WorldWideScience

Sample records for epidermal cytosolic proteins

  1. Cytosolic proteostasis through importing of misfolded proteins into mitochondria.

    Science.gov (United States)

    Ruan, Linhao; Zhou, Chuankai; Jin, Erli; Kucharavy, Andrei; Zhang, Ying; Wen, Zhihui; Florens, Laurence; Li, Rong

    2017-03-16

    Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.

  2. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

    Science.gov (United States)

    Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

    2017-08-04

    Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    OpenAIRE

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicit...

  4. The Medicago truncatula DMI1 protein modulates cytosolic calcium signaling

    DEFF Research Database (Denmark)

    Peiter, Edgar; Sun, Jongho; Heckmann, Anne Birgitte Lau

    2007-01-01

    nodulation have been cloned in model legumes. Among them, Medicago truncatula DMI1 (DOESN'T MAKE INFECTIONS1) is required for the generation of nucleus-associated calcium spikes in response to the rhizobial signaling molecule Nod factor. DMI1 encodes a membrane protein with striking similarities...... to the Methanobacterium thermoautotrophicum potassium channel (MthK). The cytosolic C terminus of DMI1 contains a RCK (regulator of the conductance of K+) domain that in MthK acts as a calcium-regulated gating ring controlling the activity of the channel. Here we show that a dmi1 mutant lacking the entire C terminus acts...... as a dominant-negative allele interfering with the formation of nitrogen-fixing nodules and abolishing the induction of calcium spikes by the G-protein agonist Mastoparan. Using both the full-length DMI1 and this dominant-negative mutant protein we show that DMI1 increases the sensitivity of a sodium...

  5. Target proteins of the cytosolic thioredoxin in Plasmodium falciparum.

    Science.gov (United States)

    Kawazu, Shin-ichiro; Takemae, Hitoshi; Komaki-Yasuda, Kanako; Kano, Shigeyuki

    2010-06-01

    The target proteins of a cytosolic Trx (PfTrx-1) in Plasmodium falciparum with Trx-affinity chromatography were examined. Based on the Trx protein reduction pathway, we generated a cysteine mutant of PfTrx-1, which captures the target protein as a mixed disulfide intermediate. A number of proteins were captured with PfTrx-1(C33S) immobilized on resin and were eluted by DTT treatment. The PfTrx-1(C33S) immobilized resin-captured proteins were trypsin-digested and analyzed on a liquid chromatography-mass spectrometry system. Analysis of the sequence data against databases assigned 20 proteins, four of which had been found previously in P. falciparum, with the remaining 16 being new targets. The potential Trx-target proteins included those in pathways such as the redox cycle, protein biosynthesis, energy metabolism and signal transduction. We captured 4 enzymes in the glycolysis pathway (hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase and L-lactate dehydrogenase (LDH)) as Trx-targets, and we found that PfTrx-1 enhanced the activity of PfGAPDH and PfLDH.

  6. Yeast and Mammals Utilize Similar Cytosolic Components to Drive Protein Transport through the Golgi Complex

    Science.gov (United States)

    Dunphy, William G.; Pfeffer, Suzanne R.; Clary, Douglas O.; Wattenberg, Binks W.; Glick, Benjamin S.; Rothman, James E.

    1986-03-01

    Vesicular transport between successive compartments of the mammalian Golgi apparatus has recently been reconstituted in a cell-free system. In addition to ATP, transport requires both membrane-bound and cytosolic proteins. Here we report that the cytosol fraction from yeast will efficiently substitute for mammalian cytosol. Mammalian cytosol contains several distinct transport factors, which we have distinguished on the basis of gel filtration and ion-exchange chromatography. Yeast cytosol appears to contain the same collection of transport factors. Resolved cytosol factors from yeast and mammals complement each other in a synergistic manner. These findings suggest that the molecular mechanisms of intracellular protein transport have been conserved throughout evolution. Moreover, this hybrid cell-free system will enable the application of yeast genetics to the identification and isolation of cytosolic proteins that sustain intracellular protein transport.

  7. Protein abundance profiling of the Escherichia coli cytosol

    Directory of Open Access Journals (Sweden)

    Mann Matthias

    2008-02-01

    Full Text Available Abstract Background Knowledge about the abundance of molecular components is an important prerequisite for building quantitative predictive models of cellular behavior. Proteins are central components of these models, since they carry out most of the fundamental processes in the cell. Thus far, protein concentrations have been difficult to measure on a large scale, but proteomic technologies have now advanced to a stage where this information becomes readily accessible. Results Here, we describe an experimental scheme to maximize the coverage of proteins identified by mass spectrometry of a complex biological sample. Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. A measure of abundance is presented for each of the identified proteins, based on the recently developed emPAI approach which takes into account the number of sequenced peptides per protein. The values of abundance are within a broad range and accurately reflect independently measured copy numbers per cell. As expected, the most abundant proteins were those involved in protein synthesis, most notably ribosomal proteins. Proteins involved in energy metabolism as well as those with binding function were also found in high copy number while proteins annotated with the terms metabolism, transcription, transport, and cellular organization were rare. The barrel-sandwich fold was found to be the structural fold with the highest abundance. Highly abundant proteins are predicted to be less prone to aggregation based on their length, pI values, and occurrence patterns of hydrophobic stretches. We also find that abundant proteins tend to be predominantly essential. Additionally we observe a significant correlation between protein and mRNA abundance in E. coli cells. Conclusion Abundance measurements for more than 1000 E. coli proteins presented in this work

  8. Metabolism of minor isoforms of prion proteins: Cytosolic prion protein and transmembrane prion protein

    Science.gov (United States)

    Song, Zhiqi; Zhao, Deming; Yang, Lifeng

    2013-01-01

    Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein configuration changes and their correlation with prion disease transmissibility and pathogenicity. In vivo and in vitro studies have shown that several cytosolic forms of prion protein with specific topological structure can destroy intracellular stability and contribute to prion protein pathogenicity. In this study, the latest molecular chaperone system associated with endoplasmic reticulum-associated protein degradation, the endoplasmic reticulum resident protein quality-control system and the ubiquitination proteasome system, is outlined. The molecular chaperone system directly correlates with the prion protein degradation pathway. Understanding the molecular mechanisms will help provide a fascinating avenue for further investigations on prion disease treatment and prion protein-induced neurodegenerative diseases. PMID:25206608

  9. Effect of sodium molybdate on the thyroxine-binding affinity of transport cytosol proteins.

    Science.gov (United States)

    Svobodová, V; Kraml, J; Schreiber, V

    1985-01-01

    The presence of sodium molybdate during tissue homogenization is known to increase the number of cytosol binding sites for glucocorticoids, progesterone, androgens and oestrogens. We wondered whether a phenomenon similar to this stabilization of steroid receptors would also occur in thyroxine-binding cytosol protein. We found that the presence of sodium molybdate (10 mmol/l) in rat adenohypophyseal cytosol increased its thyroxine-binding capacity by up to 96%. In the case of binding protein cytosol minus molybdate, Ka = 5.5 X 10(9) l.mol-1, whereas for cytosol plus molybdate Ka(1) = 6.0 X 10(9) l.mol-1 and Ka(2) = 3.0 X 10(10) l.mol-1. Cytosol prepared without molybdate did not contain a binding protein class with a higher Ka. The effect is stereo-specific and the LT4 bond is not displaced by DT4.

  10. Different in vitro and in vivo activity of low Mr phosphotyrosine protein phosphatase on epidermal growth factor receptor.

    Science.gov (United States)

    Rigacci, S; Marzocchini, R; Bucciantini, M; Berti, A

    1998-09-29

    Low Mr phosphotyrosine protein phosphatase is a cytosolic enzyme which dephosphorylates platelet-derived growth factor and insulin receptor in vivo, thus reducing cellular mitogenic response to such growth factors. Following cell stimulation with platelet-derived growth factor the phosphatase undergoes a redistribution from the citosol to the Triton X-100-insoluble fraction where its activity upon the growth factor receptor is intense. Previous research uncovered evidence that low Mr phosphotyrosine protein phosphatase dephosphorylates the epidermal growth factor receptor in vitro. Here we demonstrate that in vivo the enzyme is not active on the phosphorylated epidermal growth factor receptor and it does not influence the mitogenic response of cells. Since the enzyme distribution is not affected by epidermal growth factor stimulation, involvement of a recruitment mechanism in the definition of low Mr phosphotyrosine protein phosphatase substrate specificity is hypothesized. Copyright 1998 Academic Press.

  11. A quantitative comparison of cytosolic delivery via different protein uptake systems

    NARCIS (Netherlands)

    Verdurmen, W.P.R.; Mazlami, M.; Pluckthun, A.

    2017-01-01

    Over many years, a variety of delivery systems have been investigated that have the capacity to shuttle macromolecular cargoes, especially proteins, into the cytosol. Due to the lack of an objective way to quantify cytosolic delivery, relative delivery efficiencies of the various transport systems

  12. Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit.

    Science.gov (United States)

    Herington, A C; Ymer, S; Roupas, P; Stevenson, J

    1986-04-11

    Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.

  13. An assay for clogging the ciliary pore complex distinguishes mechanisms of cytosolic and membrane protein entry.

    Science.gov (United States)

    Takao, Daisuke; Dishinger, John F; Kee, H Lynn; Pinskey, Justine M; Allen, Ben L; Verhey, Kristen J

    2014-10-06

    As a cellular organelle, the cilium contains a unique protein composition. Entry of both membrane and cytosolic components is tightly regulated by gating mechanisms at the cilium base; however, the mechanistic details of ciliary gating are largely unknown. We previously proposed that entry of cytosolic components is regulated by mechanisms similar to those of nuclear transport and is dependent on nucleoporins (NUPs), which comprise a ciliary pore complex (CPC). To investigate ciliary gating mechanisms, we developed a system to clog the pore by inhibiting NUP function via forced dimerization. We targeted NUP62, a component of the central channel of the nuclear pore complex (NPC), for forced dimerization by tagging it with the homodimerizing Fv domain. As proof of principle, we show that forced dimerization of NUP62-Fv attenuated (1) active transport of BSA into the nuclear compartment and (2) the kinesin-2 motor KIF17 into the ciliary compartment. Using the pore-clogging technique, we find that forced dimerization of NUP62 attenuated the gated entry of cytosolic proteins but did not affect entry of membrane proteins or diffusional entry of small cytosolic proteins. We propose a model in which active transport of cytosolic proteins into both nuclear and ciliary compartments requires functional NUPs of the central pore, whereas lateral entry of membrane proteins utilizes a different mechanism that is likely specific to each organelle's limiting membrane. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    Science.gov (United States)

    Böldicke, Thomas

    2017-05-01

    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. © 2017 The Protein Society.

  15. Protein abundance profiling of the Escherichia coli cytosol

    DEFF Research Database (Denmark)

    Ishihama, Y.; Schmidt, T.; Rappsilber, J.

    2008-01-01

    proteins. Proteins involved in energy metabolism as well as those with binding function were also found in high copy number while proteins annotated with the terms metabolism, transcription, transport, and cellular organization were rare. The barrel-sandwich fold was found to be the structural fold......Background: Knowledge about the abundance of molecular components is an important prerequisite for building quantitative predictive models of cellular behavior. Proteins are central components of these models, since they carry out most of the fundamental processes in the cell. Thus far, protein...... protein and mRNA abundance in E. coli cells. Conclusion: Abundance measurements for more than 1000 E. coli proteins presented in this work represent the most complete study of protein abundance in a bacterial cell so far. We show significant associations between the abundance of a protein and its...

  16. Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver.

    Science.gov (United States)

    Hu, M L; Viljoen, A J; Tappel, A L

    1988-04-01

    Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.

  17. Enhanced proteomic analysis of Streptomyces peucetius cytosolic protein using optimized protein solubilization protocol.

    Science.gov (United States)

    Lee, Kwang Won; Song, Eunjung; Kim, June-Hyung; Lee, Hei Chan; Liou, Kwang Kyoung; Sohng, Jae Kyung; Kim, Byung-Gee

    2007-01-01

    Improvements in the dissolution of proteins in two-dimensional gel electrophoresis have greatly advanced the ability to analyze the proteomes of microorganisms under a wide variety of physiological conditions. This study examined the effect of various combinations of chaotropic agents, a reducing agent, and a detergent on the dissolution of the Streptomyces peucetius cytosolic proteins. The use of urea alone in a rehydration buffer as a chaotropic agent gave the proteome a higher solubility than any of the urea and thiourea combinations, and produced the highest resolution and clearest background in two-dimensional gel electrophoresis. Two % CHAPS, as a detergent in a rehydration buffer, improved the protein solubility. After examining the effect of several concentrations of reducing agent, 50 mM DTT in a rehydration buffer was found to be an optimal condition for the proteome analysis of Streptomyces. Using this optimized buffer condition, more than 2,000 distinct and differentially expressed soluble proteins could be resolved using two-dimensional gel electrophoresis with a pI ranging from 4-7. Under this optimized condition, 15 novel small proteins with low-level expression, which could not be analyzed under the non-optimized conditions, were identified. Overall, the optimized condition helped produce a better reference gel for Streptomyces peucetius.

  18. Diversity of cytosolic HSP70 Heat Shock Protein from decapods and their phylogenetic placement within Arthropoda.

    Science.gov (United States)

    Baringou, Stephane; Rouault, Jacques-Deric; Koken, Marcel; Hardivillier, Yann; Hurtado, Luis; Leignel, Vincent

    2016-10-10

    The 70kDa heat shock proteins (HSP70) are considered the most conserved members of the HSP family. These proteins are primordial to the cell, because of their implications in many cellular pathways (e. g., development, immunity) and also because they minimize the effects of multiple stresses (e. g., temperature, pollutants, salinity, radiations). In the cytosol, two ubiquitous HSP70s with either a constitutive (HSC70) or an inducible (HSP70) expression pattern are found in all metazoan species, encoded by 5 or 6 genes (Drosophila melanogaster or yeast and human respectively). The cytosolic HSP70 protein family is considered a major actor in environmental adaptation, and widely used in ecology as an important biomarker of environmental stress. Nevertheless, the diversity of cytosolic HSP70 remains unclear amongst the Athropoda phylum, especially within decapods. Using 122 new and 311 available sequences, we carried out analyses of the overall cytosolic HSP70 diversity in arthropods (with a focus on decapods) and inferred molecular phylogenies. Overall structural and phylogenetic analyses showed a surprisingly high diversity in cytosolic HSP70 and revealed the existence of several unrecognised groups. All crustacean HSP70 sequences present signature motifs and molecular weights characteristic of non-organellar HSP70, with multiple specific substitutions in the protein sequence. The cytosolic HSP70 family in arthropods appears to be constituted of at least three distinct groups (annotated as A, B and C), which comprise several subdivisions, including both constitutive and inducible forms. Group A is constituted by several classes of Arthropods, while group B and C seem to be specific to Malacostraca and Hexapoda/Chelicerata, respectively. The HSP70 organization appeared much more complex than previously suggested, and far beyond a simple differentiation according to their expression pattern (HSC70 versus HSP70). This study proposes a new classification of cytosolic

  19. The presence of a protein activator of sarcolemmal polyphosphoinositide phospholipase C in cardiac cytosol

    Energy Technology Data Exchange (ETDEWEB)

    Quist, E.E.; Kriewaldt, S.D.; Powell, P.B. (Texas College of Osteopathic Medicine, Fort Worth (USA))

    1989-01-01

    To study polyphosphoinositide phospholipase (PL) C, isolated sarcolemmal membranes were preincubated with Mg({sup 32}P)-ATP to label phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-diphosphate (PIP{sub 2}). After washing, PLC activity was determined by measuring the release of {sup 32}P-labeled inositol diphosphate (IP{sub 2}) and/or inositol trisphospate (IP{sub 3}) from membrane PIP and PIP{sub 2} during incubation at 25{degree}C and pH 7.4. Increasing concentrations of Ca{sup 2+} (0-100 {mu}M) increased IP{sub 2} by 100% over the 0 Ca{sup 2+} control levels. Ca{sup 2+} dependent PLC hydrolyzed both PIP and PIP{sub 2} with apparent D{sub A}'s of approximately 0.5 and 70 {mu}M. Addition of dialyzed cytosol further increased IP{sub 2} release by 250% without affecting the K{sub A}'s for Ca{sup 2+} activation. The cytosolic activator was partially purified by DEAE Sephacel chromatography was heat labile and sensitive to trypsin pretreatment identifying it as a protein. In contrast, 10 mM NaF increased the Ca{sup 2+} affinity for PLC 2-fold. These results show that cardiac sarcolemma possess a membrane bound Ca{sup 2+} dependent PLC activity which is regulated by a cytosolic protein activator and a G protein. The cytosolic activator would potentially amplify the amount of sarcolemmal polyphosphoinositides hydrolyzed by PLC in response to muscarinic receptor activation by acetylcholine. In addition, activation of PLC by NaF or other G protein activators could result from increasing the Ca{sup 2+} affinity of PLC to physiological intracellular Ca{sup 2+} levels.

  20. Bacterial cytosolic proteins with a high capacity for Cu(I) that protect against copper toxicity

    Science.gov (United States)

    Vita, Nicolas; Landolfi, Gianpiero; Baslé, Arnaud; Platsaki, Semeli; Lee, Jaeick; Waldron, Kevin J.; Dennison, Christopher

    2016-12-01

    Bacteria are thought to avoid using the essential metal ion copper in their cytosol due to its toxicity. Herein we characterize Csp3, the cytosolic member of a new family of bacterial copper storage proteins from Methylosinus trichosporium OB3b and Bacillus subtilis. These tetrameric proteins possess a large number of Cys residues that point into the cores of their four-helix bundle monomers. The Csp3 tetramers can bind a maximum of approximately 80 Cu(I) ions, mainly via thiolate groups, with average affinities in the (1-2) × 1017 M-1 range. Cu(I) removal from these Csp3s by higher affinity potential physiological partners and small-molecule ligands is very slow, which is unexpected for a metal-storage protein. In vivo data demonstrate that Csp3s prevent toxicity caused by the presence of excess copper. Furthermore, bacteria expressing Csp3 accumulate copper and are able to safely maintain large quantities of this metal ion in their cytosol. This suggests a requirement for storing copper in this compartment of Csp3-producing bacteria.

  1. Cytosolic HSP90 associates with and modulates the Arabidopsis RPM1 disease resistance protein.

    Science.gov (United States)

    Hubert, David A; Tornero, Pablo; Belkhadir, Youssef; Krishna, Priti; Takahashi, Akira; Shirasu, Ken; Dangl, Jeffery L

    2003-11-03

    The Arabidopsis protein RPM1 activates disease resistance in response to Pseudomonas syringae proteins targeted to the inside of the host cell via the bacterial type III delivery system. We demonstrate that specific mutations in the ATP-binding domain of a single Arabidopsis cytosolic HSP90 isoform compromise RPM1 function. These mutations do not affect the function of related disease resistance proteins. RPM1 associates with HSP90 in plant cells. The Arabidopsis proteins RAR1 and SGT1 are required for the action of many R proteins, and display some structural similarity to HSP90 co-chaperones. Each associates with HSP90 in plant cells. Our data suggest that (i) RPM1 is an HSP90 client protein; and (ii) RAR1 and SGT1 may function independently as HSP90 cofactors. Dynamic interactions among these proteins can regulate RPM1 stability and function, perhaps similarly to the formation and regulation of animal steroid receptor complexes.

  2. Characterization of protein quality control components via dual reporter-containing misfolded cytosolic model substrates.

    Science.gov (United States)

    Amm, Ingo; Kawan, Mona; Wolf, Dieter H

    2016-12-15

    Protein misfolding and protein aggregation are causes of severe diseases as neurodegenerative disorders, diabetes and cancer. Therefore, the cell has to constantly monitor the folding status of its proteome. Chaperones and components of the ubiquitin-proteasome system are key players in the cellular protein quality control process. In order to characterize components of the protein quality control system in a well-established model eukaryote - the yeast Saccharomyces cerevisiae - we established new cytosolic model substrates based on firefly luciferase and β-isopropylmalate dehydrogenase (Leu2). The use of these two different enzymes arranged in tandem as reporters enabled us to analyse the folding status and the degradation propensity of these new model substrates in yeast cells mutated in components of the cellular protein quality control system. The Hsp70 chaperone system known to be essential in the cellular protein quality control was chosen as a model for showing the high value of the luciferase-based model substrates in the characterization of components of the cytosolic protein quality control system in yeast. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez, M.L.; Tellez-Inon, M.T. (Instituto de Ingenieria Genetica y Biologia Molecular, Buenos Aires (Argentina)); Medrano, E.E.; Cafferatta, E.G.A. (Instituto de Investigaciones Bioquimicas Fundacion Campomar, Buenos Aires (Argentina))

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

  4. Oil and protein accumulation in developing seeds is influenced by the expression of a cytosolic pyrophosphatase in Arabidopsis.

    Science.gov (United States)

    Meyer, Knut; Stecca, Kevin L; Ewell-Hicks, Kim; Allen, Stephen M; Everard, John D

    2012-07-01

    This study describes a dominant low-seed-oil mutant (lo15571) of Arabidopsis (Arabidopsis thaliana) generated by enhancer tagging. Compositional analysis of developing siliques and mature seeds indicated reduced conversion of photoassimilates to oil. Immunoblot analysis revealed increased levels of At1g01050 protein in developing siliques of lo15571. At1g01050 encodes a soluble, cytosolic pyrophosphatase and is one of five closely related genes that share predicted cytosolic localization and at least 70% amino acid sequence identity. Expression of At1g01050 using a seed-preferred promoter recreated most features of the lo15571 seed phenotype, including low seed oil content and increased levels of transient starch and soluble sugars in developing siliques. Seed-preferred RNA interference-mediated silencing of At1g01050 and At3g53620, a second cytosolic pyrophosphatase gene that shows expression during seed filling, led to a heritable oil increase of 1% to 4%, mostly at the expense of seed storage protein. These results are consistent with a scenario in which the rate of mobilization of sucrose, for precursor supply of seed storage lipid biosynthesis by cytosolic glycolysis, is strongly influenced by the expression of endogenous pyrophosphatase enzymes. This emphasizes the central role of pyrophosphate-dependent reactions supporting cytosolic glycolysis during seed maturation when ATP supply is low, presumably due to hypoxic conditions. This route is the major route providing precursors for seed oil biosynthesis. ATP-dependent reactions at the entry point of glycolysis in the cytosol or plastid cannot fully compensate for the loss of oil content observed in transgenic events with increased expression of cytosolic pyrophosphatase enzyme in the cytosol. These findings shed new light on the dynamic properties of cytosolic pyrophosphate pools in developing seed and their influence on carbon partitioning during seed filling. Finally, our work uniquely demonstrates that

  5. Abnormal proteins of shortened length are preferentially degraded in the cytosol of cultured MRC5 fibroblasts.

    Science.gov (United States)

    Wharton, S A; Hipkiss, A R

    1984-03-12

    Puromycyl peptides were degraded in MRC5 fibroblasts more rapidly than normal proteins labelled for the corresponding length of time for both long and short labelling periods. The degradation of the puromycyl peptides occurred almost exclusively in the cytosol of the cells. Even when the half-lives of normal and puromycyl peptides were manipulated to be similar, proportionally more of the normal proteins were degraded in the lysosomes. The rapid degradation of the puromycyl peptides was not due to the inhibition of protein synthesis brought about by puromycin but was due to the structure of the substrates themselves. The degree and intracellular site of degradation of puromycyl peptides closely mimic those of abnormal (missense) proteins containing amino acid analogues.

  6. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Sasnauskas Kęstutis

    2011-05-01

    Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of

  7. Cytosolic fatty acid-binding proteins: subjects and tools in metabolic research

    Energy Technology Data Exchange (ETDEWEB)

    Binas, B. [Max Delbrueck Center for Molecular Medicine, Berlin-Buch (Germany)

    1998-12-31

    Fatty acid-binding proteins (FABPs) are major targets for specific binding of fatty acids in vivo. They constitute a widely expressed family of genetically related, small cytosolic proteins which very likely mediate intracellular transport of free long chain fatty acids. Genetic inhibition of FABP expression in vivo should therefore provide a useful tool to investigate and engineer fatty acid metabolism. (orig.) [Deutsch] Fettsaeurebindungsproteine (FABPs) sind wichtige Bindungsstellen fuer Fettsaeuren in vivo; sie bilden eine breit exprimierte Familie genetisch verwandter kleiner Zytosoleiweisse, die sehr wahrscheinlich den intrazellulaeren Transport unveresterter langkettiger Fettsaeuren vermitteln. Die genetische Hemmung der FABP-Expanssion in vivo bietet sich deshalb als Werkzeug zur Erforschung und gezielten Veraenderung des Fettsaeurestoffwechsels an. (orig.)

  8. Caerulein causes translocation of protein kinase C in rat acini without increasing cytosolic free Ca2+.

    Science.gov (United States)

    Bruzzone, R; Regazzi, R; Wollheim, C B

    1988-07-01

    We investigated the relationships between changes in cytosolic free Ca2+ ([Ca2+]i) and amylase secretion in dispersed rat pancreatic acini. Although 10 pM caerulein did not raise [Ca2+]i, higher concentrations (1 nM) of the peptide elicited a prompt, marked, but transient (2-3 min) elevation of [Ca2+]i. Both concentrations of caerulein caused an almost identical release of amylase over a 30-min period. To investigate the mechanism(s) underlying Ca2+-independent secretion, we measured the effect of the secretagogue on protein kinase C activity and found that both caerulein concentrations caused a significant translocation of protein kinase C from the cytosolic to the microsomal fraction. Because 1 nM caerulein induced a greater enzyme secretion than 10 pM caerulein during the first 2-5 min of stimulation, we explored further the role of [Ca2+]i transients during the first minutes of secretion. Addition of ionomycin in the presence of 10 pM caerulein resulted in a rise in [Ca2+]i and enhanced secretion as a result of caerulein in a near additive fashion during the first 2 min of stimulation. Second, we pretreated acini for 5 min with 1 microM 12-O-tetradecanoylphorbol-13-acetate. This maneuver inhibited both caerulein-induced inositol trisphosphate formation and [Ca2+]i elevation. These findings were paralleled by a similar inhibition of caerulein-stimulated amylase release only during the first 5 min of secretion. These results indicate that 1) caerulein can stimulate amylase secretion independently of a concomitant [Ca2+]i rise, possibly by activation of protein kinase C, and 2) an elevation of [Ca2+]i serves as a trigger to enhance amylase release only during the initial phase of secretion.

  9. Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio [Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela (Spain); Bermejo-Barrera, Pilar [Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias s/n, 15782 Santiago de Compostela (Spain)], E-mail: pbermejo@usc.es; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad [Department of Analytical Chemistry, Faculty of Sciences, University of A Coruna, Campus da Zapateira s/n, 15071 A Coruna (Spain); Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario [Department of Analytical Chemistry, Faculty of Sciences, University of A Coruna, Campus da Zapateira s/n, 15071 A Coruna (Spain); University Institute of Environment, University of A Coruna, Pazo de Longora, Lians, 15179 Oleiros (Spain)

    2007-11-05

    The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices.

  10. Acyl-CoA binding protein and epidermal barrier function

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Neess, Ditte; Færgeman, Nils J

    2014-01-01

    includes tousled and greasy fur, development of alopecia and scaling of the skin with age. Furthermore, epidermal barrier function is compromised causing a ~50% increase in transepidermal water loss relative to that of wild type mice. Lipidomic analyses indicate that this is due to significantly reduced...

  11. [Isolation of tropomyosin particles from the cytosol of cultured cells and their protein composition analysis].

    Science.gov (United States)

    Bobkov, D E; Aĭzenshtadt, A A; Kropacheva, I V; Pinaev, G P

    2012-01-01

    The presence of actin-binding protein, tropomyosin, shaped as particles or protein complexes that have no bonds with actin structures were found while the analisys of structural rearrangements of actin cytoskeleton. However, their functioning is still unknown. To study the composition and properties of these protein complexes a novel method of their separation from the cells without destroying the structures of the cytoskeleton have been developed. The protein composition of isolated tropomyosin particles has been analised by gel filtration, electrophoresis and Western blotting. They appeared to be a multimolecular complexes of about 700 kDa. Beside the tropomyosin and actin these complexes also contain the Hsp70, Hsp90 and myosin-9 identified by mass spectrometry analisys. Also, under inhibition of deacetylases by trichostatin A, changes in the number of particles and redistribution of tropomyosin between cytosol and cytoskeleton take place along with actin cytoskeleton rearrangements. The results obtained give a reason to assume that these multimolecular complexes may participate in the process of reorganization of the actin microfilaments.

  12. Amino acid starvation has opposite effects on mitochondrial and cytosolic protein synthesis.

    Directory of Open Access Journals (Sweden)

    Mark A Johnson

    Full Text Available Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.

  13. Amino Acid Starvation Has Opposite Effects on Mitochondrial and Cytosolic Protein Synthesis

    Science.gov (United States)

    Pearce, Sarah F.; Rorbach, Joanna; He, Jiuya; Brea-Calvo, Gloria; Minczuk, Michal; Reyes, Aurelio; Holt, Ian J.; Spinazzola, Antonella

    2014-01-01

    Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation. PMID:24718614

  14. Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family

    NARCIS (Netherlands)

    Titorenko, Vladimir I.; Evers, Melchior E.; Diesel, Andre; Samyn, Bart; Beeumen, Josef van; Roggenkamp, Rainer; Kiel, Jan A.K.W.; Klei, Ida J. van der; Veenhuis, Marten

    We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was

  15. Occurrence and sequence of a DnaJ protein in plant (Allium porrum) epidermal cells.

    Science.gov (United States)

    Bessoule, J J

    1993-05-24

    Antibodies raised against a purified fraction from microsomal membranes of leek epidermal cells were used to screen a lambda zap expression library from epidermal cells of leak plants. A near full-length clone was isolated. This cDNA contains an open reading-frame of 1,191 bp coding for a DnaJ protein (leek DNAJ 1 or LDJ1). Leek DnaJ1 represents the second protein of this type described in a pluricellular organism, the first being that sequenced from human cells.

  16. The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.

    Science.gov (United States)

    Patterson, Melissa A; Bandyopadhyay, Anannya; Devaraneni, Prasanna K; Woodward, Josha; Rooney, LeeAnn; Yang, Zhongying; Skach, William R

    2015-11-27

    Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Urinary excretion of Tamm-Horsfall protein and epidermal growth factor in chronic nephropathy

    DEFF Research Database (Denmark)

    Torffvit, O; Jørgensen, P E; Kamper, A L

    1998-01-01

    Tamm-Horsfall protein (THP) and epidermal growth factor (EGF) are both synthesized by tubular cells in the distal part of the nephron and excreted with the urine. The present study examines the urinary excretion rates of the two peptides in relation to functional tubular markers in patients with ...

  18. Lack of upregulation of epidermal fatty acid binding protein in dithranol induced irritation.

    NARCIS (Netherlands)

    Kucharekova, M.; Vissers, W.H.P.M.; Schalkwijk, J.; Kerkhof, P.C.M. van de; Valk, P.G.M. van der

    2003-01-01

    The exact role of epidermal fatty acid binding protein (E-FABP) in skin is unknown. A restoration of the barrier function may be associated with an upregulation of E-FABP. Moreover, E-FABP is upregulated in a variety of cells in response to oxidative stress. A recent observation that dithranol

  19. Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins.

    Science.gov (United States)

    Cesari, Stella; Moore, John; Chen, Chunhong; Webb, Daryl; Periyannan, Sambasivam; Mago, Rohit; Bernoux, Maud; Lagudah, Evans S; Dodds, Peter N

    2016-09-06

    Plants possess intracellular immune receptors designated "nucleotide-binding domain and leucine-rich repeat" (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively.

  20. [Experimental studies on seborrheic alopecia. II. Determination of the structural differences between testosterone transported cytosol protein of the bald and hairy areas].

    Science.gov (United States)

    Bassas, E

    1975-01-01

    By previous isolation of the testosterone transport cytosol protein in bald and hairy areas in patients with male pattern baldness, the author studies the differences concerning the protein citosol-testosterone complex, after fixation by means of labelled testosterone and electrophoresis, measuring the corresponding radioactivity curves. The results show that there are important structural differences for the cytosol protein between the hairy and bald regions.

  1. Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

    Science.gov (United States)

    Shi, Yunhua; Mowery, Richard A; Ashley, Jonathan; Hentz, Michelle; Ramirez, Alejandro J; Bilgicer, Basar; Slunt-Brown, Hilda; Borchelt, David R; Shaw, Bryan F

    2012-01-01

    The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of “gel shifting” has perplexed biochemists and biologists for decades. We identify a mechanism for “gel shifting” that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues ∼80–101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80–101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80–101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic). PMID:22692797

  2. Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells.

    Science.gov (United States)

    Balasubramanyam, M; Gardner, J P

    1995-12-01

    Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca2+i) extrusion and 45Ca2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca2+i extrusion were further explored. PMA significantly increased the rate of Ca2+ extrusion in TG-treated cells from 0.28 +/- 0.02 to 0.35 +/- 0.03 s-1 (mean +/- SEM) and stimulated the initial rate of 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i

  3. Tetanus toxin inhibits neurotensin-induced mobilization of cytosolic protein kinase C activity in NG-108 cells.

    Science.gov (United States)

    Considine, R V; Handler, C M; Simpson, L L; Sherwin, J R

    1991-01-01

    There is considerable literature on the pathogenesis of tetanus toxin poisoning; however, the mechanism of action and intracellular substrate of this toxin have not been defined. It was demonstrated that the NG-108 neuroblastoma x glioma cell line is a suitable model in which to study the mechanism of tetanus toxin action, from binding of the toxin to inhibition of transmitter release. Further, it has been shown that tetanus toxin pretreatment attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C (PKC) in this cell line. In the present study a 4-hr tetanus toxin pretreatment (10(-10)-10(-13) M) completely inhibited the mobilization of cytosolic PKC induced by a 30-min exposure to 10 microM neurotensin. Pretreatment with 10(-10) M tetanus toxin for periods as short as 1 hr was sufficient to attenuate the ability of neurotensin to mobilize cytosolic PKC; however, a 30-min pretreatment had no significant effect. At a concentration of 10(-11) M, it was necessary to pretreat the cells for greater than 1 hr to significantly attenuate neurotensin-mobilized PKC activity. The exact role that PKC plays in the secretory process is not yet known; however, these findings suggest that the effect of tetanus toxin on neurotransmitter release is accompanied by an alteration in PKC metabolism in differentiated NG-108 cells.

  4. Cytosol protein regulation in H295R steroidogenesis model induced by the zearalenone metabolites, α- and β-zearalenol.

    Science.gov (United States)

    Busk, Øyvind L; Frizzell, Caroline; Verhaegen, Steven; Uhlig, Silvio; Connolly, Lisa; Ropstad, Erik; Sørlie, Morten

    2012-01-01

    α- and β-zearalenol (α-ZOL and β-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of α-ZOL and β-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by α-ZOL and β-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis thaliana.

    Science.gov (United States)

    Verma, Amit K; Diwan, Danish; Raut, Sandeep; Dobriyal, Neha; Brown, Rebecca E; Gowda, Vinita; Hines, Justin K; Sahi, Chandan

    2017-06-07

    Heat shock proteins of 70 kDa (Hsp70s) partner with structurally diverse Hsp40s (J proteins), generating distinct chaperone networks in various cellular compartments that perform myriad housekeeping and stress-associated functions in all organisms. Plants, being sessile, need to constantly maintain their cellular proteostasis in response to external environmental cues. In these situations, the Hsp70:J protein machines may play an important role in fine-tuning cellular protein quality control. Although ubiquitous, the functional specificity and complexity of the plant Hsp70:J protein network has not been studied. Here, we analyzed the J protein network in the cytosol of Arabidopsis thaliana and, using yeast genetics, show that the functional specificities of most plant J proteins in fundamental chaperone functions are conserved across long evolutionary timescales. Detailed phylogenetic and functional analysis revealed that increased number, regulatory differences, and neofunctionalization in J proteins together contribute to the emerging functional diversity and complexity in the Hsp70:J protein network in higher plants. Based on the data presented, we propose that higher plants have orchestrated their "chaperome," especially their J protein complement, according to their specialized cellular and physiological stipulations. Copyright © 2017 Verma et al.

  6. Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3'-deoxyribonucleotidase and partially inhibits its activity.

    Directory of Open Access Journals (Sweden)

    Chiu-Ping Fang

    Full Text Available Infection with hepatitis C virus (HCV is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3 is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3'-deoxyribonucleotidase (cdN, dNT-1 was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

  7. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Veenhuis Marten

    2005-10-01

    Full Text Available Abstract Background Native as well as recombinant bacterial cell surface layer (S-layer protein of Geobacillus (G. stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099 of the S-layer protein and EGFP (enhanced green fluorescent protein, in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. Results Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S. cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE promoter. Conclusion The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of

  8. Cytosolic SYT/SS18 isoforms are actin-associated proteins that function in matrix-specific adhesion.

    Directory of Open Access Journals (Sweden)

    Jaehong Kim

    2009-07-01

    Full Text Available SYT (SYnovial sarcoma Translocated gene or SS18 is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

  9. Measuring protein-bound glutathioine (PSSG): Critical correction for cytosolic glutathione species

    Science.gov (United States)

    Introduction: Protein glutathionylation is gaining recognition as an important posttranslational protein modification. The common first step in measuring protein glutathionylation is the denaturation and precipitation of protein away from soluble, millimolar quantities of glutathione (GSH) and glut...

  10. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    Science.gov (United States)

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-07-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

  11. Phosphorylation of the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor is required for the interaction with a cytosolic protein

    DEFF Research Database (Denmark)

    Rosorius, O; Issinger, O G; Braulke, T

    1993-01-01

    ) and to 35- and 91-kDa proteins salt-washed from bovine brain membranes. Gel filtration suggested that TIP 35 is part of a higher molecular mass complex of approximately 130-150 kDa. Inhibition studies, using non-phosphorylated and phosphorylated MPR 300-CT and cross-linking, indicate that the interaction...... with a cytosolic protein depending on the phosphorylation by a casein kinase II-like kinase. The cross-linking with salt-washed membrane proteins, however, is inhibited by non-phosphorylated MPR 300-CT, suggesting that different structural determinants in the MPR 300-CT interact with cytosol- and membrane...

  12. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    Science.gov (United States)

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Cytosolic Phospholipase A2 Protein as a Novel Therapeutic Target for Spinal Cord Injury

    Science.gov (United States)

    Liu, Nai-Kui; Deng, Ling-Xiao; Zhang, Yi Ping; Lu, Qing-Bo; Wang, Xiao-Fei; Hu, Jian-Guo; Oakes, Eddie; Bonventre, Joseph V; Shields, Christopher B; Xu, Xiao-Ming

    2014-01-01

    Objective The objective of this study was to investigate whether cytosolic phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). Methods A combination of molecular, histological, immunohistochemical, and behavioral assessments were used to test whether blocking cPLA2 activation pharmacologically or genetically reduced cell death, protected spinal cord tissue, and improved behavioral recovery after a contusive SCI performed at the 10th thoracic level in adult mice. Results SCI significantly increased cPLA2 expression and activation. Activated cPLA2 was localized mainly in neurons and oligodendrocytes. Notably, the SCI-induced cPLA2 activation was mediated by the extracellular signal-regulated kinase signaling pathway. In vitro, activation of cPLA2 by ceramide-1-phosphate or A23187 induced spinal neuronal death, which was substantially reversed by arachidonyl trifluoromethyl ketone, a cPLA2 inhibitor. Remarkably, blocking cPLA2 pharmacologically at 30 minutes postinjury or genetically deleting cPLA2 in mice ameliorated motor deficits, and reduced cell loss and tissue damage after SCI. Interpretation cPLA2 may play a key role in the pathogenesis of SCI, at least in the C57BL/6 mouse, and as such could be an attractive therapeutic target for ameliorating secondary tissue damage and promoting recovery of function after SCI. PMID:24623140

  14. Bacteria modulate the CD8+ T cell epitope repertoire of host cytosol-exposed proteins to manipulate the host immune response.

    Directory of Open Access Journals (Sweden)

    Yaakov Maman

    2011-10-01

    Full Text Available The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.

  15. Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

    Science.gov (United States)

    Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Geo, Jini; Dilipkumar, Shilpa; Pasricha, Renu; Gulyani, Akash; Raghavan, Srikala; Palakodeti, Dasaradhi

    2017-09-01

    Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. © 2017. Published by The Company of Biologists Ltd.

  16. Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

    2012-09-01

    Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+)-transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance.

    Science.gov (United States)

    Wang, Guan-Feng; Wei, Xuening; Fan, Renchun; Zhou, Huanbin; Wang, Xianping; Yu, Chunmei; Dong, Lingli; Dong, Zhenying; Wang, Xiaojie; Kang, Zhensheng; Ling, Hongqing; Shen, Qian-Hua; Wang, Daowen; Zhang, Xiangqi

    2011-07-01

    Heat shock protein 90 (Hsp90) molecular chaperones play important roles in plant growth and responses to environmental stimuli. However, little is known about the genes encoding Hsp90s in common wheat. Here, we report genetic and functional analysis of the genes specifying cytosolic Hsp90s in this species. Three groups of homoeologous genes (TaHsp90.1, TaHsp90.2 and TaHsp90.3), encoding three types of cytosolic Hsp90, were isolated. The loci containing TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes were assigned to groups 2, 7 and 5 chromosomes, respectively. TaHsp90.1 genes exhibited higher transcript levels in the stamen than in the leaf, root and culm. TaHsp90.2 and TaHsp90.3 genes were more ubiquitously transcribed in the vegetative and reproductive organs examined. Decreasing the expression of TaHsp90.1 genes through virus-induced gene silencing (VIGS) caused pronounced inhibition of wheat seedling growth, whereas the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the hypersensitive resistance response of the wheat variety Suwon 11 to stripe rust fungus. Our work represents the first systematic determination of wheat genes encoding cytosolic Hsp90s, and provides useful evidence for the functional involvement of cytosolic Hsp90s in the control of seedling growth and disease resistance in common wheat. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  18. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.

    Directory of Open Access Journals (Sweden)

    Constantin F Urban

    2009-10-01

    Full Text Available Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs. NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

  19. Human epidermal keratinocytes death and expression of protein markers of apoptosis after ionizing radiation exposure

    Directory of Open Access Journals (Sweden)

    Sharon Wong

    2013-12-01

    Full Text Available Purpose: Knowledge of the pathophysiology of the irradiated skin is important to understand the tolerance and cosmetic response of the human skin to radiation. There are limited studies on the effect of radiotherapy dosage and fraction size in inducing apoptotic cell death in human skin. The expression of apoptotic biomarkers within a controlled population in different fractionation schemes has also never been studied. This study aims to investigate radiation induced apoptotic cell death in human skin cells after fractionated radiation exposure and the expression of unique biomarkers that reflect cell death or biology using multiplexed immunoassays.Methods: Breast skin biopsies were obtained from a single individual and divided into small pieces. Each piece was irradiated under different radiotherapy treatment fractionation schedules to a total dose of 50Gy. The irradiated skin tissues were analysed using Tunnel, immunohistochemistry and Western blot assays for expression of apoptotic keratinocytes and biomarkers (p53, p21, and PCNA. Haematoxylin and eosin (H&E immunostaining was performed to study the morphological changes in the skin cells. Results: Radiation is mostly absorbed by the epidermal layers and observed to damage the epidermal keratinocytes leading to the activation of apoptotic proteins. Apoptotic proteins (p53, p21 and PCNA were confirmed to be up-regulated in radiation exposed skin cells as compared to normal skin cells with no radiation. There is strong correlation of apoptotic protein expressions with increased radiation dosage and dose fractionation. Statistical analysis with ANOVA revealed a significant increase of PCNA and p21 expression with increased radiation dosage and dose fractionation (p < 0.05. Immunohistochemically, 14 % (range 10.71% to 17.29% of the keratinocytes were positive for PCNA and 22.5% (range 18.28% to 27.2% for p21 after 2Gy of irradiation.  The most widespread, intense and uniform staining for PCNA

  20. Cytosolic Hsp70/Hsc70 protein expression in lymphocytes exposed to low gamma-ray dose

    Energy Technology Data Exchange (ETDEWEB)

    Manzanares A, E.; Vega C, H.R.; Letechipia de Leon, C. [Unidades Academicas de Estudios Nucleares, UAZ, A.P. 336, 98000 Zacatecas (Mexico)]. E-mail: emanz@cantera.reduaz.mx; Guzman E, L.J. [Unidad Academica de Biologia Experimental, Guadalupe, Zacatecas (Mexico); Garcia T, M. [LIBRA, Centro I and D, Campus Miguel Delibes, Valladolid 47011 (Spain)

    2004-07-01

    The purpose of this study was to evaluate the effect of low gamma ray intensity upon Hsp70 expression in human Iymphocytes. The heat shock proteins (Hsp) family, are a group of proteins present in all living organism, therefore there are highly conserved and are related to adaptation and evolution. At cellular level these proteins acts as chaperones correcting denatured proteins. When a stress agent, such heavy metals, UV, heat, etc. is affecting a cell a response to this aggression is triggered only through over expression of Hsp. Several studies has been carried out in which the cellular effect are observed, mostly of these studies uses large doses, but very few studies are related with low doses. Blood of healthy volunteers was obtained and the Iymphocytes were isolated by ficoll- histopaque gradient. Experimental lots were irradiated in a {sup 137} Cs gamma-ray. Hsp70 expression was found since 0.5 c Gy, indicating a threshold to very low doses of gamma rays. (Author)

  1. Proteomic comparison of the cytosolic proteins of three Bifidobacterium longum human isolates and B. longum NCC2705

    Directory of Open Access Journals (Sweden)

    Champomier-Vergès Marie-Christine

    2010-01-01

    Full Text Available Abstract Background Bifidobacteria are natural inhabitants of the human gastrointestinal tract. In full-term newborns, these bacteria are acquired from the mother during delivery and rapidly become the predominant organisms in the intestinal microbiota. Bifidobacteria contribute to the establishment of healthy intestinal ecology and can confer health benefits to their host. Consequently, there is growing interest in bifidobacteria, and various strains are currently used as probiotic components in functional food products. However, the probiotic effects have been reported to be strain-specific. There is thus a need to better understand the determinants of the observed benefits provided by these probiotics. Our objective was to compare three human B. longum isolates with the sequenced model strain B. longum NCC2705 at the chromosome and proteome levels. Results Pulsed field electrophoresis genotyping revealed genetic heterogeneity with low intraspecies strain relatedness among the four strains tested. Using two-dimensional gel electrophoresis, we analyzed qualitative differences in the cytosolic protein patterns. There were 45 spots that were present in some strains and absent in others. Spots were excised from the gels and subjected to peptide mass fingerprint analysis for identification. The 45 spots represented 37 proteins, most of which were involved in carbohydrate metabolism and cell wall or cell membrane synthesis. Notably, the protein patterns were correlated with differences in cell membrane properties like surface hydrophobicity and cell agglutination. Conclusion These results showed that proteomic analysis can be valuable for investigating differences in bifidobacterial species and may provide a better understanding of the diversity of bifidobacteria and their potential use as probiotics.

  2. Plasma beads loaded with Candida albicans cytosolic proteins impart protection against the fungal infection in BALB/c mice.

    Science.gov (United States)

    Ahmad, Ejaj; Fatima, Munazza T; Saleemuddin, M; Owais, M

    2012-11-06

    The development of a prophylactic vaccine against systemic candidiasis, employing Candida albicans cytosolic proteins (Cp) as antigen and fibrin cross-linked plasma beads as an antigen bearing dual delivery system is described. Groups of mice were administered either with free Cp, or Cp entrapped in plasma beads, Cp entrapped in liposomes or liposome encapsulated Cp further entrapped in plasma beads. Humoral immunity was studied by measuring the anti-Cp antibody titers in the sera of the immunized animals. Induction of cell-mediated immunity was assessed by delayed type hypersensitivity (DTH), NO production, up-regulation of co-stimulatory molecules viz. CD80, CD86 on APCs on one hand and T-cells proliferation as well as induction of IFN-γ and IL-4 on the other. The efficacy of various vaccine formulations in protecting mice against a lethal challenge with C. albicans, was assessed by determining animal survival rate and fungal burden in the systemic circulation and vital organs. Among various Cp-based vaccines investigated, the preparation containing liposomized Cp entrapped in plasma beads imparted superior protection in the immunized mice as compared to other antigens delivery systems. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Epidermal hyperproliferation in mice lacking fatty acid transport protein 4 (FATP4) involves ectopic EGF receptor and STAT3 signaling

    OpenAIRE

    Lin, Meei-Hua; Chang, Kuo-Wei; Lin, Shu-Chun; Miner, Jeffrey H.

    2010-01-01

    Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective epidermal barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene-driven expression of FATP4 solely in suprabasal keratinocytes. Here we show that Fatp4 mutants exhibit epidermal hyperplasia resulting from an incre...

  4. Computational Exploration of Natural Compounds to Target Cytosolic Phospholipase A 2 Protein: A Novel Therapeutic Target for Spinal Cord Injury.

    Science.gov (United States)

    Fan, Hongwu; Wang, Shengqun; Zhao, Qiheng; Qin, Zhigang

    2018-01-22

    Cytosolic phospholipase A2 (cPLA2), an important isoform of PLA2 that mediates the release of arachidonic acid, plays a role in the pathogenesis of spinal cord injury (SCI). The expression and activation of Cpla2is significantly higher in SCI, leading to neuronal death in spinal cord tissue. Novel strategies are needed to substantially reverse the effect of cPLA2 activation; one such strategy is inhibiting cPLA2 by jamming its lipid binding C2 domain. To develop a much needed strategy to treat SCI we used a computer aided drug design (CADD) method to discover novel cPLA2 inhibitors. we used a natural chemiome database for virtual screening, from which we selected the compounds exhibiting the greatest drug-likeliness properties for molecular docking simulation analysis. We studied the interaction of lead compounds at the atomic level; the results yielded a cPLA2 inhibitor of natural origin with the potential for ameliorating secondary tissue damage and promoting recovery of function after SCI. The top compound, lead 4exibited a binding energy of -10.02 Kcal/mol and formed three hydrogen bonds with the lipid binding C2 domain of the cPLA2 protein. An evaluation of cell cytotoxicity revealed an IC50 for lead4 of 134.2 ± 6.8 µM. An in-vitro analysis of lead4 is indicated anti-apoptotic activity via a decrease in caspase-3 expression. We used the CADD method to make a novel lead discovery for the treatment of SCIusing compounds of natural origin. The selected natural compounds are non-toxic promising drugs against cPLA2 protein, allowing us to limits our focus on single compound for future in-vitro and in-vivo testing. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Forkhead box protein O3 transcription factor negatively regulates autophagy in human cancer cells by inhibiting forkhead box protein O1 expression and cytosolic accumulation.

    Directory of Open Access Journals (Sweden)

    Wan Long Zhu

    Full Text Available FoxO proteins are important regulators in cellular metabolism and are recognized to be nodes in multiple signaling pathways, most notably those involving PI3K/AKT and mTOR. FoxO proteins primarily function as transcription factors, but recent study suggests that cytosolic FoxO1 participates in the regulation of autophagy. In the current study, we find that cytosolic FoxO1 indeed stimulates cellular autophagy in multiple cancer cell lines, and that it regulates not only basal autophagy but also that induced by rapamycin and that in response to nutrient deprivation. These findings illustrate the importance of FoxO1 in cell metabolism regulation independent of its transcription factor function. In contrast to FoxO1, we find the closely related FoxO3a is a negative regulator of autophagy in multiple cancer cell lines, a previously unrecognized function for this protein, different from its function in benign fibroblast and muscle cells. The induction of autophagy by the knockdown of FoxO3a was found not to be mediated through the suppression of mTORC1 signaling; rather, the regulatory role of FoxO3a on autophagy was determined to be through its ability to transcriptionally suppress FoxO1. This complicated interplay of FoxO1 and FoxO3a suggests a complex checks- and balances-relationship between FoxO3a and FoxO1 in regulating autophagy and cell metabolism.

  6. The iron-responsive element (IRE)/iron-regulatory protein 1 (IRP1)–cytosolic aconitase iron-regulatory switch does not operate in plants

    Science.gov (United States)

    Arnaud, Nicolas; Ravet, Karl; Borlotti, Andrea; Touraine, Brigitte; Boucherez, Jossia; Fizames, Cécile; Briat, Jean-François; Cellier, Françoise; Gaymard, Frédéric

    2007-01-01

    Animal cytosolic ACO (aconitase) and bacteria ACO are able to switch to RNA-binding proteins [IRPs (iron-regulatory proteins)], thereby playing a key role in the regulation of iron homoeostasis. In the model plant Arabidopsis thaliana, we have identified three IRP1 homologues, named ACO1–3. To determine whether or not they may encode functional IRP proteins and regulate iron homoeostasis in plants, we have isolated loss-of-function mutants in the three genes. The aco1-1 and aco3-1 mutants show a clear decrease in cytosolic ACO activity. However, none of the mutants is affected in respect of the accumulation of the ferritin transcript or protein in response to iron excess. cis-acting elements potentially able to bind to the IRP have been searched for in silico in the Arabidopsis genome. They appear to be very rare sequences, found in the 5′-UTR (5′-untranslated region) or 3′-UTR of a few genes unrelated to iron metabolism. They are therefore unlikely to play a functional role in the regulation of iron homoeostasis. Taken together, our results demonstrate that, in plants, the cytosolic ACO is not converted into an IRP and does not regulate iron homoeostasis. In contrast with animals, the RNA binding activity of plant ACO, if any, would be more likely to be attributable to a structural element, rather than to a canonical sequence. PMID:17437406

  7. Epidermal hyperproliferation in mice lacking fatty acid transport protein 4 (FATP4) involves ectopic EGF receptor and STAT3 signaling.

    Science.gov (United States)

    Lin, Meei-Hua; Chang, Kuo-Wei; Lin, Shu-Chun; Miner, Jeffrey H

    2010-08-15

    Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective epidermal barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene-driven expression of FATP4 solely in suprabasal keratinocytes. Here we show that Fatp4 mutants exhibit epidermal hyperplasia resulting from an increased number of proliferating suprabasal cells. In addition, barrier formation initiates precociously but never progresses to completion. To investigate possible mechanisms whereby Fatp4 influences skin development, we identified misregulated genes in Fatp4 mutants. Remarkably, three members of the epidermal growth factor (EGF) family (Ereg, Areg, and Epgn) showed increased expression that was associated with elevated epidermal activation of the EGF receptor (EGFR) and STAT3, a downstream effector of EGFR signaling. Both Tyrphostin AG1478, an EGFR tyrosine kinase inhibitor, and curcumin, an inhibitor of both STAT3 and EGFR, attenuated STAT3 activation/nuclear translocation, reduced skin thickening, and partially suppressed the barrier abnormalities. These data identify FATP4 activity as negatively influencing EGFR activation and the resulting STAT3 signaling during normal skin development. These findings have important implications for understanding the pathogenesis of ichthyosis prematurity syndrome, a disease recently shown to be caused by FATP4 mutations. Copyright 2010 Elsevier Inc. All rights reserved.

