DOCSLIB.ORG

Antigen Presentation Pathway Adenylate Cyclase Toxin to The

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Antigen Presentation Pathway Adenylate Cyclase Toxin to The Direct Delivery of the Bordetella pertussis Adenylate Cyclase Toxin to the MHC Class I Antigen Presentation Pathway This information is current as Pierre Guermonprez, Daniel Ladant, Gouzel Karimova, of September 27, 2021. Agnes Ullmann and Claude Leclerc J Immunol 1999; 162:1910-1916; ; http://www.jimmunol.org/content/162/4/1910 Downloaded from References This article cites 46 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/162/4/1910.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Direct Delivery of the Bordetella pertussis Adenylate Cyclase Toxin to the MHC Class I Antigen Presentation Pathway1 Pierre Guermonprez,* Daniel Ladant,† Gouzel Karimova,† Agnes Ullmann,† and Claude Leclerc2* Among bacterial toxins, the adenylate cyclase toxin of Bordetella pertussis (CyaA) has a unique mechanism of entry that consists in the direct translocation of its catalytic domain across the plasma membrane of target cell, a mechanism supposed to be independent of any endocytic pathway. Here, we report that the CyaA toxin is delivered to the cytosolic pathway for MHC class I-restricted Ag presentation. Using peritoneal macrophages as APC, we show that the OVA 257–264 CD81 epitope genetically inserted into a detoxified CyaA (CyaA-OVA E5) is presented to CD81 T cells by a mechanism requiring 1) proteasome processing, 2) TAP, and 3) neosynthesis of MHC class I. We demonstrate that the presentation of CyaA-OVA E5, like the translocation of CyaA into eukaryotic cells, is dependent on extracellular Ca21 and independent of vacuolar acidification. Moreover, inhibitors of Downloaded from the phagocytic and macropinocytic endocytic pathways do not affect the CyaA-OVA E5 presentation. The absence of specific cellular receptors for CyaA correlates with the ability of various APC to present the recombinant CyaA toxin, including dendritic cells, macrophages, splenocytes, and lymphoid tumoral lines. Taken together, our results show that the CyaA presentation path- way is not cell type specific and is unrelated to a defined type of endocytic mechanism. Thus, it represents a new and unconven- tional delivery of an exogenous Ag into the conventional cytosolic pathway. The Journal of Immunology, 1999, 162: 1910–1916. http://www.jimmunol.org/ ytotoxic T lymphocytes recognize antigenic peptides peptides on post-Golgi MHC class I molecules and are indepen- from 8 to 10 amino acids derived from endogenous cy- dent of the TAP system (2, 3). Cytosolic pathways constitute a C tosolic Ag complexed with MHC class I molecules at the nonconventional access of exogenous Ag to the cytosol, resulting surface of APCs. After degradation of Ag by the proteasome, the in its introduction into the classic presentation pathway for endog- processed peptides are transported to the lumen of the endoplasmic enous Ag described above. This has been clearly demonstrated for reticulum (ER)3 by the ATP-dependent transporters associated particulate Ag internalized by phagocytosis in a restricted subset of with Ag presentation, TAP1 and TAP2. Then, peptides associate macrophages (5–7) and for soluble Ag internalized by macropino- with nascent MHC class I molecules and b -microglobulin. The 2 cytosis in both activated macrophages (8) and dendritic cells (9, by guest on September 27, 2021 trimeric complexes reach the surface using the conventional se- 10). In both cases, a defined endocytic mechanism (phagocytosis cretory pathway via the Golgi network (reviewed in Ref. 1). As a or macropinocytosis) was involved, and a membrane-disruption consequence, immunization with an exogenous Ag does not in- mechanism was suggested to explain the introduction of exoge- duce, in most cases, a CTL response. nous Ag into the cytosolic pathway. However, it has been well established that in some cases, ex- 1 Several bacterial toxins exert their toxic effect by modifying ogenous Ag can be processed and presented to CD8 T cells (re- cytosolic components. Therefore, they are equipped with a dedi- viewed in Refs. 2–4). Schematically, these pathways have been cated translocating capability, and as such, they constitute attrac- classified as either cytosolic or noncytosolic. Noncytosolic path- tive vectors for the delivery of heterologous CTL epitopes into the ways are related to the endocytic processing and involve loading of endogenous pathway. Some toxins, such as the diphtheria toxin, are internalized after receptor-mediated endocytosis in an acidic *Unite´de Biologie des Re´gulations Immunitaires