DOCSLIB.ORG

Mushrooms & Marine 2012

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mushrooms & Marine 2012 M & M Mushrooms and Marine Howard A. Greller Introduction • Mushrooms are the fruiting body of a fungus • More than 40,000 species worldwide • 5000 species in U.S., < 2% poisonous • 79% poison < 6 years old • Raw >> cooked • Allergy and anaphylaxis more common than poisoning Mushroom Anatomy and identification General properties of mushrooms. Not everyone has the “cup” (pileus) (lamella) (annulus) cup (volva) (stape) (mycelial threads) Mushroom ID • Books • Location • Size, color, shape • Spore print • Spore ID • Mycologist Melzer’s reagent (mycologist prefers) - 20 mL H2O, 1.5 g K iodide, 0.5 g iodine, 20 g chloral hydrate - Defines presence of amatoxin = dark blue Meixner reaction (reliability = doubtful) - http://namyco.org/ Several drops 10-12 N HCl on newspaper to amatoxin-containing spore - blue reaction Tricksy mushrooms yeeesssss. precious . Cortinarius speciosissimus. Nephrotoxic (non-US). Wound up with a renal transplant (from his daughter). His wife and pssst. Nicholas . the Lord he was dining eat these. they’re yummy. with are still on dialysis. Famous mushroom quote number one There are old mushroom hunters, and there are bold mushroom hunters. But there are no old, bold mushroom hunters. Mushroom Taxonomy Classification Famous mushroom quote number two Falling in love is like eating (poisonous) mushrooms. You never know if it is the real thing until it’s too late. Cyclopeptides Group I Amanita phalloides Amanita verosa Modern series have a lower case fatality rate Amanita species - cyclopeptides - A. phalloides (death cap), A. virosa (death angel) - 95% mushroom related fatalities, 50% case fatality - One mushroom enough to kill an adult Galerina autumnalis Lepiota helveola Galerina & Lepiota species - cyclopeptides - G. autumnalis, G. marginata, G. venenata - L. helveola, L. josserandi, Lbrunneoincarnata Cyclopeptides Amatoxins (cyclic octapeptides) - α-amanitin - heat stabile - poorly absorbed, enterohepatic recirculation - interferes with RNA polymerase II, protein translation - sodium-taurocholate co-transporter polypeptide - centrilobular hepatic necrosis with intact architecture Cyclopeptides Phallotoxins (cyclic heptapeptides) - Phalloidin - limited absorption - interrupts actin polymerization - impairs cell membrane function Cyclopeptides Virotoxins (cyclic heptapeptides) - Virotoxin - ? human toxicity Toxicity - cyclopeptides Phase I - not earlier than 6 hours, lasts up to 24 hours - initial cholera-like diarrhea, then nausea/vomiting Phase II - “quiescent” phase (no n&v) - hepatotoxicity Phase III - hepatic, renal, pancreatic - death 2-3 days after ingestion thioctic acid is a co-enzyme in TCA cycle, free radical scavenger Therapeutics - cyclopeptides PCN displaces amanitin from albumin, Early blocks uptake, binds circulating - volume resuscitation amatoxins, prevents amanitin from - ? MDAC - ? hemodialysis / hemoperfusion binding to RNA polymerase Late(r) Silymarin block amatnitin uptake - thioctic (α-lipoic) acid NAC is good for what ails the liver - penicillin G (1.6 million U/kg/day) Cimetidine potentially inhibits - silymarin (silibinin) from milk thistle metabolism - n-acetylcysteine - cimetidine Bioartificial Liver (“bridge” to recovery or T/P) Therapeutics Liver Transplant (EARLY > late) •Bioartificial liver •Liver transplant •Early >> late Gyrometrin Group II gyromitra esculenta morchella esculenta Gyromitra species - gyromitrin - G. esculenta, G. californica, G. brunnea, G. infula - found in spring under conifers - often confused for the edible morel Gyromitrin - N-methyl-N-formyl hydrazone - hydrolyzed to monomethylhydrazine - heat sensitive (although fumes have ? toxicity) ➜➜➜ gyromitrin monomethylhydrazine Toxicity - gyromitrin - asymptomatic for at