  8. Cytosolic delivery: Just passing through

    Science.gov (United States)

    Sánchez-Navarro, Macarena; Teixidó, Meritxell; Giralt, Ernest

    2017-08-01

    Intracellular protein delivery has been a major challenge in the field of cell biology for decades. Engineering such delivery is a key step in the development of protein- and antibody-based therapeutics. Now, two different approaches that enable the delivery of antibodies and antibody fragments into the cytosol have been developed.

  9. Forced interaction of cell surface proteins with Derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation.

    Science.gov (United States)

    Cho, Sunglim; Lee, Miriam; Jun, Youngsoo

    2013-01-11

    Aberrantly folded proteins in the endoplasmic reticulum (ER) are rapidly removed into the cytosol for degradation by the proteasome via an evolutionarily conserved process termed ER-associated protein degradation (ERAD). ERAD of a subset of proteins requires Derlin-1 for dislocation into the cytosol; however, the molecular function of Derlin-1 remains unclear. Human cytomegalovirus US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. Because US11 binds to both MHC-I molecules and Derlin-1 via its luminal and transmembrane domains (TMDs), respectively, the major role of US11 has been proposed to simply be delivery of MHC-I molecules to Derlin-1. Here, we directly tested this proposal by generating a hybrid MHC-I molecule, which contains the US11 TMD, and thus can associate with Derlin-1 in the absence of US11. Intriguingly, this MHC-I hybrid was rapidly degraded in a Derlin-1- and proteasome-dependent manner. Similarly, the vesicular stomatitis virus G protein, otherwise expressed at the cell surface, was degraded via Derlin-1-dependent ERAD when its TMD was replaced with that of US11. Thus, forced interaction of cell surface proteins with Derlin-1 is sufficient to induce their degradation via ERAD. Taken together, these results suggest that the main role of US11 is to recruit MHC-I molecules to Derlin-1, which then mediates the dislocation of MHC-I molecules into the cytosol for degradation. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Shadab Anwar

    Full Text Available Iron-Sulfur (Fe-S proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1 protein and Nucleotide binding protein 35 (Nbp35. In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151 of Nbp35 and (G5-V6, M34-D39 and G46-A52 of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins

  11. Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

    Science.gov (United States)

    Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

    2014-01-01

    Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any

  12. Profiling of cytosolic and mitochondrial H2O2production using the H2O2-sensitive protein HyPer in LPS-induced microglia cells.

    Science.gov (United States)

    Park, Junghyung; Lee, Seunghoon; Lee, Hyun-Shik; Lee, Sang-Rae; Lee, Dong-Seok

    2017-07-27

    Dysregulation of the production of pro-inflammatory mediators in microglia exacerbates the pathologic process of neurodegenerative disease. ROS actively affect microglia activation by regulating transcription factors that control the expression of pro-inflammatory genes. However, accurate information regarding the function of ROS in different subcellular organelles has not yet been established. Here, we analyzed the pattern of cytosolic and mitochondrial H 2 O 2 formation in LPS-activated BV-2 microglia using the H 2 O 2- sensitive protein HyPer targeted to specific subcellular compartments. Our results show that from an early time, cytosolic H 2 O 2 started increasing constantly, whereas mitochondrial H 2 O 2 rapidly increased later. In addition, we found that MAPK affected cytosolic H 2 O 2 , but not mitochondrial H 2 O 2 . Consequently, our study provides the basic information about subcellular H 2 O 2 generation in activated microglia, and a useful tool for investigating molecular targets that can modulate neuroinflammatory responses. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. The stress response against denatured proteins in the deletion of cytosolic chaperones SSA1/2 is different from heat-shock response in Saccharomyces cerevisiae

    Science.gov (United States)

    Matsumoto, Rena; Akama, Kuniko; Rakwal, Randeep; Iwahashi, Hitoshi

    2005-01-01

    Background A yeast strain lacking the two genes SSA1 and SSA2, which encode cytosolic molecular chaperones, acquires thermotolerance as well as the mild heat-shocked wild-type yeast strain. We investigated the genomic response at the level of mRNA expression to the deletion of SSA1/2 in comparison with the mild heat-shocked wild-type using cDNA microarray. Results Yeast cDNA microarray analysis revealed that genes involved in the stress response, including molecular chaperones, were up-regulated in a similar manner in both the ssa1/2 deletion mutant and the mild heat-shocked wild-type. Genes involved in protein synthesis were up-regulated in the ssa1/2 deletion mutant, but were markedly suppressed in the mild heat-shocked wild-type. The genes involved in ubiquitin-proteasome protein degradation were also up-regulated in the ssa1/2 deletion mutant, whereas the unfolded protein response (UPR) genes were highly expressed in the mild heat-shocked wild-type. RT-PCR confirmed that the genes regulating protein synthesis and cytosolic protein degradation were up-regulated in the ssa1/2 deletion mutant. At the translational level, more ubiquitinated proteins and proteasomes were detected in the ssa1/2 deletion mutant, than in the wild-type, confirming that ubiquitin-proteasome protein degradation was up-regulated by the deletion of SSA1/2. Conclusion These results suggest that the mechanism for rescue of denatured proteins in the ssa1/2 deletion mutant is different from that in the mild heat-shocked wild-type: Activated protein synthesis in the ssa1/2 deletion mutant supplies a deficiency of proteins by their degradation, whereas mild heat-shock induces UPR. PMID:16209719

  14. Mechanical stretch stimulates protein kinase B/Akt phosphorylation in epidermal cells via angiotensin II type 1 receptor and epidermal growth factor receptor.

    Science.gov (United States)

    Kippenberger, Stefan; Loitsch, Stefan; Guschel, Maike; Müller, Jutta; Knies, Yvonne; Kaufmann, Roland; Bernd, August

    2005-01-28

    Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-oncogene protein kinase B (PKB)/Akt at both phosphorylation sites (serine 473/threonine 308) in different epithelial cells (HaCaT, A-431, and human embryonic kidney-293). Blocking of phosphoinositide 3-OH kinase by selective inhibitors (LY-294002 and wortmannin) abrogated the stretch-induced PKB/Akt phosphorylation. Furthermore mechanical stretch stimulated phosphorylation of epidermal growth factor receptor (EGFR) and the formation of EGFR membrane clusters. Functional blocking of EGFR phosphorylation by either selective inhibitors (AG1478 and PD168393) or dominant-negative expression suppressed stretch-induced PKB/Akt phosphorylation. Finally, the angiotensin II type 1 receptor (AT1-R) was shown to induce positive transactivation of EGFR in response to cell stretch. These findings define a novel signaling pathway of mechanical stretch, namely the activation of PKB/Akt by transactivation of EGFR via angiotensin II type 1 receptor. Evidence is provided that stretch-induced activation of PKB/Akt protects cells against induced apoptosis.

  15. The Campylobacter jejuni CiaC virulence protein is secreted from the flagellum and delivered to the cytosol of host cells

    Directory of Open Access Journals (Sweden)

    Jason eNeal-McKinney

    2012-03-01

    Full Text Available Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute C. jejuni-mediated disease (campylobacteriosis involves C. jejuni invasion of host epithelial cells using a set of virulence proteins known as the Campylobacter invasion antigens (Cia. The genes encoding the Cia proteins are up-regulated upon co-culture of C. jejuni with epithelial cells. One of the Cia proteins, CiaC, is required for maximal invasion of host cells by C. jejuni. Previous work has also revealed that CiaC is, in part, responsible for host cell cytoskeletal rearrangements that result in membrane ruffling. This study was performed to test the hypothesis that CiaC is delivered to the cytosol of host cells. To detect the delivery of CiaC into cultured epithelial cells, we used the adenylate cyclase domain (ACD of Bordetella pertussis CyaA as a reporter. In this study, we found that export and delivery of the C. jejuni Cia proteins into human INT 407 epithelial cells required a functional flagellar hook complex composed of FlgE, FlgK, and FlgL. Assays performed with bacterial culture supernatants supported the hypothesis that CiaC delivery requires bacteria-host cell contact. We also found that that CiaC was delivered to host cells by cell-associated (bound bacteria, as judged by experiments performed with inhibitors that specifically target the cell signaling pathways utilized by C. jejuni for cell invasion. Interestingly, the C. jejuni flgL mutant, which is incapable of exporting and delivering the Cia proteins, did not induce INT 407 cell membrane ruffles. Complementation of the flgL mutant with plasmid-encoded flgL restored the motility and membrane ruffling. These data support the hypothesis that the C. jejuni Cia proteins, which are exported from the flagellum, are delivered to the cytosol of host cells.

  16. Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor

    DEFF Research Database (Denmark)

    Sanchez-Quiles, Virginia; Akimov, Vyacheslav; Osinalde, Nerea

    2017-01-01

    Cylindromatosis tumor suppressor protein (CYLD) is a deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within...... spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors...

  17. Impaired cellular responses to cytosolic DNA or infection with Listeria monocytogenes and vaccinia virus in the absence of the murine LGP2 protein.

    Directory of Open Access Journals (Sweden)

    Darja Pollpeter

    2011-04-01

    Full Text Available Innate immune signaling is crucial for detection of and the initial response to microbial pathogens. Evidence is provided indicating that LGP2, a DEXH box domain protein related to the RNA recognition receptors RIG-I and MDA5, participates in the cellular response to cytosolic double-stranded DNA (dsDNA. Analysis of embryonic fibroblasts and macrophages from mice harboring targeted disruption in the LGP2 gene reveals that LGP2 can act as a positive regulator of type I IFN and anti-microbial gene expression in response to transfected dsDNA. Results indicate that infection of LGP2-deficient mice with an intracellular bacterial pathogen, Listeria monocytogenes, leads to reduced levels of type I IFN and IL12, and allows increased bacterial growth in infected animals, resulting in greater colonization of both spleen and liver. Responses to infection with vaccinia virus, a dsDNA virus, are also suppressed in cells lacking LGP2, reinforcing the ability of LGP2 to act as a positive regulator of antiviral signaling. In vitro mechanistic studies indicate that purified LGP2 protein does not bind DNA but instead mediates these responses indirectly. Data suggest that LGP2 may be acting downstream of the intracellular RNA polymerase III pathway to activate anti-microbial signaling. Together, these findings demonstrate a regulatory role for LGP2 in the response to cytosolic DNA, an intracellular bacterial pathogen, and a DNA virus, and provide a plausible mechanistic hypothesis as the basis for this activity.

  18. Fluorescent pH probes, fluorescent proteins, and intrinsic cellular fluorochromes are tools to study cytosolic pH (pHcyt) in mammalian cells.

    Science.gov (United States)

    Martinez, Gloria M.; Gollahon, Lauren S.; Shafer, Keri; Oomman, Sowmini K.; Busch, Christian; Martinez-Zaguilan, Raul

    2001-07-01

    Our understanding of intracellular pH homeostatis in eukaryotic systems has been enhanced since the introduction of carboxyfluorescein diacetate as a useful pH probe more than 20 years ago. BCECF, a derivative of this earlier fluoroprobe has dominated the field. In the past 10 years, SNARF-1 has emerged as an alternative pH probe. Recently, a novel derivative of BCECF, BCPCF has been developed. Green Fluorescent Proteins (GFPs) have also been used recently to monitor pH in a non invasive manner in several cell types. Here, we report that human mammary epithelial cells can be transfected with the gene encoding for cyan (CFP), green (GFP), and yellow (YFP), to study cytosolic pH. The novel red fluorescent protein (DsRed) is not sensitive to pH. Multidrug resistance (MDR) has been associated with altered cytosolic pH homeostasis. We show that experimental maneuvers that decrease pHin enhance the efficacy of chemotherapeutic drugs. We also show that short pulses of UV-B light elicited acidosis in cells, as evaluated by ratio ion cell imaging, and confocal/spectral imaging microscopy. During the course of these experiments we noticed that cells exhibit intrinsic fluorochromes that can be used to monitor pH in living cells.

  19. Isolation and characterization of binding proteins for retinol from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens.

    Science.gov (United States)

    Rao, M R; Prasad, V R; Padmanaban, G; Ganguly, J

    1979-01-01

    Protein fractions that bind retinol were isolated from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens. The proteins isolated from the three sources showed similar elution profiles on chromatography through Sephadex G-75 and G-50 columns, and comparable mobility during electrophoresis on sodium dodecyl sulphate/polyacrylamide gels. Their molecular weights were calculated to be around 14500. When oviducts from vitamin A-depleted and vitamin A-repleted immature chicks given oestrogen injections for 6 consecutive days were incubated with [3H]retinyl acetate, uptake of the radioactivity in the nuclei of the vitamin A-depleted tissue was severalfold higher than that in the nuclei from the vitamin A-repleted tissue. Images Fig. 3. PMID:575485

  20. Overexpression of a cytosolic abiotic stress responsive universal stress protein (SbUSP mitigates salt and osmotic stress in transgenic tobacco plants

    Directory of Open Access Journals (Sweden)

    Pushpika eUdawat

    2016-04-01

    Full Text Available The Universal Stress Protein (USP is a ubiquitous protein and plays an indispensable role in plant abiotic stress tolerance. The genome of Salicornia brachiata contains two homologues of intron less SbUSP gene which encodes for salt and osmotic responsive universal stress protein. In vivo localization reveals that SbUSP is a membrane bound cytosolic protein. The role of the gene was functionally validated by developing transgenic tobacco and compared with control (wild type and vector control plants under different abiotic stress condition. Transgenic lines (T1 exhibited higher chlorophyll, relative water, proline, total sugar, reducing sugar, free amino acids, polyphenol contents, osmotic potential, membrane stability and lower electrolyte leakage and lipid peroxidation (malondialdehyde content under stress treatments than control (WT and VC plants. Lower accumulation of H2O2 and O2- radicals was also detected in transgenic lines compared to control plants under stress conditions. Present study confers that overexpression of the SbUSP gene enhances plant growth, alleviates ROS buildup, maintains ion homeostasis and improves the physiological status of the plant under salt and osmotic stresses. Principal component analysis (PCA exhibited a statistical distinction of plant response to salinity stress, and a significant response was observed for transgenic lines under stress, which provides stress endurance to the plant. A possible signaling role is proposed that some downstream genes may get activated by abiotic stress responsive cytosolic SbUSP, which leads to the protection of cell from oxidative damages. The study unveils that ectopic expression of the gene mitigates salt or osmotic stress by scavenging ROS and modulating the physiological process of the plant.

  1. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lone , Abdul G.; Atci, Erhan; Renslow, Ryan S.; Beyenal, Haluk; Noh, S.; Fransson, B.; Abu-Lail, Nehal; Park, Jeong-Jin; Gang, David R.; Call, Douglas R.

    2015-08-31

    A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using MSE mass spectrometry. We found that S. aureus biofilm grows predominantly in sebum-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2-3 fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after four days of culture. The colonized explants released significantly (P< 0.01) more anti-oxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentration found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in media from infected explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

  2. Modeling and simulation of aggregation of membrane protein LAT with molecular variability in the number of binding sites for cytosolic Grb2-SOS1-Grb2.

    Directory of Open Access Journals (Sweden)

    Ambarish Nag

    Full Text Available The linker for activation of T cells (LAT, the linker for activation of B cells (LAB, and the linker for activation of X cells (LAX form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1:1 and 2:1 complexes with the guanine nucleotide exchange factor SOS1. The 2:1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate. We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

  3. Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.

    Directory of Open Access Journals (Sweden)

    V Chandana Epa

    Full Text Available Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2. HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

  4. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR AND HUMAN PAPILLOMAVIRUS (HPV L1 CAPSID PROTEIN IN CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS

    Directory of Open Access Journals (Sweden)

    Balan Raluca

    2010-09-01

    Full Text Available We analyzed the immunohistochemical pattern of epidermal growth factor receptor (EGFR in cervical squamous intraepithelial lesions (SILs in correlation with L1 HPV capsid protein, in order to determine the relationship between EGFR expression and the infection status of human papillomavirus (HPV. The study included 40 cases, 24 LSIL (low grade SIL (CIN1, cervical intraepithelial neoplasia and 16 HSIL (high grade SIL (6 cases of CIN2 and 10 cases of CIN3. The immunoexpression of L1 HPV protein was assessed on conventional cervico-vaginal smears and EGFR was immunohistochemically evaluated on the corresponding cervical biopsies. The HPV L1 capsid protein was expressed in 45.83% of LSIL and 25% of HSIL. EGFR was overexpressed in 62,4% of HSIL (58,4% CIN2 and 41,6% CIN3 and 37,6% LSIL. The immunoexpression of L1 HPV has clinical application in the progression assessment of the cervical precancerous lesions without a correlation to the grade of the cervical SIL. EGFR is expressed by all proliferating squamous epithelial cells, thus corresponding with the grade of SIL. The evaluation of EGFR status, correlated with L1 HPV protein expression, can provide useful data of progression risk of cervical squamous intraepithelial lesions

  5. Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    González-Méndez Ricardo

    2009-05-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways. Results In this work we describe a new G protein α subunit gene in S. schenckii, ssg-2. The cDNA sequence of ssg-2 revealed a predicted open reading frame of 1,065 nucleotides encoding a 355 amino acids protein with a molecular weight of 40.9 kDa. When used as bait in a yeast two-hybrid assay, a cytoplasmic phospholipase A2 catalytic subunit was identified as interacting with SSG-2. The sspla2 gene, revealed an open reading frame of 2538 bp and encoded an 846 amino acid protein with a calculated molecular weight of 92.62 kDa. The principal features that characterize cPLA2 were identified in this enzyme such as a phospholipase catalytic domain and the characteristic invariable arginine and serine residues. A role for SSPLA2 in the control of dimorphism in S. schenckii is suggested by observing the effects of inhibitors of the enzyme on the yeast cell cycle and the yeast to mycelium transition in this fungus. Phospholipase A2 inhibitors such as AACOCF3 (an analogue of archidonic acid and isotetrandrine (an inhibitor of G protein

  6. Cytosolic YB-1 and NSUN2 are the only proteins recognizing specific motifs present in mRNAs enriched in exosomes.

    Science.gov (United States)

    Kossinova, Olga A; Gopanenko, Alexander V; Tamkovich, Svetlana N; Krasheninina, Olga A; Tupikin, Alexey E; Kiseleva, Elena; Yanshina, Darya D; Malygin, Alexey A; Ven'yaminova, Alia G; Kabilov, Marsel R; Karpova, Galina G

    2017-06-01

    Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC. RNA hairpins other than those mentioned above pull out neither YB-1 nor NSUN2. Both these proteins are found in exosomes secreted by HEK293 cells. YB-1 for all that is detected as a form having a slightly higher electrophoretic mobility than that of YB-1 associated with the above RNA hairpins, assuming changes in posttranslational modifications of the protein during its transfer from cytoplasm into exosomes. Next generation sequencing of total exosomal RNA (eRNA) reveals a large representative set of RNA species, including mRNAs containing the above-mentioned motifs. The degree of enrichment in exosomes with this kind of mRNAs strongly depends on the locations of eRNA-specific motifs within the mRNA sequences. Altogether, our findings point to YB-1 and NSUN2 as possible mediators of the process of transfer of specific mRNAs into exosomes, allowing us to speculate on an involvement of these proteins in the mRNA sorting via the recognition of the above motifs. Copyright © 2017. Published by Elsevier B.V.

  7. In Vivo Cosmetic Product Efficacy Testing by Analyzing Epidermal Proteins Extracted from Tape Strips

    Directory of Open Access Journals (Sweden)

    Marie Westman

    2014-02-01

    Full Text Available The objective of this in vivo pilot study was to investigate whether differential biomarker analysis from skin tape strips could be used, not only to evaluate the difference between treated and untreated skin, but also to evaluate the effect of different product treatments. Ten volunteers were included in the study, applying two different basic formulations on their forearms. After four weeks of product application, and also after one week of treatment remission, tape strips were collected from the different treatment sites, as well as from untreated skin. The biomarkers investigated were selected to cover different aspects of epidermal differentiation and in connection with moisturization and barrier function. Levels of Involucrin were increased in both treatments, compared to untreated skin, whereas the levels of Keratin-6 were decreased for both treatments. In addition, a pattern for increased levels of Hornerin and Claudin-1 was also detected. There were no significant differences between the two treatments, only for treatment compared to untreated, but there were tendencies for different effect on some of the biomarkers investigated, differences that may reach significance with increased sample size. The major differences between the two treatments in this study were seen after one week of product remission, although due to too small sample size these differences were not significant.

  8. ZmHSP16.9, a cytosolic class I small heat shock protein in maize (Zea mays), confers heat tolerance in transgenic tobacco.

    Science.gov (United States)

    Sun, Liping; Liu, Yang; Kong, Xiangpei; Zhang, Dan; Pan, Jiaowen; Zhou, Yan; Wang, Li; Li, Dequan; Yang, Xinghong

    2012-08-01

    Various organisms produce HSPs in response to high temperature and other stresses. The function of heat shock proteins, including small heat shock protein (sHSP), in stress tolerance is not fully explored. To improve our understanding of sHSPs, we isolated ZmHSP16.9 from maize. Sequence alignments and phylogenetic analysis reveal this to be a cytosolic class I sHSP. ZmHSP16.9 expressed in root, leaf and stem tissues under 40 °C treatment, and was up-regulated by heat stress and exogenous H₂O₂. Overexpression of ZmHSP16.9 in transgenic tobacco conferred tolerance to heat and oxidative stresses by increased seed germination rate, root length, and antioxidant enzyme activities compared with WT plants. These results support the positive role of ZmHSP16.9 in response to heat stress in plant. The overexpression of ZmHSP16.9 enhanced tolerance to heat and oxidative stress in transgenic tobacco.

  9. A mutational analysis of the cytosolic domain of the tomato Cf-9 disease-resistance protein shows that membrane-proximal residues are important for Avr9-dependent necrosis.

    Science.gov (United States)

    Chakrabarti, Apratim; Velusamy, Thilaga; Tee, Choon Yang; Jones, David A

    2016-05-01

    The tomato Cf-9 gene encodes a membrane-anchored glycoprotein that imparts race-specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N-terminal half of the extracellular leucine-rich repeat (eLRR) domain of the Cf-9 protein determines its specificity for Avr9, the C-terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf-9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9-dependent necrosis triggered by Cf-9. Our results indicate that the membrane-proximal region of the cytosolic domain of Cf-9 plays an important role in Cf-9-mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf-9 function. An alanine mutation of T835 had no effect on Cf-9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf-9 function. We therefore postulate that phosphorylation/de-phosphorylation of T835 could act as a molecular switch to determine whether Cf-9 is in a primed or inactive state. Yeast two-hybrid analysis was used to show that the cytosolic domain of Cf-9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline-induced kink in the membrane-proximal region of the cytosolic domain of Cf-9 may be important for interaction with VAP27, which may, in turn, be important for Cf-9 function. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  10. Amination of tyrosine in liver cytosol protein of male F344 rats treated with 2-nitropropane, 2-nitrobutane, 3-nitropentane, or acetoxime.

    Science.gov (United States)

    Sodum, R S; Fiala, E S

    1997-12-01

    Previously, the secondary nitroalkane 2-nitropropane, a strong hepatocarcinogen in rats, had been shown to induce the formation of 8-aminoguanine in both DNA and RNA of rat liver through a sulfotransferase-mediated pathway. This pathway was postulated to convert the carcinogen into an aminating species [Sodum, R. S., et al. (1994) Chem. Res. Toxicol. 7, 344-351]. To submit this postulate to further test, we examined liver proteins of rats treated with 2-nitropropane, other carcinogenic secondary nitroalkanes, or the related rat liver tumorigen acetoxime for the presence of 3-aminotyrosine, the expected product of tyrosine amination. Using ion-pair and/or cation-exchange high-performance liquid chromatography with electrochemical detection, we found that the liver cytosolic proteins of these animals contained 0.1-1.5 mol of 3-aminotyrosine/10(3) mol of tyrosine. Treatment with the noncarcinogenic primary nitroalkane 1-nitropropane or with other primary nitroalkanes did not produce an analogous increase in the aminated amino acid (level of detection estimated at approximately 0.01 mol/10(3) mol of tyrosine). To our knowledge, this is the first report of the modification of protein tyrosine in vivo by a carcinogen. In vitro studies with acetoxime-O-sulfonate and hydroxylamine-O-sulfonate showed that these proposed intermediates in the activation pathway of 2-nitropropane react with guanosine to give 8-aminoguanosine, N1-aminoguanosine, and 8-oxoguanosine and also react with tyrosine to give 3-aminotyrosine and 3-hydroxytyrosine. The in vitro amination and oxidation of guanosine at C8 were also produced by acetophenoxime-O-sulfonate and 2-heptanoxime-O-sulfonate. These results provide additional evidence for the production of a reactive species capable of aminating nucleic acids and proteins from 2-nitropropane and other carcinogenic secondary nitroalkanes by a pathway involving oxime- and hydroxylamine-O-sulfonates as intermediates.

  11. Analysis of Egg Cytosol Proteins in the Polyploid Ginbuna, Carassius auratus langsdorfii, by Two-Dimensional Electrophoresis.

    OpenAIRE

    土方, 誠; 梶島, 孝雄

    1984-01-01

    To investigate the mechanisms of gynogenetic development of ginbuna, Carassius auratus langsdorfii, the protein constitution of growing oocytes, full-grown oocytes, matured eggs and fertilized eggs obtained from triploid ginbuna and diploid funa were analyzed by two-dimensional gel electrophoresis. The results are as follows ; (1) the one polypeptide which exists in bisexual diploid funa and exceptional triploid ginbuna is lacked in gynogenetic triploid ginbuna which does not decondense the p...

  12. Transforming growth factor-beta and epidermal growth factor modulate basal and interleukin-6-induced amino acid uptake and acute phase protein synthesis in cultured rat hepatocytes.

    Science.gov (United States)

    Bereta, J; Szuba, K; Fiers, W; Gauldie, J; Koj, A

    1990-06-18

    Rat hepatocytes cultured for 2 days with interleukin-6 show increased synthesis of acute phase proteins and enhanced accumulation of 14C-labelled alpha-aminoisobutyric acid. Transforming growth factor-beta 1 (0.1-10 ng/ml) inhibits whereas epidermal growth factor (1-100 ng/ml) enhances both basal and interleukin-6-induced amino acid uptake by rat hepatocytes with only a slight alteration of acute phase protein synthesis.

  13. Impact of epidermal growth factor receptor protein and gene alteration on Taiwanese hepatocellular carcinomas.

    Science.gov (United States)

    Su, Yu-Hung; Ng, Kwai-Fong; Yu, Ming-Chin; Wu, Ting-Jung; Yeh, Ta-Sen; Lee, Wei-Chen; Lin, Yong-Shiang; Hsieh, Tsung-Han; Lin, Chun-Yen; Yeh, Chau-Ting; Chen, Tse-Ching

    2015-09-01

    Epidermal growth factor receptor (EGFR) overexpression is associated with disease progression and poor survival in a variety of solid tumors. The role of EGFR in hepatocellular carcinoma (HCC) remains controversial. One hundred thirty-eight HCCs were analyzed for total EGFR (t-EGFR) and phospho-EGFR (p-EGFR) expression and gene amplification using immunohistochemistry and fluorescence in situ hybridization. The role of EGFR was analyzed in relation to the clinicopathological features. Weak to strong p-EGFR immunostaining was noted in 42 of the 138 HCCs. p-EGFR expression correlated with alcoholism (P = 0.03) and chronic hepatitis B infection (P = 0.041). There was no correlation between t-EGFR expression and any of the clinicopathological features. Amplification of the EGFR gene was not identified in the 138 HCCs, but 39.1% of the HCCs showed balanced polysomy of both the EGFR gene and centromere 7. Moreover, 65 tumors showed > 2.2 copies per tumor cell. EGFR copy number gain (CNG) was significantly correlated with gender (P = 0.0491), tumor grade (P = 0.006), and vascular invasion (P = 0.005). HCCs with EGFR CNG also had a poor recurrence-free survival (RFS), as compared with HCCs without EGFR CNG (P = 0.031). When exploring the impact of gender, a significant association of EGFR CNG was found with tumor grade (P = 0.044) and cirrhosis (P = 0.015) exclusively in the male group only; however, the OS and RFS analysis show no significant difference between male and female groups. EGFR CNG was related to crucial clinicopathological features and early recurrence, indicating that EGFR CNG might be a poor prognosis factor for Taiwanese HCC. © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  14. The acyl-CoA binding protein is required for normal epidermal barrier function in mice

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Bek, Signe; Marcher, Ann-Britt

    2012-01-01

    The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling...

  15. Portraying the Effect of Calcium-Binding Proteins on Cytosolic Calcium Concentration Distribution Fractionally in Nerve Cells.

    Science.gov (United States)

    Jha, Brajesh Kumar; Joshi, Hardik; Dave, Devanshi D

    2016-11-23

    Nerve cells like neurons and astrocytes in central nervous system (CNS) take part in the signaling process which means the transformation of the information from one cell to another via signals. The signaling process is affected by various external parameters like buffers calcium-binding proteins, voltage-gated calcium channel. In the present paper, the role of buffers in the cytoplasmic calcium concentration distribution is shown. The elicitation in calcium concentration is due to the presence of lower amount calcium-binding proteins which can be shown graphically. The mathematical model is designed by keeping in mind the physiological condition taking place in CNS of mammalian brain. The thing to be noted here is that the more elicitation in the calcium concentration distribution results in the cell death which finally give neurodegenerative disease to the mammalian brain. The present paper gives a glimpse of Parkinson's diseases in particular. Computational results are performed in Wolfram Mathematica 9.0 and simulated on core(TM) i5-3210M CPU @ 2.50 GHz processing speed and 4 GB memory. It is found that the different types of buffer like ethylene glycol-bis([Formula: see text]-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and calmodulin have noteworthy effect at different fractions of time.

  16. Cytosolic delivery of materials with endosome-disrupting colloids

    Science.gov (United States)

    Helms, Brett A.; Bayles, Andrea R.

    2016-03-15

    A facile procedure to deliver nanocrystals to the cytosol of live cells that is both rapid and general. The technique employs a unique cationic core-shell polymer colloid that directs nanocrystals to the cytosol of living cells within a few hours of incubation. The present methods and compositions enable a host of advanced applications arising from efficient cytosolic delivery of nanocrystal imaging probes: from single particle tracking experiments to monitoring protein-protein interactions in live cells for extended periods.

  17. Cytosolic calcium, hydrogen peroxide and related gene expression and protein modulation in Arabidopsis thaliana cell cultures respond immediately to altered gravitation: parabolic flight data.

    Science.gov (United States)

    Hausmann, N; Fengler, S; Hennig, A; Franz-Wachtel, M; Hampp, R; Neef, M

    2014-01-01

    Callus cell cultures of Arabidopsis thaliana (cv. Columbia) were exposed to parabolic flights in order to assess molecular, short-term responses to altered gravity fields. Using transgenic cell lines, hydrogen peroxide (H2 O2 ) and cytosolic Ca(2+) were continuously monitored. In parallel, the metabolism of samples was chemically quenched (RNAlater, Ambion for RNA; acid/base for NADPH, NADP) at typical stages of a parabola [1 g before pull up; end of pull up (1.8 g), end of microgravity (20 s) and end of pull out (1.8 g)]. Cells exhibited an increase in both Ca(2+) and H2 O2 with the onset of microgravity, and a decline thereafter. This behaviour was accompanied by a decrease of the NADPH/NADP redox ratio, indicating Ca(2+) -dependent activation of a NADPH oxidase. Microarray analyses revealed concomitant expression profiles. At the end of the microgravity phase, 396 transcripts were specifically up-, while 485 were down-regulated. Up-regulation was dominated by Ca(2+) - and ROS-related gene products. The same material was also used for analysis of phosphopeptides with 2-D SDS PAGE. Relevant spots were identified by liquid chromatography-MS. With the exception of a chaperone (HSP 70-3), hypergravity (1.8 g) and microgravity modified different sets of proteins. These are partly involved in primary metabolism (glycolysis, gluconeogenesis, citrate cycle) and detoxification of ROS. Taken together, these data show that both gene expression and protein modulation jointly respond within seconds to alterations in the gravity field, with a focus on metabolic adaptation, signalling and control of ROS. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  18. Effect of oxytocin on expression of cytosolic phospholipase A2 mRNA and protein in ovine endometrial tissue in vivo.

    Science.gov (United States)

    Burns, P D; Graf, G A; Hayes, S H; Silvia, W J

    2000-11-01

    The induction of endometrial prostaglandin (PG) F2alpha synthesis by oxytocin is dependent upon activation of phospholipase (PL) A2 and mobilization of arachidonic acid. The objective of this study was to determine if oxytocin stimulates PGF2alpha synthesis by inducing synthesis of cytosolic PLA2 (cPLA2). In Experiment 1, 15 ovariectomized ewes were given progesterone and estradiol to simulate an estrous cycle. Ewes were then given an injection of oxytocin on Day 14 of the simulated estrous cycle. Jugular blood samples were collected and assayed for 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM). Uteri were collected at 0, 7.5, 25, 90, or 240 min postinjection (n = 3 ewes/time point). Total RNA was isolated from caruncular endometrium and subjected to dot-blot analysis. Oxytocin induced a rapid and transient increase in serum PGFM (P 0.10). In Experiment 2, 11 ovary-intact ewes were given oxytocin (n = 5) or saline (n = 6) on Day 15 after estrus. Jugular blood samples were collected and assayed for serum concentrations of PGFM. Uteri were collected at 15 min postinjection. Homogenates were prepared from caruncular endometrium and subjected to Western blot analysis. Concentrations of PGFM were higher in oxytocin treated ewes compared to saline treated ewes at 15 min postinjection (P 0.10). In conclusion, oxytocin did not effect expression of either cPLA2 mRNA or protein in ovine endometrium. Oxytocin may stimulate PGF2alpha synthesis by activating cPLA2 protein that is already present in an inactive form.

  19. Alternative translational initiation of ATP sulfurylase underlying dual localization of sulfate assimilation pathways in plastids and cytosol in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Anne-Sophie eBohrer

    2015-01-01

    Full Text Available Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (ATPS is the enzyme catalyzing the key entry step of the sulfate assimilation pathway in both plastids and cytosol in plants. Arabidopsis thaliana has four ATPS genes (ATPS1, -2, -3 and -4 encoding ATPS pre-proteins containing N-terminal transit peptide sequences for plastid targeting, however, the genetic identity of the cytosolic ATPS has remained unverified. Here we show that Arabidopsis ATPS2 dually encodes plastidic and cytosolic ATPS isoforms, differentiating their subcellular localizations by initiating translation at AUGMet1 to produce plastid-targeted ATPS2 pre-proteins or at AUGMet52 or AUGMet58 within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUGMet52 or AUGMet58 was verified by expressing a tandem-fused synthetic gene, ATPS2(5’UTR-His12:Renilla luciferase:ATPS2(Ile13-Val77:firefly luciferase, under a single constitutively active CaMV 35S promoter in Arabidopsis protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUGMet52 and AUGMet58 significantly reduced the firefly luciferase activity, while AUGMet52 was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUGMet52 or AUGMet58 was not modulated by changes in sulfate conditions. The dual localizations of ATPS2 in plastids and cytosol were further evidenced by expression of ATPS2-GFP fusion proteins in Arabidopsis protoplasts and transgenic lines, while they were also under control of tissue-specific ATPS2 promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots.

  20. Cytosolic glutamine synthetase

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie; Eriksson, Ulf Dennis; Møller, Inge Skrumsager

    2014-01-01

    Overexpression of the cytosolic enzyme glutamine synthetase 1 (GS1) has been investigated in numerous cases with the goal of improving crop nitrogen use efficiency. However, the outcome has generally been inconsistent. Here, we review possible reasons underlying the lack of success and conclude...

  1. PTEN Mediates Activation of Core Clock Protein BMAL1 and Accumulation of Epidermal Stem Cells

    Directory of Open Access Journals (Sweden)

    Chiara Zagni

    2017-07-01

    Full Text Available Tissue integrity requires constant maintenance of a quiescent, yet responsive, population of stem cells. In the skin, hair follicle stem cells (HFSCs that reside within the bulge maintain tissue homeostasis in response to activating cues that occur with each new hair cycle or upon injury. We found that PTEN, a major regulator of the PI3K-AKT pathway, controlled HFSC number and size in the bulge and maintained genomically stable pluripotent cells. This regulatory function is central for HFSC quiescence, where PTEN-deficiency phenotype is in part regulated by BMAL1. Furthermore, PTEN ablation led to downregulation of BMI-1, a critical regulator of adult stem cell self-renewal, and elevated senescence, suggesting the presence of a protective system that prevents transformation. We found that short- and long-term PTEN depletion followed by activated BMAL1, a core clock protein, contributed to accumulation of HFSC.

  2. Epidermal Growth Factor Receptor, Excision-Repair Cross-Complementation Group 1 Protein, and Thymidylate Synthase Expression in Penile Cancer.

    Science.gov (United States)

    Dorff, Tanya B; Schuckman, Anne K; Schwartz, Rachel; Rashad, Sadaf; Bulbul, Ajaz; Cai, Jie; Pinski, Jacek; Ma, Yanling; Danenberg, Kathleen; Skinner, Eila; Quinn, David I

    2016-10-01

    To describe the expression of tissue epidermal growth factor receptor (EGFR), excision-repair cross-complementation group 1 protein (ERCC1), and thymidylate synthase (TS) in patients with penile cancer and explore their association with stage and outcome. A total of 52 patients with penile squamous cell cancer who were treated at the University of Southern California from 1995 to 2010 were identified. Paraffin-embedded tissue underwent mRNA quantitation and immunohistochemistry for expression of EGFR, ERCC1, and TS. KRAS mutations were evaluated using polymerase chain reaction-based sequencing. EGFR overexpression was common by mRNA (median, 5.09; range, 1.92-104.5) and immunohistochemistry. EGFR expression > 7 was associated with advanced stage and poor differentiation (P = .01 and .034 respectively) but not with survival in multivariate analysis. ERCC1 mRNA expression was a median of 0.65 (range, 0.21-1.87). TS expression was a median of 1.88 (range, 0.54-6.47). ERCC1 and TS expression were not associated with grade, stage, or survival. There were no KRAS mutations identified. A total of 17 men received chemotherapy; 8 (47%) had an objective response, including 1 with a pathologic complete response. There was a trend for lower expression of EGFR corresponding to a higher likelihood of response (response rate [RR]) to chemotherapy: 67% RR in EGFR mRNA  7 (P = .31). High expression of EGFR mRNA in squamous cell carcinoma of the penis is associated with advanced stage and poor differentiation, but not survival. In our small heterogeneous subset, molecular marker expression did not show a correlation with the likelihood of chemotherapy response. A prospective evaluation of the role of the EGFR pathway and its regulatory environment in penile cancer is warranted. Given the rarity of this cancer, collaborative prospective cohort evaluations and trials need to be encouraged. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

    Science.gov (United States)

    A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants and H2O2 in media was quantified using microelectrodes. The re...

  4. Protein levels and gene expressions of the epidermal growth factor receptors, HER1, HER2, HER3 and HER4 in benign and malignant ovarian tumors

    DEFF Research Database (Denmark)

    Dahl Steffensen, Karina; Waldstrøm, Marianne; Fredslund Andersen, Rikke

    2008-01-01

    The epidermal growth factor receptors, HER1, HER2, HER3 and HER4 play a key role in the growth of malignant tumors. The receptors of the EGF receptor family are not cancer-specific proteins since these receptors are expressed to some extent in both normal and benign tissue, but this is not elucid......The epidermal growth factor receptors, HER1, HER2, HER3 and HER4 play a key role in the growth of malignant tumors. The receptors of the EGF receptor family are not cancer-specific proteins since these receptors are expressed to some extent in both normal and benign tissue...... ovarian tumors. Tissue from 207 patients (101 malignant, 19 borderline, 64 benign ovarian tumors and 23 normal ovaries) were analyzed by quantitative ELISA for HER1-HER4 protein concentrations and by real-time PCR for HER1-HER4 gene expression. HER2 was also analyzed by immunohistochemistry. The HER2......-4 receptor protein content and the median gene expression level was significantly higher in ovarian cancer patients compared to patients with benign ovarian tumors and normal ovaries (pHER1 receptor was significantly lower in ovarian cancer compared to borderline...

  5. The Arabidopsis cytosolic proteome

    DEFF Research Database (Denmark)

    Ito, Jun; Parsons, Harriet Tempé; Heazlewood, Joshua L.

    2014-01-01

    of secondary metabolites, and accumulation of enzymes for defense and detoxification. This central role is highlighted by estimates indicating that the majority of eukaryotic proteins are cytosolic. Arabidopsis thaliana has been the subject of numerous proteomic studies on its different subcellular...... compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted...

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

    Directory of Open Access Journals (Sweden)

    John G Koland

    2014-01-01

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

  7. [Ca2+]i Elevation and Oxidative Stress Induce KCNQ1 Protein Translocation from the Cytosol to the Cell Surface and Increase Slow Delayed Rectifier (IKs) in Cardiac Myocytes*

    Science.gov (United States)

    Wang, Yuhong; Zankov, Dimitar P.; Jiang, Min; Zhang, Mei; Henderson, Scott C.; Tseng, Gea-Ny

    2013-01-01

    Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca2+]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes. PMID:24142691

  8. [Ca2+]i elevation and oxidative stress induce KCNQ1 protein translocation from the cytosol to the cell surface and increase slow delayed rectifier (IKs) in cardiac myocytes.

    Science.gov (United States)

    Wang, Yuhong; Zankov, Dimitar P; Jiang, Min; Zhang, Mei; Henderson, Scott C; Tseng, Gea-Ny

    2013-12-06

    Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca(2+)]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes.

  9. Evaluation of cultured human dermal- and dermo-epidermal substitutes focusing on extracellular matrix components: Comparison of protein and RNA analysis.

    Science.gov (United States)

    Oostendorp, Corien; Meyer, Sarah; Sobrio, Monia; van Arendonk, Joyce; Reichmann, Ernst; Daamen, Willeke F; van Kuppevelt, Toin H

    2017-05-01

    Treatment of full-thickness skin defects with split-thickness skin grafts is generally associated with contraction and scar formation and cellular skin substitutes have been developed to improve skin regeneration. The evaluation of cultured skin substitutes is generally based on qualitative parameters focusing on histology. In this study we focused on quantitative evaluation to provide a template for comparison of human bio-engineered skin substitutes between clinical and/or research centers, and to supplement histological data. We focused on extracellular matrix proteins since these components play an important role in skin regeneration. As a model we analyzed the human dermal substitute denovoDerm and the dermo-epidermal skin substitute denovoSkin. The quantification of the extracellular matrix proteins type III collagen and laminin 5 in tissue homogenates using western blotting analysis and ELISA was not successful. The same was true for assaying lysyl oxidase, an enzyme involved in crosslinking of matrix molecules. As an alternative, gene expression levels were measured using qPCR. Various RNA isolation procedures were probed. The gene expression profile for specific dermal and epidermal genes could be measured reliably and reproducibly. Differences caused by changes in the cell culture conditions could easily be detected. The number of cells in the skin substitutes was measured using the PicoGreen dsDNA assay, which was found highly quantitative and reproducible. The (dis) advantages of assays used for quantitative evaluation of skin substitutes are discussed. Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.

  10. The non-specific lipid transfer protein N5 of Medicago truncatula is implicated in epidermal stages of rhizobium-host interaction

    Directory of Open Access Journals (Sweden)

    Pii Youry

    2012-12-01

    Full Text Available Abstract Background The symbiotic interaction between leguminous plants and rhizobia involves two processes: bacterial infection, resulting in the penetration of bacteria in epidermal and cortical cells, and root nodule organogenesis. Root nodule symbiosis is activated by rhizobial signalling molecules, called Nodulation factors (NFs. NF perception induces the expression of several genes called early nodulins. The early nodulin N5 of Medicago truncatula is a lipid transfer protein that has been shown to positively regulate nodulation although it displays in vitro inhibitory activity against Sinorhizobium meliloti. The purpose of this work was to investigate the role of MtN5 by studying its spatial and temporal pattern of expression during the symbiotic interaction, also in relation to known components of the symbiotic signalling pathway, and by analysing the phenotypic alterations displayed by rhizobia-inoculated MtN5-silenced roots. Results We show here that MtN5 is a NF-responsive gene expressed at a very early phase of symbiosis in epidermal cells and root hairs. MtN5 expression is induced in vitro by rhizobial effector molecules and by auxin and cytokinin, phytohormones involved in nodule organogenesis. Furthermore, lipid signaling is implicated in the response of MtN5 to rhizobia, since the activity of phospholipase D is required for MtN5 induction in S. meliloti-inoculated roots. MtN5-silenced roots inoculated with rhizobia display an increased root hair curling and a reduced number of invaded primordia compared to that in wild type roots, but with no impairment to nodule primordia formation. This phenotype is associated with the stimulation of ENOD11 expression, an early marker of infection, and with the down-regulation of Flotillin 4 (FLOT4, a protein involved in rhizobial entry. Conclusions These data indicate that MtN5 acts downstream of NF perception and upstream of FLOT4 in regulating pre-infection events. The positive effect of MtN5

  11. Food extracts consumed in Mediterranean countries and East Asia reduce protein concentrations of androgen receptor, phospho-protein kinase B, and phospho-cytosolic phospholipase A(2)alpha in human prostate cancer cells.

    Science.gov (United States)

    Singh, Jaskirat; Xie, Chanlu; Yao, Mu; Hua, Sheng; Vignarajan, Soma; Jardine, Greg; Hambly, Brett D; Sved, Paul; Dong, Qihan

    2010-04-01

    Active surveillance is an emerging management option for the rising number of men with low-grade, clinically localized prostate cancer. However, 30-40% of men on active surveillance will progress to high-grade disease over 5 y. With the ultimate aim of developing a food-based chemoprevention strategy to retard cancer progression in these otherwise healthy men, we have developed a blend of food extracts commonly consumed in Mediterranean countries and East Asia. The effect of the food extracts known as Blueberry Punch (BBP) on prostate cancer cell growth and key signaling pathways were examined in vitro and in vivo. BBP reduced prostate cancer cell growth in a dose-dependent manner (0.08-2.5%) at 72 h in vitro due to the reduction in cell proliferation and viability. Prostate cancer cell xenograft-bearing mice, administered 10% BBP in drinking water for 2 wk, had a 25% reduction in tumor volume compared with the control (water only). In vitro, BBP reduced protein concentrations in 3 signaling pathways necessary for the proliferation and survival of prostate cancer cells, namely androgen receptor, phospho-protein kinase B/protein kinase B, and phospho-cytosolic phospholipase A(2)alpha. The downstream effectors of these pathways, including prostate-specific antigen and glycogen synthase kinase 3beta, were also reduced. Thus, this palatable food supplement is a potential candidate for testing in clinical trials and may ultimately prove effective in retarding the progression of low-grade, early-stage prostate cancer in men managed by active surveillance.

  12. A NIMA-related protein kinase suppresses ectopic outgrowth of epidermal cells through its kinase activity and the association with microtubules.

    Science.gov (United States)

    Motose, Hiroyasu; Tominaga, Rumi; Wada, Takuji; Sugiyama, Munetaka; Watanabe, Yuichiro

    2008-06-01

    To study cellular morphogenesis genetically, we isolated loss-of-function mutants of Arabidopsis thaliana, designated ibo1. The ibo1 mutations cause local outgrowth in the middle of epidermal cells of the hypocotyls and petioles, resulting in the formation of a protuberance. In Arabidopsis, the hypocotyl epidermis differentiates into two alternate cell files, the stoma cell file and the non-stoma cell file, by a mechanism involving TRANSPARENT TESTA GLABRA1 (TTG1) and GLABRA2 (GL2). The ectopic protuberances of the ibo1 mutants were preferentially induced in the non-stoma cell files, which express GL2. TTG1-dependent epidermal patterning is required for protuberance formation in ibo1, suggesting that IBO1 functions downstream from epidermal cell specification. Pharmacological and genetic analyses demonstrated that ethylene promotes protuberance formation in ibo1, implying that IBO1 acts antagonistically to ethylene to suppress radial outgrowth. IBO1 is identical to NEK6, which encodes a Never In Mitosis A (NIMA)-related protein kinase (Nek) with sequence similarity to Neks involved in microtubule organization in fungi, algae, and animals. The ibo1-1 mutation, in which a conserved Glu residue in the activation loop is substituted by Arg, completely abolishes its kinase activity. The intracellular localization of GFP-tagged NEK6 showed that NEK6 mainly accumulates in cytoplasmic spots associated with cortical microtubules and with a putative component of the gamma-tubulin complex. The localization of NEK6 is regulated by the C-terminal domain, which is truncated in the ibo1-2 allele. These results suggest that the role of NEK6 in the control of cellular morphogenesis is dependent on its kinase action and association with the cortical microtubules.

  13. Comprehensive analysis of single-repeat R3 MYB proteins in epidermal cell patterning and their transcriptional regulation in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Schiefelbein John

    2008-07-01

    Full Text Available Abstract Background Single-repeat R3 MYB transcription factors are critical components of the lateral inhibition machinery that mediates epidermal cell patterning in plants. Sequence analysis of the Arabidopsis genome using the BLAST program reveals that there are a total of six genes, including TRIPTYCHON (TRY, CAPRICE (CPC, TRICHOMELESS1 (TCL1, and ENHANCER of TRY and CPC 1, 2, and 3 (ETC1, ETC2 and ETC3 encoding single-repeat R3 MYB transcription factors that are approximately 50% identical to one another at the amino acid level. Previous studies indicate that these single-repeat R3 MYBs regulate epidermal cell patterning. However, each of the previous studies of these single-repeat R3 MYBs has been limited to an analysis of only a subset of these six genes, and furthermore, they have limited their attention to epidermal development in only one or two of the organs. In addition, the transcriptional regulation of these single-repeat R3 MYB genes remains largely unknown. Results By analyzing multiple mutant lines, we report here that TCL1 functions redundantly with other single-repeat R3 MYB transcription factors to control both leaf trichome and root hair formation. On the other hand, ETC1 and ETC3 participate in controlling trichome formation on inflorescence stems and pedicles. Further, we discovered that single-repeat R3 MYBs suppress trichome formation on cotyledons and siliques, organs that normally do not bear any trichomes. By using Arabidopsis protoplast transfection assays, we found that all single-repeat R3 MYBs examined interact with GL3, and that GL1 or WER and GL3 or EGL3 are required and sufficient to activate the transcription of TRY, CPC, ETC1 and ETC3, but not TCL1 and ETC2. Furthermore, only ETC1's transcription was greatly reduced in the gl3 egl3 double mutants. Conclusion Our comprehensive analysis enables us to draw broader conclusions about the role of single-repeat R3 MYB gene family than were possible in the earlier

  14. Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

    2016-05-10

    Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

  15. A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases

    DEFF Research Database (Denmark)

    Celis, J E; Madsen, Peder; Rasmussen, H H

    1991-01-01

    A two-dimensional (2-D) gel database of cellular proteins from noncultured, unfractionated normal human epidermal keratinocytes has been established. A total of 2651 [35S]methionine-labeled cellular proteins (1868 isoelectric focusing, 783 nonequilibrium pH gradient electrophoresis) were resolved......, melanocytes, fibroblasts, dermal microvascular endothelial cells, peripheral blood mononuclear cells and sweat duct cells. The keratinocyte 2-D gel protein database will be updated yearly in the November issue of Electrophoresis. Udgivelsesdato: 1991-Nov...

  16. Photoprotective Potential of Glycolic Acid by Reducing NLRC4 and AIM2 Inflammasome Complex Proteins in UVB Radiation-Induced Normal Human Epidermal Keratinocytes and Mice.

    Science.gov (United States)

    Hung, Sung-Jen; Tang, Sheau-Chung; Liao, Pei-Yun; Ge, Jheng-Siang; Hsiao, Yu-Ping; Yang, Jen-Hung

    2017-02-01

    Exposure to UVB radiation induces inflammation and free radical-mediated oxidative stress through reactive oxygen species (ROS) that play a crucial role in the induction of skin cancer. Glycolic acid (GA) is frequently used in cosmetics and dermatology. The aim of the study was to analyze the photoprotective mechanisms through which GA retards UVB-induced ROS accumulation and inflammation in normal human epidermal keratinocytes (NHEKs) and mice skin, respectively. NHEK cell line and C57BL/6J mice were treated with GA (0.1 or 5 mM) for 24 h followed by UVB irradiation. ROS accumulation, DNA damage, and expression of inflammasome complexes (NLRP3, NLRC4, ASC, and AIM2) were measured in vitro. Epidermal thickness and inflammasome complex proteins were analyzed in vivo. GA significantly prevented UVB-induced loss of skin cell viability, ROS formation, and DNA damage (single and double strands DNA break). GA suppressed the mRNA expression levels of NLRC4 and AIM2 among the inflammasome complexes. GA also blocked interleukin (IL)-1β by reducing the activity of caspase-1 in the NHEKs. Treatment with GA (2%) inhibited UVB-induced inflammation marker NLRC4 protein levels in mouse dorsal skin. The photoprotective activity of GA was ascribed to the inhibition of ROS formation and DNA damage, as well as a reduction in the activities of inflammasome complexes and IL-1β. We propose that GA has anti-inflammatory and photoprotective effects against UVB irradiation. GA is potentially beneficial to the protection of human skin from UV damage.

  17. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    Directory of Open Access Journals (Sweden)

    Wymke Ockenga

    2014-11-01

    Full Text Available Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR, which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation.

  18. The Arabidopsis cytosolic proteome: the metabolic heart of the cell

    Directory of Open Access Journals (Sweden)

    Jun eIto

    2014-02-01

    Full Text Available The plant cytosol is the major intracellular fluid that acts as the medium for inter-organellar crosstalk and where a plethora of important biological reactions take place. These include its involvement in protein synthesis and degradation, stress response signaling, carbon metabolism, biosynthesis of secondary metabolites and accumulation of enzymes for defense and detoxification. This central role is highlighted by estimates indicating that the majority of eukaryotic proteins are cytosolic. Arabidopsis thaliana has been the subject of numerous proteomic studies on its different subcellular compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted proteomic characterizations of complexes is included. Despite this, few groups are currently applying advanced proteomic approaches to this important metabolic space. This review will highlight the current state of the Arabidopsis cytosolic proteome since its initial characterization a few years ago.

  19. Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: epidermal growth factor receptor as a model system.

