a basic polymer having a molecular weight of 1,000 to 100,000, the basic polymer being obtained from 1%-40% of a hydroxyl radical containing vinyl monomer and at least 30% of (meth)acrylate monomer. One mol of the sum of hydroxyl radicals and carboxyl radicals of the primary reaction product undergoes a secondary addition reaction with at least 0.1 mol of an epoxy radical-containing vinyl monomer to form the prepolymer(a). The addition reaction product(b) is produced by reacting an epoxy radical-containing vinyl monomer with alpha-, beta-ethylene unsaturated carboxylic acids or their anhydrides. The coating material composition contains a majority of a mixture consisting of 10%-90% of (a) and 90%-10% of (b) above by weight. Four examples of the production of basic polymers, seven examples of the production of prepolymers, seven examples of the production of oligomers, and five examples of applications are given. (Iwakiri, K.)

  2. Microplasma sprayed hydroxyapatite coatings

    CERN Document Server

    Dey, Arjun

    2015-01-01

    ""This unique book on development of microplasma sprayed HAp coating has been organized in a very compact yet comprehensive manner. This book also highlights the horizons of future research that invites the attention of global community, particularly those in bio-medical materials and bio-medical engineering field. This book will surely act as a very useful reference material for both graduate/post-graduate students and researchers in the field of biomedical, orthopedic and manufacturing engineering and research. I truly believ that this is the first ever effort which covers almost all the

  3. SPS: scrubbing or coating ?

    CERN Document Server

    Jimenez, J M

    2012-01-01

    The operation of the SPS with high intensity bunched beams is limited by the electron cloud building-up in both the arcs and long straight sections. Two consolidation options have been considered: mitigation of the electron cloud using coatings or relying, as before, on the scrubbing runs. A status report on both options will be given with a particular emphasis on measurements plans for 2012 and pending issues. The testing needs, corresponding beam parameters and MD time in 2012 will be addressed. The criteria for the decision making and the corresponding schedule will be discussed.

  4. SPS: scrubbing or coating?

    International Nuclear Information System (INIS)

    Jimenez, J.M.

    2012-01-01

    The operation of the SPS with high intensity bunched beams is limited by the electron cloud building-up in both the arcs and long straight sections. Two consolidation options have been considered: suppression of the electron cloud build-up using coatings or relying, as before, on the scrubbing mitigation. A status report on both options will be given with a particular emphasis on measurements plans for 2012 and pending issues. The testing needs, corresponding beam parameters and MD time in 2012 will be addressed. The criteria for the decision making and the corresponding schedule will be discussed. (author)

  5. Radiation hardenable coating mixture

    International Nuclear Information System (INIS)

    Howard, D.D.

    1977-01-01

    This invention relates to coatings that harden under radiation and to their compositions. Specifically, this invention concerns unsaturated urethane resins polymerisable by addition and to compositions, hardening under the effect of radiation, containing these resins. These resins feature the presence of at least one unsaturated ethylenic terminal group of structure CH 2 =C and containing the product of the reaction of an organic isocyanate compound with at least two isocyanate groups and one polyester polyol with at least two hydroxyl groups, and one unsaturated monomer compound polymerisable by addition having a single active hydrogen group reacting with the isocyanate [fr

  6. Andrographolide inhibits TNFα-induced ICAM-1 expression via suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 pathways in human endothelial cells.

    Science.gov (United States)

    Lu, Chia-Yang; Yang, Ya-Chen; Li, Chien-Chun; Liu, Kai-Li; Lii, Chong-Kuei; Chen, Haw-Wen

    2014-09-01

    Andrographolide, the major bioactive component of Andrographis paniculata, has been demonstrated to have various biological properties including anti-inflammation, antioxidation, and anti-hepatotoxicity. Oxidative stress is considered a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is a major source of endogenous reactive oxygen species (ROS). In this study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. Andrographolide attenuated TNFα-induced ROS generation, Src phosphorylation, membrane translocation of the NADPH oxidase subunits p47(phox) and p67(phox), and ICAM-1 gene expression. In the small hairpin RNA interference assay, shp47(phox) abolished TNFα-induced p65 nuclear translocation, ICAM-1 gene expression, and adhesion of HL-60 cells. Andrographolide induced the gene expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) in a time-dependent manner. Cellular glutathione (GSH) content was increased by andrographolide. shGCLM attenuated the andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed a similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. The mechanism underlying the up-regulation of HO-1 and GCLM by andrographolide was dependent on the PI3K/Akt pathway, and both the Nrf2 and AP-1 transcriptional factors were involved. Our results suggest that andrographolide attenuates TNFα-induced ICAM-1 expression at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression, which is PI3K/Akt pathway-dependent. Copyright © 2014. Published by Elsevier Inc.

  7. Federal Highway Administration 100-year coating study.

    Science.gov (United States)

    2012-11-01

    The Federal Highway Administration 100-Year Coating Study was initiated in August 2009 to search for durable : coating systems at a reasonable cost. The objective of the study was to identify and evaluate coating materials that can : provide 100 year...

  8. Decoding white coat hypertension

    Science.gov (United States)

    Bloomfield, Dennis A; Park, Alex

    2017-01-01

    There is arguably no less understood or more intriguing problem in hypertension that the “white coat” condition, the standard concept of which is significantly blood pressure reading obtained by medical personnel of authoritative standing than that obtained by more junior and less authoritative personnel and by the patients themselves. Using hospital-initiated ambulatory blood pressure monitoring, the while effect manifests as initial and ending pressure elevations, and, in treated patients, a low daytime profile. The effect is essentially systolic. Pure diastolic white coat hypertension appears to be exceedingly rare. On the basis of the studies, we believe that the white coat phenomenon is a common, periodic, neuro-endocrine reflex conditioned by anticipation of having the blood pressure taken and the fear of what this measurement may indicate concerning future illness. It does not change with time, or with prolonged association with the physician, particularly with advancing years, it may be superimposed upon essential hypertension, and in patients receiving hypertensive medication, blunting of the nighttime dip, which occurs in about half the patients, may be a compensatory mechanisms, rather than an indication of cardiovascular risk. Rather than the blunted dip, the morning surge or the widened pulse pressure, cardiovascular risk appears to be related to elevation of the average night time pressure. PMID:28352632

  9. Ceramic protective coating

    International Nuclear Information System (INIS)

    Harbach, F.; Nicoll, A.