least 6 hours - initial GI symptoms - seizures GAD - glutamic acid decarboxylase, PPK - pyridoxal phosphokinase hydrazines ! - inactivate pyridoxal 5’ phosphate ! - inhibit pyridoxal phosphokinase ! - complex with and lead to increased urinary excretion of pyridoxine GAD glutamic acid GABA pyridoxal 5’ phosphate PPK hydrazines pyridoxine Therapeutics - gyromitrin Early - ? GI decontamination Seizures - benzodiazepines - pyridoxine (vit B6) - 70 mg/kg up to 5 grams - anesthetic barbiturates pyridoxine Muscarine Group III clitocybe clavipes inocybe geophylia Clitocybe species - muscarine - C. dealbata (‘the sweater’),C. illudens Inocybe species - muscarine - I. Iacera, I. geophylla Muscarine Quarternary ammonium compound - Does not cross BBB (no CNS) - Not nicotinic - Not susceptible to ACHase hydrolysis acetylcholine muscarine When you think of rain, always bring a “sweater” Toxicity - muscarine - Rapid onset of toxicity (within 30 minutes) - SLUDGE - NO nicotinic findings (i.e. mydriasis, tachycardia, weak) Therapeutics - muscarine - Atropine (infrequently required) - ACLS doses to start - titrate until relief of symptoms, dry secretions - NO oximes (i.e. 2-PAM) Coprine Group IV Coprinus atramentarius Coprinus comatus Coprinus - coprine - C. comatus, C. atramentarius - Undergo “autodigestion” - inky caps - pleasant tasting, edible mushrooms - problematic with ethanol (disulfiram like reaction) Coprine Toxicity comes from primary metabolite - 1-aminocyclopropanol - ethanol present or ingested within 24-48 hours - usually okay if done together (delayed toxicity) ➜➜➜ coprine 1-aminocyclopropanol Toxicity - coprine - rapid onset of toxicity (within 1 hour) - inhibits aldehyde dehydrogenase - disulfiram reaction - flushing - nausea & vomiting - tachycardia - headache disulfiram Therapeutics - coprine - intravenous fluids - antiemetics - analgesics - time (for ethanol to be metabolized) + = Ibotenic acid / Muscimol Group V Amanita muscaria Amanita gemmata Modern series have a lower case fatality rate Amanita muscaria - ibotenic acid / muscimol - A. muscaria (fly agaric), A. pantherina, A. gemmata - popular children’s books and games - Babar, Super Mario Bros. NOT MUSCARINIC, NOT ANTI MUSCARINIC Ibotenic acid / muscimol Ibotenic acid - structurally similar to glutamic acid Muscimol - decarboxylated metabolite of ibotenic acid - similar to GABA ibotenic acid muscimol glutamic acid GABA Toxicity - ibotenic acid / musicmol - rapid onset (0.5 - 2 hours), resolves 6-12 hours - mild GI upset - ibotenic acid stimulates glutamic acid receptors - hallucinosis, excitation - musicmol acts as a GABA agonist - somnolence, ataxia - seizures rare (GABAB agonism) - adults manifest more GABA-ergic symptoms - pediatrics excitatory transmission predominates Therapeutics - ibotenic acid / muscimol - supportive care - benzodiazepines for seizures, excitation - no role for flumazenil for sedation Psilocybin / Psilocin Group VI Psilocybe cubensis Psilocybe cyanescens Gymnopilus spectabilis, Psathyrella foenisecii Psilocybe species - psilocybin / psilocin - P. cyanescens, P. cubensis, G. spectabilis, P. foenisecii - “magic mushrooms” - Native american religious customs, drug culture - when handled, develop blue bruises Psilocybin / psilocin Structural similarity to serotonin - interact with serotonin receptors, no adrenergic effects - rapid onset, medium duration (1 - 6 hours) - psilocybin is rapidly hydrolyzed to psilocin psilocybin serotonin psilocin illusion hallucination pallinopsia synesthesia Toxicity - psilocybin / psilocin - Early GI effects (0.5 - 1 hour) - illusions - synesthesias - “heightened perceptions” - true hallucinations are rare and dose dependent - autonomic response to altered perceptions Therapeutics - psilocybin / psilocin Primary supportive care - benzodiazepines - avoid neuroleptics - risk hallucinogen persistent