and †Unite´de Biochimie Cellulaire, compartment where they translocate to the cytosol (11). Other tox- Institut Pasteur, Paris, France ins use a receptor-mediated anterograde transport to the ER via the Received for publication July 29, 1998. Accepted for publication October 29, 1998. Golgi network, where they translocate to the cytosol (reviewed in The costs of publication of this article were defrayed in part by the payment of page Ref. 12). The adenylate cyclase toxin of Bordetella pertussis charges. This article must therefore be hereby marked advertisement in accordance (CyaA) is able to invade numerous mammalian cell types, in with 18 U.S.C. Section 1734 solely to indicate this fact. which, upon activation by the endogenous calmodulin, it catalyzes 1 This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (URA1129), Pasteur-Weizmann, and Association pour la Re- the production of supraphysiological levels of cAMP (13–19). The cherche sur le Cancer (ARC) and Agence Nationale de Recherches sur le SIDA invasivity of CyaA seems to be independent of any known cellular (ANRS) grants to C.L. receptor: it does not involve any acidic compartment and can take 2 Address correspondence and reprint requests to Dr. Claude Leclerc, Unite´de Bi- place in erythrocytes devoid of vesicular transport (20–27). The ologie des Re´gulations Immunitaires, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail address: [email protected] precise mechanism of translocation is not fully understood but 3 Abbreviations used in this paper: ER, endoplasmic reticulum; CyaA, adenylate cy- might occur in two steps. After binding of CyaA to the eukaryotic clase toxin of Bordetella pertussis; CyaA-OVA E5, genetically detoxified CyaA car- cell surface mediated by the C-terminal domain (residues 400- rying the OVA CTL epitope; LCMV, lymphocytic choriomeningitis virus; CyaA- 1706), the N-terminal catalytic domain (residues 1–400) is directly LCMV E5, genetically detoxified CyaA carrying the lymphocytic choriomeningitis virus CTL epitope; BMM, bone marrow-derived macrophage; BMDC, bone marrow- translocated through the plasma membrane into the cell. derived dendritic cell; Thio-PEC, thioglycollate-induced peritoneal exudate cells; We showed previously that a CyaA toxin genetically tagged CM, complete medium; M-CSF, macrophage CSF; GM-CSF, granulocyte-macro- phage CSF; CCB, cytochalasine B; DMA, dimethyl amiloride; BFA, brefeldin A; with a CTL epitope from the nucleoprotein of lymphocytic cho- LLnL, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal; LF, lethal factor. riomeningitis virus (LCMV) into its catalytic domain remained Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 1911 cell invasive (28). Moreover, this toxin was able to generate, in Cultures were realized in bacteriology petri dishes (Becton Dickinson, vivo, a protective CTL response against LCMV (29–30). In this Mountain View, CA). At the end of the culture period, nonadherent cells report, we asked whether the CyaA toxin, taken as an exogenous were removed, and adherent cells were harvested with 2 mM trypsin- EDTA (NEN Life Science, Boston, MA). Ag, can be delivered to the cytosolic pathway for MHC class I BMDC were obtained with a protocol adapted from Inaba et al. (35). presentation without the contribution of any endocytic mecha- Briefly, bone marrow cells were washed twice and cultured for 2 days in nisms. To address this question, we have analyzed, in vitro, the DC medium (CM supplemented with recombinant granulocyte-macro- 6 pathway leading to the presentation of the Kb-restricted CD81 phage CSF (GM-CSF; Peprotech, Rocky Hill, NJ) at 40 ng/ml) at 10 cells/ml in 100-mm culture-treated petri dishes. Then, nonadherent cells OVA epitope from OVA, which was inserted in a genetically de- were removed, and fresh DC medium was added. After a total of 5–6 days toxified form of CyaA (CyaA-OVA E5). We used the stimulation of culture, BMDC were visible as clumps of loosely adherent cells. The 1 of a specific CD8 T cell hybridoma to monitor the appearance of nonadherent cells were harvested by vigorous pipetting, washed, and used Kb/OVA complexes on the surface of APC. for presentation experiments. For the preparation of splenocytes, spleen cells from naive C57BL/6 mice were washed once with RPMI 1640 supplemented with antibiotics Materials and Methods (100 U/ml penicillin and 100 mg/ml streptomycin), resuspended in CM, Mice and used for presentation assays. Female C57BL/6 (H-2b) mice from Iffa Credo (L’Arbresle, France) were Ag presentation assays used between 6 and 8 wk of age. Female TAP1 knockout mice (31) bred 5 onto a C57BL/6 background were a kind gift from Dr.