    1987-01-01

    The basic material of the above-mentioned layer consists of pure aluminium oxide or essentially aluminium oxide. To improve this protective layer metal oxides from the groups IIA, IIIA, IIIB, VB, VIB, VIIB or VIII of the periodic system are added to its basic material before the said protective coating is applied. In this way a corundum structure is formed in the case of aluminium oxide. Gallium oxide, vanadium oxide, chromium oxide or iron oxide are particularly suited for the correlation of such a corundum structure. The formation of the corundum structure increases the resistance of the protective coating to the corrosive effects of vanadium pentoxide and sodium sulfate. By the addition of a specific quantity of magnesium oxide it is possible not only to stimulate the formation of corundum but also to reduce the increase in grain size in the case of the aluminium oxide. The other metallic oxides are especially favorable to the formation of the corundum structure, so that preferably magnesium oxide is to be added to these metallic oxides in order to reduce the increase in grain size. (author)

  10. Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains*

    Science.gov (United States)

    Guo, Emily Z.; Xu, Zhaohui

    2015-01-01

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. PMID:25657007

  11. Distinct mechanisms of recognizing endosomal sorting complex required for transport III (ESCRT-III) protein IST1 by different microtubule interacting and trafficking (MIT) domains.

    Science.gov (United States)

    Guo, Emily Z; Xu, Zhaohui

    2015-03-27

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein

    Directory of Open Access Journals (Sweden)

    Israel López-Reyes

    2010-01-01

    Full Text Available Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT. Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

  13. The cellular endosomal sorting complex required for transport pathway is not involved in avian metapneumovirus budding in a virus-like-particle expression system.

    Science.gov (United States)

    Weng, Yuejin; Lu, Wuxun; Harmon, Aaron; Xiang, Xiaoxiao; Deng, Qiji; Song, Minxun; Wang, Dan; Yu, Qingzhong; Li, Feng

    2011-05-01

    Avian metapneumovirus (AMPV) is a paramyxovirus that principally causes respiratory disease and egg production drops in turkeys and chickens. Together with its closely related human metapneumovirus (HMPV), they comprise the genus Metapneumovirus in the family Paramyxoviridae. Little is currently known about the mechanisms involved in the budding of metapneumovirus. By using AMPV as a model system, we showed that the matrix (M) protein by itself was insufficient to form virus-like-particles (VLPs). The incorporation of M into VLPs was shown to occur only when both the viral nucleoprotein (N) and the fusion (F) proteins were co-expressed. Furthermore, we provided evidence indicating that two YSKL and YAGL segments encoded within the M protein were not a functional late domain, and the endosomal sorting complex required for transport (ESCRT) machinery was not involved in metapneumovirus budding, consistent with a recent observation that human respiratory syncytial virus, closely related to HMPV, uses an ESCRT-independent budding mechanism. Taken together, these results suggest that metapneumovirus budding is independent of the ESCRT pathway and the minimal budding machinery described here will aid our future understanding of metapneumovirus assembly and egress.

  14. Rapidly curable electrically conductive clear coatings

    Energy Technology Data Exchange (ETDEWEB)

    Bowman, Mark P.; Anderson, Lawrence G.; Post, Gordon L.

    2018-01-16

    Rapidly curable electrically conductive clear coatings are applied to substrates. The electrically conductive clear coating includes to clear layer having a resinous binder with ultrafine non-stoichiometric tungsten oxide particles dispersed therein. The clear coating may be rapidly cured by subjecting the coating to infrared radiation that heats the tungsten oxide particles and surrounding resinous binder. Localized heating increases the temperature of the coating to thereby thermally cure the coating, while avoiding unwanted heating of the underlying substrate.

  15. White coat hypertension in pediatrics.

    Science.gov (United States)

    Jurko, Alexander; Minarik, Milan; Jurko, Tomas; Tonhajzerova, Ingrid

    2016-01-15

    The article summarizes current information on blood pressure changes in children during clinic visit. White coat as a general dressing of physicians and health care personnel has been widely accepted at the end of the 19th century. Two problems can be associated with the use of white coat: white coat phenomenon and white coat hypertension. Children often attribute pain and other unpleasant experience to the white coat and refuse afterwards cooperation with examinations. Definition of white coat hypertension in the literature is not uniform. It has been defined as elevated blood pressure in the hospital or clinic with normal blood pressure at home measured during the day by ambulatory blood pressure monitoring system. White coat effect is defined as temporary increase in blood pressure before and during visit in the clinic, regardless what the average daily ambulatory blood pressure values are. Clinical importance of white coat hypertension is mainly because of higher risk for cardiovascular accidents that are dependent on end organ damage (heart, vessels, kidney). Current data do not allow any clear recommendations for the treatment. Pharmacological therapy is usually started in the presence of hypertrophic left ventricle, changes in intimal/medial wall thickness of carotic arteries, microalbuminuria and other cardiovascular risk factors. Nonpharmacological therapy is less controversial and certainly more appropriate. Patients have to change their life style, need to eliminate as much cardiovascular risk factors as possible and sustain a regular blood pressure monitoring.

  16. External coating of colonic anastomoses

    DEFF Research Database (Denmark)

    Pommergaard, Hans-Christian; Achiam, Michael Patrick; Rosenberg, Jacob

    2012-01-01

    Colon anastomotic leakage remains both a frequent and serious complication in gastrointestinal surgery. External coating of colonic anastomoses has been proposed as a means to lower the rate of this complication. The aim of this review was to evaluate existing studies on external coating of colonic...

  17. Moisture transport in coated wood

    NARCIS (Netherlands)

    Meel, P.A. van; Erich, S.J.F.; Huinink, H.P.; Kopinga, K.; Jong, J. DE; Adan, O.C.G.

    2011-01-01

    Moisture accumulation inside wood causes favorable conditions for decay. Application of a coating alters the moisture sorption of wood and prevents accumulation of moisture. This paper presents the results of a nuclear magnetic resonance (NMR) study on the influence of a coating on the moisture

  18. Electroless alloy/composite coatings

    Indian Academy of Sciences (India)

    The market for these coatings is expanding fast as the potential applications are on the rise. In the present article, an attempt has been made to review different electroless alloy/composite coatings with respect to bath types and their composition, properties and applications. Different characterisation studies have been ...

  19. Lignin based controlled release coatings

    NARCIS (Netherlands)

    Mulder, W.J.; Gosselink, R.J.A.; Vingerhoeds, M.H.; Harmsen, P.F.H.; Eastham, D.

    2011-01-01

    Urea is a commonly used fertilizer. Due to its high water-solubility, misuse easily leads to excess nitrogen levels in the soil. The aim of this research was to develop an economically feasible and biodegradable slow-release coating for urea. For this purpose, lignin was selected as coating

  20. Foundry Coating Technology: A Review

    DEFF Research Database (Denmark)

    Nwaogu, Ugochukwu Chibuzoh; Tiedje, Niels Skat

    2011-01-01

    is important. In this review, a detailed description of these topics and examples are provided where necessary. A potential area of research in foundry coating development, using sol-gel process is suggested. The application of sol-gel technology in the development of foundry coatings is a novel approach....

  1. Intumescent coatings under fast heating

    DEFF Research Database (Denmark)

    Nørgaard, Kristian Petersen; Dam-Johansen, Kim; Català, Pere

    2012-01-01

    Intumescent coatings are widely used to delay or minimise the destructive effects of fire. They are usually tested under conditions that simulate the relatively slow build-up of heat in a normal fire. Here, the effects of damage during a fire causing sudden heating of the coating were studied....