perceptive disorder (HPPD) Little Brown Mushrooms Group VII russula emetica Chlorophyllum molybdites Little Brown Mushrooms (LBM) - GI toxins - Boletes, Lactarius sp., Omphalotus sp., Tricholoma sp. - Lycoperdon sp., Agaricus, Entoloma, Chlorophyllum sp. - Largest group of mushrooms - Often mistaken for edible or hallucinogenic LBM - GI toxins Heterogenous toxins - terpenes and lactones - unknown specific mechanism or toxin - direct irritant? allergic phenomenon? Toxicity - LBM - Early GI effects (0.5 - 3 hours) - rapid resolution without significant consequence - Paxillus Syndrome (rare) - P. involutus, Clitocybe claviceps, Boletus luridus - involutin? (diphenylcyclopenteneone) - immune-mediated hemolytic anemia, renal failure - repeated long term exposure Therapeutics - LBM Primary supportive care - intravenous hydration - antiemetics - antidiarrheals Prevention Orelline / Orellanine Group VIII cortinarius speciosissimus cortinarius archeri Cortinarius sp. - orelline / orellanine - C. orellanus, C. speciosissimus, C.rainierensis (NA) - Only recent recognition of toxicity Oreline / Orellanine Bipyridyl agents - similar to paraquat/diquat - heat stable - direct irritant? allergic phenomenon? orellanine paraquat Toxicity - orelline / orellanine - asymptomatic for 24 - 26 hours - headache, chills, anorexia, n/v (flu-like) - oliguric renal failure - ATN - similar prognosis to other causes of ATN Therapeutics - LBM Primary supportive care - intravenous hydration - hemodialysis - transplantation reported, but very rare Prevention Allenic norleucine Group IX amanita smithiana tricholoma magnivelare Amanita smithiana - allenic norleucine - mistaken for tricholoma magnivelare (matsutake) - Pacific N.W. US Allenic norleucine 2-amino-4,5-hexadienoic acid - limited mechanistic information - may be the compound Toxicity - allenic norleucine Phase I - early GI distress (0.5 - 12 hours) - malaise, diaphoresis, dizzy Phase II - 4-6 days after ingestion - acute renal
Load more

Recommended publications
  • Characterization of a Domoic Acid Binding Site from Pacific Razor Clam
    Aquatic Toxicology 69 (2004) 125–132 Characterization of a domoic acid binding site from Pacific razor clam Vera L. Trainer∗, Brian D. Bill NOAA Fisheries, Northwest Fisheries Science Center, Marine Biotoxin Program, 2725 Montlake Blvd. E., Seattle, WA 98112, USA Received 5 November 2003; received in revised form 27 April 2004; accepted 27 April 2004 Abstract The Pacific razor clam, Siliqua patula, is known to retain domoic acid, a water-soluble glutamate receptor agonist produced by diatoms of the genus Pseudo-nitzschia. The mechanism by which razor clams tolerate high levels of the toxin, domoic acid, in their tissues while still retaining normal nerve function is unknown. In our study, a domoic acid binding site was solubilized from razor clam siphon using a combination of Triton X-100 and digitonin. In a Scatchard analysis using [3H]kainic acid, the partially-purified membrane showed two distinct receptor sites, a high affinity, low capacity site with a KD (mean ± S.E.) of 28 ± 9.4 nM and a maximal binding capacity of 12 ± 3.8 pmol/mg protein and a low affinity, high capacity site with a mM affinity for radiolabeled kainic acid, the latter site which was lost upon solubilization. Competition experiments showed that the rank order potency for competitive ligands in displacing [3H]kainate binding from the membrane-bound receptors was quisqualate > ibotenate > iodowillardiine = AMPA = fluorowillardiine > domoate > kainate > l-glutamate. At high micromolar concentrations, NBQX, NMDA and ATPA showed little or no ability to displace [3H]kainate. In contrast, Scatchard analysis 3 using [ H]glutamate showed linearity, indicating the presence of a single binding site with a KD and Bmax of 500 ± 50 nM and 14 ± 0.8 pmol/mg protein, respectively.