Load more

Recommended publications
  • Biological Toxins Fact Sheet
    Work with FACT SHEET Biological Toxins The University of Utah Institutional Biosafety Committee (IBC) reviews registrations for work with, possession of, use of, and transfer of acute biological toxins (mammalian LD50 <100 µg/kg body weight) or toxins that fall under the Federal Select Agent Guidelines, as well as the organisms, both natural and recombinant, which produce these toxins Toxins Requiring IBC Registration Laboratory Practices Guidelines for working with biological toxins can be found The following toxins require registration with the IBC. The list in Appendix I of the Biosafety in Microbiological and is not comprehensive. Any toxin with an LD50 greater than 100 µg/kg body weight, or on the select agent list requires Biomedical Laboratories registration. Principal investigators should confirm whether or (http://www.cdc.gov/biosafety/publications/bmbl5/i not the toxins they propose to work with require IBC ndex.htm). These are summarized below. registration by contacting the OEHS Biosafety Officer at [email protected] or 801-581-6590. Routine operations with dilute toxin solutions are Abrin conducted using Biosafety Level 2 (BSL2) practices and Aflatoxin these must be detailed in the IBC protocol and will be Bacillus anthracis edema factor verified during the inspection by OEHS staff prior to IBC Bacillus anthracis lethal toxin Botulinum neurotoxins approval. BSL2 Inspection checklists can be found here Brevetoxin (http://oehs.utah.edu/research-safety/biosafety/ Cholera toxin biosafety-laboratory-audits). All personnel working with Clostridium difficile toxin biological toxins or accessing a toxin laboratory must be Clostridium perfringens toxins Conotoxins trained in the theory and practice of the toxins to be used, Dendrotoxin (DTX) with special emphasis on the nature of the hazards Diacetoxyscirpenol (DAS) associated with laboratory operations and should be Diphtheria toxin familiar with the signs and symptoms of toxin exposure.
    [Show full text]
  • Targeted Neuronal Cell Ablation in the Drosophila Embryo: Pathfinding by Follower Growth Cones in the Absence of Pioneers
    Neuron, Vol. 14, 707-715, April, 1995, Copyright© 1995 by Cell Press Targeted Neuronal Cell Ablation in the Drosophila Embryo: Pathfinding by Follower Growth Cones in the Absence of Pioneers David M. Lin,* Vanessa J. Auld,*t reach their targets in the CNS (Wigglesworth, 1953). The and Corey S. Goodman ability of the initial axons to guide later growth cones has Howard Hughes Medical Institute led to the suggestion that pioneering growth cones might Division of Neurobiology be endowed with special pathfinding abilities. Do subse- Department of Molecular and Cell Biology quent growth cones possess the same pathfinding abili- Life Science Addition, Room 519 ties, or are they different from the pioneers? University of California, Berkeley Ablation experiments in a variety of organisms thus far Berkeley, California 94720 have provided a range of conflicting answers to this ques- tion. In the grasshopper embryo CNS, the G growth cone turns anteriorly along the P axons in the NP fascicle, a Summary pathway formed by the three descending P and the two ascending A axons. Although the A and P axons normally We developed a rapid method that uses diphtheria fasciculate and follow one another, either can pioneer the toxin, the flp recognition target sequences, and the complete pathway on its own. Furthermore, any one of GAL4-UAS activation system, to ablate specific neu- the P axons will suffice, since when any two of them are rons in the Drosophila embryo and to examine the con- ablated the remaining P pioneers the pathway. When the sequences in large numbers of embryos at many time three P axons are ablated, the G growth cone stalls, and points.