  2. Steam initiated hydrotalcite conversion coatings

    DEFF Research Database (Denmark)

    Zhou, Lingli; Friis, Henrik; Roefzaad, Melanie

    2018-01-01

    A facile process of exploiting high-temperature steam to deposit nvironmentally friendly hydrotalcite (HT) coatings on Al alloy 6060 was developed in a spray system. Scanning electron microscopy showed the formationf a continuous and conformal coating comprised of a compact mass of crystallites. ...

  3. Evanescent wave assisted nanomaterial coating.

    Science.gov (United States)

    Mondal, Samir K; Pal, Sudipta Sarkar; Kumbhakar, Dharmadas; Tiwari, Umesh; Bhatnagar, Randhir

    2013-08-01

    In this work we present a novel nanomaterial coating technique using evanescent wave (EW). The gradient force in the EW is used as an optical tweezer for tweezing and self-assembling nanoparticles on the source of EW. As a proof of the concept, we have used a laser coupled etched multimode optical fiber, which generates EW for the EW assisted coating. The section-wise etched multimode optical fiber is horizontally and superficially dipped into a silver/gold nanoparticles solution while the laser is switched on. The fiber is left until the solution recedes due to evaporation leaving the fiber in air. The coating time usually takes 40-50 min at room temperature. The scanning electron microscope image shows uniform and thin coating of self-assembled nanoparticles due to EW around the etched section. A coating thickness optical fiber probes and other plasmonic circuits.

  4. Functional Plasma-Deposited Coatings

    Directory of Open Access Journals (Sweden)

    Mykhaylo Pashechko

    2017-12-01

    Full Text Available The paper focuses on the problem of low adhesion of plasma sprayed coatings to the substrate. The subsequent laser treatment modes and their influence on the coating-substrate interface were studied. This allows to decrease the level of metstability of the coating, thus decreasing its hardness down to 11-12 GPa on the surface and to about 9 GPa on depth of 400 µm. The redistribution of alloying elements through solid and liquid diffusion improves mechanical properties and rises the adhesion up to 450 MPa after remelting and up to 90-110 MPa after laser-aided thermal cycling. At he same time, remelting of coating helps to decrease its porosity down to 1%. Obtained complex of properties also allows to improve wear resistance of coatings and to decrease friction factor.

  5. Self-Healing anticorrosive coatings

    DEFF Research Database (Denmark)

    Nesterova, Tatyana

    %. The number is lower than anticipated and needs to be confirmed. Finally, a 3-D model, based on Monte-Carlo simulations, has been developed for prediction of healing efficiency of a microcapsule-based anticorrosive coating. Two kinds of cracks were considered: cracks accommodated within the bulk coating...... associated with development and testing of this type of coating. A laboratory investigation, to identify the most suitable method for production of mechanically stable (filled with industrially relevant core materials) and forming a free-flowing powder upon drying microcapsules, has been performed. Four...... reduces the intensity of crack formation (both in number and length) compared to filler-containing coatings and prevents the coating from flaking upon damage. Based on specular gloss measurements, a preliminary critical pigment (microcapsule) concentration (CPVC) value was estimated to about 30 vol...

  6. Understanding particulate coating microstructure development

    Science.gov (United States)

    Roberts, Christine Cardinal

    How a dispersion of particulates suspended in a solvent dries into a solid coating often is more important to the final coating quality than even its composition. Essential properties like porosity, strength, gloss, particulate order, and concentration gradients are all determined by the way the particles come together as the coating dries. Cryogenic scanning electron microscopy (cryoSEM) is one of the most effective methods to directly visualize a drying coating during film formation. Using this method, the coating is frozen, arresting particulate motion and solidifying the sample so that it be imaged in an SEM. In this thesis, the microstructure development of particulate coatings was explored with several case studies. First, the effect of drying conditions was determined on the collapse of hollow latex particles, which are inexpensive whiteners for paint. Using cryoSEM, it was found that collapse occurs during the last stages of drying and is most likely to occur at high drying temperatures, humidity, and with low binder concentration. From these results, a theoretical model was proposed for the collapse of a hollow latex particle. CryoSEM was also used to verify a theoretical model for the particulate concentration gradients that may develop in a coating during drying for various evaporation, sedimentation and particulate diffusion rates. This work created a simple drying map that will allow others to predict the character of a drying coating based on easily calculable parameters. Finally, the effect of temperature on the coalescence and cracking of latex coatings was explored. A new drying regime for latex coatings was identified, where partial coalescence of particles does not prevent cracking. Silica was shown to be an environmentally friendly additive for preventing crack formation in this regime.

  7. Optical coating preparation

    International Nuclear Information System (INIS)

    Belleville, P.; Sabary, F.; Marcel, C.

    2003-01-01

    In order to optimize the properties of optical components, thin film deposition with controlled thickness and refractive index is often needed. Two different deposition techniques are proposed in this article and illustrated with examples: physical vapor deposition (PVD) and liquid sol-gel process (LSG). PVD and LSG techniques are equivalent as far as the following topics are concerned: elaboration of oxide or composite coated material, optical performance, mechanical performance, and laser performance. PVD is better for the elaboration of metallic films, the design of multi-layers or complex pile-up of layers. LSG is better for the treatment of large surfaces, for substrates with complicated shapes and for its low cost. PVD technique has been widely used so it benefited from an industrial maturity and a clean technology concerning wastes and effluents. On the contrary LSG is a new technique not yet widely used in industrial processes but that looks promising. (A.C.)

  8. Novel coating compositions

    International Nuclear Information System (INIS)

    Kimura, Tadashi; Kobayashi, Juichi; Nakamoto, Hideo.

    1969-01-01

    An acrylic coating composition rapidly hardenable by irradiating with ionizing radiations or light beams is given using hydroxyl group-containing vinyl monomers, polycarboxylic acid anhydrides, epoxy group-containing vinyl monomers and an organic solvent having a boiling point of at least 120 0 C. The process comprises the steps of first and second reactions. The first reaction takes place between one mol of a hydroxyl group of a basic polymer and at least 0.1 mol of polycarboxylic acid anhydride, wherein the basic polymer has a molecular weight ranging from 5,000 to 100,000 and consists of 1-40% by weight of vinyl monomer containing hydroxyl group, at least 30% of (meth)acrylic monomer and other vinyl monomers if required. The second reaction takes place between one mol of hydroxyl plus a carboxyl group of the thus obtained basic polymer and at least 0.1 mol of an epoxy group-containing vinyl monomer to produce a prepolymer. The prepolymer is mixed with a solvent such as ethyl benzene to produce the coating material. The electron beam accelerator energy level may be 0.1-2.0 MeV. In light beam polymerization, benzoin is particularly utilized as an intensifying substance. In one example, a basic polymer is produced by reacting 39 parts of styrene, 37 parts of ethyl acrylate, 24 parts of 2-hydroxyl ethyl acrylate, 4 parts of dimethyl amino ethyl methacrylate and others. A prepolymer is produced by reacting this basic polymer with 30 parts of glycidyl acrylate and others. (Iwakiri, K.)