    [Show full text]
  • Perth Urban Bushland Fungi Field Book
    Perth Urban Bushland Fungi Field Book (A Self-Managed Format) Author Neale L. Bougher Format and Electronic Design John R. Weaver Publisher: Perth Urban Bushland Fungi 3rd Edition, 2007 Foundation 1st Edition May 2005 2nd Edition November 2005 3rd Edition February 2007 This book is Copyright. Approval is granted to reproduce this Field Book in whole or in part, for personal and educational purposes only. The Field Book may be downloaded from the Perth Urban Bushland Fungi web site at: http://www.fungiperth.org.au/fieldbook/cat_index.html With the exception of its use for personal and/or educational purposes, electronic storage of data or images from the printed or web site versions of this book and retrieval or transmission in any form from such storage is not permitted. Written permission is required prior to any potential commercial applications or non- personal reproduction or distribution. Enquiries should be made to Perth Urban Bushland Fungi, Western Australian Herbarium, Department of Environment and Conservation, Locked Bag 104, Bentley Delivery Centre, Western Australia 6983. Copyright © text: Neale L. Bougher Copyright © photographs: Neale L. Bougher (unless otherwise stated). Copyright © electronic & printed layout & design: John R. Weaver This book may be cited as: Bougher N.L. (2006). Perth Urban Bushland Fungi Field Book. Perth Urban Bushland Fungi, Perth Western Australia. (Online), from: http://www.fungiperth.org.au/fieldbook/cat_index.html (2 February 2007). © Perth Urban Bushland Fungi - Field Book / Last updated 2/02/2007 Page ii Acknowledgements PUBF activities are the result of a core team comprising Neale Bougher (Mycologist), John Weaver (Formatting and Electronic Presentation and Data Management), Roz Hart (Community Education Officer) and Sarah de Bueger (Project Officer, 2006) with past assistance from Jac Keelan-Wake (Administrative Support 2004-2005).
    [Show full text]
  • Six Domoic Acid Related Compounds from the Red Alga, Chondria Armata
    www.nature.com/scientificreports OPEN Six domoic acid related compounds from the red alga, Chondria armata, and domoic acid biosynthesis Received: 1 September 2017 Accepted: 15 December 2017 by the diatom, Pseudo-nitzschia Published: xx xx xxxx multiseries Yukari Maeno1, Yuichi Kotaki2, Ryuta Terada3, Yuko Cho1, Keiichi Konoki1 & Mari Yotsu-Yamashita1 Domoic acid (DA, 1), a potent neurotoxin that causes amnesic shellfsh poisoning, has been found in diatoms and red algae. While biosynthetic pathway towards DA from geranyl diphosphate and l-glutamate has been previously proposed, its late stage is still unclear. Here, six novel DA related compounds, 7′-methyl-isodomoic acid A (2) and B (3), N-geranyl-l-glutamic acid (4), 7′-hydroxymethyl- isodomoic acid A (5) and B (6), and N-geranyl-3(R)-hydroxy-l-glutamic acid (7), were isolated from the red alga, Chondria armata, and their structures were determined. The compounds 4 and 7, linear compounds, are predictable as the precursors to form the DA pyrrolidine ring. The compounds 2 and 3 are thought as the cyclized products of 7; therefore, dehydration and electron transfer from the internal olefn of 7 is a possible mechanism for the pyrrolidine ring formation. One terminal methyl group of the side chain of 2 and 3 is predicted to be oxidized to hydroxymethyl (5, 6), and then to carboxylic acids, forming isodomoic acids A and B. Finally, the terminal olefn of isodomoic acid A would be isomerized to form DA. In addition, [15N, D]-labeled 4 was incorporated into DA using the diatom, Pseudo-nitzschia multiseries, demonstrating that 4 is the genuine precursor of DA.