    Science.gov (United States)

    Lowe, Aaron M; Ozer, Byram H; Wiepz, Gregory J; Bertics, Paul J; Abbott, Nicholas L

    2008-08-01

    Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was (32)P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (clones H11 and 111.6) and one phosphospecific EGF receptor antibody (clone pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82 : 1, exceeding the signal-to-background measured on the ELISA plate (<48 : 1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the

  20. CUB domain-containing protein 1 and the epidermal growth factor receptor cooperate to induce cell detachment.

    Science.gov (United States)

    Law, Mary E; Ferreira, Renan B; Davis, Bradley J; Higgins, Paul J; Kim, Jae-Sung; Castellano, Ronald K; Chen, Sixue; Luesch, Hendrik; Law, Brian K

    2016-08-05

    While localized malignancies often respond to available therapies, most disseminated cancers are refractory. Novel approaches, therefore, are needed for the treatment of metastatic disease. CUB domain-containing protein1 (CDCP1) plays an important role in metastasis and drug resistance; the mechanism however, is poorly understood. Breast cancer cell lines were engineered to stably express EGFR, CDCP1 or phosphorylation site mutants of CDCP1. These cell lines were used for immunoblot analysis or affinity purification followed by immunoblot analysis to assess protein phosphorylation and/or protein complex formation with CDCP1. Kinase activity was evaluated using phosphorylation site-specific antibodies and immunoblot analysis in in vitro kinase assays. Protein band excision and mass spectrometry was utilized to further identify proteins complexed with CDCP1 or ΔCDCP1, which is a mimetic of the cleaved form of CDCP1. Cell detachment was assessed using cell counting. This paper reports that CDCP1 forms ternary protein complexes with Src and EGFR, facilitating Src activation and Src-dependent EGFR transactivation. Importantly, we have discovered that a class of compounds termed Disulfide bond Disrupting Agents (DDAs) blocks CDCP1/EGFR/Src ternary complex formation and downstream signaling. CDCP1 and EGFR cooperate to induce detachment of breast cancer cells from the substratum and to disrupt adherens junctions. Analysis of CDCP1-containing complexes using proteomics techniques reveals that CDCP1 associates with several proteins involved in cell adhesion, including adherens junction and desmosomal cadherins, and cytoskeletal elements. Together, these results suggest that CDCP1 may facilitate loss of adhesion by promoting activation of EGFR and Src at sites of cell-cell and cell-substratum contact.

  1. Cytosolic glutamine synthetase in barley

    DEFF Research Database (Denmark)

    Thomsen, Hanne Cecilie

    fertilizer requirement. The enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N...

  2. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    Science.gov (United States)

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBA's effects on fused BSC cultures.

  3. Combined treatment with troglitazone and lovastatin inhibited epidermal growth factor-induced migration through the downregulation of cysteine-rich protein 61 in human anaplastic thyroid cancer cells.

    Directory of Open Access Journals (Sweden)

    Li-Han Chin

    Full Text Available Our previous studies have demonstrated that epidermal growth factor (EGF can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61 in human anaplastic thyroid cancer (ATC cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB and extracellular signal-regulated kinase (ERK, were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.

  4. Antioxidant properties of small proline-rich proteins : from epidermal cornification to global ROS detoxification and wound healing

    NARCIS (Netherlands)

    Vermeij, Wilbert Peter

    2011-01-01

    The small proline-rich (SPRR) proteins are generally known for their involvement in the formation and adaptation of the skin’s barrier. During the cornification process, they are cross-linked within the cornified cell envelope (CE) and as such they are responsible for the physical and permeability

  5. Epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting S100A4 protein

    DEFF Research Database (Denmark)

    Klingelhöfer, Jörg; Møller, Henrik D.; Sumer, Eren U

    2009-01-01

    The function of S100A4, a member of the calcium-binding S100 protein family, has been associated with tumor invasion and metastasis. Although an essential pro-metastatic role of extracellular S100A4 in tumor progression has been demonstrated, the identification of the precise underlying mechanisms...

  6. A stable cytosolic expression of VH antibody fragment directed against PVY NIa protein in transgenic potato plant confers partial protection against the virus.

    Science.gov (United States)

    Bouaziz, Donia; Ayadi, Malika; Bidani, Amira; Rouis, Souad; Nouri-Ellouz, Oumèma; Jellouli, Raïda; Drira, Noureddine; Gargouri-Bouzid, Radhia

    2009-04-01

    The expression of recombinant antibodies in transgenic plants has been proved to be an efficient approach for large-scale production. However, the stability of these molecules and their accumulation level depend on their molecular properties and cellular targeting. The expression of single-domain antibody fragment (VH) can be advantageous since it offers small length, high expression, solubility and stability. It can therefore be preferred to other antibody derivatives avoiding the expression difficulties related to immunoglobulin domain folding via the formation of disulfide bridge. This report describes the production of transgenic potato plants expressing a VH antibody directed against the NIa protease of potato virus Y. The antibody was driven by the constitutive CaMV 35S RNA promoter. The expression cassette was transferred into potato plants via Agrobacterium tumefaciens mediated transformation. All transgenic lines showed detectable levels of VH protein confirming the efficient translation and stability of this protein. The cellular localisation of the VH antibody was investigated. Transgenic and control plants were transferred in the greenhouse and mechanically inoculated by PVY(o) suspension. Some of the transgenic lines showed delayed symptoms at the first period post inoculation and then displayed a recovery phenomenon while the virions were still detected in the leaves. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

  7. Low co-expression of epidermal growth factor receptor and its chaperone heat shock protein 90 is associated with worse prognosis in primary glioblastoma, IDH-wild-type.

    Science.gov (United States)

    Sartori, Elsa; Langer, Rupert; Vassella, Erik; Hewer, Ekkehard; Schucht, Philippe; Zlobec, Inti; Berezowska, Sabina

    2017-10-01

    Epidermal growth factor receptor (EGFR) is a major oncogenic driver in glioblastoma (GBM) without mutations in the isocitrate dehydrogenase gene (IDH-wildtype). Heat shock protein 90 (HSP90) is a regulator of the stability of oncogenic proteins including EGFR, thereby acting as a molecular chaperone. We investigated the expression of EGFR and its chaperone HSP90 in GBM, IDH-wildtype. Tissue availability permitted analysis of 237/449 consecutive GBM cases, among them 214 IDH-wildtype (90.3%). The expression of EGFR and HSP90 was analysed by immunohistochemistry on a tissue microarray containing various tumour regions. The expression intensity (EI), and an expression score (ES) combining the percentage of stained cells with EI were determined for both markers. Overall, there was a positive correlation between EGFR and HSP90 expression (EI; r=0.275, PIDH-wildtype cases, and high expression of EGFR (ES only) was in trend associated with better outcome, but failed to meet statyistical significance (P=0.061). A combination of EGFR and HSP90, however, discriminated between different prognostic groups, with EGFRlow/HSP90low tumours showing the worst prognosis in univariate analysis (P=0.001), and in multivariate analysis including the other relevant prognostic factors age, MGMT status and postoperative treatment [n=76; hazard ratio (HR)=0.571; 95% confidence interval (CI) 0.328-0.996; P=0.048]. EGFR expression stratified most pronounced among HSP90low tumours, where the EGFRhigh phenotype was associated with longer survival. Our results reveal a variable reliance on the signalling pathway by EGFR in GBM, IDH-wildtype. Low co-expression was associated with worse prognosis.

  8. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis

    DEFF Research Database (Denmark)

    Palmer, Colin N A; Irvine, Alan D; Terron-Kwiatkowski, Ana

    2006-01-01

    Atopic disease, including atopic dermatitis (eczema), allergy and asthma, has increased in frequency in recent decades and now affects approximately 20% of the population in the developed world. Twin and family studies have shown that predisposition to atopic disease is highly heritable. Although...... dermatitis. This work establishes a key role for impaired skin barrier function in the development of atopic disease....... most genetic studies have focused on immunological mechanisms, a primary epithelial barrier defect has been anticipated. Filaggrin is a key protein that facilitates terminal differentiation of the epidermis and formation of the skin barrier. Here we show that two independent loss-of-function genetic...

  9. Epidermal hairs of Acanthaceae

    NARCIS (Netherlands)

    Ahmad, Khwaja J.

    1978-01-01

    Structure and distribution of the foliar epidermal hairs of 109 species and two varieties belonging to 39 genera of the family Acanthaceae have been studied. Both glandular and non-glandular epidermal hairs have been recorded in the investigated taxa. The glandular hairs may be subsessile or

  10. The AMP-Dependent Protein Kinase (AMPK Activator A-769662 Causes Arterial Relaxation by Reducing Cytosolic Free Calcium Independently of an Increase in AMPK Phosphorylation

    Directory of Open Access Journals (Sweden)

    Yi Huang

    2017-10-01

    Full Text Available Although recent studies reveal that activation of the metabolic and Ca2+ sensor AMPK strongly inhibits smooth muscle contraction, there is a paucity of information about the potential linkage between pharmacological AMPK activation and vascular smooth muscle (VSM contraction regulation. Our aim was to test the general hypothesis that the allosteric AMPK activator A-769662 causes VSM relaxation via inhibition of contractile protein activation, and to specifically determine which activation mechanism(s is(are affected. The ability of A-769662 to cause endothelium-independent relaxation of contractions induced by several contractile stimuli was examined in large and small musculocutaneous and visceral rabbit arteries. For comparison, the structurally dissimilar AMPK activators MET, SIM, and BBR were assessed. A-769662 displayed artery- and agonist-dependent differential inhibitory activities that depended on artery size and location. A-769662 did not increase AMPK-pT172 levels, but did increase phosphorylation of the downstream AMPK substrate, acetyl-CoA carboxylase (ACC. A-769662 did not inhibit basal phosphorylation levels of several contractile protein regulatory proteins, and did not alter the activation state of rhoA. A-769662 did not inhibit Ca2+- and GTPγS-induced contractions in β-escin-permeabilized muscle, suggesting that A-769662 must act by inhibiting Ca2+ signaling. In intact artery, A-769662 immediately reduced basal intracellular free calcium ([Ca2+]i, inhibited a stimulus-induced increase in [Ca2+]i, and inhibited a cyclopiazonic acid (CPA-induced contraction. MET increased AMPK-pT172, and caused neither inhibition of contraction nor inhibition of [Ca2+]i. Together, these data support the hypothesis that the differential inhibition of stimulus-induced arterial contractions by A-769662 was due to selective inhibition of a Ca2+ mobilization pathway, possibly involving CPA-dependent Ca2+ entry via an AMPK-independent pathway. That

  11. In vitro replication of plasmids containing human ribosomal gene sequences: origin localization and dependence on an aprotinin-binding cytosolic protein.

    Science.gov (United States)

    Coffman, F D; Georgoff, I; Fresa, K L; Sylvester, J; Gonzalez, I; Cohen, S

    1993-11-01

    We previously investigated the role of an aprotinin-binding protein (ADR) in the initiation of DNA replication in isolated quiescent nuclei. In the present study, we have used a cell-free DNA replication system to test the ability of plasmid vectors which contain sequences from the human ribosomal RNA gene to serve as replicative templates in vitro when exposed to ADR-containing preparations. Significant dTTP incorporation was seen using DNA from either a 7-kb sequence in the 5' spacer region (CHE) or a 7-kb sequence which begins near the end of the 28S coding region and extends into the 3' spacer region (ADBB), while sequences from other regions of the rRNA gene mediated little or no dTTP incorporation. The characteristics of plasmid-directed dTTP incorporation indicate that most incorporation is due to DNA replication and not repair or damage-initiated processes. To conclusively demonstrate origin-dependent replication in the plasmid system and to further map replication origins, an approach was developed using ddGTP to restrict the length of daughter strands followed by hybridization of these replication products to restriction fragments spanning the putative origin region. This approach allowed us to identify replication origin activity apart from parent strand repair or synthesis initiated at random damaged sites. One of the origins was localized to a 1375-bp fragment within the 5' spacer region, and this fragment contains sequences homologous to those found in other replication origins.

  12. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  13. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

    Science.gov (United States)

    Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

    2017-08-01

    One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

  14. The effect of cytosolic extract of Alternaria aternata fungus on Monocyte-derived dendritic cell maturation and T-lymphocyte polarization in the presence of myelin basic protein

    Directory of Open Access Journals (Sweden)

    Loghmanni A

    2013-03-01

    Full Text Available Background: Multiple Sclerosis (MS is an autoimmune disease with impairment in function of central nervous system. Macrophages and dendritic cells play important roles in alleviating or progression of the disease. These cells can cause inflammation and damage to the myelin of nerve cells by realizing of harmful substances when these cells get matured. We studied the effect of Alternaria alternata extract on maturation of monocyte- derived dendritic cell (modc and T-cell responses in the presence of Myelin Basic Protein (MBP as a laboratory model of multiple sclerosis (MS. The purpose of this study is suitable dendritic cells production for usage in MS immunotherapy.Methods: For this study plastic adherent monocytes were cultured with granulocyte/ macrophage- colony stimulating factor (GM-CSF and interleukin -4 for converting these cells to modc and pulsed with MBP and matured in the presence of monocyte-conditioned medium (MCM in control group and MCM + Alternaria alternata extract in treatment groups. Anti-CD14, anti-CD83, anti-human leukocyte antigen-DR (anti HLA-DR monoclonal antibody were carried out for phenotyping. Autologos T cell responses and cytokine production were evaluated.Results: The results showed that the expression of CD14 decreased and CD83, HLA-DR increased in treatment groups in comparison with control groups. The production amount of IL-10 overcame IL-12 and in T cell the production of cytokines, IL-17 and Interferon-γ (IFN-γ decreased and IL-4 was increased (P<0.05. These effects escalated with increasing of dosage from 50 to 100 (mg/ml (P<0.001.Conclusion: Alternaria alternata extract can cause maturation of MBP-pulsed modc and skewing of T- lymphocyte toward Th2 and thereby can evolve into a new strategy in immunotherapy of MS.

  15. Cytosol-Localized Heat Shock Factor-Binding Protein, AtHSBP, Functions as a Negative Regulator of Heat Shock Response by Translocation to the Nucleus and Is Required for Seed Development in Arabidopsis1[C][W][OA

    Science.gov (United States)

    Hsu, Shih-Feng; Lai, Hui-Chuan; Jinn, Tsung-Luo

    2010-01-01

    Heat shock response (HSR) is a universal mechanism in all organisms. It is under tight regulation by heat shock factors (HSFs) and heat shock proteins (HSPs) after heat shock (HS) to prevent stress damage. On the attenuation of HSR, HSP70 and HSF Binding Protein1 (HSBP1) interact with HSF1 and thus dissociate trimeric HSF1 into an inert monomeric form in humans. However, little is known about the effect of HSBP with thermal stress in plants. This report describes our investigation of the role of AtHSBP in Arabidopsis (Arabidopsis thaliana) by genetic and molecular approaches. AtHSBP was heat inducible and ubiquitously expressed in all tissues; AtHSBP was also crucial for seed development, as demonstrated by AtHSBP-knockout lines showing seed abortion. Thermotolerance results showed that AtHSBP participates in acquired thermotolerance but not basal thermotolerance and is a negative regulator of HSR. Subcellular localization revealed that the cytosol-localized AtHSBP translocated to the nucleus in response to HS. Protoplast two-hybrid assay results confirmed that AtHSBP interacts with itself and with the HSFs, AtHSFA1a, AtHSFA1b, and AtHSFA2. AtHSBP also negatively affected AtHSFA1b DNA-binding capacity in vitro. Quantitative polymerase chain reaction and western-blot analysis demonstrated that altered levels of AtHSBP lead to differential HSP expression, mainly during the recovery from HS. These studies provide a new insight into HSBP in plants and reveal that AtHSBP is a negative regulator of HSR and required for seed development. PMID:20388662

  16. Cytosol-localized heat shock factor-binding protein, AtHSBP, functions as a negative regulator of heat shock response by translocation to the nucleus and is required for seed development in Arabidopsis.

    Science.gov (United States)

    Hsu, Shih-Feng; Lai, Hui-Chuan; Jinn, Tsung-Luo

    2010-06-01

    Heat shock response (HSR) is a universal mechanism in all organisms. It is under tight regulation by heat shock factors (HSFs) and heat shock proteins (HSPs) after heat shock (HS) to prevent stress damage. On the attenuation of HSR, HSP70 and HSF Binding Protein1 (HSBP1) interact with HSF1 and thus dissociate trimeric HSF1 into an inert monomeric form in humans. However, little is known about the effect of HSBP with thermal stress in plants. This report describes our investigation of the role of AtHSBP in Arabidopsis (Arabidopsis thaliana) by genetic and molecular approaches. AtHSBP was heat inducible and ubiquitously expressed in all tissues; AtHSBP was also crucial for seed development, as demonstrated by AtHSBP-knockout lines showing seed abortion. Thermotolerance results showed that AtHSBP participates in acquired thermotolerance but not basal thermotolerance and is a negative regulator of HSR. Subcellular localization revealed that the cytosol-localized AtHSBP translocated to the nucleus in response to HS. Protoplast two-hybrid assay results confirmed that AtHSBP interacts with itself and with the HSFs, AtHSFA1a, AtHSFA1b, and AtHSFA2. AtHSBP also negatively affected AtHSFA1b DNA-binding capacity in vitro. Quantitative polymerase chain reaction and western-blot analysis demonstrated that altered levels of AtHSBP lead to differential HSP expression, mainly during the recovery from HS. These studies provide a new insight into HSBP in plants and reveal that AtHSBP is a negative regulator of HSR and required for seed development.

  17. The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration.

    Science.gov (United States)

    Dong, Ying; He, Yaowu; de Boer, Leonore; Stack, M Sharon; Lumley, John W; Clements, Judith A; Hooper, John D

    2012-03-23

    Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

  18. Endoplasmic reticulum calcium regulates the retrotranslocation of Trypanosoma cruzi calreticulin to the cytosol.

    Directory of Open Access Journals (Sweden)

    Carlos A Labriola

    2010-10-01

    Full Text Available For most secretory pathway proteins, crossing the endoplasmic reticulum (ER membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD, is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C, since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.

  19. A new view of the bacterial cytosol environment.

    Directory of Open Access Journals (Sweden)

    Benjamin P Cossins

    2011-06-01

    Full Text Available The cytosol is the major environment in all bacterial cells. The true physical and dynamical nature of the cytosol solution is not fully understood and here a modeling approach is applied. Using recent and detailed data on metabolite concentrations, we have created a molecular mechanical model of the prokaryotic cytosol environment of Escherichia coli, containing proteins, metabolites and monatomic ions. We use 200 ns molecular dynamics simulations to compute diffusion rates, the extent of contact between molecules and dielectric constants. Large metabolites spend ∼80% of their time in contact with other molecules while small metabolites vary with some only spending 20% of time in contact. Large non-covalently interacting metabolite structures mediated by hydrogen-bonds, ionic and π stacking interactions are common and often associate with proteins. Mg(2+ ions were prominent in NIMS and almost absent free in solution. Κ(+ is generally not involved in NIMSs and populates the solvent fairly uniformly, hence its important role as an osmolyte. In simulations containing ubiquitin, to represent a protein component, metabolite diffusion was reduced owing to long lasting protein-metabolite interactions. Hence, it is likely that with larger proteins metabolites would diffuse even more slowly. The dielectric constant of these simulations was found to differ from that of pure water only through a large contribution from ubiquitin as metabolite and monatomic ion effects cancel. These findings suggest regions of influence specific to particular proteins affecting metabolite diffusion and electrostatics. Also some proteins may have a higher propensity for associations with metabolites owing to their larger electrostatic fields. We hope that future studies may be able to accurately predict how binding interactions differ in the cytosol relative to dilute aqueous solution.

  20. Vitamin B(12) dependent changes in mouse spinal cord expression of vitamin B(12) related proteins and the epidermal growth factor system

    DEFF Research Database (Denmark)

    Mutti, Elena; Lildballe, Dorte L; Kristensen, Lise

    2013-01-01

    Chronic vitamin B(12) (cobalamin) deficiency in the mammalian central nervous system causes degenerative damage, especially in the spinal cord. Previous studies have shown that cobalamin status alters spinal cord expression of epidermal growth factor (EGF) and its receptor in rats. Employing...

  1. Artificial Loading of ASC Specks with Cytosolic Antigens.

    Directory of Open Access Journals (Sweden)

    Ali Can Sahillioğlu

    Full Text Available Inflammasome complexes form upon interaction of Nod Like Receptor (NLR proteins with pathogen associated molecular patterns (PAPMS inside the cytosol. Stimulation of a subset of inflammasome receptors including NLRP3, NLRC4 and AIM2 triggers formation of the micrometer-sized spherical supramolecular complex called the ASC speck. The ASC speck is thought to be the platform of inflammasome activity, but the reason why a supramolecular complex is preferred against oligomeric platforms remains elusive. We observed that a set of cytosolic proteins, including the model antigen ovalbumin, tend to co-aggregate on the ASC speck. We suggest that co-aggregation of antigenic proteins on the ASC speck during intracellular infection might be instrumental in antigen presentation.

  2. Epidermal Stem Cells

    Directory of Open Access Journals (Sweden)

    Osman Köse

    2015-03-01

    Full Text Available The epidermis is the outermost layer of the human skin and comprises a multilayered epithelium, the interfollicular epidermis, with associated hair follicles, sebaceous glands, and eccrine sweat glands. There are many origins of stem cells in the skin and skin appendages. These stem cells are localized in different part of the pilosebaseous units and also express many different genes. Epidermal stem cells in the pilosebaseous units not only ensure the maintenance of epidermal homeostasis and hair regeneration, but also contribute to repair of the epidermis after injury. In recent years, human induced pluripotent skin stem cells are produced from the epidermal cells such as keratinocytes, fibroblasts and melanocytes. These cells can be transdifferentiated to embriyonic stem cells. Human induced pluripotent stem cells have potential applications in cell replacement therapy and regenerative medicine. These cells provide a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. In this review, we aimed an overview of epidermal stem cells for better understanding their functions in the skin. Skin will be main organ for using the epidermal cells for regenerative medicine in near future.

  3. Eradication of damaged keratinocytes in cutaneous lichen planus forms demonstrated by evaluation of epidermal and follicular expression of CK15, indices of apoptosis and regulatory protein S100.

    Science.gov (United States)

    Upeniece, Ilze; Groma, Valerie; Skuja, Sandra; Cauce, Vinita

    The study of cytoskeleton arrangement and its contribution to survival of cell-to-cell contacts appears to be essential for understanding of numerous cellular and tissue processes. Applying CK15, S100 labeling and TUNEL reaction to cutaneous lichen planus subtypes, we found CK15 expression in the outer and inner root sheath of hair follicles, the basal epidermal layer, and eccrine glands. Its follicular expression was decreased in nearby inflammatory infiltrates. The CK15 immunopositivity was mostly described as weak (92.3%) for lichen planus but equally subdivided into weak, moderate and strong in lichen planopilaris (2 = 32.514; df = 4; p lichen planopilaris involving the scalp: 81.2 ±10.7; 87.8 ±10.7 and 88.0 ±10.5 for the basal, spinous and upper epidermal layers, respectively. S100 positive epidermal and follicular cells did not differ in the lesions demonstrated in the study groups; still immunoreactivity was more pronounced in the scalp region of lichen planopilaris. Damage of cell-to-cell contacts was confirmed by electron microscopy. Apart from immunocyte-mediated keratinocyte death, cytoskeleton-based injury and loss of cell-to-cell and matrix contacts may be of great importance, leading to eradication of degrading cells and thus contributing to the pathogenesis of lichen planus.

  4. Eradication of damaged keratinocytes in cutaneous lichen planus forms demonstrated by evaluation of epidermal and follicular expression of CK15, indices of apoptosis and regulatory protein S100

    Directory of Open Access Journals (Sweden)

    Ilze Upeniece

    2016-11-01

    Full Text Available The study of cytoskeleton arrangement and its contribution to survival of cell-to-cell contacts appears to be essential for understanding of numerous cellular and tissue processes. Applying CK15, S100 labeling and TUNEL reaction to cutaneous lichen planus subtypes, we found CK15 expression in the outer and inner root sheath of hair follicles, the basal epidermal layer, and eccrine glands. Its follicular expression was decreased in nearby inflammatory infiltrates. The CK15 immunopositivity was mostly described as weak (92.3% for lichen planus but equally subdivided into weak, moderate and strong in lichen planopilaris (2 = 32.514; df = 4; p < 0.001. The greatly varying apoptotic index was the highest in the lichen planopilaris involving the scalp: 81.2 ±10.7; 87.8 ±10.7 and 88.0 ±10.5 for the basal, spinous and upper epidermal layers, respectively. S100 positive epidermal and follicular cells did not differ in the lesions demonstrated in the study groups; still immunoreactivity was more pronounced in the scalp region of lichen planopilaris. Damage of cell-to-cell contacts was confirmed by electron microscopy. Apart from immunocyte-mediated keratinocyte death, cytoskeleton-based injury and loss of cell-to-cell and matrix contacts may be of great importance, leading to eradication of degrading cells and thus contributing to the pathogenesis of lichen planus.

  5. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.

    Science.gov (United States)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

  7. The autoimmune regulator (AIRE), which is defective in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients, is expressed in human epidermal and follicular keratinocytes and associates with the intermediate filament protein cytokeratin 17.

    Science.gov (United States)

    Kumar, Vipul; Pedroza, Luis A; Mace, Emily M; Seeholzer, Steven; Cotsarelis, George; Condino-Neto, Antonio; Payne, Aimee S; Orange, Jordan S

    2011-03-01

    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, which is caused by mutation of the autoimmune regulator (AIRE) gene, is a highly variable disease characterized by multiple endocrine failure, chronic mucocutaneous candidiasis, and various ectodermal defects. AIRE is a transcriptional regulator classically expressed in medullary thymic epithelial cells, monocytes, macrophages, and dendritic cells. Previous studies have suggested that AIRE can shuttle between the nucleus and cytoplasm of cells, although its cytoplasmic functions are poorly characterized. Through mass spectrometry analysis of proteins co-immunoprecipitating with cytoplasmic AIRE, we identified a novel association of AIRE with the intermediate filament protein cytokeratin 17 (K17) in the THP-1 monocyte cell line. We confirmed AIRE expression in HaCaT epidermal keratinocytes, as well as its interaction with K17. Confocal microscopy of human fetal and adult scalp hair follicles demonstrated a cytoplasmic pattern of AIRE staining that moderately colocalized with K17. The cytoplasmic association of AIRE with the intermediate filament network in human epidermal and follicular keratinocytes may provide a new path to understanding the ectodermal abnormalities associated with the APECED syndrome. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. The structure of an LIM-only protein 4 (LMO4 and Deformed epidermal autoregulatory factor-1 (DEAF1 complex reveals a common mode of binding to LMO4.

    Directory of Open Access Journals (Sweden)

    Soumya Joseph

    Full Text Available LIM-domain only protein 4 (LMO4 is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1, with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1 and C-terminal binding protein interacting protein (CtIP/RBBP8. Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways.

  9. Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

    Directory of Open Access Journals (Sweden)

    Helena H. Chowdhury

    2014-10-01

    Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  10. Formation of disulfide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol

    NARCIS (Netherlands)

    Schouten, A.; Roosien, J.; Bakker, J.; Schots, A.

    2002-01-01

    Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody

  11. AUTOIMMUNE EPIDERMAL BLISTERING DISEASES

    OpenAIRE

    Ana Maria Abreu Velez; Juliana Calle; Howard, Michael S.

    2013-01-01

    Autoimmune bullous skin diseases (ABDs) are uncommon, potentially fatal diseases of skin and mucous membranes which are associated with deposits of autoantibodies and complement against distinct molecules of the epidermis and dermal/epidermal basement membrane zone (BMZ). These autoantibodies lead to a loss in skin molecular integrity, which manifests clinically as formation of blisters or erosions. In pemphigus vulgaris, loss of adhesion occurs within the epidermis. The pioneering work of Er...

  12. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*

    Science.gov (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.

    2015-01-01

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. PMID:25623070

  13. RNA polymerase III regulates cytosolic RNA:DNA hybrids and intracellular microRNA expression.

    Science.gov (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J

    2015-03-20

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Epigallocatechin-3-gallate attenuates the AIM2-induced secretion of IL-1β in human epidermal keratinocytes.

    Science.gov (United States)

    Yun, Mihee; Seo, Gimoon; Lee, Ji-Young; Chae, Gue Tae; Lee, Seong-Beom

    2015-11-27

    The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a central role in the pathogenesis of psoriasis. Keratinocytes are a major source of IL-1β and express absent in melanoma 2 (AIM2). AIM2 recognizes a double-stranded DNA and initiates the IL-1β-processing of inflammasome. The AIM2 inflammasome is a cytosolic multiprotein complex composed of AIM2, an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Epigallocatechin-3-Gallate (EGCG), a major polyphenolic component of green tea, has anti-inflammatory properties. In the current study, we investigated the issue of whether or how EGCG suppresses AIM2 inflammasome in human epidermal keratinocytes, neonatal (HEKn). Treatment with EGCG, before or after IFN-γ priming, attenuated poly(dA:dT)-induced IL-1β secretion in HEKn cells. Pre-treatment with EGCG reduced the level of IFN-γ-induced priming signal via the down-regulation of pro-IL-1β and pro-capspase-1 in HEKn cells. Furthermore, treatment with EGCG attenuated poly(dA:dT)-induced ASC oligomerization and caspase-1 activation in IFN-γ-primed HEKn cells. These results suggest that EGCG attenuates AIM2-induced IL-1β secretion by suppressing both IFN-γ-mediated priming and poly(dA:dT)-induced ASC oligomerization of inflammasomes in human epidermal keratinocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    Science.gov (United States)

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  16. Both genome and cytosol dynamics change in E. coli challenged with sublethal rifampicin

    Science.gov (United States)

    Wlodarski, Michal; Raciti, Bianca; Kotar, Jurij; Cosentino Lagomarsino, Marco; Fraser, Gillian M.; Cicuta, Pietro

    2017-02-01

    While the action of many antimicrobial drugs is well understood at the molecular level, a systems-level physiological response to antibiotics remains largely unexplored. This work considers fluctuation dynamics of both the chromosome and cytosol in Escherichia coli, and their response to sublethal treatments of a clinically important antibiotic, rifampicin. We precisely quantify the changes in dynamics of chromosomal loci and cytosolic aggregates (a rheovirus nonstructural protein known as μNS-GFP), measuring short time-scale displacements across several hours of drug exposure. To achieve this we develop an empirical method correcting for photo-bleaching and loci size effects. This procedure allows us to characterize the dynamic response to rifampicin in different growth conditions, including a customised microfluidic device. We find that sub-lethal doses of rifampicin cause a small but consistent increase in motility of both the chromosomal loci and cytosolic aggregates. Chromosomal and cytosolic responses are consistent with each other and between different growth conditions.

  17. Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2

    DEFF Research Database (Denmark)

    Jensen, Charlotte Harken; Krogh, Thomas N; Højrup, Peter

    1994-01-01

    The present paper describes the primary structure, glycosylation and tissue localization of fetal antigen 1 (FA1) isolated from second-trimester human amniotic fluid. FA1 is a single-chained, heterogeneous glycoprotein of 225-262 amino acid residues. FA1 has six well conserved epidermal...

  18. Comparison of the epidermal growth factor receptor protein expression between primary non-small cell lung cancer and paired lymph node metastases: implications for targeted nuclide radiotherapy

    Directory of Open Access Journals (Sweden)

    Zhang Chen

    2010-01-01

    Full Text Available Abstract Background The knowledge of Epidermal growth factor receptor (EGFR expression in metastases of NSCLC was limited. In receptor-mediated targeted nuclide radiotherapy, tumor cells are killed with delivered radiation and therapeutic efficiency is mainly dependent on the receptor expression. Thus, the level and stability of receptor expression in both primary tumors and corresponding metastases is crucial in the assessment of a receptor as target. The goal of this study was to evaluate whether EGFR is suitable as target for clinical therapy. Methods Expression of EGFR was investigated immunohistochemically in paired samples of lymph node metastases and corresponding NSCLC primary lesions (n = 51. EGFR expression was scored as 0, 1+, 2+ or 3+. Results Positive (1+, 2+ or 3+ EGFR immunostaining was evident in 36 of 47 (76.6% analysed NSCLC primary tumors, and in 78.7% of the corresponding lymph node metastases. When EGFR expression is classified as positive or negative, discordance between the primary tumors and the corresponding metastases was observed in 5 cases (10.6%. EGFR overexpression (2+ or 3+ was found in 53.2% (25/47 of the NSCLC primary tumors and 59.6% of the corresponding metastases. Nine out of the 47 paired samples (19.2% were discordant: Only three patients who had EGFR overexpression in the primary tumors showed EGFR downregulation (0 or 1+ in lymph node metastases, while six patients changed the other way around. Conclusions The EGFR expression in the primary tumor and the corresponding metastasis is discordant in about 10% of the patients. When overexpression is considered, the discordance is observed in about 20% of the cases. However, concerning EGFR overexpression in the primary tumors, similar expression in the metastases could be predicted with a reasonably high probability, which is encouraging for testing of EGFR targeted nuclide radiotherapy.

  19. 6-alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin type-1 repeats by protein O-fucosyltransferases 1 and 2.

    Science.gov (United States)

    Al-Shareffi, Esam; Chaubard, Jean-Luc; Leonhard-Melief, Christina; Wang, Sheng-Kai; Wong, Chi-Huey; Haltiwanger, Robert S

    2013-02-01

    Protein O-fucosyltransferase 1 (Pofut1) and protein O-fucosyltransferase 2 (Pofut2) add O-linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively. Glycan chain elongation past O-fucose can occur to yield a tetrasaccharide on EGF repeats and a disaccharide on TSRs. Elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes demonstrating that O-fucosylation of Notch is essential for its function. Similarly, elimination of Pofut2 results in an early embryonic lethal phenotype in mice, although the molecular mechanism for the lethality is unknown. The recent development of sugar analogs has revolutionized the study of glycans by providing a convenient method for labeling and tracking glycosylation. In order to study O-fucosylation, we took advantage of the recently developed reporter, 6-alkynyl fucose. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or "click" reaction, azido-biotin allows tagging and detection of 6AF-modified proteins. Here we examine whether proteins containing EGF repeats or TSRs with O-fucose consensus sequences are specifically modified with 6AF in cell culture. Using mass spectrometry (MS), we demonstrate that 6AF is efficiently incorporated onto the appropriate consensus sequences on EGF repeats and TSRs. Furthermore, the elongation of the O-fucose monosaccharide on EGF repeats and TSRs is not hampered when 6AF is used. These results show that 6AF is efficiently utilized in a truly bioorthogonal manner by Pofut1, Pofut2 and the enzymes that elongate O-fucose, providing evidence that 6AF is a significant new tool in the study of protein O-fucosylation.

  20. A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

    Science.gov (United States)

    Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

  1. Measuring Changes in Cytosolic Calcium Levels in HBV- and HBx-Expressing Cultured Primary Hepatocytes.

    Science.gov (United States)

    Casciano, Jessica C; Bouchard, Michael J

    2017-01-01

    Chronic infection with hepatitis B virus (HBV) remains a major worldwide health concern and is the leading cause of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is the only regulatory protein encoded in the HBV genome; HBx stimulates HBV replication in vivo and in vitro. HBx also regulates cytosolic Ca(2+) signaling, and altered Ca(2+) signaling is associated with the development of many diseases, including HCC. Importantly, many HBx functions, including HBx modulation of cell proliferation, apoptosis, and transcription pathways, have been linked to changes in cytosolic Ca(2+) signaling. Additionally, several stages of HBV replication, including capsid formation and activation of the HBV polymerase, are dependent on intracellular Ca(2+). Consequently, defining the molecular mechanism that underlies HBV and HBx modulation of cytosolic Ca(2+) levels is important for understanding HBV pathogenesis and the role of HBx in HBV replication. Here, we describe a single-cell Ca(2+)-imaging protocol that we use to investigate HBV and HBx effects on the level of cytosolic Ca(2+). We specifically outline two methods that we use to evaluate HBV and HBx regulation of cytosolic Ca(2+) levels in cultured primary hepatocytes. This protocol can also be adapted for use in liver cell lines.

  2. Structural characterization of coatomer in its cytosolic state

    Directory of Open Access Journals (Sweden)

    Shengliu Wang

    2016-07-01

    Full Text Available Abstract Studies on coat protein I (COPI have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

  3. Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells.

    Science.gov (United States)

    Xie, Yi; Nakanishi, Takeo; Natarajan, Karthika; Safren, Lowell; Hamburger, Anne W; Hussain, Arif; Ross, Douglas D

    2015-03-01

    Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways. Published by Elsevier B.V.

  4. Ki-67 staining in benign, borderline, malignant primary and metastatic ovarian tumors: Correlation with steroid receptors, epidermal-growth-factor receptor and cathepsin D

    NARCIS (Netherlands)

    S.C. Henzen-Logmans; E.J.H. Fieret (E. J H); P.M.J.J. Berns (Els); M.E.L. van der Burg (Maria); J.G.M. Klijn (Jan); J.A. Foekens (John)

    1994-01-01

    textabstractKi-67 immunoreactivity was studied in relation to immunohistochemically assessed expression of epidermal-growth-factor receptor (EGFR), estrogen receptor (ER) progesterone receptor (PgR) and cytosolic levels of cathepsin D in advanced human ovarian adenocarcinomas, borderline and benign

  5. Silencing of reversion-inducing cysteine-rich protein with Kazal motifs stimulates hyperplastic phenotypes through activation of epidermal growth factor receptor and hypoxia-inducible factor-2α.

    Directory of Open Access Journals (Sweden)

    You Mie Lee

    Full Text Available Reversion-inducing cysteine-rich protein with Kazal motifs (RECK, a tumor suppressor is down-regulated by the oncogenic signals and hypoxia, but the biological function of RECK in early tumorigenic hyperplastic phenotypes is largely unknown. Knockdown of RECK by small interfering RNA (siRECK or hypoxia significantly promoted cell proliferation in various normal epithelial cells. Hypoxia as well as knockdown of RECK by siRNA increased the cell cycle progression, the levels of cyclin D1 and c-Myc, and the phosphorylation of Rb protein (p-pRb, but decreased the expression of p21(cip1, p27(kip1, and p16(ink4A. HIF-2α was upregulated by knockdown of RECK, indicating HIF-2α is a downstream target of RECK. As knockdown of RECK induced the activation of epidermal growth factor receptor (EGFR and treatment of an EGFR kinase inhibitor, gefitinib, suppressed HIF-2α expression induced by the silencing of RECK, we can suggest that the RECK silenicng-EGFR-HIF-2α axis might be a key molecular mechanism to induce hyperplastic phenotype of epithelial cells. It was also found that shRNA of RECK induced larger and more numerous colonies than control cells in an anchorage-independent colony formation assay. Using a xenograft assay, epithelial cells with stably transfected with shRNA of RECK formed a solid mass earlier and larger than those with control cells in nude mice. In conclusion, the suppression of RECK may promote the development of early tumorigenic hyperplastic characteristics in hypoxic stress.

  6. Epidermal Growth Factor Cytoplasmic Domain Affects ErbB Protein Degradation by the Lysosomal and Ubiquitin-Proteasome Pathway in Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Aleksandra Glogowska

    2012-05-01

    Full Text Available The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt, have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22–23-encoded peptide (EGF22.23 enhanced procathepsin B (procathB expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1. This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

  7. the significance of epidermal growth factor receptor and survivin

    African Journals Online (AJOL)

    2013-01-01

    Jan 1, 2013 ... traditional staging techniques to assist in the staging of urothelial cancer. The 2 markers are epidermal growth factor receptor (EGFR) and survivin (14-22). EGFR protein expression in bladder cancer is associated with increasing pathological grade and stage and higher rates of recurrence and disease.

  8. Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus.

    Science.gov (United States)

    Dang, Michelle; Dubbin, Karen; D'Aiello, Antonio; Hartmann, Monika; Lodish, Harvey; Herrlich, Andreas

    2011-05-20

    The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Cytosolic nucleic acid sensors and innate immune regulation.

    Science.gov (United States)

    Ori, Daisuke; Murase, Motoya; Kawai, Taro

    2017-03-04

    During viral and bacterial infections, pathogen-derived cytosolic nucleic acids are recognized by the intracellular RNA sensors retinoic acid-inducible gene I and melanoma-differentiated gene 5 and intracellular DNA sensors, including cyclic-di-GMP-AMP synthase, absent in melanoma 2, interferon (IFN)-gamma inducible protein 16, polymerase III, and so on. Binding of intracellular nucleic acids to these sensors activates downstream signaling cascades, resulting in the production of type I IFNs and pro-inflammatory cytokines to induce appropriate systematic immune responses. While these sensors also recognize endogenous nucleic acids and activate immune responses, they can discriminate between self- and non-self-nucleic acids. However, dysfunction of these sensors or failure of regulatory mechanisms causes aberrant activation of immune response and autoimmune disorders. In this review, we focus on how intracellular immune sensors recognize exogenous nucleic acids and activate the innate immune system, and furthermore, how autoimmune diseases result from dysfunction of these sensors.

  10. Differential regulation of SC1/PRDM4 and PRMT5 mediated protein arginine methylation by the nerve growth factor and the epidermal growth factor in PC12 cells.

    Science.gov (United States)

    Chittka, Alexandra

    2013-08-29

    During neuronal development, the neuroepithelial stem cells (NSCs) initially undergo proliferative divisions, later switching to neurogenic ones whereby one NSC and a post-mitotic neuron are generated. We recently showed that a member of the PRDM family of transcriptional regulators, PRDM4/SC1, recruits a type II protein arginine methyltransferase, PRMT5, to maintain the "stem-like" cellular state of the embryonic mouse cortical NSCs. However, little is known about the regulation of activity of this complex under proliferation- or differentiation-inducing growth conditions. In the present work I investigate the regulation of SC1/PRMT5-mediated methylation activity in PC12 cells treated with EGF or NGF. I present evidence that NGF down-regulates SC1/PRMT5 methyltransferase (MTase) activity and that the reduction in SC1/PRMT5 MTase activity occurs mainly in the nucleus. I suggest that high levels of SC1/PRMT5 activity are associated with the proliferative state of the cells. Copyright © 2013 The Author. Published by Elsevier Ireland Ltd.. All rights reserved.

  11. AUTOIMMUNE EPIDERMAL BLISTERING DISEASES

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2013-11-01

    Full Text Available Autoimmune bullous skin diseases (ABDs are uncommon, potentially fatal diseases of skin and mucous membranes which are associated with deposits of autoantibodies and complement against distinct molecules of the epidermis and dermal/epidermal basement membrane zone (BMZ. These autoantibodies lead to a loss in skin molecular integrity, which manifests clinically as formation of blisters or erosions. In pemphigus vulgaris, loss of adhesion occurs within the epidermis. The pioneering work of Ernst H. Beutner, Ph.D. and Robert E. Jordon, M.D. confirmed the autoimmune nature of these diseases. Walter F. Lever, M.D. contributed significantly to our understanding of the histopathologic features of these diseases. Walter Lever, M.D. and Ken Hashimoto, M.D. contributed electron microscopic studies of these diseases, especially in pemphigus vulgaris and bullous pemphigoid. In bullous pemphigoid (BP, linear IgA bullous dermatosis, epidermolysis bullosa acquisita (EBA and dermatitis herpetiformis (DH, loss of adhesion takes place within or underneath the BMZ. Classic EBA demonstrates extensive skin fragility; DH is commonly associated with gluten-sensitive enteropathy, and manifests clinically with pruritic papulovesicles on the extensor surfaces of the extremities and the lumbosacral area. The clinical spectrum of bullous pemphigoid includes tense blisters, urticarial plaques, and prurigo-like eczematous lesions. Pemphigoid gestationis mostly occurs during the last trimester of pregnancy, and mucous membrane pemphigoid primarily involves the oral mucosa and conjunctivae and leads to scarring. Linear IgA bullous dermatosis manifests with tense blisters in a „cluster of jewels”-like pattern in childhood (chronic bullous disease of childhood and is more clinically heterogeneous in adulthood. Many of the autoantigens in these disorders are known and have been well characterized. ABDs may be influenced by both genetic and exogenous factors. The diagnoses of

  12. The N-terminal region of mature mitochondrial aspartate aminotransferase can direct cytosolic dihydrofolate reductase into mitochondria in vitro.

    Science.gov (United States)

    Giannattasio, S; Azzariti, A; Marra, E; Quagliariello, E

    1994-06-30

    Two fused genes were constructed which encode for two chimeric proteins in which either 10 or 191 N-terminal amino acids of mature mitochondrial aspartate aminotransferase had been attached to the entire polypeptide chain of cytosolic dihydrofolate reductase. The precursor and mature form of mitochondrial aspartate aminotransferase, dihydrofolate reductase and both chimeric proteins were synthesized in vitro and their import into isolated mitochondria was studied. Both chimeric proteins were taken up by isolated organelles, where they became protease resistant, thus indicating the ability of the N-terminal portion of the mature moiety of the precursor of mitochondrial aspartate aminotransferase to direct cytosolic dihydrofolate reductase into mitochondria.

  13. Epidermal growth factor causes hypocalcemia in sheep.

    Science.gov (United States)

    Moore, G P; Wilkinson, M; Panaretto, B A; Delbridge, L W; Posen, S

    1986-04-01

    During iv infusions of epidermal growth factor into sheep, serum calcium concentrations fell, whereas serum magnesium and serum immunoreactive PTH levels increased. Urinary calcium and magnesium decreased significantly. The role of epidermal growth factor in calcium homeostasis is discussed.

  14. Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa

    Directory of Open Access Journals (Sweden)

    Yasuhide Osaki

    2017-09-01

    Full Text Available Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb (Allium cepa as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts, thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant Egeria densa, and showed that it is a useful system for subcellular localization analysis of higher plant proteins. E. densa leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in E. densa leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed E. densa cells, and even chloroplastic structures could be clearly visualized.

  15. Micropatterned dermal-epidermal regeneration matrices create functional niches that enhance epidermal morphogenesis.

    Science.gov (United States)

    Clement, Amanda L; Moutinho, Thomas J; Pins, George D

    2013-12-01

    Although tissue engineered skin substitutes have demonstrated some clinical success for the treatment of chronic wounds such as diabetic and venous ulcers, persistent graft take and stability remain concerns. Current bilayered skin substitutes lack the characteristic microtopography of the dermal-epidermal junction that gives skin enhanced mechanical stability and creates cellular microniches that differentially promote keratinocyte function to form skin appendages and enhance wound healing. We developed a novel micropatterned dermal-epidermal regeneration matrix (μDERM) which incorporates this complex topography and substantially enhances epidermal morphology. Here, we describe the use of this three-dimensional (3-D) in vitro culture model to systematically evaluate different topographical geometries and to determine their relationship to keratinocyte function. We identified three distinct keratinocyte functional niches: the proliferative niche (narrow geometries), the basement membrane protein synthesis niche (wide geometries) and the putative keratinocyte stem cell niche (narrow geometries and corners). Specifically, epidermal thickness and keratinocyte proliferation is significantly (p<0.05) increased in 50 and 100 μm channels while laminin-332 deposition is significantly (p<0.05) increased in 400 μm channels compared to flat controls. Additionally, β1(bri)p63(+) keratinocytes, putative keratinocyte stem cells, preferentially cluster in channel geometries (similar to clustering observed in native skin) compared to a random distribution on flats. This study identifies specific target geometries to enhance skin regeneration and graft performance. Furthermore, these results suggest the importance of μDERM microtopography in designing the next generation of skin substitutes. Finally, we anticipate that 3-D organotypic cultures on μDERMS will provide a novel tissue engineered skin substitute for in vitro investigations of skin morphogenesis, wound healing and

  16. Inhibiting cytosolic translation and autophagy improves health in mitochondrial disease.

    Science.gov (United States)

    Peng, Min; Ostrovsky, Julian; Kwon, Young Joon; Polyak, Erzsebet; Licata, Joseph; Tsukikawa, Mai; Marty, Eric; Thomas, Jeffrey; Felix, Carolyn A; Xiao, Rui; Zhang, Zhe; Gasser, David L; Argon, Yair; Falk, Marni J

    2015-09-01

    Mitochondrial respiratory chain (RC) disease therapies directed at intra-mitochondrial pathology are largely ineffective. Recognizing that RC dysfunction invokes pronounced extra-mitochondrial transcriptional adaptations, particularly involving dysregulated translation, we hypothesized that translational dysregulation is itself contributing to the pathophysiology of RC disease. Here, we investigated the activities, and effects from direct inhibition, of a central translational regulator (mTORC1) and its downstream biological processes in diverse genetic and pharmacological models of RC disease. Our data identify novel mechanisms underlying the cellular pathogenesis of RC dysfunction, including the combined induction of proteotoxic stress, the ER stress response and autophagy. mTORC1 inhibition with rapamycin partially ameliorated renal disease in B6.Pdss2(kd/kd) mice with complexes I-III/II-III deficiencies, improved viability and mitochondrial physiology in gas-1(fc21) nematodes with complex I deficiency, and rescued viability across a variety of RC-inhibited human cells. Even more effective was probucol, a PPAR-activating anti-lipid drug that we show also inhibits mTORC1. However, directly inhibiting mTORC1-regulated downstream activities yielded the most pronounced and sustained benefit. Partial inhibition of translation by cycloheximide, or of autophagy by lithium chloride, rescued viability, preserved cellular respiratory capacity and induced mitochondrial translation and biogenesis. Cycloheximide also ameliorated proteotoxic stress via a uniquely selective reduction of cytosolic protein translation. RNAseq-based transcriptome profiling of treatment effects in gas-1(fc21) mutants provide further evidence that these therapies effectively restored altered translation and autophagy pathways toward that of wild-type animals. Overall, partially inhibiting cytosolic translation and autophagy offer novel treatment strategies to improve health across the diverse array

  17. Cytosolic Fc receptor TRIM21 inhibits seeded tau aggregation.

    Science.gov (United States)

    McEwan, William A; Falcon, Benjamin; Vaysburd, Marina; Clift, Dean; Oblak, Adrian L; Ghetti, Bernardino; Goedert, Michel; James, Leo C

    2017-01-17

    Alzheimer's disease (AD) and other neurodegenerative disorders are associated with the cytoplasmic aggregation of microtubule-associated protein tau. Recent evidence supports transcellular transfer of tau misfolding (seeding) as the mechanism of spread within an affected brain, a process reminiscent of viral infection. However, whereas microbial pathogens can be recognized as nonself by immune receptors, misfolded protein assemblies evade detection, as they are host-derived. Here, we show that when misfolded tau assemblies enter the cell, they can be detected and neutralized via a danger response mediated by tau-associated antibodies and the cytosolic Fc receptor tripartite motif protein 21 (TRIM21). We developed fluorescent, morphology-based seeding assays that allow the formation of pathological tau aggregates to be measured in situ within 24 h in the presence of picomolar concentrations of tau seeds. We found that anti-tau antibodies accompany tau seeds into the cell, where they recruit TRIM21 shortly after entry. After binding, TRIM21 neutralizes tau seeds through the activity of the proteasome and the AAA ATPase p97/VCP in a similar manner to infectious viruses. These results establish that intracellular antiviral immunity can be redirected against host-origin endopathogens involved in neurodegeneration.

  18. Epidermal expression of neuropilin 1 protects murine keratinocytes from UVB-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Anna Riese

    Full Text Available BACKGROUND: Neuropilin 1 (NRP1 is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear. METHODS AND RESULTS: To elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (-/- mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher. CONCLUSION: NRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.

  19. Cloning and characterization of human liver cytosolic beta-glycosidase

    NARCIS (Netherlands)

    De Graaf, M; Van Veen, IC; Van Der Meulen-Muileman, IH; Gerritsen, WR; Pinedo, HM; Haisma, HJ

    2001-01-01

    Cytosolic beta -glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of beta -D-glycosides. including beta -D-glucoside acid beta -D-galactoside. We therefore refer to this enzyme as cytosolic beta

  20. Evolution of dinosaur epidermal structures.

    Science.gov (United States)

    Barrett, Paul M; Evans, David C; Campione, Nicolás E

    2015-06-01

    Spectacularly preserved non-avian dinosaurs with integumentary filaments/feathers have revolutionized dinosaur studies and fostered the suggestion that the dinosaur common ancestor possessed complex integumentary structures homologous to feathers. This hypothesis has major implications for interpreting dinosaur biology, but has not been tested rigorously. Using a comprehensive database of dinosaur skin traces, we apply maximum-likelihood methods to reconstruct the phylogenetic distribution of epidermal structures and interpret their evolutionary history. Most of these analyses find no compelling evidence for the appearance of protofeathers in the dinosaur common ancestor and scales are usually recovered as the plesiomorphic state, but results are sensitive to the outgroup condition in pterosaurs. Rare occurrences of ornithischian filamentous integument might represent independent acquisitions of novel epidermal structures that are not homologous with theropod feathers. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  1. Human epidermal growth factor receptor 2-positive breast cancer: heat shock protein 90 overexpression, Ki67 proliferative index, and topoisomerase II-α co-amplification as predictors of pathologic complete response to neoadjuvant chemotherapy with trastuzumab and docetaxel.

    Science.gov (United States)

    Bria, Emilio; Furlanetto, Jenny; Carbognin, Luisa; Brunelli, Matteo; Caliolo, Chiara; Nortilli, Rolando; Massari, Francesco; Pedron, Serena; Manfrin, Erminia; Pellini, Francesca; Bonetti, Franco; Sperduti, Isabella; Pollini, Giovanni Paolo; Scarpa, Aldo; Tortora, Giampaolo

    2015-02-01

    The combination of trastuzumab and chemotherapy is currently considered the standard of care for patients with locally advanced/operable human epidermal growth factor receptor 2 (HER2)-positive breast cancer. The potential correlation between the pathologic complete response (pCR) and the overexpression of heat shock protein 90 (Hsp90), Ki67, and the amplification of topoisomerase II-α (TOPO2A) was investigated in a series of patients who received neoadjuvant treatment. HER2-amplified patients who received neoadjuvant trastuzumab-docetaxel were gathered. Baseline and postsurgical Hsp90 immunoscore, Ki67 proliferation index, and TOPO2A amplification were determined together with classic clinical-pathologic predictors and correlated with pCR and imaging data. A total of 24 patients were evaluated for response; pCR, clinical, and radiologic response were found in 4 patients (16.7%; 95% confidence interval [CI], 1.7-31.5), 9 patients (37.5%; 95% CI, 18.1-56.8), and 6 patients (25.0%; 95% CI, 7.6-42.3) patients, respectively. pCR was significantly higher in premenopausal (60.0% vs. 5.3%, P = .02) and negative hormonal receptor patients (50.0% vs. 5.6%, P = .03). A trend for patients with high Ki67 and TOPO2A/HER2 co-amplification was found (21.1% vs. none, P = .54; 50.0% vs. 12%, P = .16). pCR was significantly higher in patients with Hsp90 score 3+, in comparison with score 2+ and score 1+ (50.0% vs. 14.3% vs. none, P = .05). After treatment, a statistically significant lower Ki67 staining (30.0% vs. 17.5%, P = .005) and a trend for the decreased expression of high (score 3+) and moderate (score 2+) Hsp90 immunostaining (McNemar P = .25, Wilcoxon-Mann-Whitney P = .08) were found. Although underpowered, our data suggest that patients with HER2-positive breast cancer overexpressing Hsp90 should be investigated as a "newer" molecular subtype with a significantly higher chance of pCR when receiving anti-Her2 agents. Copyright © 2015 Elsevier Inc. All rights

  2. Toxic Epidermal Necrolysis Clinical Guidelines

    Science.gov (United States)

    2008-10-01

    compli- cations and identify at-risk patients for post- discharge mortality 8) Ophthalmologic consultation is highly recommended for patients with...gastrointestinal tract, vaginal mucosa, anal canal and the eyes and mouth. TEN does not affect columnar or cuboidal epithelium. Sloughing of the epidermal-dermal...proven infection may select for resistant organisms and may contribute to increased mortality.24 The principal of skin care in TENS is to prevent

  3. Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase.