  9. How PE tape performs under concrete coating

    International Nuclear Information System (INIS)

    Dritt, H.J.

    1984-01-01

    The program objectives were to evaluate the performance of polyethylene tape plant coating and fusion bonded epoxy powder systems with particular respect to the following: 1. Concrete coating application procedures; 2. The shear resistance during laying and retrieving operations of the coating at the various interfaces (a) Pipe and anti-corrosion coating; (b) Anti-corrosion coating and outerwrap; (c) Overlap areas of the anti-corrosion and outerwrap layers; (d) Between concrete and the various corrosion coatings during laying and retrieving operations. 3. Resistance to damage of the coating as a consequence of cracking or slippage of the concrete weight coating. 4. Ability of various coatings to withstand the damage during concrete application by both impact and compression methods; 5. Evaluation of tape and shrink sleeve joint coatings at the cut-back area as well as performance of tape under hot asphalt coating

  10. Avian Egg and Egg Coat.

    Science.gov (United States)

    Okumura, Hiroki

    2017-01-01

    An ovulated egg of vertebrates is surrounded by unique extracellular matrix, the egg coat or zona pellucida, playing important roles in fertilization and early development. The vertebrate egg coat is composed of two to six zona pellucida (ZP) glycoproteins that are characterized by the evolutionarily conserved ZP-domain module and classified into six subfamilies based on phylogenetic analyses. Interestingly, investigations of biochemical and functional features of the ZP glycoproteins show that the roles of each ZP-glycoprotein family member in the egg-coat formation and the egg-sperm interactions seemingly vary across vertebrates. This might be one reason why comprehensive understandings of the molecular basis of either architecture or physiological functions of egg coat still remain elusive despite more than 3 decades of intensive investigations. In this chapter, an overview of avian egg focusing on the oogenesis are provided in the first section, and unique features of avian egg coat, i.e., perivitelline layer, including the morphology, biogenesis pathway, and physiological functions are discussed mainly on chicken and quail in terms of the characteristics of ZP glycoproteins in the following sections. In addition, these features of avian egg coat are compared to mammalian zona pellucida, from the viewpoint that the structural and functional varieties of ZP glycoproteins might be associated with the evolutionary adaptation to their reproductive strategies. By comparing the egg coat of birds and mammals whose reproductive strategies are largely different, new insights into the molecular mechanisms of vertebrate egg-sperm interactions might be provided.

  11. Intumescent Coatings as Fire Retardants

    Science.gov (United States)

    Parker, J. A.; Fohlen, G. M.; Sawko, P. M.; Fish, R. H.

    1970-01-01

    The development of fire-retardant coatings to protect surfaces which may be exposed to fire or extreme heat is a subject of intense interest to many industries. A fire-retardant paint has been developed which represents a new chemical approach for preparing intumescent coatings, and potentially, is very important to fire-prevention authorities. The requirements for a superior coating include ease of application, suitability to a wide variety of surfaces and finishes, and stability over an extended period of time within a broad range of ambient temperature and humidity conditions. These innovative coatings, when activated by the heat of a fire, react to form a thick, low-density, polymeric coating or char layer. Water vapor and sulphur dioxide are released during the intumescent reaction. Two fire-protection mechanisms thus become available: (1) the char layer retards the flow of heat, due to the extremely low thermal conductivity; and (2) water vapor and sulfur dioxide are released, providing fire quenching properties. Still another mechanism functions in cases where the char, by virtue of its high oxidation resistance and low thermal conductivity, reaches a sufficiently high temperature to re-radiate much of the incident heat load. The coatings consist of dispersions of selective salts of a nitro-amino-arornatic compound. Specifically, para-nitroaniline bisulfate and the ammonium salt of para-nitroaniline-ortho sulphuric acid (2-amino-5-nitrobenzenesulphuric acid) are used. Suitable vehicles are cellulose nitrate of lacquer grade, a nitrite-phenolic modified rubber, or epoxy-polysulfide copolymer. Three separate formulations have been developed. A solvent is usually employed, such as methylethyl ketone, butyl acetate, or toluene, which renders the coatings suitably thin and which evaporates after the coatings are applied. Generally, the intumescent material is treated as insoluble in the vehicle, and is ground and dispersed in the vehicle and solvent like an

  12. Integrated Glass Coating Manufacturing Line

    Energy Technology Data Exchange (ETDEWEB)

    Brophy, Brenor [Enki Technology Inc., San Jose, CA (United States)

    2015-09-30

    This project aims to enable US module manufacturers to coat glass with Enki’s state of the art tunable functionalized AR coatings at the lowest possible cost and highest possible performance by encapsulating Enki’s coating process in an integrated tool that facilitates effective process improvement through metrology and data analysis for greater quality and performance while reducing footprint, operating and capital costs. The Phase 1 objective was a fully designed manufacturing line, including fully specified equipment ready for issue of purchase requisitions; a detailed economic justification based on market prices at the end of Phase 1 and projected manufacturing costs and a detailed deployment plan for the equipment.

  13. Laser-based coatings removal

    International Nuclear Information System (INIS)

    Freiwald, J.G.; Freiwald, D.A.

    1995-01-01

    Over the years as building and equipment surfaces became contaminated with low levels of uranium or plutonium dust, coats of paint were applied to stabilize the contaminants in place. Most of the earlier paint used was lead-based paint. More recently, various non-lead-based paints, such as two-part epoxy, are used. For D ampersand D (decontamination and decommissioning), it is desirable to remove the paints or other coatings rather than having to tear down and dispose of the entire building. This report describes the use of pulse-repetetion laser systems for the removal of paints and coatings

  14. Hydrogen permeation resistant phosphate coatings

    International Nuclear Information System (INIS)

    McGuire, J.C.

    1979-01-01

    A method for reducing hydrogen diffusion through metal wherein the metal is coated with a phosphate-radical-containing, phosphate-glass-forming material on at least one surface thereof. The coating is then heated to at least 350 0 C to form a phosphate glass. This method is especially applicable to nuclear reactors to minimize tritium diffusion. The coating is preferably formed with a solution of phosphoric acid which may also contain compounds such as MnSO 4 , SiO 2 and Na 2 Cr 2 0 7 . (author)

  15. Hydrogen permeation resistant phosphate coatings

    International Nuclear Information System (INIS)

    McGuire, J.C.