    [Show full text]
  • Fungi of North East Victoria Online
    Agarics Agarics Agarics Agarics Fungi of North East Victoria An Identication and Conservation Guide North East Victoria encompasses an area of almost 20,000 km2, bounded by the Murray River to the north and east, the Great Dividing Range to the south and Fungi the Warby Ranges to the west. From box ironbark woodlands and heathy dry forests, open plains and wetlands, alpine herb elds, montane grasslands and of North East Victoria tall ash forests, to your local park or backyard, fungi are found throughout the region. Every fungus species contributes to the functioning, health and An Identification and Conservation Guide resilience of these ecosystems. Identifying Fungi This guide represents 96 species from hundreds, possibly thousands that grow in the diverse habitats of North East Victoria. It includes some of the more conspicuous and distinctive species that can be recognised in the eld, using features visible to the Agaricus xanthodermus* Armillaria luteobubalina* Coprinellus disseminatus Cortinarius austroalbidus Cortinarius sublargus Galerina patagonica gp* Hypholoma fasciculare Lepista nuda* Mycena albidofusca Mycena nargan* Protostropharia semiglobata Russula clelandii gp. yellow stainer Australian honey fungus fairy bonnet Australian white webcap funeral bell sulphur tuft blewit* white-crowned mycena Nargan’s bonnet dung roundhead naked eye or with a x10 magnier. LAMELLAE M LAMELLAE M ■ LAMELLAE S ■ LAMELLAE S, P ■ LAMELLAE S ■ LAMELLAE M ■ ■ LAMELLAE S ■ LAMELLAE S ■ LAMELLAE S ■ LAMELLAE S ■ LAMELLAE S ■ LAMELLAE S ■ When identifying a fungus, try and nd specimens of the same species at dierent growth stages, so you can observe the developmental changes that can occur. Also note the variation in colour and shape that can result from exposure to varying weather conditions.
    [Show full text]
  • Amnesic Shellfish Poisoning: Emergency Medical Management
    nce: Res ie ea c rc S h e & n i Schroeder et al., J Marine Sci Res Dev 2015, 6:1 D r e Journal of a v M DOI; 10.4172/2155-9910.1000179 e f l o o p l m a ISSN:n 2155-9910 e r n u t o J Marine Science: Research & Development ResearchShort Communication Article OpenOpen Access Access Amnesic Shellfish Poisoning: Emergency Medical Management George Schroeder1*, Stephen S. Bates2 and John Spallino3 1American Academy of Urgent Care Medicine 2813 Hiawassee Road, Suite 206 Orlando, FL USA 32835 2Fisheries and Oceans Canada Gulf Fisheries Centre P.O. Box 5030 Moncton, NB E1C 9B6, Canada 3Laser Spine Institute 3001 N Rocky Point Dr. # 185 Tampa, FL 33607 USA Keywords: Amnesic shellfish poisoning; Diatom; Domoic acid; Blooms of toxigenic Pseudo-nitzschia have become more prevalent Excitotoxicity; Neurotoxin; Pseudo-nitzschia along coastal waters worldwide. The 2015 toxic bloom along the entire west coast of North America resulted in numerous harvesting closures Introduction and human health concerns. It is not known why this diatom produces Human consumption of shellfish and certain finfish contaminated domoic acid, as this biotoxin does not appear to harm its immediate with the neurotoxin domoic acid causes Amnesic Shellfish Poisoning predators. (ASP), a syndrome that results in preventable morbidity and mortality Humans become poisoned after consuming molluscan shellfish [1-5]. Although the incidence of ASP is rare around the world due to (e.g., mussels, clams, oysters, scallops, cockles) that have filtered the careful monitoring by government agencies since the original incident toxic diatom cells out of the water, therefore concentrating the toxin in in 1987, patients can still present with clinical symptoms (Table 1) that their digestive system (Figure 2).