    Science.gov (United States)

    Byrne, P C; Sanders, P G; Snell, K

    1992-08-15

    A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template. A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5' end and 653 bp at the 3' end. The full-length cDNA was cloned into an expression vector and transfected into COS-1 cells. Serine hydroxymethyltransferase activity was increased by 33% in the transfected cells and a new protein band of the appropriate size was seen by SDS/PAGE analysis of proteins extracted from transfected cells. The protein sequence for rabbit cytosolic serine hydroxymethyltransferase derived from the cDNA nucleotide sequence was compared with three other derived or known prokaryotic and eukaryotic sequences. An overall sequence similarity of 34% was noted between all four sequences, whereas the similarity between the rabbit cytosolic and mitochondrial isoforms was 62%.

  4. Mitochondrial Iron-Sulfur Cluster Activity and Cytosolic Iron Regulate Iron Traffic in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wofford, Joshua D; Lindahl, Paul A

    2015-11-06

    An ordinary differential equation-based mathematical model was developed to describe trafficking and regulation of iron in growing fermenting budding yeast. Accordingly, environmental iron enters the cytosol and moves into mitochondria and vacuoles. Dilution caused by increasing cell volume is included. Four sites are regulated, including those in which iron is imported into the cytosol, mitochondria, and vacuoles, and the site at which vacuolar Fe(II) is oxidized to Fe(III). The objective of this study was to determine whether cytosolic iron (Fecyt) and/or a putative sulfur-based product of iron-sulfur cluster (ISC) activity was/were being sensed in regulation. The model assumes that the matrix of healthy mitochondria is anaerobic, and that in ISC mutants, O2 diffuses into the matrix where it reacts with nonheme high spin Fe(II) ions, oxidizing them to nanoparticles and generating reactive oxygen species. This reactivity causes a further decline in ISC/heme biosynthesis, which ultimately gives rise to the diseased state. The ordinary differential equations that define this model were numerically integrated, and concentrations of each component were plotted versus the concentration of iron in the growth medium and versus the rate of ISC/heme biosynthesis. Model parameters were optimized by fitting simulations to literature data. The model variant that assumed that both Fecyt and ISC biosynthesis activity were sensed in regulation mimicked observed behavior best. Such "dual sensing" probably arises in real cells because regulation involves assembly of an ISC on a cytosolic protein using Fecyt and a sulfur species generated in mitochondria during ISC biosynthesis and exported into the cytosol. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Reduction of dimethylarsinic acid to the highly toxic dimethylarsinous acid by rats and rat liver cytosol.

    Science.gov (United States)

    Németi, Balázs; Gregus, Zoltán

    2013-03-18

    Dimethylarsinic acid (DMAs(V)), the major urinary metabolite of inorganic arsenic, is weakly cytotoxic, whereas its reduced form, dimethylarsinous acid (DMAs(III)), is highly toxic. Although glutathione S-transferase omega 1 (GSTO1) and arsenic methyltransferase have been shown or thought to catalyze DMAs(V) reduction, their role in DMAs(V) reduction in vivo, or in cell extracts is uncertain. Therefore, the reduction of DMAs(V) to DMAs(III) in rats and in rat liver cytosol was studied to better understand its mechanism. To assess DMAs(V) reduction in rats, a novel procedure was devised based on following the accumulation of red blood cell (RBC)-bound dimethylarsenic (DMAs), which represents DMAs(III), in the blood of DMAs(V)-injected anesthetized rats. These studies indicated that rats reduced DMAs(V) to DMAs(III) to a significant extent, as in 90 min 31% of the injected 50 μmol/kg DMAs(V) dose was converted to DMAs(III) that was sequestered by the circulating erythrocytes. Pretreatment of rats with glutathione (GSH) depletors (phorone or BSO) delayed the elimination of DMAs(V) and the accumulation of RBC-bound DMAs, whereas the indirect methyltransferase inhibitor periodate-oxidized adenosine was without effect. Assessment of DMAs(V)-reducing activity of rat liver cytosol revealed that reduction of DMAs(V) required cytosolic protein and GSH and was inhibited by thiol reagents, GSSG and dehydroascorbate. Although thioredoxin reductase (TRR) inhibitors (aurothioglucose and Sb(III)) inhibited cytosolic DMAs(V) reduction, recombinant rat TRR plus NADPH, alone or when added to the cytosol, failed to support DMAs(V) reduction. On ultrafiltration of the cytosol through a 3 kDa filter, the reducing activity in the retentate was lost but was largely restored by NADPH. Such experiments also suggested that the reducing enzyme was larger than 100 kDa and was not GSTO1. In summary, reduction of DMAs(V) to the highly toxic DMAs(III) in rats and rat liver cytosol is a GSH

  6. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Leigh A Knodler

    Full Text Available Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV. We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1, but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.

  7. Epidermal growth factor in rat milk is dependent on insulin

    DEFF Research Database (Denmark)

    Thulesen, J; Poulsen, Steen Seier; Nexo, E

    1993-01-01

    Epidermal growth factor (EGF) was measured in milk from four groups of rats: untreated diabetic, insulin-treated diabetic, insulin-treated normal and control rats. In the untreated diabetic group the volume of milk, and the concentration of EGF and the total output of EGF were significantly decre...... of EGF from the mammary glands is dependent on insulin and that the decrement in milk-EGF from diabetic rats is selective when compared to the content of protein in milk....

  8. Cytosolic activity of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1A.

    Science.gov (United States)

    Livne, Sivan; Weiss, David

    2014-10-01

    Arabidopsis has three gibberellin receptors, GIBBERELLIN INSENSITIVE DWARF1 A (GID1A), GID1B and GID1C. All GID1s are localized to the nucleus but have also been detected in the cytoplasm. Since other major gibberellin signaling components have been localized to the nucleus, the current model suggests that gibberellin perception and signaling is nuclear. In this work we investigated whether GID1A can perceive gibberellin and initiate a signaling cascade in the cytosol. We have generated transgenic plants expressing GID1A fused to green florescence protein (GFP) and either a nuclear export signal (NES) or a nuclear localization signal (NLS), in the background of the gid1a gid1c (gid1ac) double mutant. Analyses by confocal microscopy confirmed the localization of GFP-GID1A-NES only in the cytosol and of GFP-GID1A-NLS only in the nucleus. Surprisingly, both recombinant proteins were able to complement the gid1ac mutation and to restore growth and responsiveness to gibberellin. However, transgenic seeds and seedlings expressing GFP-GID1A-NLS were more sensitive to gibberellin than those expressing GFP-GID1A-NES. The results of this study suggest that GID1A can bind gibberellin in the cytosol to initiate gibberellin signaling and responses. It is possible that activated cytosolic GID1A interacts with DELLA proteins before they enter the nucleus and induces a conformational transition that leads to DELLA's deactivation or degradation in the nucleus. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  9. Epidermal nevus syndrome and dysplatic kidney disease.

    Directory of Open Access Journals (Sweden)

    Azar Nickavar

    2014-08-01

    Full Text Available Epidermal nevus syndrome is a rare congenital disorder, characterized by epidermal nevi and multiple organ involvement. Multicystic kidney disease has been very rarely reported in this syndrome. Here is the report of a boy presented with multiple epidermal nevi, cardiac anomaly, seizure attack, hemi hypertrophy, and multicystic dysplastic kidney complicated with Wilms' tumor. According to this association, it is suggested to search for dysplastic kidney disease in patients with neurocutaneous disorders.

  10. Zebrafish scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF (epidermal growth factor) domain-containing protein 1) is involved in primitive hematopoiesis.

    Science.gov (United States)

    Tsao, Ku-Chi; Tu, Cheng-Fen; Lee, Shyh-Jye; Yang, Ruey-Bing

    2013-02-15

    scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.

  11. Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

    Science.gov (United States)

    Mellouk, Nora; Enninga, Jost

    2016-01-01

    Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

  12. The maize DWARF1 encodes a gibberellin 3-oxidase and is dual localized to the nucleus and cytosol.

    Science.gov (United States)

    Chen, Yi; Hou, Mingming; Liu, Lijuan; Wu, Shan; Shen, Yun; Ishiyama, Kanako; Kobayashi, Masatomo; McCarty, Donald R; Tan, Bao-Cai

    2014-12-01

    The maize (Zea mays) gibberellin (GA)-deficient mutant dwarf1 (d1) displays dwarfism and andromonoecy (i.e. forming anthers in the female flower). Previous characterization indicated that the d1 mutation blocked three steps in GA biosynthesis; however, the locus has not been isolated and characterized. Here, we report that D1 encodes a GA 3-oxidase catalyzing the final step of bioactive GA synthesis. Recombinant D1 is capable of converting GA20 to GA1, GA20 to GA3, GA5 to GA3, and GA9 to GA4 in vitro. These reactions are widely believed to take place in the cytosol. However, both in vivo GFP fusion analysis and western-blot analysis of organelle fractions using a D1-specific antibody revealed that the D1 protein is dual localized in the nucleus and cytosol. Furthermore, the upstream gibberellin 20-oxidase1 (ZmGA20ox1) protein was found dual localized in the nucleus and cytosol as well. These results indicate that bioactive GA can be synthesized in the cytosol and the nucleus, two compartments where GA receptor Gibberellin-insensitive dwarf protein1 exists. Furthermore, the D1 protein was found to be specifically expressed in the stamen primordia in the female floret, suggesting that the suppression of stamen development is mediated by locally synthesized GAs. © 2014 American Society of Plant Biologists. All Rights Reserved.

  13. [Staphylococcal epidermal exfoliation (Ritter's disease)].

    Science.gov (United States)

    Ruiz Maldonado, R; Tamayo, L; Vazquez, V; Dominguez, J

    1976-01-01

    According to the authors the best designation of Ritter's disease would be "staphilococcic epidermal exfoliation" SEE. The physiopathological and agnoslogical basis for this denomination could be the following: 1st The "S. aureus" is the ehtiological agent of the SSE in man. The Koch postulates necessary to confirm this hypothesis have been accomplished. 2nd "Staphylococcus aureus" produces a thermostable toxin that is active indepently of the staphilococcus and gives rise to the separation of the cells of the stratum granulosus of the epidermis and eventually exfoliation in suckling babies and in the newborn mouse. 3rd The "Staphylococcus aureus" may be present on the skin or in other localisations such as the bowel or pharinx. 4th The viable "S. aureus" when administered subcutaneously to the adult mice gives rise to lesions clinically and histologically similar to the impetigo observed in children. 5th The "S. aureus" killed by means of autoclave (that is, the staphylococcic toxine by itself does not give rise to any lesion when administered to the healthy adult mouse). Neijther has the SEE been observed in healthy adult man. The authors reach the conclusion that the SSE and the toxic epidermal necrolysis are basically different according to the histopathology therapeutic response and prognosis and they must be considered as independant entities.

  14. Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing.

    Science.gov (United States)

    Mout, Rubul; Ray, Moumita; Yesilbag Tonga, Gulen; Lee, Yi-Wei; Tay, Tristan; Sasaki, Kanae; Rotello, Vincent M

    2017-03-28

    Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.

  15. Epidermal Stem Cells in Orthopaedic Regenerative Medicine

    Science.gov (United States)

    Li, Jin; Zhen, Gehua; Tsai, Shin-Yi; Jia, Xiaofeng

    2013-01-01

    In the last decade, great advances have been made in epidermal stem cell studies at the cellular and molecular level. These studies reported various subpopulations and differentiations existing in the epidermal stem cell. Although controversies and unknown issues remain, epidermal stem cells possess an immune-privileged property in transplantation together with easy accessibility, which is favorable for future clinical application. In this review, we will summarize the biological characteristics of epidermal stem cells, and their potential in orthopedic regenerative medicine. Epidermal stem cells play a critical role via cell replacement, and demonstrate significant translational potential in the treatment of orthopedic injuries and diseases, including treatment for wound healing, peripheral nerve and spinal cord injury, and even muscle and bone remodeling. PMID:23727934

  16. Staphylococcus hyicus virulence in relation to exudative epidermitis in pigs

    DEFF Research Database (Denmark)

    Wegener, Henrik Caspar; Andresen, Lars Ole; Bille-Hansen, Vivi

    1993-01-01

    Staphylococcus hyicus strains with different phage types, plasmid profiles, and antibiotic resistance patterns were isolated from piglets with exudative epidermitis. The strains could be divided into virulent strains, producing exudative epidermitis, and avirulent strains, producing no dermal...... changes when injected in experimental piglets. The results showed that both virulent and avirulent strains were present simultaneously on diseased piglets. This constitutes a diagnostic problem. Concentrated culture supernatants from nine virulent strains injected in the skin of healthy piglets produced...... a crusting reaction in all piglets. Acanthosis was observed in the histopathological examination of the crustaceous skin. Concentrated culture supernatants from nine avirulent strains produced no macroscopic or microscopic skin changes. Protein profiles from all virulent strains and seven out of nine...

  17. Cellular, ultrastructural and molecular analyses of epidermal cell development in the planarian Schmidtea mediterranea.

    Science.gov (United States)

    Cheng, Li-Chun; Tu, Kimberly C; Seidel, Chris W; Robb, Sofia M C; Guo, Fengli; Sánchez Alvarado, Alejandro

    2017-09-11

    The epidermis is essential for animal survival, providing both a protective barrier and cellular sensor to external environments. The generally conserved embryonic origin of the epidermis, but the broad morphological and functional diversity of this organ across animals is puzzling. We define the transcriptional regulators underlying epidermal lineage differentiation in the planarian Schmidtea mediterranea, an invertebrate organism that, unlike fruitflies and nematodes, continuously replaces its epidermal cells. We find that Smed-p53, Sox and Pax transcription factors are essential regulators of epidermal homeostasis, and act cooperatively to regulate genes associated with early epidermal precursor cell differentiation, including a tandemly arrayed novel gene family (prog) of secreted proteins. Additionally, we report on the discovery of distinct and previously undescribed secreted organelles whose production is dependent on the transcriptional activity of soxP-3, and which we term Hyman vesicles. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Microtubules CLASP to Adherens Junctions in epidermal progenitor cells

    DEFF Research Database (Denmark)

    Shahbazi, Marta N; Perez-Moreno, Mirna

    2014-01-01

    and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT...... of epithelial tissues. We hypothesize the existence of adaptation mechanisms that regulate the formation and stability of AJs in different cellular contexts to allow the dynamic behavior of these complexes during tissue homeostasis and remodeling....

  19. A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.

    Science.gov (United States)

    Fresa, K L; Autieri, M V; Coffman, F D; Georgoff, I; Cohen, S

    1993-04-01

    Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself.

  20. Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol

    Directory of Open Access Journals (Sweden)

    Harmen M. van Rossum

    2016-05-01

    Full Text Available In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo. This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1, nuclear-mitochondrial communication (RTG2, and encoding a carnitine acetyltransferase (YAT2. Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle.

  1. Changes in cytosolic pH and calcium of guard cells precede stomatal movements.

    Science.gov (United States)

    Irving, H R; Gehring, C A; Parish, R W

    1992-03-01

    Stomatal opening is induced by indoleacetic acid (IAA), cytokinins, and fusicoccin (FC), whereas stomatal closure is induced by abscisic acid (ABA). To test the effect of these growth regulators on guard cell cytosolic Ca2+ ([Ca2+]cyt) and pH (pHcyt), epidermal strips were taken from the lower side of leaves of the orchid Paphiopedilum tonsum and were loaded with acetomethoxy-esterified forms of the Ca2+ indicator fluo-3 or the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)carboxyfluorescein. Basal [Ca2+]cyt ranged from 0.05 to 0.3 M and was 0.22 +/- 0.015 (n = 21). Increases in both [Ca2+]cyt and pHcyt were observed in guard cells after application of 10-100 M ABA to open stomata, and these preceded stomatal closure. The increase in [Ca2+]cyt ranged from 1.5- to 3-fold and was seen in 7 of 10 experiments. Guard cell alkalinization began within 2 min of ABA treatment and continued for the next 8 min. The increase ranged from 0.04 to 0.3 pH unit and was seen in 13 of 14 experiments. Guard cell [Ca2+]cyt increased, whereas pHcyt decreased after treatment of closed stomata with IAA, kinetin, or FC. In response to 50-100 M IAA, [Ca2+]cyt increased 1.5- to 2-fold in all cases, and pHcyt decreased 0.2-0.4 unit within 5 min in 7 experiments. Within 12 min, 10-100 M kinetin caused [Ca2+]cyt to increase in 28 of 34 experiments (1.3- to 2.5-fold) and pHcyt fell 0.1-0.4 unit in 15 of 17 treatments. The response to 10-50 M FC was similar in both time and magnitude. These results show that stomatal opening is accompanied by an increase in [Ca2+]cyt and cytosolic acidification in the guard cells, whereas stomatal closure is preceded by an increase in [Ca2+]cyt and cytosolic alkalinization in the guard cells. The order of these events is still uncertain, but changes in pHcyt are correlated with stomatal movement, and these changes may be an important factor in the regulation of guard cell movement.

  2. Epidermal electronic systems for sensing and therapy

    Science.gov (United States)

    Lu, Nanshu; Ameri, Shideh K.; Ha, Taewoo; Nicolini, Luke; Stier, Andrew; Wang, Pulin

    2017-04-01

    Epidermal electronic system is a class of hair thin, skin soft, stretchable sensors and electronics capable of continuous and long-term physiological sensing and clinical therapy when applied on human skin. The high cost of manpower, materials, and photolithographic facilities associated with its manufacture limit the availability of disposable epidermal electronics. We have invented a cost and time effective, completely dry, benchtop "cut-and-paste" method for the green, freeform and portable manufacture of epidermal electronics within minutes. We have applied the "cut-and-paste" method to manufacture epidermal electrodes, hydration and temperature sensors, conformable power-efficient heaters, as well as cuffless continuous blood pressure monitors out of metal thin films, two-dimensional (2D) materials, and piezoelectric polymer sheets. For demonstration purpose, we will discuss three examples of "cut-and-pasted" epidermal electronic systems in this paper. The first will be submicron thick, transparent epidermal graphene electrodes that can be directly transferred to human skin like a temporary transfer tattoo and can measure electrocardiogram (ECG) with signal-to-noise ratio and motion artifacts on par with conventional gel electrodes. The second will be a chest patch which houses both electrodes and pressure sensors for the synchronous measurements of ECG and seismocardiogram (SCG) such that beat-to-beat blood pressure can be inferred from the time interval between the R peak of the ECG and the AC peak of the SCG. The last example will be a highly conformable, low power consumption epidermal heater for thermal therapy.

  3. ELT-1, a GATA-like transcription factor, is required for epidermal cell fates in Caenorhabditis elegans embryos.

    Science.gov (United States)

    Page, B D; Zhang, W; Steward, K; Blumenthal, T; Priess, J R

    1997-07-01

    Epidermal cells are generated during Caenorhabditis elegans embryogenesis by several distinct lineage patterns. These patterns are controlled by maternal genes that determine the identities of early embryonic blastomeres. We show that the embryonically expressed gene elt-1, which was shown previously to encode a GATA-like transcription factor, is required for the production of epidermal cells by each of these lineages. Depending on their lineage history, cells that become epidermal in wild-type embryos become either neurons or muscle cells in elt-1 mutant embryos. The ELT-1 protein is expressed in epidermal cells and in their precursors. We propose that elt-1 functions at an early step in the specification of epidermal cell fates.

  4. Cytosolic superoxide dismutase can provide protection against Fasciola gigantica.

    Science.gov (United States)

    Jaikua, Wipaphorn; Kueakhai, Pornanan; Chaithirayanon, Kulathida; Tanomrat, Rataya; Wongwairot, Sirima; Riengrojpitak, Suda; Sobhon, Prasert; Changklungmoa, Narin

    2016-10-01

    Superoxide dismutases (SOD), antioxidant metallo-enzymes, are a part of the first line of defense in the trematode parasites which act as the chief scavengers for reactive oxygen species (ROS). A recombinant Fasciola gigantica cytosolic SOD (FgSOD) was expressed in Escherichia coli BL21 (DE3) and used for immunizing rabbits to obtain polyclonal antibodies (anti-rFgSOD). This rabbit anti-rFgSOD reacted with the native FgSOD at a molecular weight of 17.5kDa. The FgSOD protein was expressed at high level in parenchyma, caecal epithelium and egg of the parasite. The rFgSOD reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 2, 5, and 7 weeks after infection, and reacted with sera of infected mice. Anti-rFgSOD exhibited cross reactivity with the other parasites' antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. A vaccination was performed in imprinting control region (ICR) mice by subcutaneous injection with 50μg of rFgSOD combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 15 metacercariae by oral route. IgG1 and IgG2a in the immune sera were determined to indicate Th2 and Th1 immune responses. It was found that the parasite burden was reduced by 45%, and both IgG1 and IgG2a levels showed correlation with the numbers of worm recoveries. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Psoralens and coumarins for receptor targeting on epidermal cells

    Energy Technology Data Exchange (ETDEWEB)

    Jetter, M.M.

    1989-01-01

    Specific binding sites have been identified for the psoralens, discrete from DNA, in different epidermal cell lines. These receptors are saturable and are alkylated by the action of psoralens + UVA light. A psoralen receptor has been partially purified and established to be a protein of approximately 20,000 daltons. Inhibition of the binding of epidermal growth factor to its receptor and inhibition of the tyrosine kinase activity of the EGF receptor has been associated with PUVA treatment. These findings conflict with the general assumption that the biological effects of psoralens as photoactive compounds are associated with their ability to covalently bind to and crosslink DNA. In collaboration with Laskin's,laboratory, several classes of psoralen agonists were synthesized. These compounds include coumarins, furocoumarin and benzodipyran-2-one derivatives. The methods of preparation were varied and include variants of the Claisen rearrangement, acid and base-catalyzed condensations. The synthesized compounds were tested for their potential inhibition of {sup 125}I-EGF receptor binding. It was discovered that many of these agents showed potent inhibition activity similar to the psoralens. This data offers the possibility that sites of action, other than DNA, are involved in the mechanism by which photoactivated psoralens modulate epidermal cell lines.

  6. Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Yira Bermudez

    Full Text Available Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells.Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional G(i-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional.The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s of nicotinic acid receptors in human skin homeostasis.

  7. Simvastatin-induced Toxic Epidermal Necrolysis

    Directory of Open Access Journals (Sweden)

    Emmanouil Petrou

    2014-01-01

    Full Text Available Toxic epidermal necrolysis comprises a severe immune-complex mediated hypersensitivity reaction that typically involves the skin and mucous membranes. Herein, we describe a 68-year -old man who presented with the condition after simvastatin administration.

  8. Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans

    OpenAIRE

    Černila, Boštjan; Črešnar, Bronislava; Breskvar, Katja

    2003-01-01

    Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any addit...

  9. Improved prediction of peroxisomal PTS1 proteins from genome sequences based on experimental subcellular targeting analyses as exemplified for protein kinases from Arabidopsis.

    Science.gov (United States)

    Ma, Changle; Reumann, Sigrun

    2008-01-01

    Due to current experimental limitations in peroxisome proteome research, the identification of low-abundance regulatory proteins such as protein kinases largely relies on computational protein prediction. To test and improve the identification of regulatory proteins by such a prediction-based approach, the Arabidopsis genome was screened for genes that encode protein kinases with predicted type 1 or type 2 peroxisome targeting signals (PTS1 or PTS2). Upon transient expression in onion epidermal cells, the predicted PTS1 domains of four of the seven protein kinases re-directed the reporter protein, enhanced yellow green fluorescent (EYFP), to peroxisomes and were thus verified as functional PTS1 domains. The full-length fusions, however, remained cytosolic, suggesting that PTS1 exposure is induced by specific signals. To investigate why peroxisome targeting of three other kinases was incorrectly predicted and ultimately to improve the prediction algorithms, selected amino acid residues located upstream of PTS1 tripeptides were mutated and the effect on subcellular targeting of the reporter protein was analysed. Acidic residues in close proximity to major PTS1 tripeptides were demonstrated to inhibit protein targeting to plant peroxisomes even in the case of the prototypical PTS1 tripeptide SKL>, whereas basic residues function as essential auxiliary targeting elements in front of weak PTS1 tripeptides such as SHL>. The functional characterization of these inhibitory and essential enhancer-targeting elements allows their consideration in predictive algorithms to improve the prediction accuracy of PTS1 proteins from genome sequences.

  10. Epidermal growth factor in the rat prostate

    DEFF Research Database (Denmark)

    Tørring, Niels; Jørgensen, P E; Poulsen, Steen Seier

    1998-01-01

    Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate.......Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate....

  11. Review of Toxic Epidermal Necrolysis

    Directory of Open Access Journals (Sweden)

    Victoria Harris

    2016-12-01

    Full Text Available Toxic epidermal necrolysis (TEN is a rare but life threatening mucocutaneous reaction to drugs or their metabolites. It is characterised by widespread keratinocyte apoptosis and sloughing of the skin, erosions of the mucous membranes, painful blistering, and severe systemic disturbance. The pathophysiology of TEN is incompletely understood. Historically, it has been regarded as a drug-induced immune reaction initiated by cytotoxic lymphocytes via a human leukocyte antigen (HLA-restricted pathway. Several mediators have been identified as contributors to the cell death seen in TEN, including; granulysin, soluble Fas ligand, perforin/granzyme, tumour necrosis factor-α (TNF-α, and TNF-related apoptosis-inducing ligand. Currently, granulysin is accepted as the most important mediator of T cell proliferation. There is uncertainty around the accepted management of TEN. The lack of definitive management guidelines for TEN is explained in part by the rarity of the disease and its high mortality rate, which makes it difficult to conduct randomised control trials on emerging therapies. Developments have been made in pharmacogenomics, with numerous HLA alleles identified; however, these have largely been ethnically specific. These associations have translated into screening recommendations for Han Chinese.

  12. Rapidly Developing Toxic Epidermal Necrolysis

    Directory of Open Access Journals (Sweden)

    Viktoria Oline Barrios Poulsen

    2013-01-01

    Full Text Available Severe cutaneous reactions with potentially fatal outcomes can have many different causes. The Stevens-Johnson syndrome (SJS and toxic epidermal necrolysis (TEN are rare. They are characterized by a low incidence but high mortality, and drugs are most commonly implicated. Urgent active therapy is required. Prompt recognition and withdrawal of suspect drug and rapid intervention can result in favourable outcome. No further international guidelines for treatment exist, and much of the treatment relies on old or experimental concepts with no scientific evidence. We report on a 54-year-old man experiencing rapidly developing drug-induced severe TEN and presented multiorgan failure involving the respiratory and circulatory system, coagulopathy, and renal insufficiency. Detachment counted 30% of total body surface area (TBSA. SCORTEN = 5, indicating a mortality rate >90%. The patient was sedated and mechanically ventilated, supported with fluids and inotropes to maintain a stable circulation. Component therapy was guided by thromboelastography (TEG. The patient received plasmapheresis, and shock reversal treatment was initiated. He was transferred to a specialized intensive care burn unit within 24 hours from admittance. The initial care was continued, and hemodialysis was started. Pulmonary, circulatory, and renal sequelae resolved with intensive care, and re-epithelialization progressed slowly. The patient was discharged home on hospital day 19.

  13. Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol.

    Science.gov (United States)

    van Rossum, Harmen M; Kozak, Barbara U; Niemeijer, Matthijs S; Dykstra, James C; Luttik, Marijke A H; Daran, Jean-Marc G; van Maris, Antonius J A; Pronk, Jack T

    2016-05-03

    In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle. This study demonstrates, for the first time, that Saccharomyces cerevisiae can be engineered to employ the carnitine shuttle for export of acetyl moieties from the mitochondria and, thereby, to act as the sole source of cytosolic acetyl-CoA. Further optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient

  14. Targeted liposomes for cytosolic drug delivery to tumor cells

    NARCIS (Netherlands)

    Mastrobattista, E.

    2001-01-01

    In this thesis, a Trojan horse strategy with antibody-targeted liposomes has been followed to obtain cytosolic delivery of biotherapeutics to tumor cells in vitro. This strategy involves targeting of immunoliposomes to specific receptors on tumor cells that result in receptor-mediated uptake of the

  15. Calcium-independent phospholipase A2 in rat tissue cytosols

    NARCIS (Netherlands)

    Pierik, A.J.; Nijssen, J.G.; Aarsman, A.J.; Bosch, H. van den

    1988-01-01

    Cytosols (105000 X g supernatant) from seven rat tissues were assayed for Ca²⁺-independent phospholipase A₂ activity with either 1-acyl-2-[1-¹⁴C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[l-¹⁴C]linoleoyl-snglycero- 3-phosphoethanohunine or

  16. Monitoring disulfide bond formation in the eukaryotic cytosol

    DEFF Research Database (Denmark)

    Østergaard, Henrik; Tachibana, Christine; Winther, Jakob R.

    2004-01-01

    Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green flu...

  17. Differential changes in the activity of cytosolic and vacuolar trehalases along the growth cycle of Saccharomyces cerevisiae.

    Science.gov (United States)

    San Miguel, P F; Argüelles, J C

    1994-07-06

    Saccharomyces cerevisiae cells contain two intracellular and soluble trehalases with distinct subcellular location (cytosol and vacuoles, respectively). Both enzymes showed an opposite pattern of activity along the growth cycle. Activity of the cytosolic trehalase was high in cells growing exponentially on fermentable sugars (glucose, mannose or galactose) and sharply decayed as the cultures enter stationary phase coinciding with the beginning of trehalose biosynthesis. By contrast, vacuolar trehalase was only detectable in glucose-grown resting cells or in cultures growing on respiratory substrates (glycerol or ethanol). This enzyme was partially derepressed in the mutant hex2, which is deficient in glucose repression. Addition of fresh YPD medium to stationary-phase cultures induced the sudden reactivation of cytosolic trehalase with the concomitant slower inactivation of vacuolar trehalase. However, addition of glucose or various nitrogen sources alone had only a minor effect on both activities. The presence of cycloheximide had no effect on cytosolic trehalase, whereas completely blocked the appearance of vacuolar trehalase suggesting the requirement of protein synthesis 'de novo'.

  18. Mechanotransduction in epidermal Merkel cells.

    Science.gov (United States)

    Nakatani, Masashi; Maksimovic, Srdjan; Baba, Yoshichika; Lumpkin, Ellen A

    2015-01-01

    The cellular and molecular basis of vertebrate touch reception remains least understood among the traditional five senses. Somatosensory afferents that innervate the skin encode distinct tactile qualities, such as flutter, slip, and pressure. Gentle touch is thought to be transduced by somatosensory afferents whose tactile end organs selectively filter mechanical stimuli. These tactile end organs comprise afferent terminals in association with non-neuronal cell types such as Merkel cells, keratinocytes, and Schwann cells. An open question is whether these non-neuronal cells serve primarily as passive mechanical filters or whether they actively participate in mechanosensory transduction. This question has been most extensively studied in Merkel cells, which are epidermal cells that complex with sensory afferents in regions of high tactile acuity such as fingertips, whisker follicles, and touch domes. Merkel cell-neurite complexes mediate slowly adapting type I (SAI) responses, which encode sustained pressure and represent object features with high fidelity. How Merkel cells contribute to unique SAI firing patterns has been debated for decades; however, three recent studies in rodent models provide some direct answers. First, whole-cell recordings demonstrate that Merkel cells are touch-sensitive cells with fast, mechanically activated currents that require Piezo2. Second, optogenetics and intact recordings show that Merkel cells mediate sustained SAI firing. Finally, loss-of-function studies in transgenic mouse models reveal that SAI afferents are also touch sensitive. Together, these studies identify molecular mechanisms of mechanotransduction in Merkel cells, reveal unexpected functions for these cells in touch, and support a revised, two-receptor site model of mechanosensory transduction.

  19. Cloning and Expression of a Cytosolic HSP90 Gene in Chlorella vulgaris

    Directory of Open Access Journals (Sweden)

    Zhengyi Liu

    2014-01-01

    Full Text Available Heat shock protein 90 (HSP90, a highly conserved molecular chaperone, plays essential roles in folding, keeping structural integrity, and regulating the subset of cytosolic proteins. We cloned the cDNA of Chlorella vulgaris HSP90 (named CvHSP90 by combining homology cloning with rapid amplification of cDNA ends (RACE. Sequence analysis indicated that CvHSP90 is a cytosolic member of the HSP90 family. Quantitative RT-PCR was applied to determine the expression level of messenger RNA (mRNA in CvHSP90 under different stress conditions. C. vulgaris was kept in different temperatures (5–45°C for 1 h. The mRNA expression level of CvHSP90 increased with temperature from 5 to 10°C, went further from 35 to 40°C, and reached the maximum at 40°C. On the other hand, for C. vulgaris kept at 35°C for different durations, the mRNA expression level of CvHSP90 increased gradually and reached the peak at 7 h and then declined progressively. In addition, the expression level of CvHSP90 at 40 or 45 in salinity (‰ was almost fourfold of that at 25 in salinity (‰ for 2 h. Therefore, CvHSP90 may be a potential biomarker to monitor environment changes.

  20. Chronic treatment with epidermal growth factor stimulates growth of the urinary tract in the rat

    DEFF Research Database (Denmark)

    Vinter-Jensen, Lars; Smerup, Morten Holdgaard; Jørgensen, P E

    1996-01-01

    Twenty-four male Wistar rats, 8 weeks old, were allocated into three groups and treated with human recombinant epidermal growth factor (EGF) administered subcutaneously in doses of 0, 30, and 150 micrograms/kg per day for 4 weeks. Blood sampling was done every 2nd week and urine sampling was done...... on the urinary excretion of electrolytes, proteins, and endogenous EGF....

  1. Problem-Solving Test: The Role of Ubiquitination in Epidermal Growth Factor Receptor Trafficking

    Science.gov (United States)

    Szeberenyi, Jozsef

    2012-01-01

    Terms to be familiar with before you start to solve the test: growth factor signaling, epidermal growth factor, tyrosine protein kinase, tyrosine phosphorylation, ubiquitin, monoubiquitination, polyubiquitination, site-directed mutagenesis, transfection, expression vector, cDNA, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, Western…

  2. Mechanisms of epidermal growth factor receptor signaling as characterized by patterned ligand activation and mutational analysis.

    Science.gov (United States)

    Holowka, David; Baird, Barbara

    2017-09-01

    The cell surface receptor for epidermal growth factor (EGFR), a receptor tyrosine kinase, is a key player in normal cell growth and proliferation. Mutations in this receptor often lead to oncological transformation and other pathologies. Because of its representation of the receptor tyrosine kinase family and its important role in health and disease, a broad range of studies have been carried out in many laboratories to investigate the structural basis for transmembrane receptor activation and the resulting assembly of cytosolic signaling components. This review highlights two approaches our laboratory has taken to gain more detailed information about both aspects: Surface patterned ligands to examine recruitment of the signaling machinery, and mutational analysis to examine the regulatory role of EGFR's juxtamembrane segment. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein

    DEFF Research Database (Denmark)

    Liu, Xiangdong; Martens, Helle; Schulz, Alexander

    Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein.......Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein....

  4. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  5. Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

    Directory of Open Access Journals (Sweden)

    Corinna Hiller

    2014-04-01

    Full Text Available African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective

  6. A cytosolic juxtamembrane interface modulates plexin A3 oligomerization and signal transduction.

    Directory of Open Access Journals (Sweden)

    Rachael Barton

    Full Text Available Plexins (plxns are transmembrane (TM receptors involved in the guidance of vascular, lymphatic vessel, and neuron growth as well as cancer metastasis. Plxn signaling results in cytosolic GTPase-activating protein activity, and previous research implicates dimerization as important for activation of plxn signaling. Purified, soluble plxn extracellular and cytosolic domains exhibit only weak homomeric interactions, suggesting a role for the plxn TM and juxtamembrane regions in homooligomerization. In this study, we consider a heptad repeat in the Danio rerio PlxnA3 cytosolic juxtamembrane domain (JM for its ability to influence PlxnA3 homooligomerization in TM-domain containing constructs. Site-directed mutagenesis in conjunction with the AraTM assay and bioluminescent energy transfer (BRET² suggest an interface involving a JM heptad repeat, in particular residue M1281, regulates PlxnA3 homomeric interactions when examined in constructs containing an ectodomain, TM and JM domain. In the presence of a neuropilin-2a co-receptor and semaphorin 3F ligand, disruption to PlxnA3 homodimerization caused by an M1281F mutation is eliminated, suggesting destabilization of the PlxnA3 homodimer in the JM is not sufficient to disrupt co-receptor complex formation. In contrast, enhanced homodimerization of PlxnA3 caused by mutation M1281L remains even in the presence of ligand semaphorin 3F and co-receptor neuropilin-2a. Consistent with this pattern of PlxnA3 dimerization in the presence of ligand and co-receptor, destabilizing mutations to PlxnA3 homodimerization (M1281F are able to rescue motor patterning defects in sidetracked zebrafish embryos, whereas mutations that enhance PlxnA3 homodimerization (M1281L are not. Collectively, our results indicate the JM heptad repeat, in particular residue M1281, forms a switchable interface that modulates both PlxnA3 homomeric interactions and signal transduction.

  7. DMPD: Cytosolic DNA recognition for triggering innate immune responses. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18280611 Cytosolic DNA recognition for triggering innate immune responses. Takaoka ...A, Taniguchi T. Adv Drug Deliv Rev. 2008 Apr 29;60(7):847-57. Epub 2007 Dec 31. (.png) (.svg) (.html) (.csml) Show Cytosolic DNA reco...gnition for triggering innate immune responses. PubmedID 18280611 Title Cytosolic DNA reco

  8. Mechanical forces drive neuroblast morphogenesis and are required for epidermal closure.

    Science.gov (United States)

    Wernike, Denise; Chen, Yun; Mastronardi, Karina; Makil, Neetha; Piekny, Alisa

    2016-04-15

    Tissue morphogenesis requires myosin-dependent events such as cell shape changes and migration to be coordinated between cells within a tissue, and/or with cells from other tissues. However, few studies have investigated the simultaneous morphogenesis of multiple tissues in vivo. We found that during Caenorhabditis elegans ventral enclosure, when epidermal cells collectively migrate to cover the ventral surface of the embryo, the underlying neuroblasts (neuronal precursor cells) also undergo morphogenesis. We found that myosin accumulates as foci along the junction-free edges of the ventral epidermal cells to form a ring, whose closure is myosin-dependent. We also observed the accumulation of myosin foci and the adhesion junction proteins E-cadherin and α-catenin in the underlying neuroblasts. Myosin may help to reorganize a subset of neuroblasts into a rosette-like pattern, and decrease their surface area as the overlying epidermal cells constrict. Since myosin is required in the neuroblasts for ventral enclosure, we propose that mechanical forces in the neuroblasts influence constriction of the overlying epidermal cells. In support of this model, disrupting neuroblast cell division or altering their fate influences myosin localization in the overlying epidermal cells. The coordination of myosin-dependent events and forces between cells in different tissues could be a common theme for coordinating morphogenetic events during metazoan development. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Identification and partial characterization of cytosolic progesterone-binding sites in the filamentous fungus Rhizopus nigricans.

    Science.gov (United States)

    Lenasi, H; Hudnik-Plevnik, T

    1996-06-01

    Progesterone and some other steroids have been shown to induce a steroid 11alpha-hydroxylating enzyme system requiring cytochrome P450 in the filamentous fungus Rhizopus nigricans. In the present work, we attempted to find out whether the mycelial cytosol contained progesterone-binding sites (PBS) which could function as receptors for P450-inducing steroids and might, therefore, be included in the induction process. Two types of constitutive PBS, PBS-I and PBS-II, were identified in the cytosol pretreated with dextran-coated charcoal which removed the endogenous ligand. The protein nature of these binding activities was indicated by their susceptibility to trypsin and proteinase K digestion, heat denaturation, and their resistance to DNase. Progesterone binding was rapid, the maximal level being reached after 45 min of incubation at 22 degrees C. At this temperature, dissociation of progesterone from PBS-I proceeded with a t1/2 of 17 min and that from PBS-II with a t1/2 of 133 min. The apparent Kd of PBS-I determined by Scatchard analysis was 2.1-7.0 x 10(-9)M, and Bmax 36-218 fmol/mg protein. Bmax for PBS-II was >400 fmol/mg protein, whereas the value of Kd could not be determined accurately due to the sigmoidal nature of the association kinetics. The biological role of PBS-I in transcriptional regulation is suggested by the observation that this receptor-like protein contains a functional DNA-binding domain. A specific function of PBS-I in the induction of 11alpha-hydroxylase seems to be, however, questionable because of poor correlation between the affinity and the inducing capability of corresponding steroids.

  10. Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals.

    Directory of Open Access Journals (Sweden)

    Hiroko Mochizuki-Kawai

    Full Text Available In the petals of some species of flowers, programmed cell death (PCD begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP and S1/P1 nuclease (LoNUC genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12 drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.

  11. Toxic epidermal necrolysis, DRESS, AGEP : Do overlap cases exist?

    NARCIS (Netherlands)

    Bouvresse, Sophie; Valeyrie-Allanore, Laurence; Ortonne, Nicolas; Konstantinou, Marie Pauline; Kardaun, Sylvia H.; Bagot, Martine; Wolkenstein, Pierre; Roujeau, Jean-Claude

    2012-01-01

    Background: Severe cutaneous adverse reactions to drugs (SCARs) include acute generalized exanthematous pustulosis (AGEP), drug reaction with eosinophilia and systemic symptoms (DRESS) and epidermal necrolysis (Stevens-Johnson syndrome-toxic epidermal necrolysis [SJS-TEN]). Because of the varied

  12. Particle bombardment-mediated transient expression to identify localization signals in plant disease resistance proteins and target sites for the proteolytic activity of pathogen effectors.

    Science.gov (United States)

    Takemoto, Daigo; Jones, David A

    2014-01-01

    Plant pathogens, including fungi, oomycetes, bacteria, aphids, and nematodes, produce a variety of effector proteins to counter plant disease resistance mechanisms. After delivery into the cytosol of the plant cell, effectors may target proteins localized to different compartments within the plant cell. Plants, in turn, have evolved disease resistance (R) proteins to recognize the action of effectors. Elucidation of the subcellular localization of pathogen effectors, the plant proteins they target, and plant disease resistance proteins is essential to fully understand their interactions during pathogen challenge. In recent years, expression of fluorescent protein fusions has been widely used to determine the subcellular localization of plant proteins and pathogen effectors. Use of fluorescent proteins enables researchers to monitor the dynamic behavior of proteins in living cells. Among various methods available for the introduction of genes into plant cells, particle bombardment-mediated transient expression is the most rapid method suitable for both the identification of localization signals in proteins of interest and their dissection via amino acid substitutions generated using site-directed mutagenesis. This chapter describes a rapid procedure for particle bombardment-mediated transient expression in leaf epidermal cells. This method is also applicable to detection of pathogen effector protease activities directed against target proteins in the plant cell and analysis of protease recognition sites within these target proteins.

  13. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    Science.gov (United States)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  14. Cytosolic glutamine synthetase Gln1;2 is the main isozyme contributing to GS1 activity and can be up-regulated to relieve ammonium toxicity

    DEFF Research Database (Denmark)

    Guan, Miao; de Bang, Thomas Christian; Pedersen, Carsten

    2016-01-01

    Cytosolic GS1 (Gln synthetase) is central for ammonium assimilation in plants. High ammonium treatment enhanced the expression of the GS1 isogene Gln-1;2 encoding a low-affinity high-capacity GS1 protein in Arabidopsis (Arabidopsis thaliana) shoots. Under the same conditions, the expression of th...

  15. Epidermal naevi and bullous aplasia cutis congenita in a neonate.

    Science.gov (United States)

    Fryburg, J S; Greer, K E

    1993-01-01

    The case of a neonate with cutaneous lesions consistent with epidermal naevi is presented. In addition to typical epidermal naevi, this baby had an unusual, bullous form of aplasia cutis congenita. Although aplasia cutis has been described as bullous and has been found in association with the epidermal naevus syndrome, both of these occurrences are rare in medical publications. This case illustrates an unusual presentation of epidermal naevi with bullous aplasia cutis congenita and raises difficult diagnostic and counselling issues. Images PMID:8301655

  16. Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Se Jeong [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Gu, Dong Ryun [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Jin, Su Hyun [Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Park, Keun Ha [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Lee, Seoung Hoon, E-mail: leesh2@wku.ac.kr [Department of Oral Microbiology and Immunology, College of Dentistry, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Center for Metabolic Function Regulation (CMFR), School of Medicine, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of); Wonkwang Institute of Biomaterials and Implant, Wonkwang University, Iksan, Jeonbuk 54538 (Korea, Republic of)

    2016-06-17

    Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

  17. Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Sherley, J.L.; Kelly, T.J.

    1988-01-05

    The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity.

  18. Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis.

    Science.gov (United States)

    Ogawa, Takahisa; Kimura, Ayako; Sakuyama, Harumi; Tamoi, Masahiro; Ishikawa, Takahiro; Shigeoka, Shigeru

    2015-01-01

    Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The Km and Vmax values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min(-1) mg protein(-1), respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.

  19. Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans.

    Science.gov (United States)

    Cernila, Bostjan; Cresnar, Bronislava; Breskvar, Katja

    2003-01-01

    Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any additional highly homologous copies of the cytosolic hsp70 genes in the R nigricans genome. Sequence analyses showed that all 3 genes contain introns within the open reading frame. The dynamics of the R nigricans molecular response to progesterone, 21-OH progesterone, and testosterone, as well as to heat shock, copper ions, hydrogen peroxide, and ethanol was studied by temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA accumulation. Northern blot experiments revealed that the Rnhsp70-2 transcript level is not affected by testosterone, whereas mRNA levels of both genes are rapidly increased with all the other stressors studied. Moreover, the decrease of transcript levels is notably delayed in ethanol stress, and a difference is observed between the profiles of Rnhsp70-1 and Rnhsp70-2 transcripts during heat stress.

  20. Cytosolic RNA:DNA hybrids activate the cGAS-STING axis.

    Science.gov (United States)

    Mankan, Arun K; Schmidt, Tobias; Chauhan, Dhruv; Goldeck, Marion; Höning, Klara; Gaidt, Moritz; Kubarenko, Andrew V; Andreeva, Liudmila; Hopfner, Karl-Peter; Hornung, Veit

    2014-12-17

    Intracellular recognition of non-self and also self-nucleic acids can result in the initiation of potent pro-inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2'-5'), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP-1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS-STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA. © 2014 The Authors.

  1. Expression and characterization of a cytosolic glucose 6 phosphate dehydrogenase isoform from barley (Hordeum vulgare) roots.

    Science.gov (United States)

    Castiglia, Daniela; Cardi, Manuela; Landi, Simone; Cafasso, Donata; Esposito, Sergio

    2015-08-01

    In plant cells, glucose 6 phosphate dehydrogenase (G6PDH-EC 1.1.1.49) regulates the oxidative pentose phosphate pathway (OPPP), a metabolic route involved in the production of NADPH for various biosynthetic processes and stress response. In this study, we report the overexpression of a cytosolic G6PDH isoform from barley (Hordeum vulgare) roots in bacteria, and the biochemical characterization of the purified recombinant enzyme (HvCy-G6PDH). A full-length cDNA coding for a cytosolic isoform of G6PDH was isolated, and the sequence was cloned into pET3d vector; the protein was overexpressed in Escherichia coli BL21 (DE3) and purified by anion exchange and affinity chromatography. The kinetic properties were calculated: the recombinant HvCy-G6PDH showed KMs and KINADPH comparable to those observed for the enzyme purified from barley roots; moreover, the analysis of NADPH inhibition suggested a competitive mechanism. Therefore, this enzyme could be utilised for the structural and regulatory characterization of this isoform in higher plants. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans

    Science.gov (United States)

    Černila, Boštjan; Črešnar, Bronislava; Breskvar, Katja

    2003-01-01

    Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any additional highly homologous copies of the cytosolic hsp70 genes in the R nigricans genome. Sequence analyses showed that all 3 genes contain introns within the open reading frame. The dynamics of the R nigricans molecular response to progesterone, 21-OH progesterone, and testosterone, as well as to heat shock, copper ions, hydrogen peroxide, and ethanol was studied by temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA accumulation. Northern blot experiments revealed that the Rnhsp70-2 transcript level is not affected by testosterone, whereas mRNA levels of both genes are rapidly increased with all the other stressors studied. Moreover, the decrease of transcript levels is notably delayed in ethanol stress, and a difference is observed between the profiles of Rnhsp70-1 and Rnhsp70-2 transcripts during heat stress. PMID:15115284

  3. Solution NMR of MPS-1 reveals a random coil cytosolic domain structure.

    Directory of Open Access Journals (Sweden)

    Pan Li

    Full Text Available Caenorhabditis elegans MPS1 is a single transmembrane helical auxiliary subunit that co-localizes with the voltage-gated potassium channel KVS1 in the nematode nervous system. MPS-1 shares high homology with KCNE (potassium voltage-gated channel subfamily E member auxiliary subunits, and its cytosolic domain was reported to have a serine/threonine kinase activity that modulates KVS1 channel function via phosphorylation. In this study, NMR spectroscopy indicated that the full length and truncated MPS-1 cytosolic domain (134-256 in the presence or absence of n-dodecylphosphocholine detergent micelles adopted a highly flexible random coil secondary structure. In contrast, protein kinases usually adopt a stable folded conformation in order to implement substrate recognition and phosphoryl transfer. The highly flexible random coil secondary structure suggests that MPS-1 in the free state is unstructured but may require a substrate or binding partner to adopt stable structure required for serine/threonine kinase activity.

  4. Stevens Johnsons syndrom og toksisk epidermal nekrolyse

    DEFF Research Database (Denmark)

    Kaur-Knudsen, Diljit; Zachariae, Claus; Thomsen, Simon Francis

    2013-01-01

    Stevens-Johnson syndrome and toxic epidermal necrolysis are acute mucocutaneous diseases primarily due to drug intake. The diseases are characterised by the separation of epidermis from dermis which can be life-threatening. Mortality is often caused by sepsis and multiple organ failure. The most...

  5. FOLIAR EPIDERMAL AND PHYTOCHEMICAL STUDIES OF THE ...

    African Journals Online (AJOL)

    Administrator

    alkaloid, saponin, inulin, cellulose, tannin and lignin; Eragrostis tremula tested negative for lignin and positive for cellulose, saponin and alkaloids while Axonopus compressus tested negative for lignin, but positive for alkaloid, saponin, inulin, cellulose and tannin respectively. Leaf epidermal studies help to determine ...

  6. Estrogen receptor, progesterone receptor, and human epidermal ...

    African Journals Online (AJOL)

    Current clinical practice employs the use of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), as biomarkers to appropriately select patients that would benefit from targeted therapy against these major molecular pathways of the disease. This study aims at ...

  7. Human epidermal growth factor receptor (HER 2)/neu expression ...

    African Journals Online (AJOL)

    use

    2011-11-23

    Nov 23, 2011 ... 3Department of Pathology, Renmin Hospital of Wuhan University, Wuhan, Hubei Province, 430060, China. 4Department of ... To investigate the relationship between the expression/amplification of human epidermal growth factor receptor ... epidermal growth factor receptor family; HER 2, human epidermal ...

  8. Taxonomic significance of leaf epidermal anatomy of selected ...

    African Journals Online (AJOL)

    Leaf epidermal anatomy of selected Persicaria Mill. species of the family Polygonaceae revealed variation in size and shape of epidermal cells, stomata, glandular and non glandular trichomes. This study proves to be taxonomically important tool in the delimitation of taxa. Epidermal cell shapes are variable but mostly ...

  9. Gastric luminal epidermal growth factor is affected by diet

    African Journals Online (AJOL)

    intragastric epidermal growth factor level, diet and intragastric pH. Setting and subjects. A dietary survey was co-ordinated with studies of gastric luminal epidermal growth factor and gastric fluid pH in 120 rural Transkeians. Results. Gastric .... Playford RJ, Marchbank T, Calnan DP, et at. Epidermal growth factor is digested to ...

  10. Taxonomic significance of leaf epidermal anatomy of selected ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Jun 21, 2010 ... Leaf epidermal anatomy of selected Persicaria Mill. species of the family Polygonaceae revealed variation in size and shape of epidermal cells, stomata, glandular and non glandular trichomes. This study proves to be taxonomically important tool in the delimitation of taxa. Epidermal cell shapes are.

  11. Epidermal Transglutaminase (TGase 3) Is Required for Proper Hair Development, but Not the Formation of the Epidermal Barrier

    Science.gov (United States)

    John, Susan; Thiebach, Lars; Frie, Christian; Mokkapati, Sharada; Bechtel, Manuela; Nischt, Roswitha; Rosser-Davies, Sally; Paulsson, Mats; Smyth, Neil

    2012-01-01

    Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members. PMID:22496784

  12. Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling

    National Research Council Canada - National Science Library

    Wong, Esther Sook Miin; Fong, Chee Wai; Yusoff, Permeen; Guy, Graeme R; Langdon, Wallace Y; Low, Boon Chuan; Lim, Jormay

    2002-01-01

    ... of epidermal growth factor receptor (EGFR), where it acts to attenuate downstream signalling. Casci . (1999) identified dSpry to be an intracellular protein that interacts in vitro with Drk ( Drosophila homologue of Grb2) and Gap‐1, a Ras GTPase‐activating protein, to potentially inhibit Ras signalling. Currently, four mammalian genes ha...

  13. Yeast Interacting Proteins Database: YDL239C, YKL103C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available that belongs to the peptidase family M18; often used as a marker protein in studies of autophagy and cytosol...ily M18; often used as a marker protein in studies of autophagy and cytosol to vacuole targeting (CVT) pathw

  14. Yeast Interacting Proteins Database: YKL103C, YOL082W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available he peptidase family M18; often used as a marker protein in studies of autophagy and cytosol to vacuole targe... as a marker protein in studies of autophagy and cytosol to vacuole targeting (CVT) pathway Rows with this b...on Vacuolar aminopeptidase yscI; zinc metalloproteinase that belongs to the peptidase family M18; often used

  15. A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death.

    Science.gov (United States)

    Wang, Xiaowen; Chen, Xin Jie

    2015-08-27

    Mitochondria are multifunctional organelles whose dysfunction leads to neuromuscular degeneration and ageing. The multi-functionality poses a great challenge for understanding the mechanisms by which mitochondrial dysfunction causes specific pathologies. Among the leading mitochondrial mediators of cell death are energy depletion, free radical production, defects in iron-sulfur cluster biosynthesis, the release of pro-apoptotic and non-cell-autonomous signalling molecules, and altered stress signalling. Here we identify a new pathway of mitochondria-mediated cell death in yeast. This pathway was named mitochondrial precursor over-accumulation stress (mPOS), and is characterized by aberrant accumulation of mitochondrial precursors in the cytosol. mPOS can be triggered by clinically relevant mitochondrial damage that is not limited to the core machineries of protein import. We also discover a large network of genes that suppress mPOS, by modulating ribosomal biogenesis, messenger RNA decapping, transcript-specific translation, protein chaperoning and turnover. In response to mPOS, several ribosome-associated proteins were upregulated, including Gis2 and Nog2, which promote cap-independent translation and inhibit the nuclear export of the 60S ribosomal subunit, respectively. Gis2 and Nog2 upregulation promotes cell survival, which may be part of a feedback loop that attenuates mPOS. Our data indicate that mitochondrial dysfunction contributes directly to cytosolic proteostatic stress, and provide an explanation for the association between these two hallmarks of degenerative diseases and ageing. The results are relevant to understanding diseases (for example, spinocerebellar ataxia, amyotrophic lateral sclerosis and myotonic dystrophy) that involve mutations within the anti-degenerative network.