    [Show full text]
  • ADP-Ribosylation with Clostridium Perfringens Iota Toxin
    Biochem. J. (1990) 266, 335-339 (Printed in Great Britain) 335 Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylation with Clostridium perfringens iota toxin Udo GEIPEL, Ingo JUST and Klaus AKTORIES* Rudolf-Buchheim-Institut fur Pharmakologie der Universitat GieBen, Frankfurterstr. 107, D-6300 GieBen, Federal Republic of Germany Clostridium perfringens iota toxin belongs to a novel family of actin-ADP-ribosylating toxins. The effects of ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin on cytochalasin D- stimulated actin ATPase activity was studied. Cytochalasin D stimulated actin-catalysed ATP hydrolysis maximally by about 30-fold. ADP-ribosylation of actin completely inhibited cytochalasin D-stimulated ATP hydrolysis. Inhibition of ATPase activity occurred at actin concentrations below the critical concentration (0.1 /iM), at low concentrations of Mg2" (50 ItM) and even in the actin-DNAase I complex, indicating that ADP-ribosylation of actin blocks the ATPase activity of monomeric actin and -that the inhibitory effect is not due to inhibition of the polymerization of actin. INTRODUCTION perfringens type E strain CN5063, which was kindly donated by Dr. S. Thorley (Wellcome Biotech, Various bacterial ADP-ribosylating toxins modify Beckenham, Kent, U.K.) essentially according to the pro- regulatory GTP-binding proteins, thereby affecting cedure described (Stiles & Wilkens, 1986). Cytochalasin eukaryotic cell function. Pertussis toxin and cholera D was obtained from Sigma (Deisenhofen, Germany). toxin ADP-ribosylate GTP-binding proteins involved in DNAase I was a gift from Dr. H. G. Mannherz transmembrane signal transduction (for a review, see (Marburg, Germany). [oc-32P]ATP, [y-32P]ATP and Pfeuffer & Helmreich, 1988).
    [Show full text]
  • Impact of Bacterial Toxins in the Lungs
    toxins Review Impact of Bacterial Toxins in the Lungs 1,2,3, , 4,5, 3 2 Rudolf Lucas * y, Yalda Hadizamani y, Joyce Gonzales , Boris Gorshkov , Thomas Bodmer 6, Yves Berthiaume 7, Ueli Moehrlen 8, Hartmut Lode 9, Hanno Huwer 10, Martina Hudel 11, Mobarak Abu Mraheil 11, Haroldo Alfredo Flores Toque 1,2, 11 4,5,12,13, , Trinad Chakraborty and Jürg Hamacher * y 1 Pharmacology and Toxicology, Medical College of Georgia at Augusta University, Augusta, GA 30912, USA; hfl[email protected] 2 Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA 30912, USA; [email protected] 3 Department of Medicine and Division of Pulmonary Critical Care Medicine, Medical College of Georgia at Augusta University, Augusta, GA 30912, USA; [email protected] 4 Lungen-und Atmungsstiftung, Bern, 3012 Bern, Switzerland; [email protected] 5 Pneumology, Clinic for General Internal Medicine, Lindenhofspital Bern, 3012 Bern, Switzerland 6 Labormedizinisches Zentrum Dr. Risch, Waldeggstr. 37 CH-3097 Liebefeld, Switzerland; [email protected] 7 Department of Medicine, Faculty of Medicine, Université de Montréal, Montréal, QC H3T 1J4, Canada; [email protected] 8 Pediatric Surgery, University Children’s Hospital, Zürich, Steinwiesstrasse 75, CH-8032 Zürch, Switzerland; [email protected] 9 Insitut für klinische Pharmakologie, Charité, Universitätsklinikum Berlin, Reichsstrasse 2, D-14052 Berlin, Germany; [email protected] 10 Department of Cardiothoracic Surgery, Voelklingen Heart Center, 66333
    [Show full text]
  • Diphtheria Toxin
    Duke University / Duke University Health System Policy on working with: Diphtheria Toxin I. Revision Page II. Background III. Risk Assessment IV. Policy Statement V. Emergency Information VI. Appendix I: EOHW Exposure/Injury Response Protocol VII. Appendix II: OESO SOP Template VIII. References This policy was first approved on December, 2018. Occupational and Environmental Safety Office (OESO) Employee Occupational Health and Wellness (EOHW) Page | 1 I. Revision Page Date Revision(s) / Comment(s) 12/06/2018 New policy document Page | 2 II. Background Diphtheria toxin (DT) is a biological toxin and is secreted by the bacterium Corynebacterium diphtheriae. DT is useful in biomedical research using mice because it can be used to selectively target and kill cells or organs without requiring surgery. Wild type mice do not have DT receptors, so they are relatively resistant to DT. The median lethal dose (LD50) for mice has been estimated at 1.6 mg/kg by subcutaneous injection compared to an estimated human LD50 of less than 100 ng/kg by IM injection. The gene for DT receptors can be inserted into a mouse genome so that the transgenic mice will express DT receptors only on specific cells. For example, a transgenic mouse engineered to express DT receptors only on hepatocytes can be injected with DT, which will only kill the hepatocytes, creating a nonsurgical mouse model without a functional liver. Diphtheria toxin doses used in transgenic mice range from 0.5 µg/kg to 50 µg/kg depending on the scientific goal (10 to 1000 ng for a 20-gram mouse).[Saito et al, 2001; Cha et al, 2003; Cerpa et al, 2014] A common dose is 100 ng per injection, sometimes administered in repeated doses to achieve a cumulative effect.