    [Show full text]
  • A Human Stem Cell-Derived Test System for Agents Modifying Neuronal N
    Archives of Toxicology (2021) 95:1703–1722 https://doi.org/10.1007/s00204-021-03024-0 IN VITRO SYSTEMS A human stem cell‑derived test system for agents modifying neuronal 2+ N‑methyl‑D‑aspartate‑type glutamate receptor Ca ‑signalling Stefanie Klima1,2 · Markus Brüll1 · Anna‑Sophie Spreng1,3 · Ilinca Suciu1,3 · Tjalda Falt1 · Jens C. Schwamborn4 · Tanja Waldmann1 · Christiaan Karreman1 · Marcel Leist1,5 Received: 28 October 2020 / Accepted: 4 March 2021 / Published online: 13 March 2021 © The Author(s) 2021 Abstract Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neu- roepithelial stem cell (NESC) population, we generate “mixed cortical cultures” (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell- derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intra- 2+ cellular free calcium ion concentration ([Ca ]i) were measured by fuorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range.
    [Show full text]
  • Draft Order No. R3-2018-0017 Npdes No. Ca0048551 Waste Discharge Requirements for the Monterey One Water Regional Wastewater Tr
    DRAFT ORDER NO. R3-2018-0017 NPDES NO. CA0048551 WASTE DISCHARGE REQUIREMENTS FOR THE MONTEREY ONE WATER REGIONAL WASTEWATER TREATMENT PLANT AND ADVANCED WATER PURIFICATION FACILITY DISCHARGE TO THE PACIFIC OCEAN The following Discharger is subject to waste discharge requirements (WDRs) set forth in this Order: Table 1. Discharger Information Discharger Monterey One Water1 Regional Wastewater Treatment Plant (WWTP) and Advanced Water Name of Facility Purification Facility (AWPF), Marina, Monterey County 14811 Del Monte Boulevard Facility Address Marina, CA 93933 Monterey County Table 2. Discharge Location Discharge Effluent Discharge Point Discharge Point Receiving Water Point Description Latitude (North) Longitude (West) Secondary Treated Wastewater, 001 Saline Waste, 36.72778º -121.83750º Pacific Ocean and Reverse Osmosis (RO) Concentrate Disinfected Tertiary 002 Recycled _ _ Reclamation Use Municipal Wastewater 1 Monterey One Water (abbreviated M1W) was formerly called the “Monterey Regional Water Pollution Control Agency.” Prior orders issued for this facility used this name for the Discharger. 1 / 133 Item No. 8 Attachment 1 December 6-7, 2018 Proposed Order No. R3-2018-0017 Table 3. Administrative Information This Order was adopted on: December 6, 2018 This Order shall become effective on: April 1, 2019 This Order shall expire on: November 30, 2023 The Discharger shall file a Report of Waste Discharge as an application for reissuance of WDRs in accordance with title 23, California Code of June 3, 2023 Regulations, and an application for reissuance of a National Pollutant Discharge Elimination System (NPDES) permit no later than: The U.S. Environmental Protection Agency (U.S. EPA) and the California Regional Water Quality Control Board, Central Coast Region have classified Major discharge this discharge as follows: I, John M.
    [Show full text]
  • Toxic Fungi of Western North America
    Toxic Fungi of Western North America by Thomas J. Duffy, MD Published by MykoWeb (www.mykoweb.com) March, 2008 (Web) August, 2008 (PDF) 2 Toxic Fungi of Western North America Copyright © 2008 by Thomas J. Duffy & Michael G. Wood Toxic Fungi of Western North America 3 Contents Introductory Material ........................................................................................... 7 Dedication ............................................................................................................... 7 Preface .................................................................................................................... 7 Acknowledgements ................................................................................................. 7 An Introduction to Mushrooms & Mushroom Poisoning .............................. 9 Introduction and collection of specimens .............................................................. 9 General overview of mushroom poisonings ......................................................... 10 Ecology and general anatomy of fungi ................................................................ 11 Description and habitat of Amanita phalloides and Amanita ocreata .............. 14 History of Amanita ocreata and Amanita phalloides in the West ..................... 18 The classical history of Amanita phalloides and related species ....................... 20 Mushroom poisoning case registry ...................................................................... 21 “Look-Alike” mushrooms .....................................................................................