  16. Upregulation of epidermal growth factor receptor 4 in oral leukoplakia

    Science.gov (United States)

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

    2013-01-01

    In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP). The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP. PMID:23492901

  17. A novel role of RASSF9 in maintaining epidermal homeostasis.

    Directory of Open Access Journals (Sweden)

    Chiou-Mei Lee

    Full Text Available The physiological role of RASSF9, a member of the Ras-association domain family (RASSF, is currently unclear. Here, we report a mouse line in which an Epstein-Barr virus Latent Membrane Protein 1 (LMP1 transgene insertion has created a 7.2-kb chromosomal deletion, which abolished RASSF9 gene expression. The RASSF9-null mice exhibited interesting phenotypes that resembled human ageing, including growth retardation, short lifespan, less subcutaneous adipose layer and alopecia. In the wild-type mice, RASSF9 is predominantly expressed in the epidermal keratinocytes of skin, as determined by quantitative reverse-transcription PCR, immunofluorescence and in situ hybridization. In contrast, RASSF9-/- mice presented a dramatic change in epithelial organization of skin with increased proliferation and aberrant differentiation as detected by bromodeoxyuridine incorporation assays and immunofluorescence analyses. Furthermore, characteristic functions of RASSF9-/- versus wild type (WT mouse primary keratinocytes showed significant proliferation linked to a reduction of p21Cip1 expression under growth or early differentiation conditions. Additionally, in RASSF9-/- keratinocytes there was a drastic down-modulation of terminal differentiation markers, which could be rescued by infection with a recombinant adenovirus, Adv/HA-RASSF9. Our results indicate a novel and significant role of RASSF9 in epidermal homeostasis.

  18. Cytosolic proteome profiling of aminoglycosides resistant Mycobacterium tuberculosis clinical isolates using MALDI-TOF/MS

    Directory of Open Access Journals (Sweden)

    Divakar Sharma

    2016-11-01

    Full Text Available Emergence of extremely drug resistant tuberculosis (XDR-TB is the consequence of the failure of second line TB treatment. Aminoglycosides are the important second line anti-TB drugs used to treat the multi drug resistant tuberculosis (MDR-TB. Main known mechanism of action of aminoglycosides is to inhibit the protein synthesis by inhibiting the normal functioning of ribosome. Primary target of aminoglycosides are the ribosomal RNA and its associated proteins. Various mechanisms have been proposed for aminoglycosides resistance but still some are unsolved. As proteins are involved in most of the biological processes, these act as a potential diagnostic markers and drug targets. In the present study we analyzed the purely cytosolic proteome of amikacin (AK and kanamycin (KM resistant Mycobacterium tuberculosis isolates by proteomic and bioinformatic approaches. Twenty protein spots were found to have over expressed in resistant isolates and were identified. Among these Rv3208A, Rv2623, Rv1360, Rv2140c, Rv1636 and Rv2185c are six proteins with unknown functions or undefined role. Docking results showed that AK and KM binds to the conserved domain (DUF, USP-A, Luciferase, PEBP and Polyketidecyclase/dehydrase domain of these hypothetical proteins and over expression of these proteins might neutralize/modulate the effect of drug molecules. TBPred and GPS-PUP predicted cytoplasmic nature and potential pupylation sites within these identified proteins respectively. String analysis also suggested that over expressed proteins along with their interactive partners might be involved in aminoglycosides resistance. Cumulative effect of these over expressed proteins could be involved in AK and KM resistance by mitigating the toxicity, repression of drug target and neutralizing affect. These findings need further exploitation for the expansion of newer therapeutics or diagnostic markers against AK and KM resistance so that an extreme condition like XDR-TB can

  19. Epidermal growth factor inhibits cysteamine-induced duodenal ulcers

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1983-01-01

    mg/kg, s.c.). Furthermore, measurements on tissue extracts of the pouches revealed that 5 h after cysteamine treatment, Brunner's glands were depleted of epidermal growth factor. The effect on ulcer development of intraduodenally applied exogenous epidermal growth factor (1 micrograms/kg . h) also...... was studied. Luminal epidermal growth factor significantly inhibited the formation of cysteamine-induced duodenal ulcer, compared with controls receiving saline. The effect was not due to inhibition of gastric acid secretion or stimulation of duodenal bicarbonate secretion since the dose of epidermal growth...... of synthesis and secretion of endogenous epidermal growth factor may be a pathogenetic factor in cysteamine-induced duodenal ulcer....

  20. LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany); Brosens, Jan [Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX (United Kingdom); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany)

    2015-05-08

    Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

  1. Spectral components of cytosolic [Ca2+] spiking in neurons

    DEFF Research Database (Denmark)

    Kardos, J; Szilágyi, N; Juhász, G

    1998-01-01

    into evolutionary spectra of a characteristic set of frequencies. Non-delayed small spikes on top of sustained [Ca2+]c were synthesized by a main component frequency, 0.132+/-0.012 Hz, showing its maximal amplitude in phase with the start of depolarization (25 mM KCI) combined with caffeine (10 mM) application......We show here, by means of evolutionary spectral analysis and synthesis of cytosolic Ca2+ ([Ca2+]c) spiking observed at the single cell level using digital imaging fluorescence microscopy of fura-2-loaded mouse cerebellar granule cells in culture, that [Ca2+]c spiking can be resolved...

  2. Identification and comparative analysis of the epidermal differentiation complex in snakes

    Science.gov (United States)

    Brigit Holthaus, Karin; Mlitz, Veronika; Strasser, Bettina; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

    2017-01-01

    The epidermis of snakes efficiently protects against dehydration and mechanical stress. However, only few proteins of the epidermal barrier to the environment have so far been identified in snakes. Here, we determined the organization of the Epidermal Differentiation Complex (EDC), a cluster of genes encoding protein constituents of cornified epidermal structures, in snakes and compared it to the EDCs of other squamates and non-squamate reptiles. The EDC of snakes displays shared synteny with that of the green anole lizard, including the presence of a cluster of corneous beta-protein (CBP)/beta-keratin genes. We found that a unique CBP comprising 4 putative beta-sheets and multiple cysteine-rich EDC proteins are conserved in all snakes and other squamates investigated. Comparative genomics of squamates suggests that the evolution of snakes was associated with a gene duplication generating two isoforms of the S100 fused-type protein, scaffoldin, the origin of distinct snake-specific EDC genes, and the loss of other genes that were present in the EDC of the last common ancestor of snakes and lizards. Taken together, our results provide new insights into the evolution of the skin in squamates and a basis for the characterization of the molecular composition of the epidermis in snakes. PMID:28345630

  3. Dictyostelium acetoacetyl-CoA thiolase is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol.

    Science.gov (United States)

    Isezaki, Nana; Sekiba, Atsushi; Itagaki, Shoko; Nagayama, Koki; Ochiai, Hiroshi; Ohmachi, Tetsuo

    2015-07-01

    Acetoacetyl-CoA thiolase is an enzyme that catalyses both the CoA-dependent thiolytic cleavage of acetoacetyl-CoA and the reverse condensation reaction. In Dictyostelium discoideum, acetoacetyl-CoA thiolase (DdAcat) is encoded by a single acat gene. The aim of this study was to assess the localization of DdAcat and to determine the mechanism of its cellular localization. Subcellular localization of DdAcat was investigated using a fusion protein with GFP, and it was found to be localized to peroxisomes. The findings showed that the targeting signal of DdAcat to peroxisomes is a unique nonapeptide sequence (15RMYTTAKNL23) similar to the conserved peroxisomal targeting signal-2 (PTS-2). Cell fractionation experiments revealed that DdAcat also exists in the cytosol. Distribution to the cytosol was caused by translational initiation from the second Met codon at position 16. The first 18 N-terminal residues also exhibited function as a mitochondrial targeting signal (MTS). These results indicate that DdAcat is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol using both PTS-2 and MTS signals, which overlap each other near the N-terminus, and the alternative utilization of start codons.

  4. Arf and RhoA regulate both the cytosolic and the membrane-bound phospholipase D from human placenta

    DEFF Research Database (Denmark)

    Vinggaard, Anne Marie; Hansen, Harald S.; Provost, J.J.

    1997-01-01

    In this paper we demonstrate for the first time that human placenta contains a cytosolic phospholipase D (PLD) activity. This activity had a pH optimum of 7.0 and was stimulated by PIP and inhibited by oleate. Furthermore, cytosolic PLD was stimulated by 30 µM GTP¿S (6-14-fold) and by the small G...... proteins 1 µM mArf3 (2-fold) and 0.37 nM RhoA (2-fold). This is the first report to show RhoA activation of a cytosolic PLD. The activation by mArf3 was maintained after partial purification on DEAE Sepharose of the enzyme. We have previously reported the existence of a membrane-bound PLD from human...... placenta, which is stimulated by PIP, but not by oleate. Here we show that oleic acid and a-linolenic acid both dose-dependently inhibited solubilized membrane PLD (65% inhibition at 4 mM), whereas stearic acid (4 mM) had no effect. Thus, the presence of double bonds in the fatty acid is important...

  5. Cytosolic Pellino-1-Mediated K63-Linked Ubiquitination of IRF5 in M1 Macrophages Regulates Glucose Intolerance in Obesity

    Directory of Open Access Journals (Sweden)

    Donghyun Kim

    2017-07-01

    Full Text Available IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization.

  6. Expression of novel cytosolic malate dehydrogenases (cMDH) in Lupinus angustifolius nodules during phosphorus starvation.

    Science.gov (United States)

    Le Roux, Marcellous; Phiri, Ethel; Khan, Wesaal; Sakiroğlu, Muhammet; Valentine, Alex; Khan, Sehaam

    2014-11-01

    During P deficiency, the increased activity of malate dehydrogenase (MDH, EC 1.1.1.37) can lead to malate accumulation. Cytosolic- and nodule-enhanced MDH (cMDH and neMDH, respectively) are known isoforms, which contribute to MDH activity in root nodules. The aim of this study was to investigate the role of the cMDH isoforms in nodule malate supply under P deficiency. Nodulated lupins (Lupinus angustifolius var. Tanjil) were hydroponically grown at adequate P (+P) or low P (-P). Total P concentration in nodules decreased under P deficiency, which coincided with an increase in total MDH activity. A consequence of higher MDH activity was the enhanced accumulation of malate derived from dark CO2 fixation via PEPC and not from pyruvate. Although no measurable neMDH presence could be detected via PCR, gene-specific primers detected two 1kb amplicons of cMDH, designated LangMDH1 (corresponding to +P, HQ690186) and LangMDH2 (corresponding to -P, HQ690187), respectively. Sequencing analyses of these cMDH amplicons showed them to be 96% identical on an amino acid level. There was a high degree of diversification between proteins detected in this study and other known MDH proteins, particularly those from other leguminous plants. Enhanced malate synthesis in P-deficient nodules was achieved via increased anaplerotic CO2 fixation and subsequent higher MDH activities. Novel isoforms of cytosolic MDH may be involved, as shown by gene expression of specific genes under P deficiency. Copyright © 2014 Elsevier GmbH. All rights reserved.

  7. Epidermal Growth Factor and Intestinal Barrier Function

    Directory of Open Access Journals (Sweden)

    Xiaopeng Tang

    2016-01-01

    Full Text Available Epidermal growth factor (EGF is a 53-amino acid peptide that plays an important role in regulating cell growth, survival, migration, apoptosis, proliferation, and differentiation. In addition, EGF has been established to be an effective intestinal regulator helping to protect intestinal barrier integrity, which was essential for the absorption of nutrients and health in humans and animals. Several researches have demonstrated that EGF via binding to the EGF receptor and subsequent activation of Ras/MAPK, PI3K/AKT, PLC-γ/PKC, and STATS signal pathways regulates intestinal barrier function. In this review, the relationship between epidermal growth factor and intestinal development and intestinal barrier is described, to provide a better understanding of the effects of EGF on intestine development and health.

  8. Abnormal epidermal changes after argon laser treatment

    Energy Technology Data Exchange (ETDEWEB)

    Neumann, R.A.; Knobler, R.M.; Aberer, E.; Klein, W.; Kocsis, F.; Ott, E. (Univ. of Vienna (Austria))

    1991-02-01

    A 26-year-old woman with a congenital port-wine stain on the forehead was treated three times at 2-month intervals with an argon laser. Six months after the last treatment, moderate blanching and mild scaling confined to the treated area was observed. A biopsy specimen of the treated area revealed a significant decrease in ectatic vessels. However, epidermal changes similar to those of actinic keratosis with disorganized cell layers and marked cytologic abnormalities were seen. Analysis of peripheral blood lymphocytes for a defect in DNA repair was negative. Multiple, argon laser-induced photothermal effects may be responsible for the changes observed in our case and may lead to premalignant epidermal transformation.

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

    Directory of Open Access Journals (Sweden)

    David E. Heppner

    2016-08-01

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

  10. Beyond expectations: novel insights into epidermal keratin function and regulation.

    Science.gov (United States)

    Homberg, Melanie; Magin, Thomas M

    2014-01-01

    The epidermis is a stratified epithelium that relies on its cytoskeleton and cell junctions to protect the body against mechanical injury, dehydration, and infections. Keratin intermediate filament proteins are involved in many of these functions by forming cell-specific cytoskeletal scaffolds crucial for the maintenance of cell and tissue integrity. In response to various stresses, the expression and organization of keratins are altered at transcriptional and posttranslational levels to restore tissue homeostasis. Failure to restore tissue homeostasis in the presence of keratin gene mutations results in acute and chronic skin disorders for which currently no rational therapies are available. Here, we review the recent progress on the role of keratins in cytoarchitecture, adhesion, signaling, and inflammation. By focusing on epidermal keratins, we illustrate the contribution of keratin isotypes to differentiated epithelial functions. © 2014 Elsevier Inc. All rights reserved.

  11. Collagen sheet dressings for cutaneous lesions of toxic epidermal necrolysis

    Directory of Open Access Journals (Sweden)

    S Bhattacharya

    2011-01-01

    Full Text Available Toxic epidermal necrolysis (TEN is associated with a significant mortality of 30-50% and long-term sequelae. Treatment includes early admission to a burn unit, where management with precise fluid, electrolyte, protein, and energy supplementation, moderate mechanical ventilation, and expert wound care can be provided. Specific treatment with immunosuppressive drugs or immunoglobulins did not show an improved outcome in most studies and remains controversial. We have treated the cutaneous lesions of seven patients of TEN with collagen sheet dressings and have found a significant reduction in morbidity. The sheets are a one-time dressing, easy to apply and they reduce fluid loss, prevent infection, reduce pain, avoid repeated dressings and gradually peal off as the underlying lesions heal.

  12. Metabolism of antitumor acylfulvene by rat liver cytosol.

    Science.gov (United States)

    McMorris, T C; Elayadi, A N; Yu, J; Kelner, M J

    1999-01-01

    Illudins are novel compounds from which a potent class of antitumor agents, called acylfulvenes, have been synthesized. The model illudin, illudin S, has marked in vitro and in vivo toxicity but displays a poor therapeutic index. The toxicity of illudin S is believed to involve a combination of enzymatic reduction and chemical reaction. Enzymatic reduction by a cytosolic NADPH-dependent enzyme produces an aromatic metabolite, as does reaction with thiols. Acylfulvene is formed from illudin S by reverse Prins reaction. Acylfulvene is 100-fold less toxic in vitro and in vivo than illudin S but possesses marked antitumor efficacy in vivo, thus displaying opposite properties from illudin S. For this reason we investigated the in vitro metabolism of acylfulvene. Incubation of acylfulvene with NADPH and rat liver cytosol yielded two metabolites. One metabolite, the aromatic product, is similar to that obtained with illudin S in this in vitro system and was anticipated. The other metabolite, the hydroxylated product, was not expected and no corresponding metabolite for illudin S could be detected. The production of this hydroxylated metabolite from acylfulvene may explain, in part, the increased antitumor activity of novel acylfulvenes as compared with the illudins.

  13. Toxic Epidermal Necrolysis: A Case Report

    Directory of Open Access Journals (Sweden)

    Seher Erdogan

    2016-09-01

    Full Text Available Stevens-Johnson syndrome (SJS and toxic epidermal necrolysis (TEN are severe, acute reactions of the skin and mucosa that can result in serious clinical outcomes and morbidity. We report a case of TEN resulting in mortality, following the use of tablets containing paracetamol, codeine phosphate, and chlorpheniramine for an upper respiratory tract infection. Conditions such as SJS and TEN that can lead to serious clinical outcomes should be considered in juvenile patients, and unnecessary drug use should be avoided.

  14. Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering

    OpenAIRE

    Zhao, Xin; Lang, Qi; Yildirimer, Lara; Lin, Zhi Yuan; Cui, Wenguo; Annabi, Nasim; Ng, Kee Woei; Dokmeci, Mehmet R.; Ghaemmaghami, Amir M.; Khademhosseini, Ali

    2015-01-01

    Natural hydrogels are promising scaffolds to engineer epidermis. Currently, natural hydrogels used to support epidermal regeneration are mainly collagen- or gelatin-based, which mimic the natural dermal extracellular matrix but often suffer from insufficient and uncontrollable mechanical and degradation properties. In this study, a photocrosslinkable gelatin (i.e., gelatin methacrylamide (GelMA)) with tunable mechanical, degradation, and biological properties is used to engineer the epidermis...

  15. Epidermal cell death in frogs with chytridiomycosis

    Directory of Open Access Journals (Sweden)

    Laura A. Brannelly

    2017-02-01

    Full Text Available Background Amphibians are declining at an alarming rate, and one of the major causes of decline is the infectious disease chytridiomycosis. Parasitic fungal sporangia occur within epidermal cells causing epidermal disruption, but these changes have not been well characterised. Apoptosis (planned cell death can be a damaging response to the host but may alternatively be a mechanism of pathogen removal for some intracellular infections. Methods In this study we experimentally infected two endangered amphibian species Pseudophryne corroboree and Litoria verreauxii alpina with the causal agent of chytridiomycosis. We quantified cell death in the epidermis through two assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL and caspase 3/7. Results Cell death was positively associated with infection load and morbidity of clinically infected animals. In infected amphibians, TUNEL positive cells were concentrated in epidermal layers, correlating to the localisation of infection within the skin. Caspase activity was stable and low in early infection, where pathogen loads were light but increasing. In animals that recovered from infection, caspase activity gradually returned to normal as the infection cleared. Whereas, in amphibians that did not recover, caspase activity increased dramatically when infection loads peaked. Discussion Increased cell death may be a pathology of the fungal parasite, likely contributing to loss of skin homeostatic functions, but it is also possible that apoptosis suppression may be used initially by the pathogen to help establish infection. Further research should explore the specific mechanisms of cell death and more specifically apoptosis regulation during fungal infection.

  16. Topical Hesperidin Improves Epidermal Permeability Barrier Function and Epidermal Differentiation in Normal Murine Skin

    Science.gov (United States)

    Hou, Maihua; Man, Mona; Man, Wenyan; Zhu, Wenyuan; Hupe, Melanie; Park, Kyungho; Crumrine, Debra; Elias, Peter M.; Man, Mao-Qiang

    2012-01-01

    Orange peel extract appears to exhibit beneficial effects on skin whitening, inflammation, UVB protection, as well as keratinocyte proliferation. In the present study, we determine whether topical hesperidin influences epidermal permeability barrier function and its underlying mechanisms. Hairless mice were treated topically with 2% hesperidin or 70% ethanol alone twice daily for 6 days. At the end of treatment, basal barrier function as well as transepidermal water loss (TEWL) was measured 2 and 4 hours post barrier disruption. Epidermal proliferation and differentiation were evaluated by immunohistochemical staining and Western blot analysis. Additionally, lamellar body density and secretion were assessed by electron microscopy. Although there were no significant differences in basal barrier function, in comparison to control animals, topical hesperidin significantly accelerated barrier recovery at both 2 and 4 hours after acute barrier abrogation. Enhanced barrier function in hesperidin-treated skin correlated with stimulation of both epidermal proliferation and differentiation, as well as enhanced lamellar body secretion. These results indicate that topical hesperidin enhances epidermal permeability barrier homeostasis at least in part due to stimulation of epidermal proliferation, differentiation, as well as lamellar body secretion. PMID:22509829

  17. Restricting glutamylcysteine synthetase activity to the cytosol or glutathione biosynthesis to the plastid is sufficient for normal plant development and stress tolerance.

    Science.gov (United States)

    Lim, B; Pasternak, M; Meyer, A J; Cobbett, C S

    2014-01-01

    The tripeptide glutathione (GSH) is an important metabolite with a broad spectrum of functions, and its homeostasis is essential to maintain cellular redox poise and effective responses to stress in plants. In Arabidopsis GSH is synthesised in two successive enzymatic steps by γ-glutamylcysteine synthetase (GSH1), localised exclusively in plastids, forming the pathway intermediate γ-glutamylcysteine (γ-EC), and then by glutathione synthetase (GSH2), which is located in both plastids and cytosol. This suggests a mechanism for γ-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is speculated that the cytosol may be the main compartment for GSH biosynthesis. With the availability of knockout lethal mutants of GSH1 and GSH2 in Arabidopsis, we were able to manipulate the GSH biosynthetic pathway within cells through transgenic techniques. We successfully complemented the gsh1 and gsh2 null mutants with a cytosol-targeted bacterial EcGSHA and plastid-targeted Arabidopsis GSH2 protein, respectively, to wild-type phenotypes. These transgenics were little affected under heavy metal (cadmium) or oxidative stress (H2 O2 ) when compared to the wild type. Collectively, our data show that redirecting GSH1 activity exclusively to the cytosol or restricting GSH biosynthesis to the plastids has no significant impact on development or stress resistance, suggesting efficient exchange of γ-EC and GSH between the plastid and cytosol compartments within cells. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  18. Surface-Enhanced Raman Spectroscopy (SERS Tracking of Chelerythrine, a Na+/K+ Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Kevin M. Dorney

    2013-12-01

    Full Text Available Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA chelerythrine (CET is a pro-apoptotic drug and Na+/K+ pump (NKP inhibitor in human lens epithelial cells (HLECs. In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS. Methods: Silver nanoparticles (AgNPs prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+ accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

  19. Surface-enhanced Raman spectroscopy (SERS) tracking of chelerythrine, a Na(+)/K(+) pump inhibitor, into cytosol and plasma membrane fractions of human lens epithelial cell cultures.

    Science.gov (United States)

    Dorney, Kevin M; Sizemore, Ioana E P; Alqahtani, Tariq; Adragna, Norma C; Lauf, Peter K

    2013-01-01

    The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na(+)/K(+) pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm(-1) marker band as a function of CET concentration. SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET(+)) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect. © 2014 S. Karger AG, Basel.

  20. Yeast Interacting Proteins Database: YML064C, YKL103C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available he peptidase family M18; often used as a marker protein in studies of autophagy a... to the peptidase family M18; often used as a marker protein in studies of autophagy and cytosol to vacuole

  1. DNA-Encoded Flagellin Activates Toll-Like Receptor 5 (TLR5), Nod-like Receptor Family CARD Domain-Containing Protein 4 (NRLC4), and Acts as an Epidermal, Systemic, and Mucosal-Adjuvant

    Science.gov (United States)

    Nyström, Sanna; Bråve, Andreas; Falkeborn, Tina; Devito, Claudia; Rissiek, Björn; Johansson, Daniel X.; Schröder, Ulf; Uematsu, Satoshi; Akira, Shizuo; Hinkula, Jorma; Applequist, Steven E.

    2013-01-01

    Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes. PMID:26344341

  2. Decreased levels of salivary prostaglandin E2 and epidermal growth factor in recurrent aphthous stomatitis.

    Science.gov (United States)

    Wu-Wang, C Y; Patel, M; Feng, J; Milles, M; Wang, S L

    1995-12-01

    Prostaglandin E2 and epidermal growth factor are two important cytoprotective compounds in saliva. This study investigated their salivary levels in controls and individuals with minor recurrent aphthous stomatitis. The development of recurrent aphthous stomatitis was divided into three stages: (1) early active stage (mucosal redness); (2) active stage (mucosal ulceration); (3) convalescent stage. Unstimulated mixed saliva was collected from each volunteer. Salivary prostaglandin E2 and epidermal growth factor concentrations were determined by radioimmunoassay. Their levels (mean +/- SEM) were significantly lower during the active stage of ulceration as compared to the control: (a) for prostaglandin E2, 200 +/- 55 versus 73 +/- 11 pg/mg salivary protein (p stomatitis. The prostaglandin E2 concentration decreased significantly during the active stage of ulceration, and then increased significantly during the convalescent stage. However, the recovery of salivary epidermal growth factor after the ulceration was slower than that of the prostaglandin E2. It is suggested that the diminution of prostaglandin E2 and epidermal growth factor in the saliva may be associated with the ulcer development.

  3. Metabolism of antitumor hydroxymethylacylfulvene by rat liver cytosol.

    Science.gov (United States)

    McMorris, T C; Elayadi, A N; Yu, J; Hu, Y; Kelner, M J

    1999-09-01

    Acylfulvenes are a potent class of antitumor agents derived from illudin S, a fungal sesquiterpene. Illudin S possesses antitumor activity but has a poor therapeutic index. Acylfulvene is 100-fold less toxic against human lung adenocarcinoma cells than illudin S, but inhibits tumor growth in human xenografts, opposite to illudin S. An analog of acylfulvene, MGI 114 (hydroxymethylacylfulvene), shows much greater efficacy, producing complete tumor regression in xenograft models. MGI 114 is currently in phase II clinical trials. Cytotoxicity of MGI 114, like that of illudin S, is believed to involve both chemical reaction and enzymatic reduction. Enzymatic reduction by a cytosolic NADPH-dependent enzyme (from rat liver) produced an aromatic metabolite similar to that formed from illudin S. However, the reaction occurred more slowly. In addition, four new metabolites were isolated, two hydroxylated derivatives and two in which the primary allylic hydroxyl was replaced by hydride. All retained the reactive centers of the parent MGI 114.

  4. Nod-like receptors: cytosolic watchdogs for immunity against pathogens.

    Directory of Open Access Journals (Sweden)

    Jean-Claude Sirard

    2007-12-01

    Full Text Available In mammals, tissue-specific sets of pattern-recognition molecules, including Nod-like receptors (NLR, enable concomitant and sequential detection of microbial-associated molecular patterns from both the extracellular and intracellular microenvironment. Repressing and de-repressing the cytosolic surveillance machinery contributes to vital immune homeostasis and protective responses within specific tissues. Conversely, defective biology of NLR drives the development of recurrent infectious, autoimmune and/or inflammatory diseases by failing to mount barrier functions against pathogens, to tolerate commensals, and/or to instruct the adaptive immune response against microbes. Better decoding microbial strategies that are evolved to circumvent NLR sensing will provide clues for the development of rational therapies aimed at curing and/or preventing common and emerging immunopathologies.

  5. The status of epidermal growth factor receptor in borderline ovarian tumours

    OpenAIRE

    Showeil, R; Romano, C; Valganon, M; Lambros, M.; Trivedi, P; Van Noorden, S; Sriraksa, R; El-Kaffash, D; El-Etreby, N; Natrajan, R.; Foroni, L; Osborne, R.; El-Bahrawy, M

    2016-01-01

    The majority of borderline ovarian tumours (BOTs) behave in a benign fashion, but some may show aggressive behavior. The reason behind this has not been elucidated. The epidermal growth factor receptor (EGFR) is known to contribute to cell survival signals as well as metastatic potential of some tumours. EGFR expression and gene status have not been thoroughly investigated in BOTs as it has in ovarian carcinomas. In this study we explore protein expression as well as gene mutations and amplif...

  6. Increased group IV cytosolic phospholipase A2 activity in lungs of sheep after smoke inhalation injury.

    Science.gov (United States)

    Fukuda, T; Kim, D K; Chin, M R; Hales, C A; Bonventre, J V

    1999-09-01

    Increased phospholipase A2 (PLA2) activity was measured in cytosolic fractions of lungs from sheep exposed to smoke from burning cotton or to synthetic smoke consisting of carbon and acrolein, a cotton smoke toxin. Three peaks of PLA2 activity were identified by heparin-Sepharose chromatography. The heparin-nonbinding PLA2 activity was twofold higher in the extracts from lungs exposed to smoke than in normal lungs. This activity was identified as the group IV 85-kDa cytosolic PLA2 (cPLA2). The activities of the forms of PLA2 that bound to heparin did not change after smoke exposure. Those activities showed a pH optimum of 9.0, required a millimolar Ca2+ concentration for full activity, and were inhibited by 5 mM dithiothreitol. One activity eluted at an NaCl concentration typical for group Ib and V PLA2 and had the expected substrate specificity. The other form of lung PLA2 that bound heparin was a group II PLA2. Lung myeloperoxidase activity increased progressively with increased exposure to smoke. cPLA2 was identified in sheep neutrophils. With 30 breaths of smoke exposure, there was an increase in cPLA2 activity without a difference in immunoreactivity on Western blot, indicating that the increased activity was not due to increased amounts of protein. In conclusion, smoke induces increases in resident lung cell cPLA2 activity that is likely responsible for eicosanoid production, leading to lung inflammation and bronchoconstriction.

  7. Yeast Interacting Proteins Database: YKL103C, YKL103C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available he peptidase family M18; often used as a marker protein in studies of autophagy and cytosol to vacuole targe...; often used as a marker protein in studies of autophagy and cytosol to vacuole targeting (CVT) pathway Rows...e yscI; zinc metalloproteinase that belongs to the peptidase family M18; often used as a marker protein in studies...t belongs to the peptidase family M18; often used as a marker protein in studies of autophagy and cytosol to

  8. Late onset epidermal nevus with hypertrichosis and facial hemihypertrophy

    Directory of Open Access Journals (Sweden)

    M Saritha

    2014-01-01

    Full Text Available Epidermal nevus syndromes are rare conditions, characterized by different types of keratinocytic or organoid epidermal nevi in association with ocular, neurological, and skeletal manifestations. We present a case of late onset epidermal nevus with hypertrichosis and hemihypertrophy of face. Genetic analysis did not reveal presence of FGFR3 or PIK3CA mutations. The patient has features that cannot be categorized into the present well-known syndromes.

  9. Circadian rhythms of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), cortisol, and melatonin in women with breast cancer.

    Science.gov (United States)

    Haus, E; Dumitriu, L; Nicolau, G Y; Bologa, S; Sackett-Lundeen, L

    2001-07-01

    Circadian rhythms in plasma concentrations of many hormones and cytokines determine their effects on target cells. Circadian variations were studied in cortisol, melatonin, cytokines (basic fibroblast growth factor IbFGF], EGF, insulin-like growth factor-1 [IGF-1]), and a cytokine receptor (insulin-like growth factor binding protein-3 [IGFBP-3]) in the plasma of 28 patients with metastatic breast cancer. All patients followed a diurnal activity pattern. Blood was drawn at 3h intervals during waking hours and once during the night, at 03:00. The plasma levels obtained by enzyme-linked immunoassay (ELISA) or radioimmunoassay (RIA) were evaluated by population mean cosinor (using local midnight as the phase reference) and by one-way analysis of variance (ANOVA). Cortisol and melatonin showed a high-amplitude circadian rhythm and a superimposed 12h frequency. bFGF showed a circadian rhythm with an acrophase around 13:00 with a peak-to-trough interval (double amplitude) of 18.2% and a superimposed 12h frequency. EGF showed a circadian rhythm with an acrophase around 14:20, a peak-to-trough interval of 25.8%, and a superimposed 12h frequency. IGF-1 showed a high value in the morning, which is statistically different (t test) from the low value at 10:00, but a regular circadian or ultradian rhythm was not recognizable as a group phenomenon. IGFBP-3 showed a low-amplitude (peak-to-trough difference 8.4%) circadian rhythm with the acrophase around 11:00 and low values during the night. (1) Circadian periodicity is maintained in hospitalized patients with metastatic breast cancer. (2) Ultradian (12h) variations were superimposed on the circadian rhythms of the hormones and several of the cytokines measured. (3) Studies of hormones and cytokines in cancer patients have to take their biologic rhythms into consideration. (4) The circadian periodicity of tumor growth stimulating or restraining factors raises questions about circadian and/or ultradian variations in the

  10. Giant epidermal cyst of the tarsal plate

    Directory of Open Access Journals (Sweden)

    Mohana Majumdar

    2012-01-01

    Full Text Available A 35-year-old male patient presented with a right upper eyelid mass with mechanical ptosis. The patient gave no history of trauma or surgery. On examination, there was a huge cystic mass fixed to the tarsal plate. Excisional biopsy with tarsectomy was done. Histopathology sections demonstrated a keratin-filled cyst arising from the tarsus. A thorough Pubmed search did not reveal an epidermal cyst of the tarsal plate of this size which was successfully managed. The incision was made in such a way that postoperative ptosis would be avoided. Excess skin was removed during the surgery.

  11. [Bilateral segmental neurofibromatosis simulating epidermal nevus].

    Science.gov (United States)

    Gambichler, T; Küster, W; Wolter, M; Rapp, S; Altmeyer, P; Hoffmann, K

    2000-11-01

    Neurofibromatosis is a neuroectodermal systemic disease. A rare variant of this condition is bilateral segmental neurofibromatosis. A 29-year-old man presented with bilateral papillomatous plaques in the lumbar dermatomes. Clinically, the lesions were very similar to an epidermal nevus but histologic examination revealed superficial neurofibromas. Family history, ophthalmologic and neurologic investigations were unremarkable. The unusual morphologic presentation of bilateral segmental neurofibromas in this case points to the wide clinical spectrum of the disease and the significance of histologic examination in systematic nevoid lesions.

  12. A Cytosolic Acyltransferase Contributes to Triacylglycerol Synthesis in Sucrose-Rescued Arabidopsis Seed Oil Catabolism Mutants1[W][OA

    Science.gov (United States)

    Hernández, M. Luisa; Whitehead, Lynne; He, Zhesi; Gazda, Valeria; Gilday, Alison; Kozhevnikova, Ekaterina; Vaistij, Fabián E.; Larson, Tony R.; Graham, Ian A.

    2012-01-01

    Triacylglycerol (TAG) levels and oil bodies persist in sucrose (Suc)-rescued Arabidopsis (Arabidopsis thaliana) seedlings disrupted in seed oil catabolism. This study set out to establish if TAG levels persist as a metabolically inert pool when downstream catabolism is disrupted, or if other mechanisms, such as fatty acid (FA) recycling into TAG are operating. We show that TAG composition changes significantly in Suc-rescued seedlings compared with that found in dry seeds, with 18:2 and 18:3 accumulating. However, 20:1 FA is not efficiently recycled back into TAG in young seedlings, instead partitioning into the membrane lipid fraction and diacylglycerol. In the lipolysis mutant sugar dependent1and the β-oxidation double mutant acx1acx2 (for acyl-Coenzyme A oxidase), levels of TAG actually increased in seedlings growing on Suc. We performed a transcriptomic study and identified up-regulation of an acyltransferase gene, DIACYLGLYCEROL ACYLTRANSFERASE3 (DGAT3), with homology to a peanut (Arachis hypogaea) cytosolic acyltransferase. The acyl-Coenzyme A substrate for this acyltransferase accumulates in mutants that are blocked in oil breakdown postlipolysis. Transient expression in Nicotiana benthamiana confirmed involvement in TAG synthesis and specificity toward 18:3 and 18:2 FAs. Double-mutant analysis with the peroxisomal ATP-binding cassette transporter mutant peroxisomal ABC transporter1 indicated involvement of DGAT3 in the partitioning of 18:3 into TAG in mutant seedlings growing on Suc. Fusion of the DGAT3 protein with green fluorescent protein confirmed localization to the cytosol of N. benthamiana. This work has demonstrated active recycling of 18:2 and 18:3 FAs into TAG when seed oil breakdown is blocked in a process involving a soluble cytosolic acyltransferase. PMID:22760209

  13. Interplay Between Cytosolic Free Zn2+ and Mitochondrion Morphological Changes in Rat Ventricular Cardiomyocytes.

    Science.gov (United States)

    Billur, Deniz; Tuncay, Erkan; Okatan, Esma Nur; Olgar, Yusuf; Durak, Aysegul Toy; Degirmenci, Sinan; Can, Belgin; Turan, Belma

    2016-11-01

    The Zn2+ in cardiomyocytes is buffered by structures near T-tubulus and/or sarcoplasmic/endoplasmic reticulum (S(E)R) while playing roles as either an antioxidant or a toxic agent, depending on the concentration. Therefore, we aimed first to examine a direct effect of ZnPO4 (extracellular exposure) or Zn2+ pyrithione (ZnPT) (intracellular exposure) application on the structure of the mitochondrion in ventricular cardiomyocytes by using histological investigations. The light microscopy data demonstrated that Zn2+ exposure induced marked increases on cellular surface area, an indication of hypertrophy, in a concentration-dependent manner. Furthermore, a whole-cell patch-clamp measurement of cell capacitance also supported the hypertrophy in the cells. We observed marked increases in mitochondrial matrix/cristae area and matrix volume together with increased lysosome numbers in ZnPO4- or ZnPT-incubated cells by using transmission electron microscopy, again in a concentration-dependent manner. Furthermore, we observed notable clustering and vacuolated mitochondrion, markedly disrupted and damaged myofibrils, and electron-dense small granules in Zn2+-exposed cells together with some implications of fission-fusion defects in the mitochondria. Moreover, we observed marked depolarization in mitochondrial membrane potential during 1-μM ZnPT minute applications by using confocal microscopy. We also showed that 1-μM ZnPT incubation induced significant increases in the phosphorylation levels of GSK3β (Ser21 and Ser9), Akt (Ser473), and NFκB (Ser276 and Thr254) together with increased expression levels in ER stress proteins such as GRP78 and calregulin. Furthermore, a new key player at ER-mitochondria sites, promyelocytic leukemia protein (PML) level, was markedly increased in ZnPT-incubated cells. As a summary, our present data suggest that increased cytosolic free Zn2+ can induce marked alterations in mitochondrion morphology as well as depolarization in mitochondrion

  14. Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle

    DEFF Research Database (Denmark)

    Satrústegui, Jorgina; Bak, Lasse K

    2015-01-01

    that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium...

  15. Improving scFv antibody expression levels in the plant cytosol.

    NARCIS (Netherlands)

    Schouten, A.; Roosien, J.; Boer, de J.M.; Wilmink, A.; rosso, M.N.; Bosch, D.; Stiekema, W.J.; gommers, F.J.; Bakker, J.; Schots, A.

    1997-01-01

    Expression of single-chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C-terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv-CK was

  16. Eph-2B, acting as an extracellular ligand, induces differentiation markers in epidermal keratinocytes.

    Science.gov (United States)

    Walsh, Rebecca; Blumenberg, Miroslav

    2012-06-01

    In the bi-directional signaling system comprising ephrins (EFNs) and ephrin receptors (Ephs), both EFNs and Ephs simultaneously function both as ligands and as receptors. Importantly, the EFN/Eph system is deregulated in human cancers and has been implicated in the metastatic processes because of its effects on the adhesion and migration of epithelial cells. The idiosyncratic function of Ephs, membrane-bound receptor kinases, as extracellular signaling ligands, has not been extensively studied. This prompted us to explore the transcriptional targets regulated by Ephs acting solely as ligands. To define the ligand function of EphB2 in human epidermal keratinocytes, we treated these cells with EphB2 as Fc-conjugate dimmers, which thus act exclusively as extracellular ligands. We compared the EphB2 and EFNA4 effects during a 48 h time course, using transcriptional profiling. We found that EphB2, acting as a ligand, promotes epidermal differentiation. For example, EphB2 induces expression of markers of epidermal differentiation, including keratins KRT1 and KRT10, SPRRs, desmosomal proteins and cell cycle inhibitors, while suppressing basal layer markers, integrins and cell cycle proteins. The effects of EphB2 are delayed relative to those of EFNA4. Unlike EFNA4, EphB2 did not induce lipid metabolism proteins, this particular aspect of epidermal differentiation seems not to be regulated by EphB2. Our results define the transcriptional targets of the reverse signaling by EphB2 acting exclusively as a ligand and begin to characterize this intriguing function of Ephs. Copyright © 2011 Wiley Periodicals, Inc.

  17. Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering.

    Science.gov (United States)

    Zhao, Xin; Lang, Qi; Yildirimer, Lara; Lin, Zhi Yuan; Cui, Wenguo; Annabi, Nasim; Ng, Kee Woei; Dokmeci, Mehmet R; Ghaemmaghami, Amir M; Khademhosseini, Ali

    2016-01-07

    Natural hydrogels are promising scaffolds to engineer epidermis. Currently, natural hydrogels used to support epidermal regeneration are mainly collagen- or gelatin-based, which mimic the natural dermal extracellular matrix but often suffer from insufficient and uncontrollable mechanical and degradation properties. In this study, a photocrosslinkable gelatin (i.e., gelatin methacrylamide (GelMA)) with tunable mechanical, degradation, and biological properties is used to engineer the epidermis for skin tissue engineering applications. The results reveal that the mechanical and degradation properties of the developed hydrogels can be readily modified by varying the hydrogel concentration, with elastic and compressive moduli tuned from a few kPa to a few hundred kPa, and the degradation times varied from a few days to several months. Additionally, hydrogels of all concentrations displayed excellent cell viability (>90%) with increasing cell adhesion and proliferation corresponding to increases in hydrogel concentrations. Furthermore, the hydrogels are found to support keratinocyte growth, differentiation, and stratification into a reconstructed multilayered epidermis with adequate barrier functions. The robust and tunable properties of GelMA hydrogels suggest that the keratinocyte laden hydrogels can be used as epidermal substitutes, wound dressings, or substrates to construct various in vitro skin models. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Cytosolic Ca2+, exocytosis, and endocytosis in single melanotrophs of the rat pituitary.

    Science.gov (United States)

    Thomas, P; Surprenant, A; Almers, W

    1990-11-01

    We have monitored cytosolic [Ca2+] with fura-2 and exocytosis by measuring the membrane capacitance, and we have studied the influence of cytosolic [Ca2+] on secretion in single endocrine cells. As in neurons, cytosolic Ca2+ is sufficient to trigger exocytosis. The rate of secretion grows with the fourth or fifth power of cytosolic [Ca2+], and paired stimuli reveal facilitation. Ca2+ influx through voltage-sensitive Ca2+ channels can stimulate secretion 1000-fold over the basal levels measured biochemically. Unlike neurons, however, melanotrophs continue to secrete for seconds afer a depolarizing pulse, while they extrude or sequester the Ca2+ that has entered through Ca2+ channels. Following episodes of secretion, pituitary cells can retrieve membrane with half-times around 30 s at 32 degrees C, even in the absence of cytosolic K+.

  19. Cytosolic Ca(2+) as a multifunctional modulator is required for spermiogenesis in Ascaris suum.

    Science.gov (United States)

    Shang, Yunlong; Chen, Lianwan; Liu, Zhiyu; Wang, Xia; Ma, Xuan; Miao, Long

    2013-06-01

    The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.

  20. Holocarboxylase Synthetase: A Moonlighting Transcriptional Coregulator of Gene Expression and a Cytosolic Regulator of Biotin Utilization.

    Science.gov (United States)

    León-Del-Río, Alfonso; Valadez-Graham, Viviana; Gravel, Roy A

    2017-08-21

    The vitamin biotin is an essential nutrient for the metabolism and survival of all organisms owing to its function as a cofactor of enzymes collectively known as biotin-dependent carboxylases. These enzymes use covalently attached biotin as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In human cells, biotin-dependent carboxylases catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Biotin is attached to apocarboxylases by a biotin ligase: holocarboxylase synthetase (HCS) in mammalian cells and BirA in microbes. Despite their evolutionary distance, these proteins share structural and sequence similarities, underscoring their importance across all life forms. However, beyond its role in metabolism, HCS participates in the regulation of biotin utilization and acts as a nuclear transcriptional coregulator of gene expression. In this review, we discuss the function of HCS and biotin in metabolism and human disease, a putative role for the enzyme in histone biotinylation, and its participation as a nuclear factor in chromatin dynamics. We suggest that HCS be classified as a moonlighting protein, with two biotin-dependent cytosolic metabolic roles and a distinct biotin-independent nuclear coregulatory function.

  1. Bioengineering a Human Plasma-Based Epidermal Substitute With Efficient Grafting Capacity and High Content in Clonogenic Cells

    Science.gov (United States)

    Alexaline, Maia M.; Trouillas, Marina; Nivet, Muriel; Bourreau, Emilie; Leclerc, Thomas; Duhamel, Patrick; Martin, Michele T.; Doucet, Christelle; Fortunel, Nicolas O.

    2015-01-01

    Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. Significance This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach. PMID:25848122

  2. Flightless-I, a gelsolin family member and transcriptional regulator, preferentially binds directly to activated cytosolic CaMK-II.

    Science.gov (United States)

    Seward, Matthew E; Easley, Charles A; McLeod, Jamie J; Myers, Alexandra L; Tombes, Robert M

    2008-07-23

    In order to evaluate links between Ca2+/calmodulin (CaM)-dependent protein kinase type II (CaMK-II) and cell cycle progression, CaMK-II binding partners were sought in proliferating cells by epitope-tag tandem mass spectrometry. One protein identified was the gelsolin family member, flightless-I (Fli-I). Fli-I is not a CaMK-II substrate, but binds directly and preferentially to constitutively active (T287D) CaMK-II over inactive CaMK-II. Fli-I gradually enters the nucleus upon CaMK-II inhibition and is retained in the cytosol by T287D CaMK-II. CaMK-II inhibition and Fli-I overexpression suppress transcription of beta-catenin dependent transcriptional reporters, whereas Fli-I suppression enhances their transcription. These findings support a novel mechanism whereby cytosolic CaMK-II influences beta-catenin dependent gene expression through Fli-I.

  3. Glutaredoxin 5 deficiency causes sideroblastic anemia by specifically impairing heme biosynthesis and depleting cytosolic iron in human erythroblasts.

    Science.gov (United States)

    Ye, Hong; Jeong, Suh Young; Ghosh, Manik C; Kovtunovych, Gennadiy; Silvestri, Laura; Ortillo, Danilo; Uchida, Naoya; Tisdale, John; Camaschella, Clara; Rouault, Tracey A

    2010-05-01

    Glutaredoxin 5 (GLRX5) deficiency has previously been identified as a cause of anemia in a zebrafish model and of sideroblastic anemia in a human patient. Here we report that GLRX5 is essential for iron-sulfur cluster biosynthesis and the maintenance of normal mitochondrial and cytosolic iron homeostasis in human cells. GLRX5, a mitochondrial protein that is highly expressed in erythroid cells, can homodimerize and assemble [2Fe-2S] in vitro. In GLRX5-deficient cells, [Fe-S] cluster biosynthesis was impaired, the iron-responsive element-binding (IRE-binding) activity of iron regulatory protein 1 (IRP1) was activated, and increased IRP2 levels, indicative of relative cytosolic iron depletion, were observed together with mitochondrial iron overload. Rescue of patient fibroblasts with the WT GLRX5 gene by transfection or viral transduction reversed a slow growth phenotype, reversed the mitochondrial iron overload, and increased aconitase activity. Decreased aminolevulinate delta, synthase 2 (ALAS2) levels attributable to IRP-mediated translational repression were observed in erythroid cells in which GLRX5 expression had been downregulated using siRNA along with marked reduction in ferrochelatase levels and increased ferroportin expression. Erythroblasts express both IRP-repressible ALAS2 and non-IRP-repressible ferroportin 1b. The unique combination of IRP targets likely accounts for the tissue-specific phenotype of human GLRX5 deficiency.

  4. Identification of host cytosolic sensors and bacterial factors regulating the type I interferon response to Legionella pneumophila.

    Directory of Open Access Journals (Sweden)

    Kathryn M Monroe

    2009-11-01

    Full Text Available Legionella pneumophila is a gram-negative bacterial pathogen that replicates in host macrophages and causes a severe pneumonia called Legionnaires' Disease. The innate immune response to L. pneumophila remains poorly understood. Here we focused on identifying host and bacterial factors involved in the production of type I interferons (IFN in response to L. pneumophila. It was previously suggested that the delivery of L. pneumophila DNA to the host cell cytosol is the primary signal that induces the type I IFN response. However, our data are not easily reconciled with this model. We provide genetic evidence that two RNA-sensing proteins, RIG-I and MDA5, participate in the IFN response to L. pneumophila. Importantly, these sensors do not seem to be required for the IFN response to L. pneumophila DNA, whereas we found that RIG-I was required for the response to L. pneumophila RNA. Thus, we hypothesize that bacterial RNA, or perhaps an induced host RNA, is the primary stimulus inducing the IFN response to L. pneumophila. Our study also identified a secreted effector protein, SdhA, as a key suppressor of the IFN response to L. pneumophila. Although viral suppressors of cytosolic RNA-sensing pathways have been previously identified, analogous bacterial factors have not been described. Thus, our results provide new insights into the molecular mechanisms by which an intracellular bacterial pathogen activates and also represses innate immune responses.

  5. Epidermal Inclusion Cyst Embedded in a Recurrent Benign Fibrous Histiocytoma

    OpenAIRE

    Mani Makhija

    2014-01-01

    Epidermal infundibular cysts have been known to arise from implantation of epidermis in the dermis or subcutaneous tissues. The present case illustrates the theory of implantation in an epidermal inclusion cyst in a previously operated case of recurrent benign fibrous histiocytoma.

  6. Epidermal inclusion cyst embedded in a recurrent benign fibrous histiocytoma

    Directory of Open Access Journals (Sweden)

    Mani Makhija

    2014-01-01

    Full Text Available Epidermal infundibular cysts have been known to arise from implantation of epidermis in the dermis or subcutaneous tissues. The present case illustrates the theory of implantation in an epidermal inclusion cyst in a previously operated case of recurrent benign fibrous histiocytoma.

  7. Epidermal Inclusion Cyst Embedded in a Recurrent Benign Fibrous Histiocytoma

    Science.gov (United States)

    Makhija, Mani

    2014-01-01

    Epidermal infundibular cysts have been known to arise from implantation of epidermis in the dermis or subcutaneous tissues. The present case illustrates the theory of implantation in an epidermal inclusion cyst in a previously operated case of recurrent benign fibrous histiocytoma. PMID:25284857

  8. Post-circumcision giant epidermal clitoral inclusion cyst: Case report ...

    African Journals Online (AJOL)

    Background: A large epidermal clitoral inclusion cyst may pose a diagnostic dilemma involving several differential diagnoses. Case: We describe a case of post-circumcision giant epidermal clitoral inclusion cyst including the aetiology, presentation, diagnosis, differential diagnosis, management and reason for its late ...

  9. Foliar epidermal morphology of the genera Aneilema and ...

    African Journals Online (AJOL)

    The aim was to study the structure of the foliar epidermis of five species belonging to the two genera and thus provide anatomical information that will help to possibly eliminate identification problems that exist among species of the genera using diagnostic characters of the foliar epidermal morphology. Epidermal peels of ...

  10. Determination of the exact molecular requirements for type 1 angiotensin receptor epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy.

    Science.gov (United States)

    Smith, Nicola J; Chan, Hsiu-Wen; Qian, Hongwei; Bourne, Allison M; Hannan, Katherine M; Warner, Fiona J; Ritchie, Rebecca H; Pearson, Richard B; Hannan, Ross D; Thomas, Walter G

    2011-05-01

    Major interest surrounds how angiotensin II triggers cardiac hypertrophy via epidermal growth factor receptor transactivation. G protein-mediated transduction, angiotensin type 1 receptor phosphorylation at tyrosine 319, and β-arrestin-dependent scaffolding have been suggested, yet the mechanism remains controversial. We examined these pathways in the most reductionist model of cardiomyocyte growth, neonatal ventricular cardiomyocytes. Analysis with [(32)P]-labeled cardiomyocytes, wild-type and [Y319A] angiotensin type 1 receptor immunoprecipitation and phosphorimaging, phosphopeptide analysis, and antiphosphotyrosine blotting provided no evidence for tyrosine phosphorylation at Y319 or indeed of the receptor, and mutation of Y319 (to A/F) did not prevent either epidermal growth factor receptor transactivation in COS-7 cells or cardiomyocyte hypertrophy. Instead, we demonstrate that transactivation and cardiomyocyte hypertrophy are completely abrogated by loss of G-protein coupling, whereas a constitutively active angiotensin type 1 receptor mutant was sufficient to trigger transactivation and growth in the absence of ligand. These results were supported by the failure of the β-arrestin-biased ligand SII angiotensin II to transactivate epidermal growth factor receptor or promote hypertrophy, whereas a β-arrestin-uncoupled receptor retained these properties. We also found angiotensin II-mediated cardiomyocyte hypertrophy to be attenuated by a disintegrin and metalloprotease inhibition. Thus, G-protein coupling, and not Y319 phosphorylation or β-arrestin scaffolding, is required for epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy via the angiotensin type 1 receptor.

  11. Deletion of K1/K10 does not impair epidermal stratification but affects desmosomal structure and nuclear integrity.

    Science.gov (United States)

    Wallace, Lee; Roberts-Thompson, Leiann; Reichelt, Julia

    2012-04-01

    Keratins K1 and K10 are the most abundant proteins in the upper epidermis where they polymerize to form intermediate filaments (IFs). In addition to their well-established function in providing epidermal stability, K1/K10 (i.e. the dimer between K1 and K10) IFs are supposed to be important for terminal epidermal differentiation and barrier formation. It was previously shown that the imbalanced deletion of one of the partner keratins, K10, disturbed epidermal homoeostasis, although stability was provided by compensatory upregulation of K5/K14, which formed IFs together with the remaining K1. Here, we show that deletion of both partner keratins, K1 and K10, results in lethal postnatal skin fragility in mice. Krt1(-/-);Krt10(-/-) mice revealed that K1/K10 IFs are unexpectedly dispensable for epidermal stratification. Although the stratum corneum was less compact and cornified envelope differentiation was impaired, a dye exclusion assay showed that the development of a functional water barrier was surprisingly independent from the presence of K1/K10 IFs. The deletion of K1/K10 was not compensated by any other keratin pair such as the basal epidermal keratins K5/K14, and electron microscopy revealed total absence of IFs in the suprabasal epidermis. Although plakoglobin was unchanged, the expression of the desmosomal proteins desmoplakin, desmocollin 1 and desmoglein 1 were altered and suprabasal desmosomes were smaller in Krt1(-/-);Krt10(-/-) than in wild-type epidermis suggesting an involvement of K1/K10 IFs in desmosome dynamics. Furthermore, Krt1(-/-);Krt10(-/-) mice showed premature loss of nuclei during epidermal differentiation and lower levels of emerin, lamin A/C and Sun1, revealing a previously unknown function for IFs in maintaining nuclear integrity in the upper epidermis.