    1979-01-01

    A method for reducing hydrogen diffusion through metal is described. The metal is coated with a phosphate-radical-containing, phosphate-glass-forming material on at least one surface. The coating is then heated to at least 350 0 C to form a phosphate glass. This method is especially applicable to nuclear reactors to minimize tritium diffusion. The coating is preferably formed with a solution of phosphoric acid which may also contain compounds such as MnSO 4 , SiO 2 and Na 2 Cr 2 O 7 . (author)

  16. Laser-based coatings removal

    Energy Technology Data Exchange (ETDEWEB)

    Freiwald, J.G.; Freiwald, D.A. [F2 Associates, Inc., Albuquerque, NM (United States)

    1995-10-01

    Over the years as building and equipment surfaces became contaminated with low levels of uranium or plutonium dust, coats of paint were applied to stabilize the contaminants in place. Most of the earlier paint used was lead-based paint. More recently, various non-lead-based paints, such as two-part epoxy, are used. For D&D (decontamination and decommissioning), it is desirable to remove the paints or other coatings rather than having to tear down and dispose of the entire building. This report describes the use of pulse-repetetion laser systems for the removal of paints and coatings.

  17. Experimental evaluation of coating delamination in vinyl coated metal forming

    International Nuclear Information System (INIS)

    Son, Young Ki; Lee, Chan Joo; Kim, Byung Min; Lee, Jung Min; Byoen, Sang Doek; Lee, Soen Bong

    2012-01-01

    In this paper, a new evaluation and prediction method for coating delamination during sheet metal forming is presented. On the basis of the forming limit diagram (FLD), the current study evaluates the delamination of PET coating by using a cross cut specimen, dome test, and rectangular cup drawing test. Dome test specimens were subjected to biaxial, plane strain, and uniaxial deformation modes. Rectangular cup drawing test specimens were subjected to the deep drawing deformation mode, and compression deformation mode. A vinyl coated metal (VCM) sheet consists of three layers of polymer on the sheet metals: a protective film, a PET layer and a PVC layer. The areas with coating delamination were identified, and the results of the evaluation were plotted according to major and minor strain values, depicting coating delamination. The constructed delamination limit diagram (DLD) can be used to determine the forming limit of VCM during the complex press forming process. ARGUS (GOM) was employed to identify the strain value and deformation mode of the delaminated surface after the press forming. After identifying the areas of delamination, the DLD of the PET coating can be constructed in a format similar to that of the FLD. The forming limit of the VCM sheet can be evaluated using the superimposition of the delamination limit strain of the coating onto the FLD of VCM sheet. The experimental results showed that the proposed test method will support the sheet metal forming process design for VCM sheets. The assessment method presented in this study can be used to determine the delamination limit strain under plastic deformation of other polymer coated metals. The experimental results suggested that the proposed testing method is effective in evaluating delamination for specific applications

  18. Experimental evaluation of coating delamination in vinyl coated metal forming

    Energy Technology Data Exchange (ETDEWEB)

    Son, Young Ki; Lee, Chan Joo; Kim, Byung Min [Pusan National Univ., Busan (Korea, Republic of); Lee, Jung Min [Korea Institute of Industrial Technology, Busan (Korea, Republic of); Byoen, Sang Doek [HA Digital Engineering Gr., Seongsan Gu (Korea, Republic of); Lee, Soen Bong [Keimyung Univ., Daegu (Korea, Republic of)

    2012-10-15

    In this paper, a new evaluation and prediction method for coating delamination during sheet metal forming is presented. On the basis of the forming limit diagram (FLD), the current study evaluates the delamination of PET coating by using a cross cut specimen, dome test, and rectangular cup drawing test. Dome test specimens were subjected to biaxial, plane strain, and uniaxial deformation modes. Rectangular cup drawing test specimens were subjected to the deep drawing deformation mode, and compression deformation mode. A vinyl coated metal (VCM) sheet consists of three layers of polymer on the sheet metals: a protective film, a PET layer and a PVC layer. The areas with coating delamination were identified, and the results of the evaluation were plotted according to major and minor strain values, depicting coating delamination. The constructed delamination limit diagram (DLD) can be used to determine the forming limit of VCM during the complex press forming process. ARGUS (GOM) was employed to identify the strain value and deformation mode of the delaminated surface after the press forming. After identifying the areas of delamination, the DLD of the PET coating can be constructed in a format similar to that of the FLD. The forming limit of the VCM sheet can be evaluated using the superimposition of the delamination limit strain of the coating onto the FLD of VCM sheet. The experimental results showed that the proposed test method will support the sheet metal forming process design for VCM sheets. The assessment method presented in this study can be used to determine the delamination limit strain under plastic deformation of other polymer coated metals. The experimental results suggested that the proposed testing method is effective in evaluating delamination for specific applications.

  19. Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes.

    Directory of Open Access Journals (Sweden)

    Mohammad F Saeed

    2010-09-01

    Full Text Available Zaire ebolavirus (ZEBOV, a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new

  20. Host cell entry of powdery mildew is correlated with endosomal transport of antagonistically acting VvPEN1 and VvMLO to the papilla.

    Science.gov (United States)

    Feechan, A; Jermakow, A M; Ivancevic, A; Godfrey, D; Pak, H; Panstruga, R; Dry, I B

    2013-10-01

    Challenge by a nonadapted powdery mildew fungal pathogen leads to the formation of a local cell-wall apposition (papilla) beneath the point of attempted penetration. Several plasma membrane (PM) proteins with opposing roles in powdery mildew infection, including Arabidopsis thaliana PENETRATION1 (PEN1) and barley (Hordeum vulgare) MILDEW RESISTANCE LOCUS O (MLO), are localized to the site of powdery mildew attack. PEN1 contributes to penetration resistance to nonadapted powdery mildews, whereas MLO is a susceptibility factor required by adapted powdery mildew pathogens for host cell entry. Our previous studies have demonstrated that the vesicle and endosomal trafficking inhibitors, brefeldin A and wortmannin, have opposite effects on the penetration rates of adapted and nonadapted powdery mildews on grapevine. These findings prompted us to study the pathogen-induced intracellular trafficking of grapevine variants of MLO and PEN1. We first identified grapevine (Vitis vinifera) VvPEN1 and VvMLO orthologs that rescue Arabidopsis Atpen1 and Atmlo2 mlo6 mlo12 null mutants, respectively. By using endomembrane trafficking inhibitors in combination with fluorescence microscopy, we demonstrate that VvMLO3/VvMLO4 and VvPEN1 are co-trafficked together from the PM to the site of powdery mildew challenge. This focal accumulation of VvMLO3/VvMLO4 and VvPEN1 to the site of attack seems to be required for their opposing functions during powdery mildew attack, because their subcellular localization is correlated with the outcome of attempted powdery mildew penetration.

  1. The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome.

    Directory of Open Access Journals (Sweden)

    Henrick Schomacker

    Full Text Available Interferons (IFNs play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1 inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.