    [Show full text]
  • Biosynthesis of Cyclic Peptide Natural Products in Mushrooms
    BIOSYNTHESIS OF CYCLIC PEPTIDE NATURAL PRODUCTS IN MUSHROOMS By Robert Michael Sgambelluri A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry & Molecular Biology – Doctor of Philosophy 2017 ABSTRACT BIOSYNTHESIS OF CYCLIC PEPTIDE NATURAL PRODUCTS IN MUSHROOMS By Robert Michael Sgambelluri Cyclic peptide compounds possess properties that make them attractive candidates in the development of new drugs and therapeutics. Mushrooms in the genera Amanita and Galerina produce cyclic peptides using a biosynthetic pathway that is combinatorial by nature, and involves an unidentified, core set of tailoring enzymes that synthesize cyclic peptides from precursor peptides encoded in the genome. The products of this pathway are collectively referred to as cycloamanides, and include amatoxins, phallotoxins, peptides with immunosuppressant activities, and many other uncharacterized compounds. This work aims to describe cycloamanide biosynthesis and its capacity for cyclic peptide production, and to harness the pathway as a means to design and synthesize bioactive peptides and novel compounds. The genomes of Amanita bisporigera and A. phalloides were sequenced and genes encoding cycloamanides were identified. Based on the number of genes identified and their sequences, the two species are shown to have a combined capacity to synthesize at least 51 unique cycloamanides. Using these genomic data to predict the structures of uncharacterized cycloamanides, two new cyclic peptides, CylE and CylF, were identified in A. phalloides by mass spectrometry. Two species of Lepiota mushrooms, previously not known to produce cycloamanides, were also analyzed and shown to contain amatoxins, the toxic cycloamanides responsible for fatal mushroom poisonings. The mushroom Galerina marginata, which also produces amatoxins, was used as a model orgasnism for studying cycloamanide biosynthesis due to its culturability.
    [Show full text]
  • Automated Tissue Culture Cell Fixation and Staining in Microplates
    Application Note Cell Imaging Automated Tissue Culture Cell Fixation and Staining in Microplates Using the EL406™ Combination Washer Dispenser to Prepare Samples for Imaging with the Cytation™3 Cell Imaging Multi-Mode Reader Paul Held Ph. D., Bridget Bishop, and Peter Banks, Ph. D., Applications Department, BioTek Instruments, Inc., Winooski, VT Fluorescence microscopy has traditionally been performed on microscope slides, but there is a growing trend towards the use of 96- and 384-well microplates as this allows greater number of samples to be easily processed and automated. This is certainly true in the field of High Content Screening. Here we describe the use of the EL406™ Combination Washer Dispenser to automate the fixation and staining processes typically used prior to fluorescence imaging. Introduction The hallmark of fluorescent microscopy is the Phalloidin binds to actin at the junction between use of specific mouse monoclonal antibodies subunits; and because this is not a site at which to recognize and bind to cellular structures. many actin-binding proteins bind, most of the The antibody is a marvelous tool of remarkable F-actin in cells is available for phalloidin labeling [1]. selectivity. It can be used to identify and quantify Because fluorescent phalloidin conjugates are not almost any protein in complex biological matrices. permeant to most live cells, like antibodies, It’s use as a fluorescent marker in microscopy dates they require that cells be either fixed and/or back to the pioneering work of Albert Coons permeablized. Labeled phalloidins have similar directly after World War II, where with colleagues, affinity for both large and small filaments and bind he labeled antipneumococcus antibodies with in a stoichiometric ratio of about one phalloidin anthracene isocyanate and thereby made an molecule per actin subunit.