    [Show full text]
  • 2015 Biotoxin Illness
    Understanding Toxins Jyl Burgener Midwest Area Biosafety Network (MABioN) MABioN February 18, 2016 Jyl Burgener, M.S. MBA, RBP, CBSP Topics 1. Definition and Characteristics of Toxins 2. Regulations Regarding Toxins 3. Inactivation of Toxins 4. Human Health Considerations of Toxins 2 Jyl Burgener, M.S. MBA, RBP, CBSP Definition of Toxins Simple definition of toxin: a poisonous substance produced by a living thing Full definition of toxin: • a poisonous substance • a specific product of the metabolic activities of a living organism • usually very unstable • notably toxic when introduced into the tissues, and • typically capable of inducing antibody formation 3 Jyl Burgener, M.S. MBA, RBP, CBSP Political Definition North Atlantic Treaty Organization (NATO) vs Warsaw Pact Biological or chemical agent? NATO opted for biological agent, and the Warsaw Pact, like most other countries in the world, for chemical agent. According to an International Committee of the Red Cross review of the Biological Weapons Convention: "Toxins are poisonous products of organisms; unlike biological agents, they are inanimate and not capable of reproducing themselves", "Since the signing of the Convention, there have been no disputes among the parties regarding the definition of biological agents or toxins" 4 Jyl Burgener, M.S. MBA, RBP, CBSP Biotoxin The term "biotoxin" is sometimes used to explicitly confirm the biological origin. Biotoxins can be further classified into: fungal biotoxins, or short mycotoxins, microbial biotoxins, plant biotoxins, short phytotoxins and animal biotoxins. 5 Jyl Burgener, M.S. MBA, RBP, CBSP Characteristics of Toxins Extremely potent Often mixtures Directly damage host tissues Disable the immune and nervous systems 6 Jyl Burgener, M.S.
    [Show full text]
  • Diphtheria. In: Epidemiology and Prevention of Vaccine
    Diphtheria Anna M. Acosta, MD; Pedro L. Moro, MD, MPH; Susan Hariri, PhD; and Tejpratap S.P. Tiwari, MD Diphtheria is an acute, bacterial disease caused by toxin- producing strains of Corynebacterium diphtheriae. The name Diphtheria of the disease is derived from the Greek diphthera, meaning ● Described by Hippocrates in ‘leather hide.’ The disease was described in the 5th century 5th century BCE BCE by Hippocrates, and epidemics were described in the ● Epidemics described in 6th century AD by Aetius. The bacterium was first observed 6th century in diphtheritic membranes by Edwin Klebs in 1883 and cultivated by Friedrich Löffler in 1884. Beginning in the early ● Bacterium first observed in 1900s, prophylaxis was attempted with combinations of toxin 1883 and cultivated in 1884 and antitoxin. Diphtheria toxoid was developed in the early ● Diphtheria toxoid developed 7 1920s but was not widely used until the early 1930s. It was in 1920s incorporated with tetanus toxoid and pertussis vaccine and became routinely used in the 1940s. Corynebacterium diphtheria Corynebacterium diphtheriae ● Aerobic gram-positive bacillus C. diphtheriae is an aerobic, gram-positive bacillus. ● Toxin production occurs Toxin production (toxigenicity) occurs only when the when bacillus is infected bacillus is itself infected (lysogenized) by specific viruses by corynebacteriophages (corynebacteriophages) carrying the genetic information for carrying tox gene the toxin (tox gene). Diphtheria toxin causes the local and systemic manifestations of diphtheria. ● Four biotypes: gravis, intermedius, mitis, and belfanti C. diphtheriae has four biotypes: gravis, intermedius, mitis, ● All isolates should be tested and belfanti. All biotypes can become toxigenic and cause for toxigenicity severe disease.