  12. Herbal medicines that benefit epidermal permeability barrier function

    Directory of Open Access Journals (Sweden)

    Lizhi Hu

    2015-06-01

    Full Text Available Epidermal permeability barrier function plays a critical role in regulating cutaneous functions. Hence, researchers have been searching for effective and affordable regimens to enhance epidermal permeability barrier function. In addition to topical stratum corneum lipids, peroxisome proliferator-activated receptor, and liver X receptor ligands, herbal medicines have been proven to benefit epidermal permeability barrier function in both normal and diseased skin, including atopic dermatitis, glucocorticoid-induced skin damage, and UVB-damaged skin. The potential mechanisms by which herbal medicines improve the permeability barrier include stimulation of epidermal differentiation, lipid production, antimicrobial peptide expression, and antioxidation. Therefore, utilization of herbal medicines could be a valuable alternative approach to enhance epidermal permeability barrier function in order to prevent and/or treat skin disorders associated with permeability barrier abnormalities.

  13. NMR structure of integrin α4 cytosolic tail and its interactions with paxillin.

    Directory of Open Access Journals (Sweden)

    Geok-Lin Chua

    Full Text Available BACKGROUND: Integrins are a group of transmembrane signaling proteins that are important in biological processes such as cell adhesion, proliferation and migration. Integrins are α/β hetero-dimers and there are 24 different integrins formed by specific combinations of 18 α and 8 β subunits in humans. Generally, each of these subunits has a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT. CTs of integrins are important in bidirectional signal transduction and they associate with a large number of intracellular proteins. PRINCIPAL FINDINGS: Using NMR spectroscopy, we determined the 3-D structure of the full-length α4 CT (Lys968-Asp999 and characterize its interactions with the adaptor protein paxillin. The α4 CT assumes an overall helical structure with a kink in its membrane proximal region. Residues Gln981-Asn997 formed a continuous helical conformation that may be sustained by potential ionic and/or hydrogen bond interactions and packing of aromatic-aliphatic side-chains. ¹⁵N-¹H HSQC NMR experiments reveal interactions of the α4 CT C-terminal region with a fragment of paxillin (residues G139-K277 that encompassed LD2-LD4 repeats. Residues of these LD repeats including their adjoining linkers showed α4 CT binding-induced chemical shift changes. Furthermore, NMR studies using LD-containing peptides showed predominant interactions between LD3 and LD4 of paxillin and α4 CT. Docked structures of the α4 CT with these LD repeats suggest possible polar and/or salt-bridge and non-polar packing interactions. SIGNIFICANCE: The current study provides molecular insights into the structural diversity of α CTs of integrins and interactions of integrin α4 CT with the adaptor protein paxillin.

  14. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  15. Epidermal homeostasis in long-term scaffold-enforced skin equivalents

    National Research Council Canada - National Science Library

    Stark, Hans-Jürgen; Boehnke, Karsten; Mirancea, Nicolae; Willhauck, Michael J; Pavesio, Alessandra; Fusenig, Norbert E; Boukamp, Petra

    2006-01-01

    Epidermal homeostasis is understood as the maintenance of epidermal tissue structure and function by a fine tuned regulatory mechanism balancing proliferation and cell loss by desquamation and apoptosis...

  16. Compromised epidermal barrier stimulates Harderian gland activity and hypertrophy in ACBP-/- mice

    DEFF Research Database (Denmark)

    Sørensen, Signe Bek; Neess, Ditte; Dixen, Karen

    2015-01-01

    Acyl-CoA binding protein (ACBP) is a small, ubiquitously expressed intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity and specificity. We have recently shown that targeted disruption of the Acbp gene leads to a compromised epidermal barrier and that this causes delayed...... adaptation to weaning, including the induction of the hepatic lipogenic and cholesterogenic gene programs. Here we show that ACBP is highly expressed in the Harderian gland, a gland that is located behind the eyeball of rodents and involved in the production of fur lipids and lipids used for lubrication...

  17. Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

    Directory of Open Access Journals (Sweden)

    Yu J. Shen

    2015-04-01

    Full Text Available The DNA damage response (DDR induces the expression of type I interferons (IFNs, but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

  18. Epidermal transglutaminase (TGase 3 is required for proper hair development, but not the formation of the epidermal barrier.

    Directory of Open Access Journals (Sweden)

    Susan John

    Full Text Available Transglutaminases (TGase, a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.

  19. Steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient

    Energy Technology Data Exchange (ETDEWEB)

    Giguere, Anik; Couillard, Yves; Campbell, Peter G.C.; Perceval, Olivier; Hare, Landis; Pinel-Alloul, Bernadette; Pellerin, Jocelyne

    2003-07-16

    The present study was designed to assess the environmental effects of metals in a field setting. We explored exposure{yields}bioaccumulation{yields}effects relationships in freshwater molluscs exposed to metals in their natural habitat. Indigenous floater mussels (Pyganodon grandis) were collected from ten limnologically similar lakes located along a Cd, Cu and Zn gradient. Ambient free-metal ion concentrations were estimated as a measure of metal exposure. Metallothionein (MT) was measured in mussel gills and metal partitioning among the various cytosolic protein pools was determined by size exclusion chromatography. Various biomarkers were also measured, including malondialdehyde (MDA) concentrations in the gills and in the digestive gland, glutathione-peroxidase and glutathione-reductase activities in the digestive gland, and lipid concentrations in the gonad. Cadmium and MT concentrations in the gill cytosol increased along the contamination gradient, but Cu and Zn levels were independent of the ambient free-metal ion concentrations. The distribution of Cd among the various cytosolic complexes remained quite constant: 80% in the MT-like pool, 7% in the low molecular weight pool (LMW<1.8 kDa) and 13% in the high molecular weight pool (HMW>18 kDa). For these chronically exposed molluscs there was thus no threshold exposure concentration above which spillover of Cd occurred from the MT pool to other cytosolic ligands. However, the presence of Cd in the LMW and HMW fractions suggests that metal detoxification was imperfect, i.e. that P. grandis was subject to some Cd-related stress at low chronic exposure concentrations. Consistent with this suggestion, MDA concentrations, an indicator of oxidative stress, increased with gill cytosolic Cd. In the digestive gland, MDA concentrations were unrelated to any of the measured metals, but glutathione-peroxidase and glutathione-reductase activities increased with gill cytosolic copper. We speculate that cytosolic Cu

  20. The Epidermal Growth Factor Receptor Is a Regulator of Epidermal Complement Component Expression and Complement Activation

    DEFF Research Database (Denmark)

    Abu-Humaidan, Anas H A; Ananthoju, Nageshwar; Mohanty, Tirthankar

    2014-01-01

    The complement system is activated in response to tissue injury. During wound healing, complement activation seems beneficial in acute wounds but may be detrimental in chronic wounds. We found that the epidermal expression of many complement components was only increased to a minor extent in skin...... components in keratinocytes and epidermis following stimulation with proinflammatory cytokines. Importantly, EGFR inhibition of cultured keratinocytes either alone or in combination with proinflammatory stimulus promoted activation of the complement system after incubation with serum. In keratinocytes...... wounds in vivo and in cultured keratinocytes after exposure to supernatant from stimulated mononuclear cells. In contrast, the epidermal expression of complement components was downregulated in ex vivo injured skin lacking the stimulation from infiltrating inflammatory cells but with intact injury...

  1. Bmal1 is an essential regulator for circadian cytosolic Ca²⁺ rhythms in suprachiasmatic nucleus neurons.

    Science.gov (United States)

    Ikeda, Masayuki; Ikeda, Masaaki

    2014-09-03

    The hypothalamic suprachiasmatic nucleus (SCN) plays a pivotal role in the mammalian circadian clock system. Bmal1 is a clock gene that drives transcriptional-translational feedback loops (TTFLs) for itself and other genes, and is expressed in nearly all SCN neurons. Despite strong evidence that Bmal1-null mutant mice display arrhythmic behavior under constant darkness, the function of Bmal1 in neuronal activity is unknown. Recently, periodic changes in the levels of intracellular signaling messengers, such as cytosolic Ca(2+) and cAMP, were suggested to regulate TTFLs. However, the opposite aspect of how clock gene TTFLs regulate cytosolic signaling remains unclear. To investigate intracellular Ca(2+) dynamics under Bmal1 perturbations, we cotransfected some SCN neurons with yellow cameleon together with wild-type or dominant-negative Bmal1 using a gene-gun applied for mouse organotypic cultures. Immunofluorescence staining for a tag protein linked to BMAL1 showed nuclear expression of wild-type BMAL1 and its degradation within 1 week after transfection in SCN neurons. However, dominant-negative BMAL1 did not translocate into the nucleus and the cytosolic signals persisted beyond 1 week. Consistently, circadian Ca(2+) rhythms in SCN neurons were inhibited for longer periods by dominant-negative Bmal1 overexpression. Furthermore, SCN neurons transfected with a Bmal1 shRNA lengthened, whereas those overexpressing wild-type Bmal1 shortened, the periods of Ca(2+) rhythms, with a significant reduction in their amplitude. BMAL1 expression was intact in the majority of neighboring neurons in organotypic cultures. Therefore, we conclude that proper intrinsic Bmal1 expression, but not passive signaling via cell-to-cell interactions, is the determinant of circadian Ca(2+) rhythms in SCN neurons. Copyright © 2014 the authors 0270-6474/14/3412029-10$15.00/0.

  2. The deposition of suberin lamellae determines the magnitude of cytosolic Ca2+ elevations in root endodermal cells subjected to cooling.

    Science.gov (United States)

    Moore, Catherine A; Bowen, Helen C; Scrase-Field, Sarah; Knight, Marc R; White, Philip J

    2002-05-01

    A transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt) is thought to be a prerequisite for an appropriate physiological response to both chilling and salt stress. The [Ca2+]cyt is raised by Ca2+ influx to the cytosol from the apoplast and/or intracellular stores. It has been speculated that different signals mobilise Ca2+ from different stores, but little is known about the origin(s) of the Ca2+ entering the cytosol in response to specific environmental challenges. We have utilised the developmentally regulated suberisation of endodermal cells, which is thought to prevent Ca2+ influx from the apoplast, to ascertain whether Ca2+ influx is required to increase [Ca2+]cyt in response to chilling or salt stress. Perturbations in [Ca2+]cyt were studied in transgenic Arabidopsis thaliana, expressing aequorin fused to a modified yellow fluorescent protein solely in root endodermal cells, during slow cooling of plants from 20 to 0.5 degrees C over 5 min and in response to an acute salt stress (0.333 m NaCl). Only in endodermal cells in the apical 4 mm of the Arabidopsis root did [Ca2+]cyt increase significantly during cooling, and the magnitude of the [Ca2+]cyt elevation elicited by cooling was inversely related to the extent of suberisation of the endodermal cell layer. No [Ca2+]cyt elevations were elicited by cooling in suberised endodermal cells. This is consistent with the hypothesis that suberin lamellae isolate the endodermal cell protoplast from the apoplast and, thereby, prevent Ca2+ influx. By contrast, acute salt stress increased [Ca2+]cyt in endodermal cells throughout the root. These results suggest that [Ca2+]cyt elevations, upon slow cooling, depend absolutely on Ca2+ influx across the plasma membrane, but [Ca2+]cyt elevations in response to acute salt stress do not. They also suggest that Ca2+ release from intracellular stores contributes significantly to increasing [Ca2+]cyt upon acute salt stress.

  3. Toxic Epidermal Necrolysis Caused by Amoxicillin

    Directory of Open Access Journals (Sweden)

    Celalettin Sever

    2011-09-01

    Full Text Available Toxic epidermal necrolysis (TEN is a severe skin reaction related to drugs and infections, characterized by fever, stomatitis and conjunctivitis. Many drug related TEN cases have been reported in literature but amoxicillin related TEN cases are rare. In this article, a case of amoxicillin related severe TEN in a female patient during treatment of tonsillitis has been reported. The increased use of amoxicillin, especially for control of infection, may be the reason for the increased incidence TEN due to the same drug. The identification of a drug as the cause for the immune related cytotoxic reaction may be difficult if the molecule is not generally known to be a classical cause of this reaction.

  4. Frontiers in epidermal barrier homeostasis--an approach to mathematical modelling of epidermal calcium dynamics.

    Science.gov (United States)

    Denda, Mitsuhiro; Denda, Sumiko; Tsutsumi, Moe; Goto, Makiko; Kumamoto, Junichi; Nakatani, Masashi; Takei, Kentaro; Kitahata, Hiroyuki; Nakata, Satoshi; Sawabu, Yusuke; Kobayashi, Yasuaki; Nagayama, Masaharu

    2014-02-01

    Intact epidermal barrier function is crucial for survival and is associated with the presence of gradients of both calcium ion concentration and electric potential. Although many molecules, including ion channels and pumps, are known to contribute to maintenance of these gradients, the mechanisms involved in epidermal calcium ion dynamics have not been clarified. We have established that a variety of neurotransmitters and their receptors, originally found in the brain, are expressed in keratinocytes and are also associated with barrier homeostasis. Moreover, keratinocytes and neurons show some similarities of electrochemical behaviour. As mathematical modelling and computer simulation have been employed to understand electrochemical phenomena in brain science, we considered that a similar approach might be applicable to describe the dynamics of epidermal electrochemical phenomena associated with barrier homeostasis. Such methodology would also be potentially useful to address a number of difficult problems in clinical dermatology, such as ageing and itching. Although this work is at a very early stage, in this essay, we discuss the background to our approach and we present some preliminary results of simulation of barrier recovery. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. DMPD: Regulation of arachidonic acid release and cytosolic phospholipase A2activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10080535 Regulation of arachidonic acid release and cytosolic phospholipase A2activ...on of arachidonic acid release and cytosolic phospholipase A2activation. PubmedID 10080535 Title Regulation ...of arachidonic acid release and cytosolic phospholipase A2activation. Authors Gij

  6. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*

    OpenAIRE

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.

    2015-01-01

    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cy...

  7. A rat brain cytosolic N-methyltransferase(s) activity converting phosphorylethanolamine into phosphorylcholine.

    Science.gov (United States)

    Andriamampandry, C; Massarelli, R; Freysz, L; Kanfer, J N

    1990-09-14

    It had been previously speculated upon but never proved that the methylation of phosphorylethanolamine could contribute to the production of choline containing compounds. However, experimental evidence obtained with neuronal cultures was interpreted as showing that the stepwise methylation of phosphobases may be an important route for this biosynthesis. We demonstrate that cytosolic fraction from rat brain possesses a N-methyltransferase activity capable of methylating phosphorylethanolamine and its mono- and dimethyl-derivatives into phosphorylcholine. The level of activity detectable in rat liver cytosol is only 18% of that found in the brain cytosol.

  8. Human Cytosolic Extracts Stabilize the HIV-1 Core

    Science.gov (United States)

    Fricke, Thomas; Brandariz-Nuñez, Alberto; Wang, Xiaozhao; Smith, Amos B.

    2013-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects on HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure the stability of in vitro-assembled HIV-1 CA-NC complexes. The assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core. Interestingly, stabilization of in vitro-assembled HIV-1 CA-NC complexes is not due solely to macromolecular crowding, suggesting the presence of specific cellular factors that stabilize the HIV-1 core. By using our novel assay, we measured the abilities of different drugs, such as PF74, CAP-1, IXN-053, cyclosporine, Bi2 (also known as BI-2), and the peptide CAI, to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes. Interestingly, we found that PF74 and Bi2 strongly stabilized HIV-1 CA-NC complexes. On the other hand, the peptide CAI destabilized HIV-1 CA-NC complexes. We also found that purified cyclophilin A destabilizes in vitro-assembled HIV-1 CA-NC complexes in the presence of cellular extracts in a cyclosporine-sensitive manner. In agreement with previous observations using the fate-of-the-capsid assay, we also demonstrated the ability of recombinant CPSF6 to stabilize HIV-1 CA-NC complexes. Overall, our findings suggested that cellular extracts specifically stabilize the HIV-1 core. We believe that our assay can be a powerful tool to assess HIV-1 core stability in vitro. PMID:23885082

  9. The S100 proteins in epidermis: Topology and function.

    Science.gov (United States)

    Leśniak, Wiesława; Graczyk-Jarzynka, Agnieszka

    2015-12-01

    S100 proteins are small calcium binding proteins encoded by genes located in the epidermal differentiation complex (EDC). Differently to other proteins encoded by EDC genes, which are indispensable for normal epidermal differentiation, the role of S100 proteins in the epidermis remains largely unknown. Particular S100 proteins differ in their distribution in epidermal layers, skin appendages, melanocytes and Langerhans cells. Taking into account that each epidermal component consists of specialized cells with well-defined functions, such differential distribution may be indicative of the function of a given S100 protein. We used this criterion together with the survey of the current experimental data pertinent to epidermis to provide a fairly comprehensive view on the possible function of individual S100 proteins in this tissue. S100 proteins are differently expressed and, despite extensive structural homology, perform diverse functions in the epidermis. Certain S100 proteins probably ensure constant epidermal renewal and support wound healing while others act in epidermal differentiation or have a protective role. As their expression is differently affected in various skin pathologies, particular S100 proteins could be valuable diagnostic markers. S100 proteins seem to be important although not yet fully recognized epidermal constituents. Better understanding of their role in the epidermis might be helpful in designing therapies to various skin diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. A 33 kDa protein band is enhanced during long-term adaptation of EUE cells to a hypertonic medium.

    Science.gov (United States)

    Giuliani, A; Ferraretto, A; Fuhrman Conti, A M; De Grada, L; Fraschini, A; Pellicciari, C; Manfredi Romanini, M G

    1991-04-01

    A cell line derived from human embryonic epithelium (EUE cells) shows an enhanced expression of a 33 kDa protein when adapted to grow in a hypertonic medium containing 0.246 M NaCl (1.8 x the isotonic concentration). The maximum amount of this protein, followed by SDS-PAGE electrophoresis, was found after 4 days of adaptation; thereafter, the protein band remained fairly constant up to 30 days. When the cells were transferred back to a medium containing 0.137 M NaCl (isotonic medium), the protein pattern reverted to that of control cells. This protein is mainly localized in the cytosol, although a small part is associated with the 150,000 g pellet and needs detergents to be extracted. The molecular weight and the cellular location suggest a possible analogy with the so-called amphitropic proteins, that are known to interact with both the epidermal growth factor receptor and hydrophobic structures, such as the membrane phospholipids and the cytoskeletal components.

  11. Evasion of innate cytosolic DNA sensing by a gammaherpesvirus facilitates establishment of latent infection1

    Science.gov (United States)

    Sun, Chenglong; Schattgen, Stefan A.; Pisitkun, Prapaporn; Jorgensen, Joan P.; Hilterbrand, Adam T.; Wang, Lucas J.; West, John A.; Hansen, Kathrine; Horan, Kristy A.; Jakobsen, Martin R.; O'Hare, Peter; Adler, Heiko; Sun, Ren; Ploegh, Hidde L.; Damania, Blossom; Upton, Jason W.; Fitzgerald, Katherine A.; Paludan, Søren R.

    2014-01-01

    Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is currently limited knowledge on viruses targeting the innate DNA sensing pathway and also on how the innate system impacts on the latent reservoir of herpesvirus infections. Here we report that murine gammaherpesvirus MHV68, in contrast to alpha- and beta-herpesviruses, induce very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA sensing pathways. Interestingly, a MHV68 mutant lacking the deubiquitinase (DUB) activity, embedded within the large tegument protein ORF64, gained the capacity to stimulate the DNA-activated STING pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and AIM2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which instead localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild type but not STING-deficient mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA sensing pathway, which in the MHV68 model facilitates establishment of infections. PMID:25595793

  12. Circadian waves of cytosolic calcium concentration and long-range network connections in rat suprachiasmatic nucleus.

    Science.gov (United States)

    Hong, Jin Hee; Jeong, Byeongha; Min, Cheol Hong; Lee, Kyoung J

    2012-05-01

    The suprachiasmatic nucleus (SCN) is the master clock in mammals governing the daily physiological and behavioral rhythms. It is composed of thousands of clock cells with their own intrinsic periods varying over a wide range (20-28 h). Despite this heterogeneity, an intact SCN maintains a coherent 24 h periodic rhythm through some cell-to-cell coupling mechanisms. This study examined how the clock cells are connected to each other and how their phases are organized in space by monitoring the cytosolic free calcium ion concentration ([Ca(2+)](c)) of clock cells using the calcium-binding fluorescent protein, cameleon. Extensive analysis of 18 different organotypic slice cultures of the SCN showed that the SCN calcium dynamics is coordinated by phase-synchronizing networks of long-range neurites as well as by diffusively propagating phase waves. The networks appear quite extensive and far-reaching, and the clock cells connected by them exhibit heterogeneous responses in their amplitudes and periods of oscillation to tetrodotoxin treatments. Taken together, our study suggests that the network of long-range cellular connectivity has an important role for the SCN in achieving its phase and period coherence. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  13. Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport

    Science.gov (United States)

    Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2013-01-01

    Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment. PMID:23350036

  14. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching (Takeda Cali)

    2016-07-01

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  15. Cytosolic phospholipase A2 activation correlates with HER2 overexpression and mediates estrogen-dependent breast cancer cell growth.

    LENUS (Irish Health Repository)

    Caiazza, Francesco

    2010-05-01

    Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) catalyzes the hydrolysis of membrane glycerol-phospholipids to release arachidonic acid as the first step of the eicosanoid signaling pathway. This pathway contributes to proliferation in breast cancer, and numerous studies have demonstrated a crucial role of cyclooxygenase 2 and prostaglandin E(2) release in breast cancer progression. The role of cPLA(2)alpha activation is less clear, and we recently showed that 17beta-estradiol (E2) can rapidly activate cPLA(2)alpha in MCF-7 breast cancer cells. Overexpression or gene amplification of HER2 is found in approximately 30% of breast cancer patients and correlates with a poor clinical outcome and resistance to endocrine therapy. This study reports the first evidence for a correlation between cPLA(2)alpha enzymatic activity and overexpression of the HER2 receptor. The activation of cPLA(2)alpha in response to E2 treatment was biphasic with the first phase dependent on trans-activation through the matrix metalloproteinase-dependent release of heparin-bound epidermal growth factor. EGFR\\/HER2 heterodimerization resulted in downstream signaling through the ERK1\\/2 cascade to promote cPLA(2)alpha phosphorylation at Ser505. There was a correlation between HER2 and cPLA(2)alpha expression in six breast cancer cell lines examined, and inhibition of HER2 activation or expression in the SKBR3 cell line using herceptin or HER2-specific small interfering RNA, respectively, resulted in decreased activation and expression of cPLA(2)alpha. Pharmacological blockade of cPLA(2)alpha using a specific antagonist suppressed the growth of both MCF-7 and SKBR3 cells by reducing E2-induced proliferation and by stimulating cellular apoptosis and necrosis. This study highlights cPLAalpha(2) as a potential target for therapeutic intervention in endocrine-dependent and endocrine-independent breast cancer.

  16. Mutational analysis of leucine 47 in human epidermal growth factor.

    Science.gov (United States)

    Matsunami, R K; Yette, M L; Stevens, A; Niyogi, S K

    1991-07-01

    Seven site-specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K1/2 (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity. Substitutions are tolerated to different degrees, depending upon hydrophobicity and size of the side chain. Substitution with ionic residues led to the most drastic reduction in activity. One-dimensional nuclear magnetic resonance spectroscopy, at physiological pH, of several of the mutants did not detect any major structural perturbations which would account for the loss of activity. The results suggest that the side chain of leucine 47, because of its charge neutrality, size, and hydrophobicity, is highly important, although not absolutely essential for the interaction of EGF with its receptor. A striking finding was the lower (compared with wild type) Vmax values of the mutants in the tyrosine kinase reaction, but these low Vmax mutants, in cell culture experiments, were able to stimulate at high concentrations a growth response equivalent to wild type EGF.

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

    Directory of Open Access Journals (Sweden)

    Geetanjali Kharmate

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

  18. ZNF750 is expressed in differentiated keratinocytes and regulates epidermal late differentiation genes.

    Directory of Open Access Journals (Sweden)

    Idan Cohen

    Full Text Available Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype reminiscent of psoriasis and seborrheic dermatitis. Here we show that ZNF750 is a nuclear protein bearing a functional C-terminal nuclear localization signal. ZNF750 was specifically expressed in the epidermal suprabasal layers and its expression was augmented during differentiation, both in human skin and in-vitro, peaking in the granular layer. Silencing of ZNF750 in Ca2+-induced HaCaT keratinocytes led to morphologically apparent arrest in the progression of late differentiation, as well as diminished apoptosis and sustained proliferation. ZNF750 knockdown cells presented with markedly reduced expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, overexpression of ZNF750 in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation and with its downstream targets can serve in future elucidation of therapeutics for common diseases of skin barrier.

  19. Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression

    Science.gov (United States)

    Grether-Beck, Susanne; Felsner, Ingo; Brenden, Heidi; Kohne, Zippora; Majora, Marc; Marini, Alessandra; Jaenicke, Thomas; Rodriguez-Martin, Marina; Trullas, Carles; Hupe, Melanie; Elias, Peter M.; Krutmann, Jean

    2012-01-01

    Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (LL-37 and β-defensin-2) expression. Urea both stimulates expression of, and is transported into keratinocytes by two urea transporters, UT-A1 and UT-A2, and by aquaporin 3, 7 and 9. Inhibitors of these urea transporters block the downstream biological effects of urea, which include increased mRNA and protein levels for: (i) transglutaminase-1, involucrin, loricrin and filaggrin; (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and β-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and antimicrobial peptide expression in a murine model of atopic dermatitis (AD). Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and antimicrobial peptide expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin. PMID:22418868

  20. The dynamic expression of the epidermal growth factor receptor and epidermal growth factor ligand family in a differentiating intestinal epithelial cell line.

    Science.gov (United States)

    Kuwada, S K; Li, X F; Damstrup, L; Dempsey, P J; Coffey, R J; Wiley, H S

    1999-01-01

    The Caco-2 intestinal epithelial cell line differentiates when cultured on plastic or permeable filters, and offers a valuable system to study events associated with enterocytic differentiation in vitro. Little is known as to whether the expression of the epidermal growth factor receptor (EGFR) and its ligands changes as intestinal epithelial cells differentiate. We found that total cellular EGFR protein and mRNA transcript levels were relatively unchanged during Caco-2 cell differentiation, but the expression of surface EGFR and patterns of steady state epidermal growth factor (EGF)-family ligand expression changed significantly. EGFR affinity, surface EGFR expression levels, and the repertoire of expressed EGF-family ligands, were different between Caco-2 cells cultured on plastic and filters. Functionally, EGFR-mediated cell proliferation and tyrosine phosphorylation of the signal transduction protein SHC could be inhibited in Caco-2 cells cultured on filters, but not on plastic. Thus, the substrate on which the cells were grown and the degree of cell differentiation strongly modulate EGFR affinity, EGFR surface expression, the steady state expression of EGF-family ligands, as well as, EGFR-mediated cellular responses. Our results suggest that the EGFR system is regulated during intestinal epithelial cell differentiation primarily at the level of ligand expression.

  1. A Novel Cytosolic Isoform of Mitochondrial Trans-2-Enoyl-CoA Reductase Enhances Peroxisome Proliferator-Activated Receptor α Activity

    Directory of Open Access Journals (Sweden)

    Dong-Gyu Kim

    2014-06-01

    Full Text Available BackgroundMitochondrial trans-2-enoyl-CoA reductase (MECR is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα. However, MECR and PPARα are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPARα.MethodsTo identify the expression pattern of MECR and the cytosolic form of MECR (cMECR, we performed reverse transcription polymerase chain reaction (RT-PCR with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPARα, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPARα activity.ResultsWe provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPARα in the nucleus and increased PPARα-dependent luciferase activity in HeLa cells.ConclusionWe found the cytosolic form of MECR (cMECR was expressed in the cytosolic and/or nuclear region, directly binds with PPARα, and enhances PPARα activity.

  2. A Novel Cytosolic Isoform of Mitochondrial Trans-2-Enoyl-CoA Reductase Enhances Peroxisome Proliferator-Activated Receptor α Activity.

    Science.gov (United States)

    Kim, Dong-Gyu; Yoo, Jae Cheal; Kim, Eunju; Lee, Young-Sun; Yarishkin, Oleg V; Lee, Da Yong; Lee, Kun Ho; Hong, Seong-Geun; Hwang, Eun Mi; Park, Jae-Yong

    2014-06-01

    Mitochondrial trans-2-enoyl-CoA reductase (MECR) is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). However, MECR and PPARα are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPARα. To identify the expression pattern of MECR and the cytosolic form of MECR (cMECR), we performed reverse transcription polymerase chain reaction (RT-PCR) with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPARα, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPARα activity. We provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPARα in the nucleus and increased PPARα-dependent luciferase activity in HeLa cells. We found the cytosolic form of MECR (cMECR) was expressed in the cytosolic and/or nuclear region, directly binds with PPARα, and enhances PPARα activity.

  3. Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

    Science.gov (United States)

    Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. In...

  4. High-resolution structure of the M14-type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB-REDO strategies

    Energy Technology Data Exchange (ETDEWEB)

    Rimsa, Vadim; Eadsforth, Thomas C. [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Joosten, Robbie P. [Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam (Netherlands); Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk [University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom)

    2014-02-01

    The structure of a bacterial M14-family carboxypeptidase determined exploiting microfocus synchrotron radiation and highly automated refinement protocols reveals its potential to act as a polyglutamylase. A potential cytosolic metallocarboxypeptidase from Burkholderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB-REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn{sup 2+}-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenocepacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn{sup 2+}, where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.

  5. Epidermal Nevus Syndrome Associated with Brain Malformations and Medulloblastoma

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2013-01-01

    Full Text Available Researchers at Juntendo University and Tokyo Women’s Medical University, Japan; and University of California, San Francisco, Ca, report a male infant with epidermal nevus syndrome associated with brainstem and cerebellar malformations and neonatal medulloblastoma.

  6. Immunohistochemical localization of epidermal growth factor in rat and man

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Nexø, Ebba

    1986-01-01

    Epidermal growth factor (EGF) is a peptide which stimulates cell mitotic activity and differentiation, has a cytoprotective effect on the gastroduodenal mucosa, and inhibits gastric acid secretion. The immunohistochemical localization of EGF in the Brunner's glands and the submandibular glands...

  7. Epidermal and dermal integumentary structures of ankylosaurian dinosaurs.

    Science.gov (United States)

    Arbour, Victoria M; Burns, Michael E; Bell, Phil R; Currie, Philip J

    2014-01-01

    Ankylosaurian dinosaurs are most notable for their abundant and morphologically diverse osteoderms, which would have given them a spiky appearance in life. Isolated osteoderms are relatively common and provide important information about the structure of the ankylosaur dermis, but fossilized impressions of the soft-tissue epidermis of ankylosaurs are rare. Nevertheless, well-preserved integument exists on several ankylosaur fossils that shows osteoderms were covered by a single epidermal scale, but one or many millimeter-sized ossicles may be present under polygonal, basement epidermal scales. Evidence for the taxonomic utility of ankylosaurid epidermal scale architecture is presented for the first time. This study builds on previous osteological work that argues for a greater diversity of ankylosaurids in the Dinosaur Park Formation of Alberta than has been traditionally recognized and adds to the hypothesis that epidermal skin impressions are taxonomically relevant across diverse dinosaur clades. Copyright © 2013 Wiley Periodicals, Inc.

  8. Stevens-Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis ...

    African Journals Online (AJOL)

    tandfonline.com/oemd. 57. S Afr Fam Pract. ISSN 2078-6190 EISSN 2078-6204 ..... Syndrome and Toxic Epidermal Necrolysis: An Analysis of Triggers and Implications for Improving Prevention. The American journal of medicine. 2016 Apr 15.

  9. Giardia intestinalis incorporates heme into cytosolic cytochrome b₅.

    Science.gov (United States)

    Pyrih, Jan; Harant, Karel; Martincová, Eva; Sutak, Robert; Lesuisse, Emmanuel; Hrdý, Ivan; Tachezy, Jan

    2014-02-01

    The anaerobic intestinal pathogen Giardia intestinalis does not possess enzymes for heme synthesis, and it also lacks the typical set of hemoproteins that are involved in mitochondrial respiration and cellular oxygen stress management. Nevertheless, G. intestinalis may require heme for the function of particular hemoproteins, such as cytochrome b5 (cytb5). We have analyzed the sequences of eukaryotic cytb5 proteins and identified three distinct cytb5 groups: group I, which consists of C-tail membrane-anchored cytb5 proteins; group II, which includes soluble cytb5 proteins; and group III, which comprises the fungal cytb5 proteins. The majority of eukaryotes possess both group I and II cytb5 proteins, whereas three Giardia paralogs belong to group II. We have identified a fourth Giardia cytb5 paralog (gCYTb5-IV) that is rather divergent and possesses an unusual 134-residue N-terminal extension. Recombinant Giardia cytb5 proteins, including gCYTb5-IV, were expressed in Escherichia coli and exhibited characteristic UV-visible spectra that corresponded to heme-loaded cytb5 proteins. The expression of the recombinant gCYTb5-IV in G. intestinalis resulted in the increased import of extracellular heme and its incorporation into the protein, whereas this effect was not observed when gCYTb5-IV containing a mutated heme-binding site was expressed. The electrons for Giardia cytb5 proteins may be provided by the NADPH-dependent Tah18-like oxidoreductase GiOR-1. Therefore, GiOR-1 and cytb5 may constitute a novel redox system in G. intestinalis. To our knowledge, G. intestinalis is the first anaerobic eukaryote in which the presence of heme has been directly demonstrated.

  10. Steven johnsons syndrome and toxic epidermal necrolysis: A review

    OpenAIRE

    Sri ram Anne; Sreya Kosanam; Lakshmi Prasanthi N

    2014-01-01

    Toxic epidermal necrolysis (TEN) and Stevens Johnson Syndrome (SJS) are severe adverse cutaneous drug reactions that predominantly involve the skin and mucous membranes. They are characterized by mucocutaneous tenderness and typically hemorrhagic erosions, erythema and more or less severe epidermal detachment presenting as blisters and areas of denuded skin. Drugs are assumed or identified as the main cause of SJS/TEN in most cases, but Mycoplasma pneumoniae and Herpes simplex virus infection...

  11. Clinical and Economic Benefits of Autologous Epidermal Grafting

    OpenAIRE

    Maderal, Andrea D; Kirsner, Robert S

    2016-01-01

    Chronic wounds are an increasingly prevalent disease with a significant healthcare burden. These wounds often do not respond to standard of care therapy alone, requiring the use of adjuvant therapies. Epidermal grafting, previously utilized primarily for correction of leukoderma, is increasingly being recognized as a beneficial therapy for wounds, both acute and chronic. Epidermal grafting has been shown to be effective in the management of chronic wounds, with successful healing in refractor...

  12. Epidermal Inorganic Optoelectronics for Blood Oxygen Measurement.

    Science.gov (United States)

    Li, Haicheng; Xu, Yun; Li, Xiaomin; Chen, Ying; Jiang, Yu; Zhang, Changxing; Lu, Bingwei; Wang, Jian; Ma, Yinji; Chen, Yihao; Huang, Yin; Ding, Minquang; Su, Honghong; Song, Guofeng; Luo, Yi; Feng, X

    2017-05-01

    Flexible and stretchable optoelectronics, built-in inorganic semiconductor materials, offer a wide range of unprecedented opportunities and will redefine the conventional rigid optoelectronics in biological application and medical measurement. However, a significant bottleneck lies in the brittleness nature of rigid semiconductor materials and the performance's extreme sensitivity to the light intensity variation due to human skin deformation while measuring physical parameters. In this study, the authors demonstrate a systematic strategy to design an epidermal inorganic optoelectronic device by using specific strain-isolation design, nanodiamond thinning, and hybrid transfer printing. The authors propose all-in-one suspension structure to achieve the stretchability and conformability for surrounding environment, and they propose a two-step transfer printing method for hybrid integrating III-V group emitting elements, Si-based photodetector, and interconnects. Owing to the excellent flexibility and stretchability, such device is totally conformal to skin and keeps the constant light transmission between emitting element and photodetector as well as the signal stability due to skin deformation. This method opens a route for traditional inorganic optoelectronics to achieve flexibility and stretchability and improve the performance of optoelectronics for biomedical application. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. [Epidermal growth factor, innovation and safety].

    Science.gov (United States)

    Esquirol Caussa, Jordi; Herrero Vila, Elisabeth

    2015-10-05

    Bioidentical recombinant human epidermal growth factor (rhEGF) is available in concentrations and purity suitable for therapeutic use in long time stable formulations. Beneficial effects in several skin pathologies and lesions have been reported (traumatic and surgical wound healing, laser induced wounds, abnormal scars, keloids, radiation or chemotherapy induced dermatitis, post inflammatory hyperpigmentation or for skin aging damage repairing) and also may be considered for the treatment of several oropharingeal and high gastroesophageal tract mucosa diseases (mouth sores, pharyngeal fistulas, ulcers), and several corneal or conjunctive mucosa lesions. rhEGF has not shown any important side or collateral effects in humans or in laboratory experimentation animals, showing optimal tolerability and safety with continuous use for months. Compounding gives advantages of versatility, individualization, personalization, molecular stability, safety and effectiveness in ideal conditions, showing good tissue penetration, both on intact skin and skin lesions that expose the lower planes to the surface. rhEGF compounds can be considered for prevention or as a treatment of diverse skin and mucosa diseases and conditions through compounding preparations. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

  14. Epidermal growth factor receptor signaling in tissue

    Energy Technology Data Exchange (ETDEWEB)

    Shvartsman, Stanislav; Wiley, H. S.; Lauffenburger, Douglas A.

    2004-08-01

    Abstract: A peptide purified from the salivary gland of a mouse was shown few years ago to accelerate incisor eruption and eyelid opening in newborn mice, and was named epidermal growth factor (EGF). The members of this family of peptide growth factors had been identified in numerous physiological and pathological contexts. EGF binds to a cell surface EGF receptor, which induces a biochemical modification (phosphorylation) of the receptor's cytoplasmic tail. There is a growing consensus in the research community that, in addition to cellular and molecular studies, the dynamics of the EGFR network and its operation must be examined in tissues. A key challenge is to integrate the existing molecular and cellular information into a system-level description of the EGFR network at the tissue and organism level. In this paper, the two examples of EGFR signaling in tissues are described, and the recent efforts to model EGFR autocrine loops, which is a predominant mode of EGFR activation in vivo, are summarized.

  15. Roxithromycin-induced toxic epidermal necrolysis.

    Science.gov (United States)

    Das, Saibal; Mondal, Somnath; Dey, Jayanta Kumar

    2012-08-01

    This case report highlights a very rare adverse drug reaction of oral roxithromycin causing toxic epidermal necrolysis (TEN). A 54-year-old male patient diagnosed with upper respiratory tract infection was prescribed oral roxithromycin 150 mg twice daily for 7 days. On the 10th day, the patient was admitted to the emergency with sore throat, redness, watering of eyes, painful micturition, and severe skin lesions. The skin lesions were multiple, severely painful, burning, coalesced, and filled with fluid-producing large blisters appearing on the lip, face, and trunk and then gradually spreading to legs, arms, palms, hands, and feet extensively involving much >30% of body surface area. Clinical examination, blood investigation, and histopathological examination of the skin confirmed the diagnosis of TEN. There was no history of any concomitant medications, drug allergy, burn injury, recent graft, or transplant or any coexisting infections such as herpes simplex. Other resembling skin diseases were eliminated after proper dermatological examination. This episode of TEN was probably drug (roxithromycin) induced. The drug was immediately stopped, and the patient was treated meticulously resulting in gradual reversal of the diseased state. Naranjo adverse drug reaction probability scale suggested the likelihood that oral administration of roxithromycin was responsible for the TEN was 'probable.'

  16. Autologous epidermal cell suspension: A promising treatment for chronic wounds.

    Science.gov (United States)

    Zhao, Hongliang; Chen, Yan; Zhang, Cuiping; Fu, Xiaobing

    2016-02-01

    Chronic wounds have become an increasing medical and economic problem of aging societies because they are difficult to manage. Skin grafting is an important treatment method for chronic wounds, which are refractory to conservative therapy. The technique involving epidermal cell suspensions was invented to enable the possibility of treating larger wounds with only a small piece of donor skin. Both uncultured and cultured autologous epidermal cell suspensions can be prepared and survive permanently on the wound bed. A systematic search was conducted of EMBASE, Cochrane Library, PubMed and web of science by using Boolean search terms, from the establishment of the database until May 31, 2014. The bibliographies of all retrieved articles in English were searched. The search terms were: (epithelial cell suspension OR keratinocyte suspension) and chronic and wound. From the included, 6 studies are descriptive interventions and discussed the use of autologous keratinocyte suspension to treat 61 patients' chronic wound. The various methods of preparation of epidermal cell suspension are described. The advantages and shortcomings of different carriers for epidermal cell suspensions are also summarised. Both uncultured and cultured autologous epidermal cell suspensions have been used to treat chronic wounds. Although the limitations of these studies include the small number of patient populations with chronic wounds and many important problems that remain to be solved, autologous epidermal cell suspension is a promising treatment for chronic wounds. Copyright © 2015 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

  17. Specification of Epidermal Cell Fate in Plant Shoots

    Directory of Open Access Journals (Sweden)

    Shinobu eTakada

    2014-02-01

    Full Text Available Land plants have evolved a single layer of epidermal cells, which are characterized by mostly anticlinal cell division patterns, formation of a waterproof coat called cuticle, and unique cell types such as stomatal guard cells and trichomes. The shoot epidermis plays important roles not only to protect plants from dehydration and pathogens but also to ensure their proper organogenesis and growth control. Extensive molecular genetic studies in Arabidopsis and maize have identified a number of genes that are required for epidermal cell differentiation. However, the mechanism that specifies shoot epidermal cell fate during plant organogenesis remains largely unknown. Particularly, little is known regarding positional information that should restrict epidermal cell fate to the outermost cell layer of the developing organs. Recent studies suggested that certain members of the HD-ZIP class IV homeobox genes are possible master regulators of shoot epidermal cell fate. Here, we summarize the roles of the regulatory genes that are involved in epidermal cell fate specification and discuss the possible mechanisms that limit the expression and/or activity of the master transcriptional regulators to the outermost cell layer in plant shoots.

  18. Toxic epidermal necrolysis: a paradigm of critical illness

    Science.gov (United States)

    Estrella-Alonso, Alfonso; Aramburu, José Antonio; González-Ruiz, Mercedes Yolanda; Cachafeiro, Lucía; Sánchez, Manuel Sánchez; Lorente, José A.

    2017-01-01

    Toxic epidermal necrolysis is an adverse immunological skin reaction secondary in most cases to the administration of a drug. Toxic epidermal necrolysis, Stevens-Johnson syndrome, and multiform exudative erythema are part of the same disease spectrum. The mortality rate from toxic epidermal necrolysis is approximately 30%. The pathophysiology of toxic epidermal necrolysis is similar in many respects to that of superficial skin burns. Mucosal involvement of the ocular and genital epithelium is associated with serious sequelae if the condition is not treated early. It is generally accepted that patients with toxic epidermal necrolysis are better treated in burn units, which are experienced in the management of patients with extensive skin loss. Treatment includes support, elimination, and coverage with biosynthetic derivatives of the skin in affected areas, treatment of mucosal involvement, and specific immunosuppressive treatment. Of the treatments tested, only immunoglobulin G and cyclosporin A are currently used in most centers, even though there is no solid evidence to recommend any specific treatment. The particular aspects of the treatment of this disease include the prevention of sequelae related to the formation of synechiae, eye care to prevent serious sequelae that can lead to blindness, and specific immunosuppressive treatment. Better knowledge of the management principles of toxic epidermal necrolysis will lead to better disease management, higher survival rates, and lower prevalence of sequelae. PMID:29340540

  19. Epidermal growth factor and insulin-like growth factor I upregulate the expression of the epidermal growth factor system in rat liver

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Vinter-Jensen, L

    2000-01-01

    BACKGROUND/AIM: Both epidermal growth factor and insulin-like growth factor I play a role in connection with the liver. In the present study, the possible interaction of these two growth factor systems was studied by investigating the effect of epidermal growth factor or insulin-like growth factor...... I treatment on the expression of the epidermal growth factor receptor, and its activating ligands, transforming growth factor-alpha and epidermal growth factor. METHODS: Fifty-five male rats received no treatment, human recombinant epidermal growth factor or human recombinant insulin-like growth...... factor I for either 3 or 7 days. The amount of epidermal growth factor receptor, transforming growth factor-alpha, and epidermal growth factor mRNA was quantitated by a calibrated user-friendly RT-PCR assay (CURT-PCR), and the expression of transforming growth factor-alpha and epidermal growth factor...

  20. Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin

    DEFF Research Database (Denmark)

    Pappinen, Sari; Hermansson, Martin; Kuntsche, Judith

    2008-01-01

    The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present......-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC...

  1. Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium.

    Directory of Open Access Journals (Sweden)

    Vivek Mishra

    Full Text Available Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825 on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated

  2. Angiotensin II-induced Akt activation through the epidermal growth factor receptor in vascular smooth muscle cells is mediated by phospholipid metabolites derived by activation of phospholipase D.

    Science.gov (United States)

    Li, Fang; Malik, Kafait U

    2005-03-01

    Angiotensin II (Ang II) activates cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), phospholipase D (PLD), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and Akt in vascular smooth muscle cells (VSMC). This study was conducted to investigate the relationship between Akt activation by Ang II and other signaling molecules in rat VSMC. Ang II-induced Akt phosphorylation was significantly reduced by the PLD inhibitor 1-butanol, but not by its inactive analog 2-butanol, and by brefeldin A, an inhibitor of the PLD cofactor ADP-ribosylation factor, and in cells infected with retrovirus containing PLD(2) siRNA or transfected with PLD(2) antisense but not control LacZ or sense oligonucleotide. Diacylglycerol kinase inhibitor II diminished Ang II-induced and diC8-phosphatidic acid (PA)-increased Akt phosphorylation, suggesting that PLD-dependent Akt activation is mediated by PA. Ang II-induced EGFR phosphorylation was inhibited by 1-butanol and PLD(2) siRNA and also by cPLA(2) siRNA. In addition, the inhibitor of arachidonic acid (AA) metabolism 5,8,11,14-eicosatetraynoic acid (ETYA) reduced both Ang II- and AA-induced EGFR transactivation. Furthermore, ETYA, cPLA(2) antisense, and cPLA(2) siRNA attenuated Ang II-elicited PLD activation. p38 MAPK inhibitor SB202190 [4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] reduced PLD activity and EGFR and Akt phosphorylation elicited by Ang II. Pyrrolidine-1, a cPLA(2) inhibitor, and cPLA(2) siRNA decreased p38 MAPK activity. These data indicate that Ang II-stimulated Akt activity is mediated by cPLA(2)-dependent, p38 MAPK regulated PLD(2) activation and EGFR transactivation. We propose the following scheme of the sequence of events leading to activation of Akt in VSMC by Ang II: Ang II-->cPLA(2)-->AA-->p38 MAPK-->PLD(2)-->PA-->EGFR-->Akt.

  3. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor.

    OpenAIRE

    Damstrup, L; Rude Voldborg, B.; Spang-Thomsen, M.; Br?nner, N; Skovgaard Poulsen, H.

    1998-01-01

    Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labe...

  4. Regulation of autophagy by cytosolic acetyl-coenzyme A

    DEFF Research Database (Denmark)

    Mariño, Guillermo; Pietrocola, Federico; Eisenberg, Tobias

    2014-01-01

    Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic p...

  5. Micrometer-Scale Membrane Transition of Supported Lipid Bilayer Membrane Reconstituted with Cytosol of Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Kei Takahashi

    2017-03-01

    Full Text Available Background: The transformation of the supported lipid bilayer (SLB membrane by extracted cytosol from living resources, has recently drawn much attention. It enables us to address the question of whether the purified phospholipid SLB membrane, including lipids related to amoeba locomotion, which was discussed in many previous studies, exhibits membrane deformation in the presence of cytosol extracted from amoeba; Methods: In this report, a method for reconstituting a supported lipid bilayer (SLB membrane, composed of purified phospholipids and cytosol extracted from Dictyostelium discoideum, is described. This technique is a new reconstitution method combining the artificial constitution of membranes with the reconstitution using animate cytosol (without precise purification at a molecular level, contributing to membrane deformation analysis; Results: The morphology transition of a SLB membrane composed of phosphatidylcholines, after the addition of cytosolic extract, was traced using a confocal laser scanning fluorescence microscope. As a result, pore formation in the SLB membrane was observed and phosphatidylinositides incorporated into the SLB membrane tended to suppress pore formation and expansion; Conclusions: The current findings imply that phosphatidylinositides have the potential to control cytoplasm activity and bind to a phosphoinositide-containing SLB membrane.

  6. Cytosolic and membrane-bound chitinases of two mucoraceous fungi: a comparative study.

    Science.gov (United States)

    Balasubramanian, R; Manocha, M S

    1992-04-01

    Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS-PAGE the chitinase from both fungi migrated as a single band of M(r) 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS-PAGE showed two bands for each fungus with M(r) of 69.5 and 55 kDa in C. cucurbitarum and M(r) 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N'-diacetylchitobiose, and N,N',N"-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but beta-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar.

  7. ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

    KAUST Repository

    Houben, Diane

    2012-05-08

    Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

  8. The cytosolic glyoxalases of Plasmodium falciparum are dispensable during asexual blood-stage development

    Directory of Open Access Journals (Sweden)

    Cletus A. Wezena

    2017-11-01

    Full Text Available The enzymes glyoxalase 1 and 2 (Glo1 and Glo2 are found in most eukaryotes and catalyze the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Four glyoxalases are encoded in the genome of the malaria parasite Plasmodium falciparum, the cytosolic enzymes PfGlo1 and PfcGlo2, the apicoplast enzyme PftGlo2, and an inactive Glo1-like protein that also carries an apicoplast-targeting sequence. Inhibition or knockout of the Plasmodium glyoxalases was hypothesized to lead to an accumulation of 2-oxoaldehydes and advanced glycation end-products (AGE in the host-parasite unit and to result in parasite death. Here, we generated clonal P. falciparum strain 3D7 knockout lines for PFGLO1 and PFcGLO2 using the CRISPR-Cas9 system. Although 3D7Δglo1 knockout clones had an increased susceptibility to external glyoxal, all 3D7Δglo1 and 3D7Δcglo2 knockout lines were viable and showed no significant growth phenotype under standard growth conditions. Furthermore, the lack of PfcGlo2, but not PfGlo1, increased gametocyte commitment in the knockout lines. In summary, PfGlo1 and PfcGlo2 are dispensable during asexual blood-stage development while the loss of PfcGlo2 may induce the formation of transmissible gametocytes. These combined data show that PfGlo1 and PfcGlo2 are most likely not suited as targets for selective drug development.

  9. Cytosolic streaming in vegetative mycelium and aerial structures of Aspergillus niger.

    Science.gov (United States)

    Bleichrodt, R; Vinck, A; Krijgsheld, P; van Leeuwen, M R; Dijksterhuis, J; Wösten, H A B

    2013-03-15

    Aspergillus niger forms aerial hyphae and conidiophores after a period of vegetative growth. The hyphae within the mycelium of A. niger are divided by septa. The central pore in these septa allows for cytoplasmic streaming. Here, we studied inter- and intra-compartmental streaming of the reporter protein GFP in A. niger. Expression of the gene encoding nuclear targeted GFP from the gpdA or glaA promoter resulted in strong fluorescence of nuclei within the vegetative hyphae and weak fluorescence in nuclei within the aerial structures. These data and nuclear run on experiments showed that gpdA and glaA are higher expressed in the vegetative mycelium when compared to aerial hyphae, conidiophores and conidia. Notably, gpdA or glaA driven expression of the gene encoding cytosolic GFP resulted in strongly fluorescent vegetative hyphae and aerial structures. Apparently, GFP streams from vegetative hyphae into aerial structures. This was confirmed by monitoring fluorescence of photo-activatable GFP (PA-GFP). In contrast, PA-GFP did not stream from aerial structures to vegetative hyphae. Streaming of PA-GFP within vegetative hyphae or within aerial structures of A. niger occurred at a rate of 10-15 μm s(-1). Taken together, these results not only show that GFP streams from the vegetative mycelium to aerial structures but it also indicates that its encoding RNA is not streaming. Absence of RNA streaming would explain why distinct RNA profiles were found in aerial structures and the vegetative mycelium by nuclear run on analysis and micro-array analysis.

  10. Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

    Science.gov (United States)

    Johnson, R. F.; Beltz, T. G.; Sharma, R. V.; Xu, Z.; Bhatty, R. A.; Johnson, A. K.

    2001-01-01

    The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.

  11. Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

    Directory of Open Access Journals (Sweden)

    Daniela GUZMÁN-URIBE

    Full Text Available Abstract Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%, meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. Objective The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Material and Methods Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group. Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. Results It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Conclusion Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

  12. Do epidermal lens cells facilitate the absorptance of diffuse light?

    Science.gov (United States)

    Brodersen, Craig R; Vogelmann, Thomas C

    2007-07-01

    Many understory plants rely on diffuse light for photosynthesis because direct light is usually scattered by upper canopy layers before it strikes the forest floor. There is a considerable gap in the literature concerning the interaction of direct and diffuse light with leaves. Some understory plants have well-developed lens-shaped epidermal cells, which have long been thought to increase the absorption of diffuse light. To assess the role of epidermal cell shape in capturing direct vs. diffuse light, we measured leaf reflectance and transmittance with an integrating sphere system using leaves with flat (Begonia erythrophylla, Citrus reticulata, and Ficus benjamina) and lens-shaped epidermal cells (B. bowerae, Colocasia esculenta, and Impatiens velvetea). In all species examined, more light was absorbed when leaves were irradiated with direct as opposed to diffuse light. When leaves were irradiated with diffuse light, more light was transmitted and more was reflected in both leaf types, resulting in absorptance values 2-3% lower than in leaves irradiated with direct light. These data suggest that lens-shaped epidermal cells do not aid the capture of diffuse light. Palisade and mesophyll cell anatomy and leaf thickness appear to have more influence in the capture and absorption of light than does epidermal cell shape.