    [Show full text]
  • Interaction of Phalloidin with Actin (Toxin/Cyclic Peptide/Liver Cell Actin/Cytochalasin B/Microfilaments) ANNELIESE M
    Proc. Nat. Acad. Sci. USA Vol. 71, No. 7, pp. 2803-2807, July 1974 Interaction of Phalloidin with Actin (toxin/cyclic peptide/liver cell actin/cytochalasin B/microfilaments) ANNELIESE M. LENGSFELD*, IRMENTRAUT LOWt, THEODOR WIELANDt, PETER DANCKER*, AND WILHELM HASSELBACH* * Departments of Physiologie and t Chemistry, Max-Planck-Institut fur Medizinische Forschung, Heidelberg, Germany Communicated by F. Lynen, April 10, 1974 ABSTRACT Phalloidin, a toxic bicyclic peptide of MATERIALS AND METHODS rapid action from the toadstool, Amanita phalloides, gives rise to polymerization of G-actin to filamentous structures Isolation of Cell Membrane Preparations. Two rats (body (Ph-actin) in a medium of low ionic strength. Ph-actin weights, about 250 g), after a fast of 48 hr, were each poisoned closely resembles the microfilaments found in liver mem- by intravenous injection with 0.4 mg of phalloidin in 1 ml of brane fractions (Ph-filaments) after in vivo or in vitro 0.15 M NaCl, and decapitated 10 min later. The livers were poisoning. Both phalloidin induced filaments are resistant to 0.6 M KI in contrast to F-actin, and become decorated homogenized in 0.25 M sucrose/5 mM Tris * HCl at pH 8.0 and by heavy meromyosin. After preincubation with cytocha- fractionated by centrifugation after the modified method of lasin B significantly fewer actin filaments are observed. Touster (5). The cell membrane fractions were washed two times and stored with either 0.25 mM sucrose per 1 mM Phalloidin, one of the main toxic components of the green Tris - HCl at pH 7.4, or with the Tris * HCl buffer alone.
    [Show full text]
  • Phalloidin, Amanita Phalloides
    Phalloidin, Amanita phalloides sc-202763 Material Safety Data Sheet Hazard Alert Code EXTREME HIGH MODERATE LOW Key: Section 1 - CHEMICAL PRODUCT AND COMPANY IDENTIFICATION PRODUCT NAME Phalloidin, Amanita phalloides STATEMENT OF HAZARDOUS NATURE CONSIDERED A HAZARDOUS SUBSTANCE ACCORDING TO OSHA 29 CFR 1910.1200. NFPA FLAMMABILITY1 HEALTH4 HAZARD INSTABILITY0 SUPPLIER Company: Santa Cruz Biotechnology, Inc. Address: 2145 Delaware Ave Santa Cruz, CA 95060 Telephone: 800.457.3801 or 831.457.3800 Emergency Tel: CHEMWATCH: From within the US and Canada: 877-715-9305 Emergency Tel: From outside the US and Canada: +800 2436 2255 (1-800-CHEMCALL) or call +613 9573 3112 PRODUCT USE Toxic bicyclic heptapeptide (a member of the family of phallotoxins) isolated from the green mushroom, Amanitra phalloides Agaricaceae (the green death cap or deadly agaric). Binds to polymeric actin, stabilising it and interfering with the function of endoplasmic reticulum and other actin-rich structures. NOTE: Advice physician prior to working with phallotoxins. Prepare Emergency procedures. SYNONYMS C35-H48-N8-O11-S, phalloidine, "Amanita phalloides Group I toxin", "Amanita phalloides Group I toxin", "mushroom (green death cap/ deadly agaric) phallotoxin/ peptide", "cyclopeptide/ bicyclic bioactive heptapeptide" Section 2 - HAZARDS IDENTIFICATION CANADIAN WHMIS SYMBOLS EMERGENCY OVERVIEW RISK Very toxic by inhalation, in contact with skin and if swallowed. POTENTIAL HEALTH EFFECTS ACUTE HEALTH EFFECTS SWALLOWED ■ Severely toxic effects may result from the accidental ingestion of the material; animal experiments indicate that ingestion of less than 5 gram may be fatal or may produce serious damage to the health of the individual. ■ At sufficiently high doses the material may be hepatotoxic(i.e.
    [Show full text]