  2. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT Machinery via Ubiquitination To Facilitate Viral Envelopment

    Directory of Open Access Journals (Sweden)

    Rina Barouch-Bentov

    2016-11-01

    Full Text Available Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate, an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses.

  3. Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

    Science.gov (United States)

    Merriam, Laura A; Baran, Caitlin N; Girard, Beatrice M; Hardwick, Jean C; May, Victor; Parsons, Rodney L

    2013-03-06

    After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

  4. Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

    Science.gov (United States)

    Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

    2014-01-01

    Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

  5. Effectiveness of a dynein team in a tug of war helped by reduced load sensitivity of detachment: evidence from the study of bidirectional endosome transport in D. discoideum.

    Science.gov (United States)

    Bhat, Deepak; Gopalakrishnan, Manoj

    2012-08-01

    Bidirectional cargo transport by molecular motors in cells is a complex phenomenon in which the cargo (usually a vesicle) alternately moves in retrograde and anterograde directions. In this case, teams of oppositely pulling motors (e.g., kinesin and dynein) bind to the cargo, simultaneously, and 'coordinate' their activity such that the motion consists of spells of positively and negatively directed segments, separated by pauses of varying duration. A set of recent experiments have analyzed the bidirectional motion of endosomes in the amoeba D. discoideum in detail. It was found that in between directional switches, a team of five to six dyneins stall a cargo against a stronger kinesin in a tug of war, which lasts for almost a second. As the mean detachment time of a kinesin under its stall load was also observed to be ∼1 s, we infer that the collective detachment time of the dynein assembly must also be similar. Here, we analyze this inference from a modeling perspective, using experimentally measured single-molecule parameters as inputs. We find that the commonly assumed exponential load-dependent detachment rate is inconsistent with observations, as it predicts that a five-dynein assembly will detach under its combined stall load in less than a hundredth of a second. A modified model where the load-dependent unbinding rate is assumed to saturate at stall-force level for super-stall loads gives results which are in agreement with experimental data. Our analysis suggests that the load-dependent detachment of a dynein in a team is qualitatively different at sub-stall and super-stall loads, a conclusion which is likely to have implications in other situations involving collective effects of many motors.

  6. Water permeability of pigmented waterborne coatings

    NARCIS (Netherlands)

    Donkers, P.A.J.; Huinink, H.P.; Erich, S.J.F.; Reuvers, N.J.W.; Adan, O.C.G.

    2013-01-01

    Coatings are used in a variety of applications. Last decades more and more coating systems are transforming from solvent to waterborne coating systems. In this study the influence of pigments on the water permeability of a waterborne coating system is studied, with special interest in the possible

  7. Radiation cured coatings for fiber optics

    International Nuclear Information System (INIS)

    Ketley, A.D.; Morgan, C.R.

    1978-01-01

    A continuous protective coating is formed on a fiber optic by coating the fiber optic in a bath of a liquid radiation curable composition at a temperature up to 90 0 C and exposing the coated conductor to ultraviolet or high energy ionizing radiation to cure the coating

  8. Hex Chrome Free Coatings for Electronics Overview

    Science.gov (United States)

    Kessel, Kurt

    2013-01-01

    The overall objective of the Hex Chrome Free Coatings for Electronics project is to evaluate and test pretreatment coating systems not containing hexavalent chrome in avionics and electronics housing applications. This objective will be accomplished by testing strong performing coating systems from prior NASA and DoD testing or new coating systems as determined by the stakeholders.

  9. Comparison of additive amount used in spin-coated and roll-coated organic solar cells

    DEFF Research Database (Denmark)

    Cheng, Pei; Lin, Yuze; Zawacka, Natalia Klaudia

    2014-01-01

    All-polymer and polymer/fullerene inverted solar cells were fabricated by spin-coating and roll-coating processes. The spin-coated small-area (0.04 cm(2)) devices were fabricated on indium tin oxide (ITO) coated glass substrates in nitrogen. The roll-coated large-area (1.0 cm(2)) devices were...

  10. Overlay metallic-cermet alloy coating systems

    International Nuclear Information System (INIS)

    Gedwill, M.A.; Glasgow, T.K.; Levine, S.R.

    1982-01-01

    A substrate, such as a turbine blade, vane, or the like, which is subjected to high temperature use is coated with a base coating of an oxide dispersed, metallic alloy (cermet). A top coating of an oxidation, hot corrosion, erosion resistant alloy of nickel, cobalt, or iron is then deposited on the base coating. A heat treatment is used to improve the bonding. The base coating serves as an inhibitor to interdiffusion between the protective top coating and the substrate. Otherwise, the protective top coating would rapidly interact detrimentally with the substrate and degrade by spalling of the protective oxides formed on the outer surface at elevated temperatures

  11. Overlay metallic-cermet alloy coating systems

    Science.gov (United States)

    Gedwill, M. A.; Levine, S. R.; Glasgow, T. K. (Inventor)

    1984-01-01

    A substrate, such as a turbine blade, vane, or the like, which is subjected to high temperature use is coated with a base coating of an oxide dispersed, metallic alloy (cermet). A top coating of an oxidation, hot corrosion, erosion resistant alloy of nickel, cobalt, or iron is then deposited on the base coating. A heat treatment is used to improve the bonding. The base coating serves as an inhibitor to interdiffusion between the protective top coating and the substrate. Otherwise, the protective top coating would rapidly interact detrimentally with the substrate and degrade by spalling of the protective oxides formed on the outer surface at elevated temperatures.

  12. Seal coat binder performance specifications.

    Science.gov (United States)

    2013-11-01

    Need to improve seal coat binder specs: replace empirical tests (penetration, ductility) with : performance-related tests applicable to both : unmodified and modified binders; consider temperatures that cover entire in service : range that are tied t...

  13. Protective coatings for commercial particulates

    DEFF Research Database (Denmark)

    Kindl, B.; Teng, Y.H.; Liu, Y.L.

    1994-01-01

    SiC/Al composites are in large-scale production with Al-Si alloy matrices. The same composites with pure Al or low Si matrices need diffusion barriers on the SiC reinforcement to control the interfacial reaction. The present paper describes various approaches taken to obtain protective coatings...... of alumina and zirconia on SiC particulates by sol-gel techniques. Aqueous and organic precursors have been used. The extent of the reaction, i.e., the Si and Al4C3 content in the matrix, was determined by differential thermal analysis and X-ray diffraction. The reaction rates of some coated particulates...... in liquid Al are decreased by as much as one order of magnitude during the first 15 min of immersion. Pretreatments of the SiC surface, the composition and thickness of the coating interphase and heat treatments of the coated materials have been studied, and are discussed in relation to their effect...