  13. Immune sensitization against epidermal antigens in polymorphous light eruption

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez-Amaro, R.; Baranda, L.; Salazar-Gonzalez, J.F.; Abud-Mendoza, C.; Moncada, B. (Univ. of San Luis Potosi (Mexico))

    1991-01-01

    To get further insight into the pathogenesis of polymorphous light eruption, we studied nine patients with polymorphous light eruption and six healthy persons. Two skin biopsy specimens were obtained from each person, one from previously ultraviolet light-irradiated skin and another one from unirradiated skin. An epidermal cell suspension, skin homogenate, or both were prepared from each specimen. Autologous cultures were made with peripheral blood mononuclear cells combined with irradiated or unirradiated skin homogenate and peripheral blood mononuclear cells combined with irradiated or unirradiated epidermal cell suspension. Cell proliferation was assessed by 3H-thymidine incorporation assay. The response of peripheral blood mononuclear cells to unirradiated epidermal cells or unirradiated skin homogenate was similar in both patients and controls. However, peripheral blood mononuclear cells from patients with polymorphous light eruption showed a significantly increased proliferative response to both irradiated epidermal cells and irradiated skin homogenate. Our results indicate that ultraviolet light increases the stimulatory capability of polymorphous light eruption epidermal cells in a unidirectional mixed culture with autologous peripheral blood mononuclear cells. This suggests that an immune sensitization against autologous ultraviolet light-modified skin antigens occurs in polymorphous light eruption.

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  15. Imaging of human epidermal growth factor receptor type 2 expression with 18F-labeled affibody molecule ZHER2:2395 in a mouse model for ovarian cancer.

    NARCIS (Netherlands)

    Heskamp, S.; Laverman, P.; Rosik, D.; Boschetti, F.; Graaf, W.T.A. van der; Oyen, W.J.G.; Laarhoven, H.W.M. van; Tolmachev, V.; Boerman, O.C.

    2012-01-01

    Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule (111)In-DOTA-Z(HER2)(:2395) can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)-expressing tumors,

  16. Imaging of human epidermal growth factor receptor type 2 expression with 18F-labeled affibody molecule ZHER2:2395 in a mouse model for ovarian cancer

    NARCIS (Netherlands)

    Heskamp, Sandra; Laverman, Peter; Rosik, Daniel; Boschetti, Frederic; van der Graaf, Winette T. A.; Oyen, Wim J. G.; van Laarhoven, Hanneke W. M.; Tolmachev, Vladimir; Boerman, Otto C.

    2012-01-01

    Affibody molecules are small (7 kDa) proteins with subnanomolar targeting affinity. Previous SPECT studies in xenografts have shown that the Affibody molecule (111)In-DOTA-Z(HER2)(:2395) can discriminate between high and low human epidermal growth factor receptor type 2 (HER2)-expressing tumors,

  17. The Cytosolic pH of Individual Saccharomyces cerevisiae Cells Is a Key Factor in Acetic Acid Tolerance

    Science.gov (United States)

    Fernández-Niño, Miguel; Marquina, Maribel; Swinnen, Steve; Rodríguez-Porrata, Boris

    2015-01-01

    It was shown recently that individual cells of an isogenic Saccharomyces cerevisiae population show variability in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the population level. In the current study, we investigated whether cell-to-cell variability in acetic acid tolerance could be explained by the observed differences in the cytosolic pHs of individual cells immediately before exposure to the acid. Results obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic pH drop after exposure to the acid. Moreover, only cells with a low initial cytosolic pH, which experienced a less severe drop in cytosolic pH, were able to proliferate. A similar correlation between initial cytosolic pH and cytosolic pH drop was also observed in the more acid-tolerant strain MUCL 11987-9. Interestingly, a fraction of cells in the MUCL 11987-9 population showed initial cytosolic pH values below the minimal cytosolic pH detected in cells of the strain CEN.PK113-7D; consequently, these cells experienced less severe drops in cytosolic pH. Although this might explain in part the difference between the two strains with regard to the number of cells that resumed proliferation, it was observed that all cells from strain MUCL 11987-9 were able to proliferate, independently of their initial cytosolic pH. Therefore, other factors must also be involved in the greater ability of MUCL 11987-9 cells to endure strong drops in cytosolic pH. PMID:26341199

  18. Effect of treatment with a colloidal oatmeal lotion on the acneform eruption induced by epidermal growth factor receptor and multiple tyrosine-kinase inhibitors.

    Science.gov (United States)

    Alexandrescu, D T; Vaillant, J G; Dasanu, C A

    2007-01-01

    Current treatment modalities for epidermal growth factor (EGFR)-positive cancers have recently included the use of antibodies and small-molecule tyrosine-kinase inhibitors (TKI). A significant limiting step in the use of these agents is dermatological toxicity, frequently in the form of an acneiform eruption. Present management modalities for this toxicity are largely ineffective. Colloidal oatmeal lotion demonstrates multiple anti-inflammatory properties with known effects on arachidonic acid, cytosolic phospholipase A2 and tumour necrosis factor-alpha pathways, along with an excellent side-effect profile. Treatment with colloidal oatmeal was applied to 11 patients with a rash induced by cetuximab, erlotinib, panitumumab and sorafenib. Of the 10 assessable patients, 6 had complete response and 4 partial response, giving a response rate of 100% with no associated toxicities. Treatment with colloidal oatmeal lotion is efficient in controlling the rash associated with EGFR and multiple TKI, and allows continuation of the antineoplastic treatment.

  19. Transfer of a redox-signal through the cytosol by redox-dependent microcompartmentation of glycolytic enzymes at mitochondria and actin cytoskeleton

    Directory of Open Access Journals (Sweden)

    Joanna eWojtera-Kwiczor

    2013-01-01

    Full Text Available The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12, GapC plays an important role in glycolysis by providing the cell with ATP and NADH. Interestingly, despite its glycolytic function in the cytosol, GAPDH was reported to possess additional non-glycolytic activities, correlating with its nuclear or cytoskeletal localization in animal cells. In transiently transformed mesophyll protoplasts from Arabidopsis. thaliana colocalization and interaction of the glycolytic enzymes with the mitochondria and with the actin cytoskeleton was visualized by confocal laser scanning microscopy (cLSM using fluorescent protein fusions and by bimolecular fluorescence complementation (BiFC, respectively. Yeast two-hybrid screens, dot-blot overlay assays, and co-sedimentation assays were used to identify potential protein-protein interactions between two cytosolic GAPDH isoforms (GapC1, At3g04120; GapC2, At1g13440 from A. thaliana with the neighbouring glycolytic enzyme, fructose 1,6-bisphosphate aldolase (FBA6, At2g36460, the mitochondrial porin (VDAC3; At5g15090, and actin in vitro. From these experiments, a mitochondrial association is suggested for both glycolytic enzymes, GAPDH and aldolase, which appear to bind to the outer mitochondrial membrane, in a redox-dependent manner. In addition, both glycolytic enzymes were found to bind to F-actin in cosedimentation assays, and lead to bundling of purified rabbit actin, as visualized by cLSM. Actin binding and bundling occurred reversibly under oxidizing conditions. We speculate that such dynamic formation of microcompartments is part of a redox-dependent retrograde signal transduction network for adaptation upon oxidative stress.

  20. Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis

    Science.gov (United States)

    Winter, Mircea; Moser, Mirjam A; Meunier, Dominique; Fischer, Carina; Machat, Georg; Mattes, Katharina; Lichtenberger, Beate M; Brunmeir, Reinhard; Weissmann, Simon; Murko, Christina; Humer, Christina; Meischel, Tina; Brosch, Gerald; Matthias, Patrick; Sibilia, Maria; Seiser, Christian

    2013-01-01

    The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage-dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue. Strikingly, deletion of a single Hdac2 allele in HDAC1 knockout mice results in severe epidermal defects, including alopecia, hyperkeratosis, hyperproliferation and spontaneous tumour formation. These mice display impaired Sin3A co-repressor complex function, increased levels of c-Myc protein, p53 expression and apoptosis in hair follicles (HFs) and misregulation of HF bulge stem cells. Surprisingly, ablation of HDAC1 but not HDAC2 in a skin tumour model leads to accelerated tumour development. Our data reveal a crucial function of HDAC1/HDAC2 in the control of lineage specificity and a novel role of HDAC1 as a tumour suppressor in the epidermis. PMID:24240174

  1. Genetic Markers and Danger Signals in Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis

    Directory of Open Access Journals (Sweden)

    Wen-Hung Chung

    2010-01-01

    Full Text Available Stevens-Johnson syndrome (SJS and toxic epidermal necrolysis (TEN are life-threatening adverse reactions, which could be induced by a variety of drugs. It was proposed that human leukocyte antigen (HLA-restricted presentation of antigens (drugs or their metabolites to T lymphocytes initiates the immune reactions of SJS/ TEN. However, the genetic susceptibility and the exact pathogenesis were not clear until the recent studies. We first identified that HLA-B*1502 is strongly associated with carbamazepine (CBZ-induced SJS/TEN and HLA-B*5801 with allopurinol-SJS/TEN in Han Chinese. The same associations had been validated across different human populations. For the downstream danger signals, Fas-Fas ligand (FasL and perforin/granzyme B had been advocated as cytotoxic mediators for keratinocyte death in SJS/TEN. However, expression levels of these cytotoxic proteins from the skin lesions were too low to explain the distinct and extensive epidermal necrosis. Our recent study identified that the granulysin, a cytotoxic protein released from cytotoxic T cells or natural killer (NK cells, is a key mediator for disseminated keratinocyte death in SJS/TEN. This article aims to provide an overview of both of the genomic and immunologic perspectives of SJS/TEN. These studies give us a better understanding of the immune mechanisms, biomarkers for disease prevention and early diagnosis, as well as providing the therapeutic targets for the treatments of SJS/TEN.

  2. Phospholipase Cβ interacts with cytosolic partners to regulate cell proliferation.

    Science.gov (United States)

    Scarlata, Suzanne; Singla, Ashima; Garwain, Osama

    2018-01-01

    Phospholipase Cβ (PLCβ) is the main effector of the Gαq signaling pathway relaying different extracellular sensory information to generate intracellular calcium signals. Besides this classic function, we have found that PLCβ plays an important but unknown role in regulating PC12 cell differentiation by interacting with components in the RNA-induced silencing machinery. In trying to understand the role of PLCβ in PC12 cell differentiation, we find that over-expressing PLCβ reduces PC12 cell proliferation while down-regulating PLCβ increases the rate of cell proliferation. However, this behavior is not seen in other cancerous cell lines. To determine the underlying mechanism, we carried out mass spectrometry analysis of PLCβ complexes in PC12 cells. We find that in unsynchronized cells, PLCβ primarily binds cyclin-dependent kinase (CDK)16 whose activity plays a key role in cell proliferation. In vitro studies show a direct association between the two proteins that result in loss in CDK16 activity. When cells are arrested in the G2/M phase, a large population of PLCβ is bound to Ago2 in a complex that contains C3PO and proteins commonly found in stress granules. Additionally, another population of PLCβ complexes with CDK18 and cyclin B1. Fluorescence lifetime imaging microscopy (FLIM) confirms cell cycle dependent associations between PLCβ and these other protein binding partners. Taken together, our studies suggest that PLCβ may play an active role in mediating interactions required to move through the cell cycle. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Accumulation and activation of epidermal γδ T cells in a mouse model of chronic dermatitis is not required for the inflammatory phenotype.

    Science.gov (United States)

    Sulcova, Jitka; Maddaluno, Luigi; Meyer, Michael; Werner, Sabine

    2015-09-01

    Chronic skin inflammation resulting from a defective epidermal barrier is a hallmark of atopic dermatitis (AD). We previously demonstrated that mice lacking FGF receptors 1 and 2 in keratinocytes (K5-R1/R2 mice) develop an AD-like chronic dermatitis as a result of an impaired epidermal barrier. Here, we show that γδ T cells, which rapidly respond to various insults, accumulate in the epidermis of K5-R1/R2 mice before the development of histological abnormalities. Their number and activation further increase as the phenotype progresses, most likely as a consequence of increased expression of Il-2 and Il-7 and the stress-induced proteins Rae-1, H60c, Mult1, PlexinB2, and Skint1. To determine the role of γδ T cells in the skin phenotype, we generated quadruple mutant K5-R1/-R2 mice lacking γδ T cells. Surprisingly, loss of γδ T cells did not or only marginally affect keratinocyte proliferation, epidermal thickness, epidermal barrier function, and accumulation and activation of different immune cells in the skin of K5-R1/R2 mice, possibly due to partial compensation by αβ T cells. These results demonstrate that γδ T cells do not contribute to the development or maintenance of chronic inflammation in response to a defect in the epidermal barrier. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The cytosolic tail of the Golgi apyrase Ynd1 mediates E4orf4-induced toxicity in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Karin Mittelman

    Full Text Available The adenovirus E4 open reading frame 4 (E4orf4 protein contributes to regulation of the progression of virus infection. When expressed individually, E4orf4 was shown to induce non-classical transformed cell-specific apoptosis in mammalian cells. At least some of the mechanisms underlying E4orf4-induced toxicity are conserved from yeast to mammals, including the requirement for an interaction of E4orf4 with protein phosphatase 2A (PP2A. A genetic screen in yeast revealed that the Golgi apyrase Ynd1 associates with E4orf4 and contributes to E4orf4-induced toxicity, independently of Ynd1 apyrase activity. Ynd1 and PP2A were shown to contribute additively to E4orf4-induced toxicity in yeast, and to interact genetically and physically. A mammalian orthologue of Ynd1 was shown to bind E4orf4 in mammalian cells, confirming the evolutionary conservation of this interaction. Here, we use mutation analysis to identify the cytosolic tail of Ynd1 as the protein domain required for mediation of the E4orf4 toxic signal and for the interaction with E4orf4. We also show that E4orf4 associates with cellular membranes in yeast and is localized at their cytoplasmic face. However, E4orf4 is membrane-associated even in the absence of Ynd1, suggesting that additional membrane proteins may mediate E4orf4 localization. Based on our results and on a previous report describing a collection of Ynd1 protein partners, we propose that the Ynd1 cytoplasmic tail acts as a scaffold, interacting with a multi-protein complex, whose targeting by E4orf4 leads to cell death.

  5. Drag of the cytosol as a transport mechanism in neurons.

    Science.gov (United States)

    Mussel, Matan; Zeevy, Keren; Diamant, Haim; Nevo, Uri

    2014-06-17

    Axonal transport is typically divided into two components, which can be distinguished by their mean velocity. The fast component includes steady trafficking of different organelles and vesicles actively transported by motor proteins. The slow component comprises nonmembranous materials that undergo infrequent bidirectional motion. The underlying mechanism of slow axonal transport has been under debate during the past three decades. We propose a simple displacement mechanism that may be central for the distribution of molecules not carried by vesicles. It relies on the cytoplasmic drag induced by organelle movement and readily accounts for key experimental observations pertaining to slow-component transport. The induced cytoplasmic drag is predicted to depend mainly on the distribution of microtubules in the axon and the organelle transport rate. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Influence of epidermal growth factor on liver regeneration after partial hepatectomy in rats

    DEFF Research Database (Denmark)

    Olsen, Peter Skov; Boesby, S.; Kirkegaard, P.

    2013-01-01

    The role of epidermal growth factor on liver regeneration after partial hepatectomy in rats was investigated. After a 70% hepatectomy in rats, the concentration of epidermal growth factor in portal venous blood was unchanged compared with unoperated controls. However, small amounts of epidermal...... growth factor could be identified in portal venous blood after intestinal instillation of epidermal growth factor. Brunner's glands and the submandibular glands secrete epidermal growth factor. Extirpation of Brunner's glands decreased liver regeneration, whereas removal of the submandibular glands had...... no effect on liver regeneration. Epidermal growth factor antiserum reduced liver regeneration significantly. Oral or s.c. administration of epidermal growth factor had no effect on liver regeneration, whereas epidermal growth factor enhanced the effect of insulin and glucagon on liver regeneration...

  7. Decreased level of epidermal growth factor in milk from diabetic rats

    DEFF Research Database (Denmark)

    Thulesen, J; Nexø, Ebba; Raaberg, Lasse

    1994-01-01

    . Furthermore, the time of eyelid opening was delayed, but no difference in the time of tooth eruption was observed. Insulin-treatment of diabetic rats restored the milk volume and the EGF concentration to values comparable to those of the controls. Pups of the insulin-treated diabetic dams were comparable......Experimental diabetes was induced in rats with streptozocin before mating, and the influence of diabetes on epidermal growth factor (EGF) in milk and on other milk components was studied. Throughout the lactation period, a significant decrease was found both in the production of milk...... and in the concentration of EGF in milk from untreated diabetic rats compared with an insulin-treated diabetic group and a control group. Thus, the total output of EGF in milk from diabetic rats was considerably decreased. The concentrations of total protein and haptocorrin, a cobalamin (vitamin B12)-binding protein...

  8. The type III epidermal growth factor receptor mutation. Biological significance and potential target for anti-cancer therapy.

    Science.gov (United States)

    Pedersen, M W; Meltorn, M; Damstrup, L; Poulsen, H S

    2001-06-01

    Mutations in the epidermal growth factor receptor occur frequently in a number of human tumours including gliomas, non-small-cell lung carcinomas, ovarian carcinomas and prostate carcinomas. The type III epidermal growth factor receptor mutation (variously named EGFRvIII, de2-7 EGFR or AEGFR), which lacks a portion of the extracellular ligand binding domain, is the most common. Here, we review the current status with regard to the role of EGFRvIII in human cancers. A detailed discussion of the formation of EGFRvIII and its structure at the protein level are likewise included along with a discussion of its more functional roles. The design and use (preclinical and clinical) of small molecule inhibitors, antibodies, and antisense oligonucleotides against wild-type EGFR are considered in detail as these strategies can be directly adapted to target EGFRvIII. Finally, the status of EGFRvIII targeted therapy is reviewed.

  9. Imunolocalização das proteínas dos genes supressores de tumores TP53 e p16CDKN2 no front invasivo do carcinoma epidermóide de cavidade bucal Immunolocalization of TP53 and p16CDKN2 tumour suppressor genes proteins in invasive front of oral epidermoid carcinoma

    Directory of Open Access Journals (Sweden)

    Alfredo Maurício Batista De-Paula

    2006-08-01

    Full Text Available INTRODUÇÃO: A carcinogênese bucal é um processo multipassos no qual eventos genéticos promovem o rompimento de vias regulatórias normais que controlam funções celulares básicas. O carcinoma epidermóide de cavidade bucal (CECB surge como conseqüência de múltiplos eventos moleculares induzidos pelos efeitos de vários carcinógenos influenciados por fatores ambientais contra um quadro de resistência ou suscetibilidade herdada geneticamente. OBJETIVO: Avaliar a importância clínica e morfológica da imunoexpressão das proteínas p53 e p16 na região do front invasivo de uma série de 35 casos rotineiramente processados de CECB. MATERIAL E MÉTODOS: Amostras de CECB primários tratados exclusivamente por cirurgia foram investigadas. O sistema TNM foi empregado para o estadiamento clínico dos pacientes. Para a gradação morfológica das lesões foi adotado o sistema de gradação do front invasivo. A técnica de imuno-histoquímica foi realizada nas lesões fixadas em formalina tamponada a 10% e emblocadas em parafina para identificação das proteínas p53 e p16. As contagens foram realizadas e submetidas a tratamentos estatísticos específicos. RESULTADOS: As taxas de imunolocalização para as proteínas p53 e p16 foram de 63% e 66%, respectivamente, nas 35 amostras de carcinoma estudadas. Não houve relação entre as expressões das proteínas p53 e p16 com os parâmetros clínico-morfológicos analisados. Não houve correlação entre a expressão imuno-histoquímica das proteínas p53/p16. CONCLUSÃO: A expressão das proteínas p53 e p16 não influenciou os parâmetros clínico-morfológicos analisados neste estudo e aparentemente não representa base molecular para o significado biológico da região do front invasivo tumoral. A ausência de forte correlação entre as expressões imuno-histoquímicas das proteínas p53 e p16 sugere que as mesmas podem participar de atividade biológicas do controle do ciclo celular por

  10. Structure and role of neutrophil cytosol factor 1 (NCF1) gene in ...

    African Journals Online (AJOL)

    Yomi

    2010-12-27

    Dec 27, 2010 ... in innate immunity and produce reactive oxygen species and reduce the severity and duration of parasitic infection and autoimmune disease. NCF1 also has a role in T cell activation. Key words: Neutrophil cytosol factor 1 (NCF1) gene, exons, T cell activation. INTRODUCTION. An immune system is a ...

  11. Changes in the level of cytosolic calcium, nitric oxide and nitric oxide ...

    Indian Academy of Sciences (India)

    Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis. In the present investigation, we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium ...

  12. Enzymatic Regulation of Cytosolic Thymidine Kinase 1 and Mitochondrial Thymidine Kinase 2

    DEFF Research Database (Denmark)

    Munch-Petersen, Birgitte

    2010-01-01

    . Apart from the complex de novo synthesis of dTTP through UDP reduction, dTTP is provided through salvage of thymidine catalyzed by the thymidine kinases, the cytosolic and cell cycle regulated TK1 and the mitochondrial and constitutively expressed TK2. The complex enzymatic regulation of TK1 and TK2...

  13. A reporter of UV intensity delivered to the cytosol during photolytic uncaging

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Dewitt, Sharon; Hallett, Maurice B

    2010-01-01

    Photolytic uncaging of biologically-active molecules within cells is a powerful technique. However, the delivery of uncaging light into the cytosol can vary between cell types, individual cells of the same type, and different loci within an individual cell because of optical differences in absorb...

  14. Cytosolic beta-glycosidases for activation of glycoside prodrugs of daunorubicin

    NARCIS (Netherlands)

    Pinedo, H.M.; Quadir, R.; Haisma, H.J.; Boven, E.

    2003-01-01

    Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA(3)) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present

  15. Structure and role of neutrophil cytosol factor 1 (NCF1) gene in ...

    African Journals Online (AJOL)

    Yomi

    2010-12-27

    Dec 27, 2010 ... The neutrophil cytosol factor 1 (NCF1) gene consists of 11 exons and is found in two forms; one is wild type gene and the other is pseudogene. It has more than 98% homology. Both genes occupy the same chromosome region. The mutation in this gene leads to various types of diseases such as chronic.

  16. The cytosolic B-glucosidase GBA3 does not influence type 1 Gaucher disease manifestation

    NARCIS (Netherlands)

    Dekker, N.; Voorn-Brouwer, T.; Verhoek, M.; Wennekes, T.; Narayan, R.S.; Speijer, D.; Hollak, C.E.M.; Overkleeft, H.S.; Boot, R.G.; Aerts, J.M.F.G.

    2011-01-01

    GBA3, also known as cytosolic ß-glucosidase, is thought to hydrolyze xenobiotic glycosides in man. Deficiency of glucocerebrosidase (GBA), a ß-glucosidase degrading glucosylceramide, underlies Gaucher disease. We examined GBA3, which recently was proposed to degrade glucosylceramide and influence

  17. The cytosolic beta-glucosidase GBA3 does not influence type 1 Gaucher disease manifestation

    NARCIS (Netherlands)

    Dekker, Nick; Voorn-Brouwer, Tineke; Verhoek, Marri; Wennekes, Tom; Narayan, Ravi S.; Speijer, Dave; Hollak, Carla E. M.; Overkleeft, Hermen S.; Boot, Rolf G.; Aerts, Johannes M. F. G.

    2011-01-01

    GBA3, also known as cytosolic beta-glucosidase, is thought to hydrolyze xenobiotic glycosides in man. Deficiency of glucocerebrosidase (GBA), a beta-glucosidase degrading glucosylteramide, underlies Gaucher disease. We examined GBA3, which recently was proposed to degrade glucosylceramide and

  18. Assay of oestrogen and progestin receptors in human meningioma cytosols using immunological methods

    NARCIS (Netherlands)

    Blankenstein, M.A.; Meulen-Dijk, C. van der; Thijssen, J.H.H.

    1987-01-01

    Oestrogen (ER) and progestin receptors (PR) were assayed in human meningioma cytosol by radioligand binding assay with Scatchard plot analysis and by monoclonal antibody based enzyme immunoassays. For comparison, human breast cancer tissues were used. Results of both assays agreed very well. For

  19. Live cell imaging of cytosolic NADH/NAD+ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  20. The Cytosolic Oligosaccharide-Degrading Proteome of Butyrivibrio Proteoclasticus

    Directory of Open Access Journals (Sweden)

    Jonathan C. Dunne

    2015-10-01

    Full Text Available The growth and productivity of ruminants depends on a complex microbial community found in their fore-stomach (rumen, which is able to breakdown plant polysaccharides and ferment the released sugars. Butyrivibrio proteoclasticus B316T is a Gram-positive polysaccharide-degrading, butyrate-producing bacterium that is present at high numbers in the rumen of animals consuming pasture or grass silage based diets. B316T is one of a small number of rumen fibrolytic microbes capable of efficiently degrading and utilizing xylan, as well as being capable of utilizing arabinose, xylose, pectin and starch. We have therefore carried out a proteomic analysis of B316T to identify intracellular enzymes that are implicated in the metabolism of internalized xylan. Three hundred and ninety four proteins were identified including enzymes that have potential to metabolize assimilated products of extracellular xylan digestion. Identified enzymes included arabinosidases, esterases, an endoxylanase, and β-xylosidase. The presence of intracellular debranching enzymes indicated that some hemicellulosic side-chains may not be removed until oligosaccharides liberated by extracellular digestion have been assimilated by the cells. The results support a model of extracellular digestion of hemicellulose to oligosaccharides that are then transported to the cytoplasm for further digestion by intracellular enzymes.

  1. Epidermal electronics for electromyography: An application to swallowing therapy.

    Science.gov (United States)

    Constantinescu, Gabriela; Jeong, Jae-Woong; Li, Xinda; Scott, Dylan K; Jang, Kyung-In; Chung, Hyun-Joong; Rogers, John A; Rieger, Jana

    2016-08-01

    Head and neck cancer treatment alters the anatomy and physiology of patients. Resulting swallowing difficulties can lead to serious health concerns. Surface electromyography (sEMG) is used as an adjuvant to swallowing therapy exercises. sEMG signal collected from the area under the chin provides visual biofeedback from muscle contractions and is used to help patients perform exercises correctly. However, conventional sEMG adhesive pads are relatively thick and difficult to effectively adhere to a patient's altered chin anatomy, potentially leading to poor signal acquisition in this population. Here, the emerging technology of epidermal electronics is introduced, where ultra-thin geometry allows for close contouring of the chin. The two objectives of this study were to (1) assess the potential of epidermal electronics technology for use with swallowing therapy and (2) assess the significance of the reference electrode placement. This study showed comparative signals between the new epidermal sEMG patch and the conventional adhesive patches used by clinicians. Furthermore, an integrated reference yielded optimal signal for clinical use; this configuration was more robust to head movements than when an external reference was used. Improvements for future iterations of epidermal sEMG patches specific to day-to-day clinical use are suggested. Copyright © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.

  2. Clinical Studies on conformal radiotherapy combined with epidermal ...

    African Journals Online (AJOL)

    Purpose: To study the effect of conformal radiotherapy combined with epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in the second-line treatment of non-small cell lung cancer (NSCLC). Methods: A total of 316 patients attending Shanghai Pulmonary Hospital affiliated to Tongji University, were divided ...

  3. Foliar epidermal study of some species of Aglaonema Schott ...

    African Journals Online (AJOL)

    The adaxial and abaxial epidermal surfaces of six species of Aglaonema Schott were studied in order to document characters of taxonomic importance among them. The species are Aglaonema brevispathum Schott (Engl.) Engl., Aglaonema commutatum Schott, Aglaonema costatum Schott, Aglaonema crispum Schott, ...

  4. Improvement of arbutin trans-epidermal delivery using ...

    African Journals Online (AJOL)

    Purpose: To assess the ability of radiofrequency (RF) microporation to promote trans-epidermal delivery of arbutin. Methods: To investigate the enhancing effect of RF microchannels on skin permeation of arbutin, in vitro skin permeability studies were performed with RF microporation-treated Hartley albino guinea pig skin ...

  5. An immunologic approach to induction of epidermal growth factor deficiency

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Poulsen, Steen Seier

    1995-01-01

    Epidermal growth factor (EGF) in pharmacologic doses is able to induce growth and development in the fetus and the newborn. To investigate the opposite situation, the effects of insufficient amounts of EGF during development, we wanted to establish an in vivo model with a state of EGF deficiency....

  6. Exudative epidermitis in pigs caused by toxigenic Staphylococcus chromogenes

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Ahrens, Peter; Daugaard, Lise

    2005-01-01

    Staphylococcus chromogenes is closely related to Staphylococcus hyicus, which is recognised as the causative agent of exudative epidermitis (EE) in pigs. S. chromogenes is part of the normal skin flora of pigs, cattle and poultry and has so far been considered non-pathogenic to pigs. A strain of S...

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

    NARCIS (Netherlands)

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

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

  8. Assessment of the Developmental Toxicity of Epidermal Growth ...

    African Journals Online (AJOL)

    Purpose: To determine whether epidermal growth factor (EGF) is involved in reproductive developmental toxicity, using the embryonic stem cell test (EST), as well as ascertain how EGF influences embryonic development. Methods: To predict developmental toxicity on the basis of reducing cell viability and inhibition of ...

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

    NARCIS (Netherlands)

    Santos Oliveira, S.

    2008-01-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  11. Epidermal growth factor in mammary glands and milk from rats

    DEFF Research Database (Denmark)

    Thulesen, J; Raaberg, Lasse; Nexø, Ebba

    1993-01-01

    Epidermal growth factor (EGF) is one of the major growth-promoting agents in milk. Using immunohistochemistry we localized EGF in the mammary glands of lactating rats to the luminal border of the secretory cells. Following proteolytic pretreatment of the histological sections, the EGF...

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    Little data exist on the expression of epidermal growth factor receptors (EGF-Rs) in human endometrial cancer. EGF-R status was studied in 65 patients with endometrial carcinomas and in 26 women with nonmalignant postmenopausal endometria, either inactive/atrophic endometrium or adenomatous...

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

    African Journals Online (AJOL)

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

  14. Foliar Epidermal Characters of Some Sterculiaceae Species in Nigeria

    African Journals Online (AJOL)

    Foliar epidermal studies were conducted on ten species in the family Sterculiaceae in search of stable taxonomic characters that could be employed in order contribute to their classification and identification. In spite of the remarkable morphological differences, the results indicated that the species are relatively uniform in ...

  15. Epidermal morphology of west african okra Abelmoschus caillei (A ...

    African Journals Online (AJOL)

    A study of the micro-morphology of 53 accessions of West African Okra was undertaken using light microscopy techniques. Results showed that epidermal cells are polygonal, isodiametric and irregularly shaped with different anticlinal cell wall patterns. Stomata type is 100% paracytic and 100% amphistomatic in distribution ...

  16. Gastric luminal epidermal growth factor is affected by diet | Iputo ...

    African Journals Online (AJOL)

    Objective. Diet is an area of major interest to those investigating the causes of cancer of the oesophagus in the Transkei. This study looked at the associations between intragastric epidermal growth factor level, diet and intragastric pH. Setting and subjects. A dietary survey was co-ordinated with studies of gastric luminal ...

  17. Identification of grazed grasses using epidermal characters | R ...

    African Journals Online (AJOL)

    The use of anatomical features of the abaxial epidermis of grasses is discussed for the identification of fragments of epidermis present in samples of rumen. The reliability of this technique, and the variation of the epidermal characters in two widely distributed species of grass, is given. A "Key" to identity certain genera of ...

  18. evaluation of the prognostic value of the expression of epidermal ...

    African Journals Online (AJOL)

    Objectives To evaluate the role and prognostic value of the expression of epidermal growth factor receptors (EGFR) in serum and urine for the detection of human bladder cancer. Patients and Methods The study comprised 30 patients with newly diagnosed transitional cell carcinoma of the bladder and 10 normal volunteers ...

  19. Inflammatory linear verrucous epidermal naevus: Report of three ...

    African Journals Online (AJOL)

    Background: Epidermal naevi are congenital harmatomas that arise from embryonal ectodermal cells. The inflammatory linear verrucous variant is rare and presents with disturbing symptoms. In blacks the classical erythema is not common but pruritus and discharge are the commonest features. Methods and results: We ...

  20. Epidermal hydration levels in rosacea patients improve after minocycline therapy.

    LENUS (Irish Health Repository)

    Ní Raghallaigh, S

    2013-12-06

    Patients with rosacea frequently report increased skin sensitivity, with features suggestive of an abnormal stratum corneum (SC) permeability barrier. Sebum, pH and hydration levels influence epidermal homeostasis. The correlation of the change in these parameters with clinically effective treatment has not been previously analysed.

  1. Foliar epidermal anatomy and its systematic implication within the ...

    African Journals Online (AJOL)

    Micro morphological investigations of the foliar epidermal anatomy, particularly the diversity and distribution of glandular and eglandular trichomes on leaves of Sida alba L., S. alii S. Abedin var. alii, S. cordata (Burm. F.) Brss, S. mysorensis Wight and Arn, S. ovata Forssk. S. spinosa L and S.yunnanensis S.Y.Hu have been ...

  2. Epidermal characteristics of some Nigerian species of Corchorus ...

    African Journals Online (AJOL)

    The three species of Corchorus used in this study were collected from Ife and Port Harcourt. The comparative study of the species were done using light microscope. The observations recorded on the epidermal architecture, stomata types, stomata distribution and frequency and the trichomes have been discussed in line ...

  3. Stevens-Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis ...

    African Journals Online (AJOL)

    Stevens-Johnson syndrome (SJS) is a form of toxic epidermal necrolysis (TEN) a rare but life-threatening hypersensitivity reactions that affect the skin and mucous membranes. The most common triggers are drugs, ... Carbamazepine, phenytoin, phenobarbitone, lamitrogine. Antigout. Allopurinol. Antimalarials. Chloroquine.

  4. Stevens-Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis ...

    African Journals Online (AJOL)

    Stevens-Johnson syndrome (SJS) is a form of toxic epidermal necrolysis (TEN) a rare but life-threatening hypersensitivity reactions that affect the skin and mucous membranes. The most common triggers are drugs, but they can also be triggered by infections. Granulysin has been recently identified as the major molecule ...

  5. Micromorphology and development of the epicuticular structure on the epidermal cell of ginseng leaves

    Directory of Open Access Journals (Sweden)

    Kyounghwan Lee

    2015-04-01

    Conclusion: The outwardly projected cuticle and epidermal cell wall (i.e., an epicuticular wrinkle acts as a major barrier to block out sunlight in ginseng leaves. The small vesicles in the peripheral region of epidermal cells may suppress the cuticle and parts of epidermal wall, push it upward, and consequently contribute to the formation of the epicuticular structure.

  6. Studies on the foliar epidermal tissues of three species of Digitaria ...

    African Journals Online (AJOL)

    Foliar epidermal studies were carried out on Digitaria iburua Stapf, Digitaria exilis Stapf and Digitaria barbinodis Henr. with the aim of determining the patterns of variation in their epidermal characteristics and assessing the value of leaf epidermal characters in the identification of these culturally important species.

  7. Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development.

    Science.gov (United States)

    Pasternak, Maciej; Lim, Benson; Wirtz, Markus; Hell, Rüdiger; Cobbett, Christopher S; Meyer, Andreas J

    2008-03-01

    Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, gamma-glutamate cysteine ligase (GSH1), responsible for synthesis of gamma-glutamylcysteine (gamma-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by gamma-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of gamma-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by gamma-EC in vitro strongly suggests export of gamma-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, gamma-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.

  8. Limits to anaerobic energy and cytosolic concentration in the living cell

    Science.gov (United States)

    Paglietti, A.

    2015-11-01

    For many physical systems at any given temperature, the set of all states where the system's free energy reaches its largest value can be determined from the system's constitutive equations of internal energy and entropy, once a state of that set is known. Such an approach is fraught with complications when applied to a living cell, because the cell's cytosol contains thousands of solutes, and thus thousands of state variables, which makes determination of its state impractical. We show here that, when looking for the maximum energy that the cytosol can store and release, detailed information on cytosol composition is redundant. Compatibility with cell's life requires that a single variable that represents the overall concentration of cytosol solutes must fall between defined limits, which can be determined by dehydrating and overhydrating the cell to its maximum capacity. The same limits are shown to determine, in particular, the maximum amount of free energy that a cell can supply in fast anaerobic processes, starting from any given initial state. For a typical skeletal muscle in normal physiological conditions this energy, i.e., the maximum anaerobic capacity to do work, is calculated to be about 960 J per kg of muscular mass. Such energy decreases as the overall concentration of solutes in the cytosol is increased. Similar results apply to any kind of cell. They provide an essential tool to understand and control the macroscopic response of single cells and multicellular cellular tissues alike. The applications include sport physiology, cell aging, disease produced cell damage, drug absorption capacity, to mention the most obvious ones.

  9. Epidermal growth factor reactivity in rat milk

    DEFF Research Database (Denmark)

    Raaberg, Lasse; Nexø, Ebba; Tollund, L

    1990-01-01

    whey elutes as a broad peak corresponding to a Stokes radius of 4.0 nm (an approximate molecular weight of 80 kDa). Almost no 6 kDa EGF is present. Judged by gel filtration of whey pre-incubated with 125I-EGF (6 kDa), no binding protein for EGF is present in rat whey. When rat milk is incubated...

  10. Purification and in vitro Chaperone Activity of a Class I Small Heat-Shock Protein Abundant in Recalcitrant Chestnut Seeds

    National Research Council Canada - National Science Library

    Carmen Collada; Luis Gomez; Rosa Casado; Cipriano Aragoncillo

    1997-01-01

    .... This protein, termed Cs smHSP 1, forms homododecameric complexes under nondenaturing conditions and appears to be homologous to cytosolic class I small heat-shock proteins (smHSPs) from plant sources...

  11. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yi Ma

    2016-01-01

    Full Text Available Human epidermal growth factor (hEGF is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  12. Cytosolic guanine nucledotide binding deficient form of transglutaminase 2 (R580a potentiates cell death in oxygen glucose deprivation.

    Directory of Open Access Journals (Sweden)

    Gozde Colak

    Full Text Available Transglutaminase 2 (TG2 is a hypoxia-responsive protein that is a calcium-activated transamidating enzyme, a GTPase and a scaffolding/linker protein. Upon activation TG2 undergoes a large conformational change, which likely affects not only its enzymatic activities but its non-catalytic functions as well. The focus of this study was on the role of transamidating activity, conformation and localization of TG2 in ischemic cell death. Cells expressing a GTP binding deficient form of TG2 (TG2-R580A with high basal transamidation activity and a more extended conformation showed significantly increased cell death in response to oxygen-glucose deprivation; however, targeting TG2-R580A to the nucleus abrogated its detrimental role in oxygen-glucose deprivation. Treatment of cells expressing wild type TG2, TG2-C277S (a transamidating inactive mutant and TG2-R580A with Cp4d, a reversible TG2 inhibitor, did not affect cell death in response to oxygen-glucose deprivation. These findings indicate that the pro-cell death effects of TG2 are dependent on its localization to the cytosol and independent of its transamidation activity. Further, the conformational state of TG2 is likely an important determinant in cell survival and the prominent function of TG2 in ischemic cell death is as a scaffold to modulate cellular processes.

  13. Cytosolic prostaglandin E2 synthase (cPGES) expression is decreased in discrete cortical regions in psychiatric disease.

    Science.gov (United States)

    Maida, Mary E; Hurley, Sean D; Daeschner, Jo Anna; Moore, Amy H; O'Banion, M Kerry

    2006-08-04

    The number of adults in the US affected by bipolar disorder, depression, or schizophrenia is approaching 15 million. Despite decades of research, etiologies of these illnesses remain elusive. Theories of aberrant brain morphology, neurotransmission, and signal conduction have provided the heuristic framework for a large body of literature, with attention focused upon hypotheses of monoamine signaling underlying psychiatric disease. More recently, attention has turned to potential contributions of other signaling pathways, including the arachidonic acid cascade and generation of prostaglandins (PG). To determine the potential involvement of the pathways leading to PGE2 synthesis in psychiatric disease, immunohistochemistry and immunoblotting were performed to measure regional expression of the cyclooxygenases (COX) and one of the terminal PGE2 synthases (PGES) in postmortem tissue provided by The Stanley Medical Research Institute. For normal, bipolar, depressed, and schizophrenic subjects, COX-1 and COX-2 protein levels did not differ across region and patient populations. In contrast, there was a significant effect of diagnosis on cytosolic PGES (cPGES) protein levels in the frontal cortex, with remarkable decreases observed in all psychiatric groups relative to normal tissue (P bipolar subjects. Evaluation of medicated vs. non-medicated subjects revealed a significant effect of medication on cPGES expression in the frontal cortex of bipolar, but not depressed or schizophrenic subjects. These novel findings further support hypotheses of abnormalities in fatty acid and phospholipid metabolism in regions associated with psychiatric disease.

  14. Cytosolic phospholipase A2 modulates TLR2 signaling in synoviocytes.

    Directory of Open Access Journals (Sweden)

    Randi M Sommerfelt

    Full Text Available Rheumatoid arthritis (RA is an autoimmune disease characterized by chronic synovitis leading to destruction of cartilage and bone. PLA2 enzymes are key players in inflammation regulating the release of unsaturated fatty acids such as arachidonic acid (AA, a precursor of pro-inflammatory eicosanoids. Several lines of evidence point to toll-like receptors (TLRs as drivers of synovitis and joint destruction in RA. However, few studies have addressed the implication of PLA2 activity downstream TLR activation in the synovium. Here, we aimed to characterize PLA2 enzyme involvement in TLR2-induced signaling in synovial fibroblast-like cells. TLRs1-7 and a range of sPLA2, iPLA2 and cPLA2 enzymes were found to be transcriptionally expressed in cultured synoviocytes. Activation of TLR2/1 and TLR2/6 led to phosphorylation of cPLA2α at Ser505, and induced AA release and PGE2 production; effects that were attenuated by cPLA2α inhibitors. In contrast, sPLA2 inhibitors did not affect AA or PGE2 release. cPLA2α inhibitors furthermore attenuated TLR-induced expression of IL-6, IL-8 and COX2. COX1/2 inhibitors attenuated TLR2/6-induced IL-6 transcription and protein production comparable to cPLA2α inhibition. Moreover, exogenously PGE2 added alone induced IL-6 production and completely rescued IL-6 transcription when added simultaneously with FSL-1 in the presence of a cPLA2α inhibitor. Our results demonstrate for the first time that cPLA2α is involved in TLR2/1- and TLR2/6-induced AA release, PGE2 production and pro-inflammatory cytokine expression in synoviocytes, possibly through COX/PGE2-dependent pathways. These findings expand our understanding of cPLA2α as a modulator of inflammatory molecular mechanisms in chronic diseases such as RA.

  15. Superficial Dsg2 Expression Limits Epidermal Blister Formation Mediated by Pemphigus Foliaceus Antibodies and Exfoliative Toxins

    Directory of Open Access Journals (Sweden)

    Donna Brennan

    2010-01-01

    Full Text Available Cell-cell adhesion mediated by desmosomes is crucial for maintaining proper epidermal structure and function, as evidenced by several severe and potentially fatal skin disorders involving impairment of desmosomal proteins. Pemphigus foliaceus (PF and staphylococcal scalded skin syndrome (SSSS are subcorneal blistering diseases resulting from loss of function of the desmosomal cadherin, desmoglein 1 (Dsg1. To further study the pathomechanism of these diseases and to assess the adhesive properties of Dsg2, we employed a recently established transgenic (Tg mouse model expressing Dsg2 in the superficial epidermis. Neonatal Tg and wild type (WT mice were injected with purified ETA or PF Ig. We showed that ectopic expression of Dsg2 reduced the extent of blister formation in response to both ETA and PF Ig. In response to PF Ig, we observed either a dramatic loss or a reorganization of Dsg1-α, Dsg1-β, and, to a lesser extent, Dsg1-γ, in WT mice. The Inv-Dsg2 Tg mice showed enhanced retention of Dsg1 at the cell-cell border. Collectively, our data support the role for Dsg2 in cell adhesion and suggest that ectopic superficial expression of Dsg2 can increase membrane preservation of Dsg1 and limit epidermal blister formation mediated by PF antibodies and exfoliative toxins.

  16. A Comparison of Two Yeast MnSODs: Mitochondrial Saccharomyces cerevisiae versus Cytosolic Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Sheng Y.; Cabelli D.; Stich, T.A.; Barnese, K.; Gralla, E.B.; Cascio, D.; Britt, R.D.; Valentine, J.S.

    2011-12-28

    Human MnSOD is significantly more product-inhibited than bacterial MnSODs at high concentrations of superoxide (O{sub 2}{sup -}). This behavior limits the amount of H{sub 2}O{sub 2} produced at high [O{sub 2}{sup -}]; its desirability can be explained by the multiple roles of H{sub 2}O{sub 2} in mammalian cells, particularly its role in signaling. To investigate the mechanism of product inhibition in MnSOD, two yeast MnSODs, one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), were isolated and characterized. ScMnSOD and CaMnSODc are similar in catalytic kinetics, spectroscopy, and redox chemistry, and they both rest predominantly in the reduced state (unlike most other MnSODs). At high [O{sub 2}{sup -}], the dismutation efficiencies of the yeast MnSODs surpass those of human and bacterial MnSODs, due to very low level of product inhibition. Optical and parallel-mode electron paramagnetic resonance (EPR) spectra suggest the presence of two Mn{sup 3+} species in yeast Mn{sup 3+}SODs, including the well-characterized 5-coordinate Mn{sup 3+} species and a 6-coordinate L-Mn{sup 3+} species with hydroxide as the putative sixth ligand (L). The first and second coordination spheres of ScMnSOD are more similar to bacterial than to human MnSOD. Gln154, an H-bond donor to the Mn-coordinated solvent molecule, is slightly further away from Mn in yeast MnSODs, which may result in their unusual resting state. Mechanistically, the high efficiency of yeast MnSODs could be ascribed to putative translocation of an outer-sphere solvent molecule, which could destabilize the inhibited complex and enhance proton transfer from protein to peroxide. Our studies on yeast MnSODs indicate the unique nature of human MnSOD in that it predominantly undergoes the inhibited pathway at high [O{sub 2}{sup -}].

  17. Biochemical parameters of epidermal aging in the hairless mouse and the relationship to UV-carcinogenesis.

    Science.gov (United States)

    Black, H S; Chiang, J; Gerguis, J; Lenger, W; Thornby, J I

    1994-05-01

    Epidemiological studies suggest that the incidence of cancer increases with age in both human and animal populations and that declining physiologic condition associated with aging might be responsible. Experimentally, the reverse has been most often observed, that is, older animals appear less susceptible to the induction of UV-carcinogenesis. Thus, we examined several biochemical parameters of epidermal macromolecular synthesis in hairless mice in an effort to gain insight into the role these processes play in physiological aging and their relationship to carcinogenesis. SKh-Hr-1 hairless mice were randomized into two groups (UV-irradiated and non-irradiated controls) and were two months of age at the start of irradiation and biochemical analyses. The UV group received 0.028 sunburn units (SBUs) daily (5 days wk-1) for 16 months from 40 watt BZS-WLG lamps. Stratum corneum turnover rates (SCR), cell label index (CLI), protein, DNA and RNA synthesis, and ornithine decarboxylase (ODC) induction were determined at monthly intervals over a period of two years. There were no age-related tendencies observed in SCR. CLI increased with age. Chronic, low-dose UV had no effect upon either of these parameters. Epidermal capacity for DNA and protein synthesis increased with age from 2 months to 12-15 months at which time both parameters peaked and then began to decline. UV significantly reduced (P < 0.04) the magnitude of DNA synthetic capacity at peak periods of synthesis but had no effect upon protein synthesis. RNA synthetic rates declined with age, reaching their lowest levels at 24 months. Further, a significant reduction (P < 0.001) in ODC inducibility occurred with advancing age.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Morphometric study of microvessels, epidermal characteristics and inflammation in psoriasis vulgaris with their correlations

    Directory of Open Access Journals (Sweden)

    Dibyajyoti Boruah

    2013-01-01

    Full Text Available Background: Vascular proliferation, inflammation and epidermal changes are important features in the pathogenesis of psoriasis. Aims: In this study we attempted an objective evaluation of these parameters using morphometry. Methods: Inflammation, microvessels and epidermal parameters were assessed in 50 newly diagnosed cases of psoriasis vulgaris (between 01 Nov 2008 and 31 Oct 2011 by morphometry. Parameters studied were microvessel density, microvessel caliber, inflammatory cell density in dermis, ratio between inner and outer epidermal length, maximum epidermal thickness, minimum epidermal thickness and difference between maximum epidermal thickness and minimum epidermal thickness. Results: Microvessel caliber showed moderate correlation (r = 0.645 and microvessel density, weak correlation (r = 0.226 with inflammatory cell density in dermis. Both these parameters also showed mild positive correlation with "ratio between inner and outer epidermal length". All parameters except minimum epidermal thickness showed mild positive correlation with inflammatory cell density in dermis. Conclusion: All microvessels and epidermal parameters showed positive correlation with dermal inflammation; and epidermal parameters exhibited positive correlation with micro-vascular dilation. It is likely that inflammation is a key factor in the pathogenesis of psoriasis.

  19. Morphometric study of microvessels, epidermal characteristics and inflammation in psoriasis vulgaris with their correlations.

    Science.gov (United States)

    Boruah, Dibyajyoti; Moorchung, Nikhil; Vasudevan, Biju; Malik, Ajay; Chatterjee, Manas

    2013-01-01

    Vascular proliferation, inflammation and epidermal changes are important features in the pathogenesis of psoriasis. In this study we attempted an objective evaluation of these parameters using morphometry. Inflammation, microvessels and epidermal parameters were assessed in 50 newly diagnosed cases of psoriasis vulgaris (between 01 Nov 2008 and 31 Oct 2011) by morphometry. Parameters studied were microvessel density, microvessel caliber, inflammatory cell density in dermis, ratio between inner and outer epidermal length, maximum epidermal thickness, minimum epidermal thickness and difference between maximum epidermal thickness and minimum epidermal thickness. Microvessel caliber showed moderate correlation (r = 0.645) and microvessel density, weak correlation (r = 0.226) with inflammatory cell density in dermis. Both these parameters also showed mild positive correlation with "ratio between inner and outer epidermal length". All parameters except minimum epidermal thickness showed mild positive correlation with inflammatory cell density in dermis. All microvessels and epidermal parameters showed positive correlation with dermal inflammation; and epidermal parameters exhibited positive correlation with micro-vascular dilation. It is likely that inflammation is a key factor in the pathogenesis of psoriasis.

  20. Epidermal and cortical roles of NFP and DMI3 in coordinating early steps of nodulation in Medicago truncatula.

    Science.gov (United States)

    Rival, Pauline; de Billy, Françoise; Bono, Jean-Jacques; Gough, Clare; Rosenberg, Charles; Bensmihen, Sandra

    2012-09-01

    Legumes have evolved the capacity to form a root nodule symbiosis with soil bacteria called rhizobia. The establishment of this symbiosis involves specific developmental events occurring both in the root epidermis (notably bacterial entry) and at a distance in the underlying root cortical cells (notably cell divisions leading to nodule organogenesis). The processes of bacterial entry and nodule organogenesis are tightly linked and both depend on rhizobial production of lipo-chitooligosaccharide molecules called Nod factors. However, how these events are coordinated remains poorly understood. Here, we have addressed the roles of two key symbiotic genes of Medicago truncatula, the lysin motif (LysM) domain-receptor like kinase gene NFP and the calcium- and calmodulin-dependent protein kinase gene DMI3, in the control of both nodule organogenesis and bacterial entry. By complementing mutant plants with corresponding genes expressed either in the epidermis or in the cortex, we have shown that epidermal DMI3, but not NFP, is sufficient for infection thread formation in root hairs. Epidermal NFP is sufficient to induce cortical cell divisions leading to nodule primordia formation, whereas DMI3 is required in both cell layers for these processes. Our results therefore suggest that a signal, produced in the epidermis under the control of NFP and DMI3, is responsible for activating DMI3 in the cortex to trigger nodule organogenesis. We integrate these data to propose a new model for epidermal/cortical crosstalk during early steps of nodulation.

  1. Genética Molecular das Epidermólises Bolhosas Molecular Genetics of Epidermolysis Bullosa

    Directory of Open Access Journals (Sweden)

    Hiram Larangeira de Almeida Jr

    2002-10-01

    Full Text Available O estudo das alterações moleculares das epidermólises bolhosas tem contribuído para que se compreenda melhor essas enfermidades. Na epidermólise bolhosa simples a maioria dos casos está associada com alteração nas citoqueratinas basais 5 (gen KRT5 e 14 (gen KRT14, o que modifica o citoesqueleto na camada basal da epiderme, levando à degeneração dessa camada, formando bolha intra-epidérmica. Mutações na plectina (gen PLEC1, componente da placa interna do hemidesmossoma, levam também à clivagem intra-epidérmica. Na epidermólise bolhosa juncional vários gens estão envolvidos, em decorrência da complexidade da zona da membrana basal, todos levando ao descolamento dos queratinócitos basais na lâmina lúcida, pela disfunção da aderência entre esses e a lâmina densa. Alterações na laminina 5 (gens LAMA3, LAMB3 e LAMC2, integrina alfa6beta4 (gens ITGA6 e ITGB4 e colágeno XVII (gen COL17A1 foram descritas. Por fim, na epidermólise bolhosa distrófica apenas um gen está mutado, alterando o colágeno VII (gen COL7A1, principal componente das fibrilas ancorantes, produzindo clivagem abaixo da lâmina densa, variando fenotipicamente de acordo com a conseqüência da mutação. Outra aplicação importante dessas informações refere-se ao diagnóstico pré-natal, com a perspectiva no futuro da terapia gênica.New data regarding the molecular aspects of the heterogeneous group of epidermolysis bullosa has brought some important information about its pathogenesis. In epidermolysis bullosa simplex the majority of mutations are localized in the genes of the basal cytokeratin 5 (gene KRT5 and 14 (gene KRT14, cytolysis at this layer with intraepidermal blister is seen under light microscopy. Mutations of plectin (gene PLEC1, a protein found in the inner hemidesmosomal plaque, leads also to intraepidermal blisters. In junctional epidermolysis bullosa many proteins from the basal membrane zone are involved, such as laminin 5 (genes

  2. The compartmentalisation of phosphorylated free oligosaccharides in cells from a CDG Ig patient reveals a novel ER-to-cytosol translocation process.