    [Show full text]
  • Cold Sensing by Nav1.8-Positive and Nav1.8-Negative Sensory Neurons
    Cold sensing by NaV1.8-positive and NaV1.8-negative sensory neurons A. P. Luiza, D. I. MacDonalda, S. Santana-Varelaa, Q. Milleta, S. Sikandara, J. N. Wooda,1, and E. C. Emerya,1 aMolecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, United Kingdom Edited by Peter McNaughton, King’s College London, London, United Kingdom, and accepted by Editorial Board Member David E. Clapham January 8, 2019 (received for review August 23, 2018) The ability to detect environmental cold serves as an important define the distribution and identity of cold-sensitive DRG neurons, survival tool. The sodium channels NaV1.8 and NaV1.9, as well as in live mice, using in vivo imaging. the TRP channel Trpm8, have been shown to contribute to cold sensation in mice. Surprisingly, transcriptional profiling shows that Results NaV1.8/NaV1.9 and Trpm8 are expressed in nonoverlapping neuro- Distribution of DRG Sensory Neurons Responsive to Noxious Cold, in nal populations. Here we have used in vivo GCaMP3 imaging to Vivo. To identify cold-sensitive neurons in vivo, we used a pre- identify cold-sensing populations of sensory neurons in live mice. viously developed in vivo imaging technique to study the responses We find that ∼80% of neurons responsive to cold down to 1 °C do of individual DRG neurons in situ (8). Mice coexpressing Pirt- not express NaV1.8, and that the genetic deletion of NaV1.8 does GCaMP3 (which enables pan-DRG GCaMP3 expression), not affect the relative number, distribution, or maximal response NaV1.8 Cre, and a Cre-dependent reporter (tdTomato) were of cold-sensitive neurons.
    [Show full text]
  • Diphtheria Toxin Effects on Human Cells in Tissue Culture1
    [CANCER RESEARCH 36, 4590-4594, December 1976] Diphtheria Toxin Effects on Human Cells in Tissue Culture1 Barbara R. Venter2 and Nathan 0. Kaplan3 Departments of Biology (B. A. V.) and Chemistry (N. 0. K.), University of California, San Diego, La Jolla, California 92093 SUMMARY component that renders the hybrids retaining chromosome 5 much more sensitive to diphtheria toxin. HeLa calls exposed to a single sublethal concentration of Several reports have appeared in the recent literature diphtheria toxin were found to have diminished sensitivity exploring the possible use of diphtheria toxin as an anhineo when subsequently reaxposad to the toxin. Three calls plastic agent (2, 8). In 1974, lglewski and Rithenbeng (5) strains exhibiting toxin resistance were developed. In the reported that diphtheria toxin produced a greater inhibitory calls that had previously been exposed to toxin at 0.015 j.@g/ effect on protein synthesisof humanand mousetumor cells ml, 50% inhibition of protein synthesis required a toxin than on normal tissues from the same species. Their paper concentration of 0.3 pg/mI, which is more than 10 times expanded upon an earlier study by Buzzi and Maistrallo (1) that required in normal HeLa cells. in which it was reported that striking results could be ob There appears to be a threshold level of diphtheria toxin tamed with diphtheria toxin treatment of experimental hu action.Concentrationsof toxingreaterthan that required momsin Swiss mica. Papenheiman and Randall (9), in 1975, for 50% inhibition of protein synthesis (0.01 @g/ml)are while confirming that diphtheria toxin causes some tempo associated with cytotoxicity, whereas those below this con nary regression of Ehrlich ascihas tumors in mice, were less cantration may not be lethal.