  14. Cementless Hydroxyapatite Coated Hip Prostheses

    Science.gov (United States)

    Herrera, Antonio; Mateo, Jesús; Gil-Albarova, Jorge; Lobo-Escolar, Antonio; Ibarz, Elena; Gabarre, Sergio; Más, Yolanda

    2015-01-01

    More than twenty years ago, hydroxyapatite (HA), calcium phosphate ceramics, was introduced as a coating for cementless hip prostheses. The choice of this ceramic is due to its composition being similar to organic apatite bone crystals. This ceramic is biocompatible, bioactive, and osteoconductive. These qualities facilitate the primary stability and osseointegration of implants. Our surgical experience includes the implantation of more than 4,000 cementless hydroxyapatite coated hip prostheses since 1990. The models implanted are coated with HA in the acetabulum and in the metaphyseal area of the stem. The results corresponding to survival and stability of implants were very satisfactory in the long-term. From our experience, HA-coated hip implants are a reliable alternative which can achieve long term survival, provided that certain requirements are met: good design selection, sound choice of bearing surfaces based on patient life expectancy, meticulous surgical technique, and indications based on adequate bone quality. PMID:25802848

  15. Black Sprayable Molecular Adsorber Coating

    Data.gov (United States)

    National Aeronautics and Space Administration — The main objective of this technology project is to develop, optimize, and flight qualify a black version of the molecular adsorber coating and a conductive version...

  16. Self-stratifying antimicrobial coatings

    NARCIS (Netherlands)

    Yagci, M.B.

    2012-01-01

    Today, antimicrobial polymers/coatings are widely used in various areas, such as biomedical devices, pharmaceuticals, hospital buildings, textiles, food processing, and contact lenses, where sanitation is needed. Such wide application facilities have made antimicrobial materials very attractive for

  17. Smart Coatings for Corrosion Protection

    Science.gov (United States)

    Calle, Luz Marina; Li, Wendy; Buhrow, Jerry W.; Johnsey, Marissa N.

    2016-01-01

    Nearly all metals and their alloys are subject to corrosion that causes them to lose their structural integrity or other critical functionality. It is essential to detect corrosion when it occurs, and preferably at its early stage, so that action can be taken to avoid structural damage or loss of function. Protective coatings are the most commonly used method of corrosion control. However, progressively stricter environmental regulations have resulted in the ban of many commercially available corrosion protective coatings due to the harmful effects of their solvents or corrosion inhibitors. This work concerns the development of a multifunctional, smart coating for the autonomous control of corrosion. This coating is being developed to have the inherent ability to detect the chemical changes associated with the onset of corrosion and respond autonomously to indicate it and control it.

  18. Dielectric coatings on metal substrates

    International Nuclear Information System (INIS)

    Glaros, S.S.; Baker, P.; Milam, D.

    1976-01-01

    Large aperture, beryllium substrate-based mirrors have been used to focus high intensity pulsed laser beams. Finished surfaces have high reflectivity, low wavefront distortion, and high laser damage thresholds. This paper describes the development of a series of metallic coatings, surface finishing techniques, and dielectric overcoatings to meet specified performance requirements. Beryllium substrates were coated with copper, diamond-machined to within 5 micro-inches to final contour, nickel plated, and abrasively figured to final contour. Bond strengths for several bonding processes are presented. Dielectric overcoatings were deposited on finished multimetallic substrates to increase both reflectivity and the damage thresholds. Coatings were deposited using both high and low temperature processes which induce varying stresses in the finished coating substrate system. Data are presented to show the evolution of wavefront distortion, reflectivity, and damage thresholds throughout the many steps involved in fabrication

  19. Cementless Hydroxyapatite Coated Hip Prostheses

    Directory of Open Access Journals (Sweden)

    Antonio Herrera

    2015-01-01

    Full Text Available More than twenty years ago, hydroxyapatite (HA, calcium phosphate ceramics, was introduced as a coating for cementless hip prostheses. The choice of this ceramic is due to its composition being similar to organic apatite bone crystals. This ceramic is biocompatible, bioactive, and osteoconductive. These qualities facilitate the primary stability and osseointegration of implants. Our surgical experience includes the implantation of more than 4,000 cementless hydroxyapatite coated hip prostheses since 1990. The models implanted are coated with HA in the acetabulum and in the metaphyseal area of the stem. The results corresponding to survival and stability of implants were very satisfactory in the long-term. From our experience, HA-coated hip implants are a reliable alternative which can achieve long term survival, provided that certain requirements are met: good design selection, sound choice of bearing surfaces based on patient life expectancy, meticulous surgical technique, and indications based on adequate bone quality.

  20. Electrically conductive polymer concrete coatings

    Science.gov (United States)

    Fontana, Jack J.; Elling, David; Reams, Walter

    1990-01-01

    A sprayable electrically conductive polymer concrete coating for vertical d overhead applications is described. The coating is permeable yet has low electrical resistivity (<10 ohm-cm), good bond strength to concrete substrates, and good weatherability. A preferred formulation contains about 60 wt % calcined coke breeze, 40 wt % vinyl ester with 3.5 wt % modified bentonite clay. Such formulations apply evenly and provide enough rigidity for vertical or overhead structures so there is no drip or sag.

  1. Nanocrystalline diamond coatings for machining

    Energy Technology Data Exchange (ETDEWEB)

    Frank, M.; Breidt, D.; Cremer, R. [CemeCon AG, Wuerselen (Germany)

    2007-07-01

    This history of CVD diamond synthesis goes back to the fifties of the last century. However, the scientific and economical potential was only gradually recognized. In the eighties, intensive worldwide research on CVD diamond synthesis and applications was launched. Industrial products, especially diamond-coated cutting tools, were introduced to the market in the middle of the nineties. This article shows the latest developments in this area, which comprises nanocrystalline diamond coating structures. (orig.)

  2. Dry and coating of powders

    International Nuclear Information System (INIS)

    Alonso, M.; Alguacil, F. J.

    1999-01-01

    This paper presents a review on the mixing and coating of powders by dry processes. The reviews surveys fundamental works on mixture characterization (mixing index definitions and sampling techniques), mixing mechanisms and models, segregation with especial emphasis on free-surface segregation, mixing of cohesive powders and interparticle forces, ordered mixing (dry coating) including mechanism, model and applications and mixing equipment selection. (Author) 180 refs

  3. Silicone nanocomposite coatings for fabrics

    Science.gov (United States)

    Eberts, Kenneth (Inventor); Lee, Stein S. (Inventor); Singhal, Amit (Inventor); Ou, Runqing (Inventor)

    2011-01-01

    A silicone based coating for fabrics utilizing dual nanocomposite fillers providing enhanced mechanical and thermal properties to the silicone base. The first filler includes nanoclusters of polydimethylsiloxane (PDMS) and a metal oxide and a second filler of exfoliated clay nanoparticles. The coating is particularly suitable for inflatable fabrics used in several space, military, and consumer applications, including airbags, parachutes, rafts, boat sails, and inflatable shelters.