    Directory of Open Access Journals (Sweden)

    Delphine Peric

    Full Text Available BACKGROUND: Biosynthesis of the dolichol linked oligosaccharide (DLO required for protein N-glycosylation starts on the cytoplasmic face of the ER to give Man(5GlcNAc(2-PP-dolichol, which then flips into the ER for further glycosylation yielding mature DLO (Glc(3Man(9GlcNAc(2-PP-dolichol. After transfer of Glc(3Man(9GlcNAc(2 onto protein, dolichol-PP is recycled to dolichol-P and reused for DLO biosynthesis. Because de novo dolichol synthesis is slow, dolichol recycling is rate limiting for protein glycosylation. Immature DLO intermediates may also be recycled by pyrophosphatase-mediated cleavage to yield dolichol-P and phosphorylated oligosaccharides (fOSGN2-P. Here, we examine fOSGN2-P generation in cells from patients with type I Congenital Disorders of Glycosylation (CDG I in which defects in the dolichol cycle cause accumulation of immature DLO intermediates and protein hypoglycosylation. METHODS AND PRINCIPAL FINDINGS: In EBV-transformed lymphoblastoid cells from CDG I patients and normal subjects a correlation exists between the quantities of metabolically radiolabeled fOSGN2-P and truncated DLO intermediates only when these two classes of compounds possess 7 or less hexose residues. Larger fOSGN2-P were difficult to detect despite an abundance of more fully mannosylated and glucosylated DLO. When CDG Ig cells, which accumulate Man(7GlcNAc(2-PP-dolichol, are permeabilised so that vesicular transport and protein synthesis are abolished, the DLO pool required for Man(7GlcNAc(2-P generation could be depleted by adding exogenous glycosylation acceptor peptide. Under conditions where a glycotripeptide and neutral free oligosaccharides remain predominantly in the lumen of the ER, Man(7GlcNAc(2-P appears in the cytosol without detectable generation of ER luminal Man(7GlcNAc(2-P. CONCLUSIONS AND SIGNIFICANCE: The DLO pools required for N-glycosylation and fOSGN2-P generation are functionally linked and this substantiates the hypothesis that

  3. E2F1 interactive with BRCA1 pathway induces HCC two different small molecule metabolism or cell cycle regulation via mitochondrion or CD4+T to cytosol.

    Science.gov (United States)

    Chen, Qingchun; Wang, Lin; Jiang, Minghu; Huang, Juxiang; Jiang, Zhenfu; Feng, Haitao; Ji, Zhili

    2018-02-01

    Breast cancer 1 (BRCA1) and E2F transcription factor 1 (E2F1) are related to metabolism and cell cycle regulation. However, the corresponding mechanism is not clear in HCC. High BRCA1 direct pathway was constructed with 11 molecules from E2F1 feedback-interactive network in HCC by GRNInfer based on 39 Pearson mutual positive corelation CC ≥0.25 molecules with E2F1. Integration of GRNInfer with GO, KEGG, BioCarta, GNF_U133A, UNIGENE_EST, Disease, GenMAPP databases by DAVID and MAS 3.0, E2F1 feedback-interactive BRCA1 indirect mitochondrion to cytosol pathway was identified as upstream LAPTM4B activation, feedback UNG, downstream BCAT1-HIST1H2AD-TK1 reflecting protein, and DNA binding with enrichment of small molecule metabolism; The corresponding BRCA1 indirect membrane to cytosol pathway as upstream CCNB2-NUSAP1 activation, feedback TTK-HIST1H2BJ-CENPF, downstream MCM4-TK1 reflecting ATP, and microtubule binding with enrichment of CD4+T-related cell cycle regulation in HCC. Therefore, we propose that E2F1 interactive with BRCA1 pathway induces HCC two different small molecule metabolism or cell cycle regulation via mitochondrion or CD4+T to cytosol. Knowledge analysis demonstrates our E2F1 feedback-interactive BRCA1 pathway wide disease distribution and reflects a novel common one of tumor and cancer. © 2017 Wiley Periodicals, Inc.

  4. Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell

    Directory of Open Access Journals (Sweden)

    Sachie Tanaka

    2017-05-01

    Full Text Available Background/Aims: Our previous study revealed that cytosolic Cl- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl-–induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. Methods: To clarify the cytosolic Cl- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. Results: The decline of cytosolic Cl- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl- condition. Conclusions: Our results strongly suggest that the upregulation of cytosolic Cl- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl- would be one of the key targets for anti-cancer therapy.

  5. Specific quantification of mouse and human keratin proteins by radioimmunoassay.

    OpenAIRE

    Yuspa, S H; Poirier, M C; Harness, J R; Olsom, D R; Steinert, P. M.

    1980-01-01

    Mouse epidermal keratin proteins were purified and labelled with 125I by chemical techniques. A radioimmunoassay method was established with a rabbit antibody elicited against the mouse keratin. This assay method was utilized to quantify keratin proteins of mouse and human epidermal extracts, both from intact tissues and cells in culture. As little as 30 ng of mouse keratin (0.5 pmol) was quantifiable.

  6. Hair Follicle and Sebaceous Gland De Novo Regeneration With Cultured Epidermal Stem Cells and Skin-Derived Precursors.

    Science.gov (United States)

    Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E; Cao, Mengjun; Wu, Yaojiong

    2016-12-01

    : Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. ©AlphaMed Press.

  7. Basis for the gain and subsequent dilution of epidermal pigmentation during human evolution: The barrier and metabolic conservation hypotheses revisited.

    Science.gov (United States)

    Elias, Peter M; Williams, Mary L

    2016-10-01

    The evolution of human skin pigmentation must address both the initial evolution of intense epidermal pigmentation in hominins, and its subsequent dilution in modern humans. While many authorities believe that epidermal pigmentation evolved to protect against either ultraviolet B (UV-B) irradiation-induced mutagenesis or folic acid photolysis, we hypothesize that pigmentation augmented the epidermal barriers by shifting the UV-B dose-response curve from toxic to beneficial. Whereas erythemogenic UV-B doses produce apoptosis and cell death, suberythemogenic doses benefit permeability and antimicrobial function. Heavily melanized melanocytes acidify the outer epidermis and emit paracrine signals that augment barrier competence. Modern humans, residing in the cooler, wetter climes of south-central Europe and Asia, initially retained substantial pigmentation. While their outdoor lifestyles still permitted sufficient cutaneous vitamin D3 (VD3) synthesis, their marginal nutritional status, coupled with cold-induced caloric needs, selected for moderate pigment reductions that diverted limited nutritional resources towards more urgent priorities (=metabolic conservation). The further pigment-dilution that evolved as humans reached north-central Europe (i.e., northern France, Germany), likely facilitated cutaneous VD3 synthesis, while also supporting ongoing, nutritional requirements. But at still higher European latitudes where little UV-B breaches the atmosphere (i.e., present-day UK, Scandinavia, Baltic States), pigment dilution alone could not suffice. There, other nonpigment-related mutations evolved to facilitate VD3 production; for example, in the epidermal protein, filaggrin, resulting in reduced levels of its distal metabolite, trans-urocanic acid, a potent UV-B chromophore. Thus, changes in human pigmentation reflect a complex interplay between latitude, climate, diet, lifestyle, and shifting metabolic priorities. © 2016 Wiley Periodicals, Inc.

  8. A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

    Directory of Open Access Journals (Sweden)

    Julie Adam

    2013-05-01

    Full Text Available The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH, predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

  9. Sulfation of afimoxifene, endoxifen, raloxifene, and fulvestrant by the human cytosolic sulfotransferases (SULTs: A systematic analysis

    Directory of Open Access Journals (Sweden)

    Ying Hui

    2015-07-01

    Full Text Available Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.

  10. Toxic epidermal necrolysis - management issues and treatment options.

    Science.gov (United States)

    Widgerow, Alan D

    2011-01-01

    Toxic epidermal necrolysis (TEN) and Steven-Johnson syndrome (SJS) are characterized by extensive necrosis and cleavage of the epidermis from the dermis akin to a superficial or partial thickness burn. Sepsis is the usual cause of mortality but much of the pathophysiologic process results from an outpouring of cytokines and matrixmetalloproteinases (MMPs) which have a destructive effect on the extracellular matrix and may play a part in the epidermal/dermal cleavage seen with this disease. Recent attention has been focused on the modulation of proteases in an attempt to decrease the MMP-mediated destruction. Nanocrystalline silver (NCS) is one such agent that has good anti-microbial efficacy, but is also effective in modulating MMP levels. Twelve cases of confirmed TEN that were treated with NCS were analyzed with a view to assessing efficacy and setting logical guidelines for managing this condition, particularly in relation to immunosupressed patients. From this study important issues have been highlighted for discussion.

  11. Acetaminophen induced Steven Johnson syndrome-toxic epidermal necrolysis overlap.

    Science.gov (United States)

    Khawaja, Ali; Shahab, Ahmed; Hussain, Syed Ather

    2012-05-01

    Steven Johnson Syndrome and Toxic Epidermal Necrolysis are rare but severe form of hypersensitivity inflammatory reactions to multiple offending agents including drugs. Acetaminophen is extensively used due to its analgesic and anti-pyretic properties. It is rendered to be relatively safe, with hepatotoxicity considered to be the major adverse effect. However, very few cases of Steven Johnson Syndrome and Toxic Epidermal Necrolysis have been reported with acetaminophen usage in the past. We present the case of a 40 years old lady who developed an overlap of the two condition after taking several doses of acetaminophen for fever. She presented with widespread maculopapular rash, stinging in the eyes, oral mucosal ulcerations and high grade fever. She was successfully treated with corticosteroid therapy along with the supportive treatment. This case addresses the fact, that severe hypersensitivity reactions can occur with acetaminophen which can be potentially life threatening.

  12. Epidermal response of rainbow trout to Ichthyobodo necator

    DEFF Research Database (Denmark)

    Chettri, Jiwan Kumar; Kuhn, Jesper Andreas; Mohammad, Rezkar Jaafar

    2014-01-01

    Infections with the parasitic flagellate Ichthyobodo necator (Henneguy, 1883) cause severe skin and gill disease in rainbow trout Oncorhynchus mykiss (Walbaum, 1792) juveniles. The epidermal disturbances including hyperplasia and mucous cell exhaustion caused by parasitization are known, but no d......Infections with the parasitic flagellate Ichthyobodo necator (Henneguy, 1883) cause severe skin and gill disease in rainbow trout Oncorhynchus mykiss (Walbaum, 1792) juveniles. The epidermal disturbances including hyperplasia and mucous cell exhaustion caused by parasitization are known...... an experimental infection of juvenile rainbow trout. The course of infection was followed by sampling for parasite enumeration, immunohistochemistry (IHC) and quantitative PCR (qPCR) on days 0, 5, 9 and 14 post-infection. IHC showed a significant increase in the occurrence of IgM-positive cells in the skin...

  13. Neonicotinoid insecticides: reduction and cleavage of imidacloprid nitroimine substituent by liver microsomal and cytosolic enzymes.

    Science.gov (United States)

    Schulz-Jander, Daniel A; Leimkuehler, William M; Casida, John E

    2002-09-01

    The major insecticide imidacloprid (IMI) is known to be metabolized by human cytochrome P450 3A4 with NADPH by imidazolidine hydroxylation and dehydrogenation to give 5-hydroxy-imidacloprid and the olefin, respectively, and by nitroimine reduction and cleavage to yield the nitrosoimine, guanidine, and urea derivatives. More extensive metabolism by human or rabbit liver microsomes with NADPH or rabbit liver cytosol without added cofactor reduces the IMI N-nitro group to an N-amino substituent, i.e., the corresponding hydrazone. A major metabolite on incubation of IMI in the human microsome-NADPH system is tentatively assigned by LC/MS as a 1,2,4-triazol-3-one derived from the hydrazone; the same product is obtained on reaction of the hydrazone with ethyl chloroformate. The hydrazone and proposed triazolone are considered here together (referred to as the hydrazone) for quantitation. Only a portion of the microsomal reduction and cleavage of the nitroimine substituent is attributable to a CYP450 enzyme. The cytosolic enzyme conversion to the hydrazone is inhibited by added cofactors (NAD > NADH > NADP > NADPH) and enhanced by an argon instead of an air atmosphere. The responsible cytosolic enzyme(s) does not appear to be DT-diaphorase (which is inhibited by several neonicotinoids), aldose reductase, aldehyde reductase, or xanthine oxidase. However, the cytosolic metabolism of IMI is inhibited by several aldo-keto-reductase inhibitors (i.e., alrestatin, EBPC, Ponalrestat, phenobarbital, and quercetin). Other neonicotinoids with nitroimine, nitrosoimine, and nitromethylene substituents are probably also metabolized by "neonicotinoid nitro reductase(s)" since they serve as competitive substrates for [(3)H]IMI metabolism.

  14. [Utilization of nitrate by bacteroids and cytosol of nodules formed by Rhizobium leguminosarum].

    Science.gov (United States)

    Fernández-López, M; Delgado, M J; Olivares, J; Bedmar, E J

    1989-06-01

    Nitrite production by nodules and roots of pea plants (Pisum sativum L., cultivar Alaska) inoculated with Rhizobium leguminosarum strain 3855 has been studied. Nitrate reductase (NR) activity and nitrite reductase (NiR) activity of the bacteroidal and cytosolic fractions of the nodules were also determined, as well as the nitrite content of the nodules cytosol. Nitrite production by nodules and roots from plants treated with 5 mM KNO3 was higher than that of nodules and roots from plants not treated with nitrate, and regardless of the nitrate treatment, nitrite production increased with the incubation period. The presence of nitrate, propanol or both compounds in the incubation mixtures significantly increased the nitrite production by nodules and roots. Nitrite reductase activity was detected in fresh by isolated bacteroids of R. leguminosarum strain 3855, although the presence of nitrate reductase activity could not be detected both in bacteroids of nodules isolated from plants treated or not with 5 mM KNO3. After isolation, when bacteroids were incubated in a mixture with nitrate, nitrate reductase activity developed after incubation for 12 h. Consequently, there was an increase in nitrite reductase activity, which resulted in the disappearance of the nitrite previously accumulated in the incubation medium. Nitrate utilization by bacteroids was not detected until 5 h from the beginning of the incubation period. Since the presence of chloramphenicol or rifampicin in the incubation medium prevented the development of the nitrate reductase activity, such activity was induced in bacteroids. Nitrite content and nitrate reductase and nitrite reductase activities of the cytosol from nodules of pea plants treated or not with 5 mM KNO3 varied with the buffer used for nodules homogenization. However, no nitrite was found when nodules were homogenized with ethanol, what indicates that nitrite accumulation in the cytosol occurs during the homogenization process of the

  15. High effective cytosolic H+ buffering in mouse cortical astrocytes attributable to fast bicarbonate transport.

    Science.gov (United States)

    Theparambil, Shefeeq M; Deitmer, Joachim W

    2015-09-01

    Cytosolic H(+) buffering plays a major role for shaping intracellular H(+) shifts and hence for the availability of H(+) for biochemical reactions and acid/base-coupled transport processes. H(+) buffering is one of the prime means to protect the cell from large acid/base shifts. We have used the H(+) indicator dye BCECF and confocal microscopy to monitor the cytosolic H(+) concentration, [H(+)]i, in cultured cortical astrocytes of wild-type mice and of mice deficient in sodium/bicarbonate cotransporter NBCe1 (NBCe1-KO) or in carbonic anhydrase isoform II (CAII-KO). The steady-state buffer strength was calculated from the amplitude of [H(+)]i transients as evoked by CO2/HCO3(-) and by butyric acid in the presence and absence of CO2/HCO3(-). We tested the hypotheses if, in addition to instantaneous physicochemical H(+) buffering, rapid acid/base transport across the cell membrane contributes to the total, "effective" cytosolic H(+) buffering. In the presence of 5% CO2/26 mM HCO3(-), H(+) buffer strength in astrocytes was increased 4-6 fold, as compared with that in non-bicarbonate, HEPES-buffered solution, which was largely attributable to fast HCO3 (-) transport into the cells via NBCe1, supported by CAII activity. Our results show that within the time frame of determining physiological H(+) buffering in cells, fast transport and equilibration of CO2/H(+)/HCO3(-) can make a major contribution to the total "effective" H(+) buffer strength. Thus, "effective" cellular H(+) buffering is, to a large extent, attributable to membrane transport of base equivalents rather than a purely passive physicochemical process, and can be much larger than reported so far. Not only physicochemical H(+) buffering, but also rapid import of HCO3(-) via the electrogenic sodium-bicarbonate cotransporter NBCe1, supported by carbonic anhydrase II (CA II), was identified to enhance cytosolic H(+) buffer strength substantially. © 2015 Wiley Periodicals, Inc.

  16. Unique hepatic cytosolic arginase evolved independently in ureogenic freshwater air-breathing teleost, Heteropneustes fossilis.

    Directory of Open Access Journals (Sweden)

    Shilpee Srivastava

    Full Text Available Hepatic cytosolic arginase (ARG I, an enzyme of the urea cycle operating in the liver of ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and N(ω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150-200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of ureotelic vertebrates.

  17. Mediator-assisted Simultaneous probing of Cytosolic and Mitochondrial Redox activity in living cells

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Spegel, Christer; Kostesha, Natalie

    2009-01-01

    This work describes an electron transfer mediator-assisted amperometric flow injection method for assessing redox enzyme activity in different subcellular compartments of the phosphoglucose isomerase deletion mutant strain of Saccharomyces cerevisiae, EBY44. The method is demonstrated using...... either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pen-rose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric...

  18. Quantitative analysis of endocytosis and turnover of epidermal growth factor (EGF) and EGF receptor.

    Science.gov (United States)

    Sorkin, Alexander; Duex, Jason E

    2010-03-01

    Binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) initiates signal transduction, ultimately leading to altered gene expression. Ligand-activated EGFR is also rapidly internalized and then targeted to lysosomes for degradation or recycled back to the plasma membrane. Endocytosis is a major regulator of EGFR signaling. Therefore, elucidation of the mechanisms of EGFR endocytosis is essential for a better understanding of EGFR biology. In order to achieve a comprehensive analysis of these mechanisms, reliable methods for measuring the rates of EGFR protein turnover and the rate parameters for individual steps of EGFR endocytic trafficking must be employed. The protocols in this unit describe methodologies to measure the rates of EGFR synthesis and degradation, to monitor EGF-induced down-regulation of surface EGFR, to measure the kinetic rate parameters of internalization, recycling, and degradation of radiolabeled EGF, and to perform radioiodination of EGF by the chloramine T method.

  19. Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

    Science.gov (United States)

    Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

    2011-10-01

    Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Delayed Hepatic Adaptation to Weaning in ACBP(-/-) Mice Is Caused by Disruption of the Epidermal Barrier

    DEFF Research Database (Denmark)

    Neess, Ditte; Bek, Signe; Bloksgaard, Maria

    2013-01-01

    in the skin rather than in the liver. Similarly to ACBP(-/-) mice, K14-ACBP(-/-) mice exhibit an increased transepidermal water loss, and we show that the hepatic phenotype is caused specifically by the epidermal barrier defect, which leads to increased lipolysis in white adipose tissue. Our data demonstrate......We previously reported that mice deficient in acyl-CoA-binding protein (ACBP) display a delayed metabolic adaptation to weaning. This includes a delayed activation of the hepatic lipogenic gene program, which may result from hepatic accumulation of triacylglycerol and/or cholesteryl esters...... in the late suckling period. To further investigate the basis for this phenotype, we generated mice deficient in ACBP in hepatocytes (Alb-ACBP(-/-)) and keratinocytes (K14-ACBP(-/-)). Surprisingly, the delayed adaptation to weaning, including hepatic lipid accumulation, is caused by ACBP deficiency...

  1. Skin Pigmentation and Pigmentary Disorders: Focus on Epidermal/Dermal Cross-Talk.

    Science.gov (United States)

    Bastonini, Emanuela; Kovacs, Daniela; Picardo, Mauro

    2016-06-01

    Variation in human skin and hair color is the most notable aspect of human variability and several studies in evolution, genetics and developmental biology contributed to explain the mechanisms underlying human skin pigmentation, which is responsible for differences in skin color across the world's populations. Despite skin pigmentation is primarily related to melanocytes functionality, the surrounding keratinocytes and extracellular matrix proteins and fibroblasts in the underlying dermal compartment actively contribute to cutaneous homeostasis. Many autocrine/paracrine secreted factors and cell adhesion mechanisms involving both epidermal and dermal constituents determine constitutive skin pigmentation and, whenever deregulated, the occurrence of pigmentary disorders. In particular, an increased expression of such mediators and their specific receptors frequently lead to hyperpigmentary conditions, such as in melasma and in solar lentigo, whereas a defect in their expression/release is related to hypopigmented disorders, as seen in vitiligo. All these interactions underline the relevant role of pigmentation on human evolution and biology.

  2. Epidermal transformation leads to increased perlecan synthesis with heparin-binding-growth-factor affinity

    DEFF Research Database (Denmark)

    Tapanadechopone, P; Tumova, S; Jiang, X

    2001-01-01

    cells was significantly increased in both mRNA and protein levels. JB6 perlecan was exclusively substituted with heparan sulphate, whereas that of RT101 contained some additional chondroitin sulphate. Detailed structural analysis of the heparan sulphate (HS) chains from perlecan of both cell types...... revealed that their overall sulphation and chain length were similar (approximately 60 kDa), but the HS chains of tumour-cell-derived perlecan were less sulphated. This resulted from reduced 2-O- and 6-O-sulphation, but not N-sulphation, and an increase in the proportion of unsulphated disaccharides....... Despite this, the heparan sulphate of RT101- and JB6-derived perlecan bound fibroblast growth factor-1, -2, -4 and -7 and heparin-binding epidermal growth factor with similar affinity. Therefore abundant tumour-derived perlecan may support the angiogenic responses seen in vivo and be a key player...

  3. [Effects of transfection of human epidermal growth factor gene with adenovirus vector on biological characteristics of human epidermal cells].

    Science.gov (United States)

    Yin, Kai; Ma, Li; Shen, Chuan'an; Shang, Yuru; Li, Dawei; Li, Longzhu; Zhao, Dongxu; Cheng, Wenfeng

    2016-05-01

    To investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs. hECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n=3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n=6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n=6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50). CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture

  4. Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Bosche, Bert, E-mail: bert.bosche@uk-essen.de [Department of Neurology, University of Duisburg-Essen (Germany); Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne (Germany); Schäfer, Matthias, E-mail: matthias.schaefer@sanofi.com [Institute of Physiology, Justus-Liebig-University Giessen (Germany); Graf, Rudolf, E-mail: rudolf.graf@nf.mpg.de [Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne (Germany); Härtel, Frauke V., E-mail: frauke.haertel@tu-dresden.de [Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden (Germany); Schäfer, Ute, E-mail: ute.schaefer@medunigraz.at [Research Unit for Experimental Neurotraumatology, Medical University of Graz (Austria); Noll, Thomas, E-mail: thomas.noll@tu-dresden.de [Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden (Germany)

    2013-05-03

    Highlights: •We investigate free calcium as a central signalling element in endothelial cells. •Inhibition of glycolysis with 2-deoxy-D-glucose reduces cellular ATP. •This manoeuvre leads to a biphasic increase and overload of free calcium. •Pre-treatment with lithium for 24 h abolishes both phases of the calcium increase. •This provides a new strategy to protect endothelial calcium homeostasis and barrier function. -- Abstract: Cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca{sup 2+}]{sub i} overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca{sup 2+}]{sub i} overload can be prevented by lithium treatment. [Ca{sup 2+}]{sub i} and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-D-glucose (5 mM; 2-DG) plus sodium cyanide (5 mM; NaCN) caused a significant decrease in cellular ATP content (14 ± 1 nmol/mg protein vs. 18 ± 1 nmol/mg protein in the control, n = 6 culture dishes, P < 0.05), an increase in [Ca{sup 2+}]{sub i} (278 ± 24 nM vs. 71 ± 2 nM in the control, n = 60 cells, P < 0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10 mM 2-DG led to a similar decrease in ATP content (14 ± 2 nmol/mg vs. 18 ± 1 nmol/mg in the control, P < 0.05) with a delay of 5 min. The [Ca{sup 2+}]{sub i} response of EC was biphasic with a peak after 1 min (183 ± 6 nM vs. 71 ± 1 nM, n = 60 cells, P < 0.05) followed by a sustained increase in [Ca{sup 2+}]{sub i}. A 24-h pre-treatment with 10 mM of lithium

  5. "Cut-and-Paste" Manufacture of Multiparametric Epidermal Sensor Systems.

    Science.gov (United States)

    Yang, Shixuan; Chen, Ying-Chen; Nicolini, Luke; Pasupathy, Praveenkumar; Sacks, Jacob; Su, Becky; Yang, Russell; Sanchez, Daniel; Chang, Yao-Feng; Wang, Pulin; Schnyer, David; Neikirk, Dean; Lu, Nanshu

    2015-11-04

    Multifunctional epidermal sensor systems (ESS) are manufactured with a highly cost and time effective, benchtop, and large-area "cut-and-paste" method. The ESS made out of thin and stretchable metal and conductive polymer ribbons can be noninvasively laminated onto the skin surface to sense electrophysiological signals, skin temperature, skin hydration, and respiratory rate. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Epidermal electronics with advanced capabilities in near-field communication.

    Science.gov (United States)

    Kim, Jeonghyun; Banks, Anthony; Cheng, Huanyu; Xie, Zhaoqian; Xu, Sheng; Jang, Kyung-In; Lee, Jung Woo; Liu, Zhuangjian; Gutruf, Philipp; Huang, Xian; Wei, Pinghung; Liu, Fei; Li, Kan; Dalal, Mitul; Ghaffari, Roozbeh; Feng, Xue; Huang, Yonggang; Gupta, Sanjay; Paik, Ungyu; Rogers, John A

    2015-02-25

    Epidermal electronics with advanced capabilities in near field communications (NFC) are presented. The systems include stretchable coils and thinned NFC chips on thin, low modulus stretchable adhesives, to allow seamless, conformal contact with the skin and simultaneous capabilities for wireless interfaces to any standard, NFC-enabled smartphone, even under extreme deformation and after/during normal daily activities. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. GH administration patterns differently regulate epidermal growth factor signaling

    OpenAIRE

    Díaz, Maria E.; Miquet, Johanna Gabriela; Rossi, Soledad Paola; Irene, Pablo E.; Sotelo, Ana Isabel; Frungieri, Monica Beatriz; Turyn, Daniel; Gonzalez, Lorena

    2016-01-01

    Current GH administration protocols imply frequent s.c. injections, resulting in suboptimal compliance. Therefore, there is interest in developing delivery systems for sustained release of the hormone. However, GH has different actions depending on its continuous or pulsatile plasma concentration pattern. GH levels and circulating concentration patterns could be involved in the regulation of epidermal growth factor receptor (EGFR) expression in liver. Aberrant expression of this receptor and/...

  8. Does hypernatremia impact mortality in Toxic Epidermal Necrolysis?

    OpenAIRE

    Mailänder, Peter; Stang, Felix H.; Stollwerck, Peter L.; Siemers, Frank; von Wild, Tobias; Namdar, Thomas; Lange, Thomas

    2010-01-01

    Introduction: In-hospital hypernatremia is associated with increased mortality rates. We want to elucidate the impact of in-hospital acquired hypernatremia in mortality of Toxic Epidermal Necrolysis (TEN).Purpose: Is there an association between hypernatremia and mortality in patients with TEN?Method: Retrospective study of 25 patients with TEN. Laboratory electrolyte results, diuresis and survival were analyzed. Patients were separated in two groups without (Group A) or with (Group B) hyp...

  9. Pseudotumoral appearance of a ruptured epidermal cyst in the foot

    Energy Technology Data Exchange (ETDEWEB)

    Harish, S.; Jan, E.; Finlay, K. [Department of Diagnostic Imaging, Henderson General Hospital, Hamilton, ON (Canada); Ghert, M. [Henderson General Hospital, Hamilton, ON (Canada). Dept. of Orthopaedic Surgery; Alowami, S. [Henderson General Hospital, Hamilton, ON (Canada). Dept. of Pathology

    2006-11-15

    We present a case of keratin granuloma due to a ruptured epidermal cyst, occurring in the foot, in a 52-year-old woman. The patient presented with a history of a slow-growing lump in the web space of the foot that had been present for over a year. Imaging appearances suggested a soft tissue neoplasm. Clinical presentation, radiological features and histopathological findings are described, and the relevant literature is reviewed.

  10. Aetiology and treatment of epidermal depigmentory disorder in humans

    OpenAIRE

    S. S. Sawhney

    2012-01-01

    The epidermal depigmentary trigger in humans at post-natal level may occur with the toxification of skin organ with the endogenously produced melanocytotoxic hydrogen peroxide and subsequent formation of hydrogen peroxide- melanolipoprotein conjugate involving the hydrogen bonding of complementary hydroxyl and carbonyl molecular surfaces of these biosignitures respectively. The condition is multifactorial but reversible. The structural and functional degeneration of melanocytes under the acqu...

  11. Phospholipase C-epsilon regulates epidermal morphogenesis in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Rafael P Vázquez-Manrique

    2008-03-01

    Full Text Available Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP(3-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP(3 production is stimulated is unknown. IP(3 is produced by the action of phospholipase C (PLC. We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-epsilon produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753 embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP(3 receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-gamma and EGL-8/PLC-beta can compensate for reduced PLC-1 activity. Our work places PLC-epsilon into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-epsilon.

  12. A review of toxic epidermal necrolysis management in Japan

    Directory of Open Access Journals (Sweden)

    Yuri Kinoshita

    2017-01-01

    Full Text Available Toxic epidermal necrolysis (TEN is a severe adverse drug reaction characterized by necrosis of the epidermis. Its incidence is approximately 1 per million a year and average mortality rate is high at 25–50%. TEN has a flu-like prodrome, followed by atypical, targetoid erythematous or purpuric macules on the skin. These macules coalesce to form flaccid blisters that slough off as areas of epidermal necrosis. Drugs such as allopurinol, sulfonamides, and carbamazepine are the most common causes. The human leukocyte antigen (HLA-B*15:02 in Asians being administered carbamazepine and the HLA-B*58:01 antigen in patients of all ethnicities being administered allopurinol are known to be high-risk factors. Rapid diagnosis, discontinuation of the causative drug, and supportive treatment are essential for better prognosis and improvement of sequelae. Till now, systemic corticosteroids and intravenous immunoglobulins have been used as the most common active interventions; however, no gold standard has been established. In Japan, physicians follow a unique diagnostic criteria and treatment guideline to improve the diagnosis rate and streamline treatments. This may be a contributing factor for the lower mortality rate (14.3%. The efficacy of systemic corticosteroids, immunoglobulins, and plasmapheresis may have been beneficial as well. In Japan, TEN is defined as an epidermal detachment of over 10% of the body surface area (BSA, while the globally accepted definition established by Bastuji-Garin describes it as an epidermal detachment of over 30% of the BSA. In Japanese individuals, HLA-A*02:06, HLA-A*02:07, HLA-A*31:01 and HLA-B*51:01 may be linked to higher risks of TEN.

  13. Sulfation of 6-Gingerol by the Human Cytosolic Sulfotransferases: A Systematic Analysis.

    Science.gov (United States)

    Luo, Lijun; Mei, Xue; Xi, Yuecheng; Zhou, Chunyang; Hui, Ying; Kurogi, Katsuhisa; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh

    2016-02-01

    Previous studies have demonstrated the presence of the sulfated form of 6-gingerol, a major pharmacologically active component of ginger, in plasma samples of normal human subjects who were administered 6-gingerol. The current study was designed to systematically identify the major human cytosolic sulfotransferase enzyme(s) capable of mediating the sulfation of 6-gingerol. Of the 13 known human cytosolic sulfotransferases examined, six (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C4, SULT1E1) displayed significant sulfating activity toward 6-gingerol. Kinetic parameters of SULT1A1, SULT1A3, SULT1C4, and SULT1E1 that showed stronger 6-gingerol-sulfating activity were determined. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-gingerol-sulfating activity than those of the lung and kidney. Moreover, sulfation of 6-gingerol was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under the metabolic setting. Collectively, these results provided useful information relevant to the metabolism of 6-gingerol through sulfation both in vitro and in vivo. Georg Thieme Verlag KG Stuttgart · New York.

  14. Metformin Restores Parkin-Mediated Mitophagy, Suppressed by Cytosolic p53

    Directory of Open Access Journals (Sweden)

    Young Mi Song

    2016-01-01

    Full Text Available Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP-kinase-independent, sirtuin 1 (SIRT1-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1 ad libitum feeding of a standard chow diet; (2 intraperitoneal injections of metformin 300 mg/kg; and (3 3 g/day caloric restriction (CR. HepG2 cells were treated with palmitate (PA plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins.

  15. Effect of Cyclosporin A and Angiotensin II on cytosolic calcium levels in primary human gingival fibroblasts

    Directory of Open Access Journals (Sweden)

    Ajitkumar Supraja

    2016-01-01

    Full Text Available Background: To evaluate the effect of Cyclosporin A (CsA and angiotensin II (Ang II on cytosolic calcium levels in cultured human gingival fibroblasts (HGFs. Materials and Methods: Healthy gingival samples from six volunteers were obtained, and primary HGFs were cultured. Cell viability and proliferation assay were performed to identify the ideal concentrations of CsA and Ang II. Cytosolic calcium levels in cultured gingival fibroblasts treated with CsA and Ang II were studied using colorimetric assay, confocal and fluorescence imaging. Statistical analyses were done using SPSS software and GraphPad Prism. Results: Higher levels of cytosolic levels were evident in cells treated with CsA and Ang II when compared to control group and was statistically significant (P < 0.05 in both colorimetric assay and confocal imaging. Fluorescent images of the cultured HGFs revealed the same. Conclusion: Thus calcium being a key player in major cellular functions, plays a major role in the pathogenesis of drug-induced gingival overgrowth.

  16. Cytosolic acidification as a signal mediating hyperosmotic stress responses in Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Klein Gérard

    2001-06-01

    Full Text Available Abstract Background Dictyostelium cells exhibit an unusual response to hyperosmolarity that is distinct from the response in other organisms investigated: instead of accumulating compatible osmolytes as it has been described for a wide range of organisms, Dictyostelium cells rearrange their cytoskeleton and thereby build up a rigid network which is believed to constitute the major osmoprotective mechanism in this organism. To gain more insight into the osmoregulation of this amoeba, we investigated physiological processes affected under hyperosmotic conditions in Dictyostelium. Results We determined pH changes in response to hyperosmotic stress using FACS or 31P-NMR. Hyperosmolarity was found to acidify the cytosol from pH 7.5 to 6.8 within 5 minutes, whereas the pH of the endo-lysosomal compartment remained constant. Fluid-phase endocytosis was identified as a possible target of cytosolic acidification, as the inhibition of endocytosis observed under hypertonic conditions can be fully attributed to cytosolic acidification. In addition, a deceleration of vesicle mobility and a decrease in the NTP pool was observed. Conclusion Together, these results indicate that hyperosmotic stress triggers pleiotropic effects, which are partially mediated by a pH signal and which all contribute to the downregulation of cellular activity. The comparison of our results with the effect of hyperosmolarity and intracellular acidification on receptor-mediated endocytosis in mammalian cells reveals striking similarities, suggesting the hypothesis of the same mechanism of inhibition by low internal pH.

  17. Tensegrity behaviour of cortical and cytosolic cytoskeletal components in twisted living adherent cells.

    Science.gov (United States)

    Laurent, Valérie M; Cañadas, Patrick; Fodil, Redouane; Planus, Emmanuelle; Asnacios, Atef; Wendling, Sylvie; Isabey, Daniel

    2002-01-01

    The present study is an attempt to relate the multicomponent response of the cytoskeleton (CSK), evaluated in twisted living adherent cells, to the heterogeneity of the cytoskeletal structure--evaluated both experimentally by means of 3D reconstructions, and theoretically considering the predictions given by two tensegrity models composed of (four and six) compressive elements and (respectively 12 and 24) tensile elements. Using magnetic twisting cytometry in which beads are attached to integrin receptors linked to the actin CSK of living adherent epithelial cells, we specifically measured the elastic CSK response at quasi equilibrium state and partitioned this response in terms of cortical and cytosolic contributions with a two-component model (i.e., a series of two Voigt bodies). These two CSK components were found to be prestressed and exhibited a stress-hardening response which both characterize tensegrity behaviour with however significant differences: compared to the cytosolic component, the cortical cytoskeleton appears to be a faster responding component, being a less prestressed and easily deformable structure. The discrepancies in elastic behaviour between the cortical and cytosolic CSK components may be understood on the basis of prestress tensegrity model predictions, given that the length and number of constitutive actin elements are taken into account.

  18. Structural Studies on Cytosolic Domain of Magnesium Transporter MgtE from Enterococcus faecalis

    Energy Technology Data Exchange (ETDEWEB)

    Ragumani, S.; Sauder, J; Burley, S; Swaminathan, S

    2009-01-01

    Magnesium (Mg{sup 2+}) is an essential element for growth and maintenance of living cells. It acts as a cofactor for many enzymes and is also essential for stability of the plasma membrane. There are two distinct classes of magnesium transporters identified in bacteria that convey Mg{sup 2+} from periplasm to cytoplasm [ATPase-dependent (MgtA and MgtB) and constitutively active (CorA and MgtE)]. Previously published work on Mg{sup 2+} transporters yielded structures of full length MgtE from Thermus thermophilus, determined at 3.5 {angstrom} resolution, and its cytoplasmic domain with and without bond Mg{sup 2+} determined at 2.3 and 3.9 {angstrom} resolution, respectively. Here, they report the crystal structure of the Mg{sup 2+} bound form of the cytosolic portion of MgtE (residues 6-262) from Enterococcus faecalis at 2.2 {angstrom} resolution. The present structure and magnesium bound cytosolic domain structure from T. thermophilus (PDB ID: 2YVY) are structurally similar. Three magnesium binding sites are common to both MgtE full length and the present structure. Their work revealed an additional Mg{sup 2+} binding site in the E. faecalis structure. In this report, they discuss the functional significance of Mg{sup 2+} binding sites in the cytosolic domains of MgtE transporters.

  19. Using human epithelial amnion cells in human de-epidermized dermis for skin regeneration.

    Science.gov (United States)

    Jiang, Lei-Wei; Chen, Hongduo; Lu, Hongguang

    2016-01-01

    staining strip located at the epidermal-dermal junction. BM component proteins (type IV collagen and laminin) and cell adhesion protein (desmoglein) were detected by immunohistochemistry in organotypic skin. Ultrastructurally, desmosomes, hemidesmosomes and BM zone (BMZ) were observed in organotypic skin. Our studies indicate that the hAECs is a promising stem cell source for tissue-engineered skin, and DED with hAECs is a potential application prospects in regenerative medicine. Copyright © 2015. Published by Elsevier Ireland Ltd.

  20. Cytosolic phospholipase A2 alpha/arachidonic acid signaling mediates depolarization-induced suppression of excitation in the cerebellum.

    Directory of Open Access Journals (Sweden)

    De-Juan Wang

    Full Text Available Depolarization-induced suppression of excitation (DSE at parallel fiber-Purkinje cell synapse is an endocannabinoid-mediated short-term retrograde plasticity. Intracellular Ca(2+ elevation is critical for the endocannabinoid production and DSE. Nevertheless, how elevated Ca(2+ leads to DSE is unclear.We utilized cytosolic phospholipase A(2 alpha (cPLA(2α knock-out mice and whole-cell patch clamp in cerebellar slices to observed the action of cPLA(2α/arachidonic acid signaling on DSE at parallel fiber-Purkinje cell synapse. Our data showed that DSE was significantly inhibited in cPLA(2α knock-out mice, which was rescued by arachidonic acid. The degradation enzyme of 2-arachidonoylglycerol (2-AG, monoacylglycerol lipase (MAGL, blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine (AEA, fatty acid amide hydrolase (FAAH, did not affect DSE. These results suggested that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+, indicating that large conductance Ca(2+-activated potassium channel (BK is sufficient to inhibit cPLA(2α/arachidonic acid-mediated DSE. In addition, we showed that the release of 2-AG was independent of soluble NSF attachment protein receptor (SNARE, protein kinase C and protein kinase A.Our data first showed that cPLA(2α/arachidonic acid/2-AG signaling pathway mediates DSE at parallel fiber-Purkinje cell synapse.

  1. NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi.

    Science.gov (United States)

    Nývltová, Eva; Šuták, Robert; Harant, Karel; Šedinová, Miroslava; Hrdy, Ivan; Paces, Jan; Vlček, Čestmír; Tachezy, Jan

    2013-04-30

    In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an ε-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.

  2. Epidermal growth factor receptor expression in urinary bladder cancer

    Directory of Open Access Journals (Sweden)

    Dayalu S.L. Naik

    2011-01-01

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

  3. Epidermal growth in the bottlenose dolphin, Tursiops truncatus

    Energy Technology Data Exchange (ETDEWEB)

    Hicks, B.D.; St. Aubin, D.J.; Geraci, J.R.; Brown, W.R.

    1985-07-01

    Epidermal growth in two mature female bottlenose dolphins, Tursiops truncatus, was investigated by following the movement of a cohort of tritiated thymidine-labeled epidermal cells for 59 days. The majority of the cells migrated in a cluster which was estimated to reach the skin surface in 73 days. The authors calculate that the outermost cell layer is sloughed 12 times per day. Turnover time and sloughing rate are estimated to be 1.7 times longer and 8.5 times faster than the respective values for epidermal cell kinetics in humans. This apparent inconsistency of slow transit time and rapid sloughing rate is reconciled by the convoluted structure of the stratum germinativum in the dolphin which results in a ratio of germinatival to superficial cells of 876:1. The stratum germinativum of dolphin epidermis appears to lack morphologically distinct, spatially segregated subpopulations of anchoring and stem cells. Dolphin epidermis has a large capacity for cell population, relatively long turnover time, and rapid sloughing rate. The adaptive advantages of these characteristics are discussed.

  4. Steven-Johnson Syndrome (SJS and Toxic Epidermal Necrolysis

    Directory of Open Access Journals (Sweden)

    Erkan Alpsoy

    2010-12-01

    Full Text Available Stevens-Johnson syndrome (SJS and toxic epidermal necrolysis (TEN are rare, life-threatening conditions with a high mortality rate. SJS and TEN are used to denote a group of disorders closely related to each other, characterized by extensive epidermal necrolysis, and usually induced by drugs. Keratinocyte apoptosis is the main reason for widespread epidermal detachment. Drugs or their methobolites can act as a hapten after binding to the keratinocyte surface and initiate cytotoxic immunological attack. Drug-specific CD8+cytotoxic T cells mediate keratinocyte apoptosis by the Fas/FasL pathway and perforin/granzyme pathway. Although numerous drugs have been noted as responsible, sulfonamide class of antibiotics, anticonvulsants, beta-lactam antibiotics, allopurinol, nonsteroidal anti-inflammatory drugs, nevirapine and thiacetazone are the most frequently reported causative ones. Early diagnosis and withdrawal of suspected drug or drugs are one of the most important steps in the treatment. Other diseases resembling SJS/TEN should be excluded as soon as possible. Although various topical and systemic treatments have recently been used, ideal supportive care is still the most important and effective therapeutic approach. SCORTEN, a scoring system used to predict mortality in TEN, has been widely used in recent years. Transfer to a burn unit or intensive care unit is recommended for patients with a SCORTEN 3 (mortality rate; 35.3% or over. In this paper, we aimed to review clinical findings, aetiopathogenesis and treatment of these syndromes in the light of current literature.

  5. Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mulholland, William J [PowderJect Centre for Gene and Drug delivery Research, Department of Engineering Science, University of Oxford OX2 6PE (United Kingdom); Kendall, Mark A F [PowderJect Centre for Gene and Drug delivery Research, Department of Engineering Science, University of Oxford OX2 6PE (United Kingdom); White, Nick [Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE (United Kingdom); Bellhouse, Brian J [PowderJect Centre for Gene and Drug delivery Research, Department of Engineering Science, University of Oxford OX2 6PE (United Kingdom)

    2004-11-21

    Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery.

  6. Stevens-Johnson syndrome and toxic epidermal necrolysis: a review

    Directory of Open Access Journals (Sweden)

    Anthony Wong

    Full Text Available SUMMARY Stevens-Johnson syndrome (SJS and toxic epidermal necrolysis (TEN are uncommon, acute and potentially life-threatening adverse cutaneous drug reactions. These pathologies are considered a hypersensitivity reaction and can be triggered by drugs, infections and malignancies. The drugs most often involved are allopurinol, some antibiotics, including sulfonamides, anticonvulsants such as carbamazepine, and some non-steroid anti-inflammatory drugs (NSAIDs. Necrosis of keratinocytes is manifested clinically by epidermal detachment, leading to scalded skin appearance. The rash begins on the trunk with subsequent generalization, usually sparing the palmoplantar areas. Macular lesions become purplish, and epidermal detachment occurs, resulting in flaccid blisters that converge and break, resulting in extensive sloughing of necrotic skin. Nikolsky's sign is positive in perilesional skin. SJS and TEN are considered to be two ends of the spectrum of one disease, differing only by their extent of skin detachment. Management of patients with SJS or TEN requires three measures: removal of the offending drug, particularly drugs known to be high-risk; supportive measures and active interventions. Early diagnosis of the disease, recognition of the causal agent and the immediate withdrawal of the drug are the most important actions, as the course of the disease is often rapid and fatal.

  7. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

    Directory of Open Access Journals (Sweden)

    Bronwyn Jane Barkla

    2015-06-01

    Full Text Available One of the remarkable adaptive features of the halophyte and facultative CAM plant Mesembryathemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples was used to identify 352 significantly differing metabolites (268 after correction for FDR. Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na and Cl ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggest large alterations in Mesembryanthemum crystallinum epidermal bladder cells.

  8. Effects of radiation on the epidermal growth factor receptor pathway in the heart.

    Science.gov (United States)

    Sridharan, Vijayalakshmi; Sharma, Sunil K; Moros, Eduardo G; Corry, Peter M; Tripathi, Preeti; Lieblong, Benjamin J; Guha, Chandan; Hauer-Jensen, Martin; Boerma, Marjan

    2013-07-01

    Radiation-induced heart disease (RIHD) is a serious side-effect of thoracic radiotherapy. The epidermal growth factor receptor (EGFR) pathway is essential for the function and survival of cardiomyocytes. Hence, agents that target the EGFR pathway are cardiotoxic. Tocotrienols protect from radiation injury, but may also enhance the therapeutic effects of EGFR pathway inhibitors in cancer treatment. This study investigated the effects of local irradiation on the EGFR pathway in the heart and tests whether tocotrienols may modify radiation-induced changes in this pathway. Male Sprague-Dawley rats received image-guided localized heart irradiation with 21 Gy. Twenty four hours before irradiation, rats received a single dose of tocotrienol-enriched formulation or vehicle by oral gavage. At time points from 2 h to 9 months after irradiation, left ventricular expression of EGFR pathway mediators was studied. Irradiation caused a decrease in the expression of epidermal growth factor (EGF) and neuregulin-1 (Nrg-1) mRNA from 6 h up to 10 weeks, followed by an upregulation of these ligands and the receptor erythroblastic leukemia viral oncogene homolog (ErbB)4 at 6 months. In addition, the upregulation of Nrg-1 was statistically significant up to 9 months after irradiation. A long-term upregulation of ErbB2 protein did not coincide with changes in transcription or post-translational interaction with the chaperone heat shock protein 90 (HSP90). Pretreatment with tocotrienols prevented radiation-induced changes at 2 weeks. Local heart irradiation causes long-term changes in the EGFR pathway. Studies have to address how radiation may interact with cardiotoxic effects of EGFR inhibitors.

  9. Asian dust storm particles induce a broad toxicological transcriptional program in human epidermal keratinocytes.

    Science.gov (United States)

    Choi, Hyun; Shin, Dong Wook; Kim, Wonnyon; Doh, Seong-Jae; Lee, Soo Hwan; Noh, Minsoo

    2011-01-15

    Exposure to airborne dust particles originated from seasonal Asian dust storms in Chinese and Mongolian deserts results in increased incidence of a range of diseases including asthma, contact dermatitis and conjunctivitis. The areas affected by Asian dust particles extend from East China to the west coast of North America. In order to study toxicological mechanisms in human skin, we evaluated the effects of dust particles collected during Asian dust storms (Asian dust particles) on gene expression in human epidermal keratinocytes (HEK). In HEK, exposure to Asian dust particles significantly increased gene expressions of cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP1B1, which is an indication of aryl hydrocarbon receptor (AHR) activation. In addition, Asian dust particles increased gene transcription of the cytokines IL-6, IL-8, and GM-CSF, which have broad pro-inflammatory and immunomodulatory properties. Asian dust particles significantly up-regulated expression of caspase 14 in HEK, suggesting that Asian dust particles directly affect keratinocyte differentiation. We also demonstrated that protein extract of pollen, a material frequently adsorbed onto Asian dust particles, potentially contributes to the increased transcription of IL-6, CYP1A1, CYP1A2, and CYP1B1. Taken together, these studies suggest that Asian dust particles can exert toxicological effects on human skin through the activation of the cellular detoxification system, the production of pro-inflammatory and immunomodulatory cytokines, and changes in the expression of proteins essential in normal epidermal differentiation. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  10. Keratins K2 and K10 are essential for the epidermal integrity of plantar skin.

    Science.gov (United States)

    Fischer, Heinz; Langbein, Lutz; Reichelt, Julia; Buchberger, Maria; Tschachler, Erwin; Eckhart, Leopold

    2016-01-01

    K1 and K2 are the main type II keratins in the suprabasal epidermis where each of them heterodimerizes with the type I keratin K10 to form intermediate filaments. In regions of the ears, tail, and soles of the mouse, only K2 is co-expressed with K10, suggesting that these keratins suffice to form a mechanically resilient cytoskeleton. To determine the effects of the suppression of both main keratins, K2 and K10, in the suprabasal plantar epidermis of the mouse. Krt2(-/-) Krt10(-/-) mice were generated by crossing Krt2(-/-) and Krt10(-/-) mice. Epidermal morphology of soles of hind-paws was examined macroscopically and histologically. Immunofluorescence analysis and quantitative PCR analysis were performed to analyze the expression of keratins in sole skin of wildtype and Krt2(-/-) Krt10(-/-) mice. Highly abundant proteins of the sole stratum corneum were determined by electrophoretic and chromatographic separation and subsequent mass spectrometry. K2 and K10 are the most prominent suprabasal keratins in normal mouse soles with the exception of the footpads where K1, K9 and K10 predominate. Mice lacking both K2 and K10 were viable and developed epidermal acanthosis and hyperkeratosis in inter-footpad epidermis of the soles. The expression of keratins K1, K9 and K16 was massively increased at the RNA and protein levels in the soles of Krt2(-/-) Krt10(-/-) mice. This study demonstrates that the loss of the main cytoskeletal components of plantar epidermis, i.e. K2 and K10, can be only partly compensated by the upregulation of other keratins. The thickening of the epidermis in the soles of Krt2(-/-) Krt10(-/-) mice may serve as a model for pathomechanistic aspects of palmoplantar keratoderma. Copyright © 2015. Published by Elsevier Ireland Ltd.

  11. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario

    2015-01-01

    One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na(+) and Cl(-) ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells.

  12. Identification of c-di-AMP-Binding Proteins Using Magnetic Beads.

    Science.gov (United States)

    Kampf, Jan; Gundlach, Jan; Herzberg, Christina; Treffon, Katrin; Stülke, Jörg

    2017-01-01

    To identify cytosolic proteins that bind to cyclic di-AMP, a biotinylated analog of the nucleotide is used for protein pull-down experiments. In this approach, biotinylated c-di-AMP is coupled to Streptactin-covered beads. After protein separation using standard SDS-PAGE, the protein(s) of interest are identified by mass spectrometric analyses.

  13. Overexpression of transforming growth factor-α and epidermal growth factor receptor, but not epidermal growth factor, in exocrine pancreatic tumours in hamsters

    NARCIS (Netherlands)

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

    1996-01-01

    Using immunohistochemistry, Northern blotting and a semi-quantitative PCR technique, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and epidermal growth factor receptor (EGFR) expression were studied in the pancreas of N-nitrosobis(2-oxopropyl)-amine (BOP)-treated hamsters.

  14. Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance

    Directory of Open Access Journals (Sweden)

    Baksh Shairaz

    2007-02-01

    Full Text Available Abstract Background The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC is associated with frequent treatment failures (~90% in metastatic cases, and these tumors frequently contain PTEN abnormalities. Results Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. Conclusion Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.

  15. Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance

    Science.gov (United States)

    Lin, Pei-Yin; Fosmire, Susan P; Park, See-Hyoung; Park, Jin-Young; Baksh, Shairaz; Modiano, Jaime F; Weiss, Robert H

    2007-01-01

    Background The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (~90% in metastatic cases), and these tumors frequently contain PTEN abnormalities. Results Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis. Conclusion Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories. PMID:17300726

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

    Science.gov (United States)

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

    1996-01-01

    In the present study, the expression of the epidermal growth factor receptor (EGFR) was investigated in putative preneoplastic and neoplastic acinar cell lesions induced in the rat pancreas by azaserine, using Northern blotting, in situ hybridisation (ISH) and immunohistochemistry. EGFR protein levels were decreased in putative preneoplastic eosinophilic acinar cell lesions (atypical acinar cell nodules, AACN) in comparison with normal acinar cells of the pancreas. However, EGFR mRNA expression correlated positively with the volume of AACN in pancreatic homogenates and ISH showed equal or stronger EGFR mRNA expression in AACN than in the surrounding normal acinar cells. Neither EGFR protein nor EGFR mRNA was detected in more advanced lesions such as acinar adenocarcinomas (in situ). Moreover, EGFR protein expression showed an inverse relationship with the mitotic rate of the acinar cells. These findings suggest that down-regulation of EGFR at the protein level may abrogate negative constraints on cell growth, which may stimulate the development of putative preneoplastic AACN to more advanced lesions and, ultimately, acinar adenocarcinomas. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8679465

  17. The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

    Science.gov (United States)

    Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

    2014-12-01

    The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

  18. Novel forms of protein glycosylation

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Casset, F.

    1998-01-01

    A large number of new glycans, derived from glycoproteins, has been characterized in the past few years. O-linked fucose was found in epidermal growth factor-like domains of several proteins. For the N-linked glycans of Helix pomatia hemocyanin, novel types of antennae were identified. The positions

  19. Nucleotide exchange between cytosolic ATP and F-actin-bound ADP may be a major energy-utilizing process in unstimulated platelets.

    Science.gov (United States)

    Daniel, J L; Molish, I R; Robkin, L; Holmsen, H

    1986-05-02

    About 40% of the cytosolic ADP of human platelets is tightly bound to protein and the complex is precipitated from the cells by 43% ethanol. We show here that this ADP is bound to F-actin by three criteria (a) copurification with F-actin, (b) specific extraction with water and (c) by specific interaction with DNase I. Platelets contain 0.3 mumol/10(11) cells of this F-actin--ADP complex compared to the total actin content of 0.8 mumol/10(11) cells, which is consistent with the view that actin is maintained in different pools (F-actin--ADP, profilactin, G-actin). In intact platelets the F-actin-bound ADP turns over rapidly and we have determined a turnover rate at 37 degrees C of 0.1 +/- 0.025 s-1 by using a double-labelling procedure. This rapid turnover indicates that F-actin in intact platelets is in a very dynamic state, which may be necessary for rapid responses to stimuli. If it is assumed that the source of the ADP bound to F-actin is cytosolic ATP, the turnover of F-actin ADP measured represents an ATP-consuming process that would account for up to 50% of total ATP consumption in resting platelets.

  20. Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.

    Directory of Open Access Journals (Sweden)

    Kathleen M Averette

    2009-11-01

    Full Text Available NOD-like receptors (NLRs are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT, is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP. The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.