    [Show full text]
  • Bio-Toxins-And-Venoms.Pdf
    Institutional Biosafety Committee Policy: Applies to: BIOSAFETY BEST PRACTICES FOR Vanderbilt University (VU) RESEARCH USE OF BIOLOGICAL Vanderbilt University Medical Center (MC) TOXINS & VENOMS This document applies to the use of biological toxins and venoms that are: 1. received in a premeasured quantity packaged in a septum vial that will not be opened prior to reconstitution with a vial adapter; and 2. meets one or more of the following criteria: toxin/venom presents a life-threatening or severe irreversible health effect risk in a single exposure incident scenario (i.e., acutely toxic); toxin/venom is included on the CDC/APHIS Select Agent List. Any toxin work that meets the two conditions above will require registration with, and approval by, the Institutional Biosafety Committee (IBC) as outlined below. Common examples of toxin use that meet the criteria above include: . tetrodotoxin used in cell culture or on animal protocols, . staphylococcal enterotoxin B (SEB) used for cell culture, and . administration of diphtheria toxin on animal protocols. NOTE: If work with acutely toxic materials is planned, but it requires manipulation of dry toxin, consult with VEHS Chemical & Laboratory Safety. Institutional Biosafety Committee Review & Approval for Possession and Use of Toxins In order to gain approval for this activity, the following actions must be completed. 1. The PI must complete and submit a Vanderbilt Biological Materials Registration (or amend an existing registration). 2. The PI must prepare and maintain a Toxin Safety Plan in the lab that includes: A copy of this document; Manufacturer’s safety data sheets (SDS) for the toxin(s) to be used; Inventory for each toxin which will allow the end user to quickly and clearly communicate how much toxin is on hand in storage.
    [Show full text]
  • Anti-Pseudomonas Exotoxin a (P2318)
    Anti-Pseudomonas Exotoxin A antibody produced in rabbit, delipidized, whole antiserum Catalog Number P2318 Product Description Storage/Stability Anti-Pseudomonas Exotoxin A is developed in rabbit For continuous use, store at 2–8 °C for up to one using purified Exotoxin A from Pseudomonas month. For extended storage, the solution may be aeruginosa as immunogen. The antiserum has been frozen in working aliquots. Repeated freezing and treated to remove lipoproteins. thawing is not recommended. Storage in "frost-free" freezers is not recommended. If slight turbidity occurs Pseudomonas aeruginosa exotoxin A (PE) exhibits a upon prolonged storage, clarify the solution by number of properties similar to those of other microbial centrifugation before use. super antigens. PE has three structural domains. The N-terminal domain (I) is responsible for the binding of Product Profile toxin to its receptor on the cells, the middle domain (II) By dot blot immunoassay, using ligands immobilized on has a role in the translocation of toxin across the nitrocellulose membrane (50–500 ng/dot), the membrane, and the C-terminal domain (III) has the antiserum reacts versus Psuedomonas exotoxin A, but ADP-ribosylation activity.1 This toxin has been used as shows no reaction versus Staphylococcal enterotoxin A, a component of recombinant toxins developed as novel Staphylococcal enterotoxin B, and Cholera toxin. The therapeutics.2 PE is lethal for cells because it has the product has not been tested for its neutralization ability to irreversibly shut down protein synthesis. In potency against active Pseudomonas exotoxin A. tissue culture, PE is most active against fibroblastic cell lines. 1.
    [Show full text]
  • Involvement of Nicotinamide Adenine Dinucleotide in the Action of Cholera Toxin in Vitro (Adenylate Cyclase/Erythrocyte) D
    Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2064-2068, June 1975 Involvement of Nicotinamide Adenine Dinucleotide in the Action of Cholera Toxin In Vitro (adenylate cyclase/erythrocyte) D. MICHAEL GILL Harvard University, Department of Biology, 16 Divinity Ave., Cambridge, Massachusetts 02138 Communicated by A. M. Pappenheimer, Jr., March 14, 1975 ABSTRACT NAD is a necessary cofactor for the activa- daltons) is needed for adenylate cyilase activation in a lysate. tion of adenylate cyclase [ATP pyrophosphate-lyase Gill and King concluded that, cholera toxin acts upon (cyclizing), EC 4.6.1.11 by cholera toxin. Lysates of certain whenR types of cell that hydrolyze their endogenous store of a whole cell, its peptide Al must penetrate at least as far as NAD after cell disruption respond poorly or not at all to the inner surface of the membrane, if not into the cytoplasm cholera toxin. Lysates of pigeon erythrocytes, which lack itself. They suggested that peptide Al might be an enzyme enzymes that degrade NAD, provide a convenient and re- that catalyzes some intracellular reaction resulting in the producible system for assaying the activity of cholera toxin in vitro and allow investigation of the mechanism modification of a component of the inner surface of the plasma of action of the toxin upon broken cells. membrane, where adenylate cyclase is located. Gill and King noticed that erythrocyte ghosts would not The protein exotoxin secreted by Vibrio cholerae increases the respond to cholera toxin once they had been separated from adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), the soluble erythrocyte cytoplasm.
    [Show full text]