  4. Coating of silicon pore optics

    DEFF Research Database (Denmark)

    Cooper-Jensen, Carsten P.; Ackermann, M.; Christensen, Finn Erland

    2009-01-01

    For the International X-ray observatory (IXO), a mirror module with an effective area of 3 m2 at 1.25 keV and at least 0.65 m2 at 6 keV has to be realized. To achieve this goal, coated silicon pore optics has been developed over the last years. One of the challenges is to coat the Si plates...

  5. Damage-resistant brittle coatings

    Energy Technology Data Exchange (ETDEWEB)

    Lawn, B.R.; Lee, K.S. [National Inst. of Stand. and Technol., Gaithersburg, MD (United States). Mater. Sci. and Eng. Lab.; Chai, H. [Tel Aviv Univ. (Israel). Faculty of Engineering; Pajares, A. [Universidad de Extremadura, Badajoz (Spain). Dept. de Fisica; Kim, D.K. [Korea Advanced Inst. of Science and Technolgy, Taejon (Korea). Dept. of Materials Science and Engineering; Wuttiphan, S. [National Metal and Materials Technology Center, Bangkok (Thailand); Peterson, I.M. [Corning Inc., NY (United States); Hu Xiaozhi [Western Australia Univ., Nedlands, WA (Australia). Dept. of Mechanical and Materials Engineering

    2000-11-01

    Laminate structures consisting of hard, brittle coatings and soft, tough substrates are important in a wide variety of engineering applications, biological structures, and traditional pottery. In this study the authors introduce a new approach to the design of damage-resistant brittle coatings, based on a combination of new and existing relations for crack initiation in well-defined contact-induced stress fields. (orig.)

  6. Corrosion resistant neutron absorbing coatings

    Science.gov (United States)

    Choi, Jor-Shan [El Cerrito, CA; Farmer, Joseph C [Tracy, CA; Lee, Chuck K [Hayward, CA; Walker, Jeffrey [Gaithersburg, MD; Russell, Paige [Las Vegas, NV; Kirkwood, Jon [Saint Leonard, MD; Yang, Nancy [Lafayette, CA; Champagne, Victor [Oxford, PA

    2012-05-29

    A method of forming a corrosion resistant neutron absorbing coating comprising the steps of spray or deposition or sputtering or welding processing to form a composite material made of a spray or deposition or sputtering or welding material, and a neutron absorbing material. Also a corrosion resistant neutron absorbing coating comprising a composite material made of a spray or deposition or sputtering or welding material, and a neutron absorbing material.

  7. Material Science Smart Coatings

    Energy Technology Data Exchange (ETDEWEB)

    Rubinstein, A. I. [Univ. of Nebraska Medical Center, Omaha, NE (United States); Sabirianov, R. F. [Univ. of Nebraska Medical Center, Omaha, NE (United States); Namavar, Fereydoon [Univ. of Nebraska Medical Center, Omaha, NE (United States)

    2014-07-01

    The contribution of electrostatic interactions to the free energy of binding between model protein and a ceramic implant surface in the aqueous solvent, considered in the framework of the nonlocal electrostatic model, is calculated as a function of the implant low-frequency dielectric constant. We show that the existence of a dynamically ordered (low-dielectric) interfacial solvent layer at the protein-solvent and ceramic-solvent interface markedly increases charging energy of the protein and ceramic implant, and consequently makes the electrostatic contribution to the protein-ceramic binding energy more favorable (attractive). Our analysis shows that the corresponding electrostatic energy between protein and oxide ceramics depends nonmonotonically on the dielectric constant of ceramic, εC. Obtained results indicate that protein can attract electrostatically to the surface if ceramic material has a moderate εC below or about 35 (in particularly ZrO2 or Ta2O5). This is in contrast to classical (local) consideration of the solvent, which demonstrates an unfavorable electrostatic interaction of protein with typical metal oxide ceramic materials (εC>10). Thus, a solid implant coated by combining oxide ceramic with a reduced dielectric constant can be beneficial to strengthen the electrostatic binding of the protein-implant complex.

  8. Silane based coating of aluminium mold

    DEFF Research Database (Denmark)

    2013-01-01

    having at least one closed cavity is provided, at least one surface of the at least one cavity being an aluminium surface coated with a silane based coating layer. The silane based anti-stiction coating improves the anti-stiction properties of the mold which may allow for molding and demolding...... of structures which would otherwise be difficult to mold. The resistance of the coated aluminium mold is significantly improved by applying a silane-based coating layer....

  9. Switchable antifouling coatings and uses thereof

    Science.gov (United States)

    Denton, Michele L. Baca; Dirk, Shawn M.; Johnson, Ross Stefan

    2017-02-28

    The present invention relates to antifouling coatings capable of being switched by using heat or ultraviolet light. Prior to switching, the coating includes an onium cation component having antimicrobial and antibacterial properties. Upon switching, the coating is converted to a conjugated polymer state, and the cationic component is released with any adsorbed biofilm layer. Thus, the coatings herein have switchable and releasable properties. Methods of making and using such coatings are also described.

  10. Graphene: corrosion-inhibiting coating.

    Science.gov (United States)

    Prasai, Dhiraj; Tuberquia, Juan Carlos; Harl, Robert R; Jennings, G Kane; Rogers, Bridget R; Bolotin, Kirill I

    2012-02-28

    We report the use of atomically thin layers of graphene as a protective coating that inhibits corrosion of underlying metals. Here, we employ electrochemical methods to study the corrosion inhibition of copper and nickel by either growing graphene on these metals, or by mechanically transferring multilayer graphene onto them. Cyclic voltammetry measurements reveal that the graphene coating effectively suppresses metal oxidation and oxygen reduction. Electrochemical impedance spectroscopy measurements suggest that while graphene itself is not damaged, the metal under it is corroded at cracks in the graphene film. Finally, we use Tafel analysis to quantify the corrosion rates of samples with and without graphene coatings. These results indicate that copper films coated with graphene grown via chemical vapor deposition are corroded 7 times slower in an aerated Na(2)SO(4) solution as compared to the corrosion rate of bare copper. Tafel analysis reveals that nickel with a multilayer graphene film grown on it corrodes 20 times slower while nickel surfaces coated with four layers of mechanically transferred graphene corrode 4 times slower than bare nickel. These findings establish graphene as the thinnest known corrosion-protecting coating.

  11. Polymeric Coatings for Combating Biocorrosion

    Science.gov (United States)

    Guo, Jing; Yuan, Shaojun; Jiang, Wei; Lv, Li; Liang, Bin; Pehkonen, Simo O.

    2018-03-01

    Biocorrosion has been considered as big trouble in many industries and marine environments due to causing great economic loss. The main disadvantages of present approaches to prevent corrosion include being limited by environmental factors, being expensive, inapplicable to field, and sometimes inefficient. Studies show that polymer coatings with anti-corrosion and anti-microbial properties have been widely accepted as a novel and effective approach to preventbiocorrosion. The main purpose of this review is to summarize up the progressive status of polymer coatings