DOCSLIB.ORG

Microcystis Aeruginosa Toxin: Cell Culture Toxicity, Hemolysis, and Mutagenicity Assays W

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Microcystis Aeruginosa Toxin: Cell Culture Toxicity, Hemolysis, and Mutagenicity Assays W APPLIED AND ENVIRONMENTAL MICROBIOLOGY. June 1982, p. 1425-1433 Vol. 43, No. 6 0099-2240/82/061425-09$02.00/0 Microcystis aeruginosa Toxin: Cell Culture Toxicity, Hemolysis, and Mutagenicity Assays W. 0. K. GRABOW,l* W. C. Du RANDT,1 O. W. PROZESKY,2 AND W. E. SCOTT1 National Institute for Water Research, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001,1 and National Institute for Virology, Johannesburg,2 South Africa Received 9 November 1981/Accepted 22 February 1982 Crude toxin was prepared by lyophilization and extraction of toxic Microcystis aeruginosa from four natural sources and a unicellular laboratory culture. The responses of cultures of liver (Mahlavu and PLC/PRF/5), lung (MRC-5), cervix (HeLa), ovary (CHO-Kl), and kidney (BGM, MA-104, and Vero) cell lines to these preparations did not differ significantly from one another, indicating that toxicity was not specific for liver cells. The results of a trypan blue staining test showed that the toxin disrupted cell membrane permeability within a few minutes. Human, mouse, rat, sheep, and Muscovy duck erythrocytes were also lysed within a few minutes. Hemolysis was temperature dependent, and the reaction seemed to follow first-order kinetics. Escherichia coli, Streptococcus faecalis, and Tetrahymena pyriformis were not significantly affected by the toxin. The toxin yielded negative results in Ames/Salmonella mutagenicity assays. Micro- titer cell culture, trypan blue, and hemolysis assays for Microcvstis toxin are described. The effect of the toxin on mammalian cell cultures was characterized by extensive disintegration of cells and was distinguishable from the effects of E. coli enterotoxin, toxic chemicals, and pesticides. A possible reason for the acute lethal effect of Microcystis toxin, based on cytolytic activity, is discussed. Some cellular components of various species number of disturbances in enzymatic systems of algae have toxic, carcinogenic, or mutagenic and metabolic processes have been observed, effects (7, 11, 23, 31, 32, 34, 37). Neither the and it has been concluded that the toxins of blue- chemical nor the toxicological properties of green algae cause degenerative changes in par- these compounds have been fully established (6, enchymatous organs and brain cells (22). Toxins 7, 11). Certain strains of the blue-green alga of blue-green algae have been reported to agglu- (cyanobacterium) Microcystis aeruginosa Kutz tinate erythrocytes (6) and to be cytopathogenic emend Elenkin produce toxins which may con- for cultures of mammalian cells (22). Primary sist of different combinations of a number of fibroblast cultures of human and rat embryo peptide or peptide-containing toxins of unde- cells have proved particularly sensitive, fol- fined structure with hepatotoxic or neurotoxic lowed, in decreasing order of sensitivity, by a rat activities (7, 11, 22). The M. aeruginosa toxin fibroblast cell line and the HeLa, A-8, and HEp- known as microcystin or fast-death factor, 2 cell lines (22). which kills white mice in 1 to 3 h, may consist of This study deals with the response of previ- more than one type of cyclic polypeptide which ously untested cells to Microcystis toxin, the may contain up to 16 amino acids and have a development and evaluation of biological assays molecular weight ranging from 654 to 19,400 (7, for Microcystis toxin, and the testing of toxin for 11, 28, 37). Death of mice and vervet monkeys mutagenic activity by means of the Ames/Sal- injected with Microcystis toxin has been as- monella microsome mutagenicity assay (Ames cribed to circulatory failure as an indirect conse- test). The results contribute to the characteriza- quence of extensive liver damage (10, 37, 38). tion of Microcystis toxin, the establishment of However, the short survival time suggests that practical methods for research on the toxin, and liver damage may not be the primary cause of the understanding of its mechanism of action. death, and indications that neurotoxic activity located in the same fraction as the hepatotoxin MATERIALS AND METHODS or in a different fraction is the cause of death Microcystis toxin. Harvests consisting predominantly have been observed (11). In white rat and mouse of toxic M. aeruginosa were collected in South Africa toxicological studies in which algal toxin prepa- from the Hartbeespoort Dam (near Pretoria) in 1978, rations that probably included microcystin but the Vaal Dam (near Johannesburg) in 1977 and 1980, possibly also other toxins have been used, a and the Roodeplaat Dam (near Pretoria) in 1978 (35, 1425 1426 GRABOW ET AL. APPL. ENVIRON. MICROBIOL. 37). Similar harvests of nontoxic Microcystis orga- of streptomycin and the other containing (per millili- nisms collected at the Hartbeespoort Dam in 1974 and ter) 1 mg each of EDTA (Titriplex III; E. Merck) and 1979 and the Rietvlei Dam (near Pretoria) in 1975 glucose in sodium chloride (8 mg/ml), 0.2 mg of served as negative controls. A unicellular laboratory potassium chloride, 2.89 mg of disodium hydrogen culture of a toxic strain of M. aeruginosa designated phosphate 12 hydrate, and 0.2 mg of potassium hydro- WR70 (35) was propagated as described elsewhere (35; gen phosphate (calcium- and magnesium-free phos- W. E. Scott, D. J. Barlow, and J. H. Hauman, Pro- phate-buffered saline), were mixed together immedi- ceedings ofthe International Conference on the Water ately before being used for the trypsinization of the Environment: Algal Toxins and Health, in press). cells. Freeze-dried samples were thoroughly ground with a Microtiter cell culture assay. The mammalian cell pestle and mortar, suspended in 0.1 M ammonium cultures were trypsinized, and the cells were suspend- bicarbonate (1 g/100 ml), stirred overnight at 10°C, and ed in appropriate volumes of growth medium supple- centrifuged (10,000 x g for 30 min); the toxin-contain- mented with 2 to 10% serum (the harvest from conflu- ing supernatant was then freeze-dried. This material ent growth in 25-cm2 flasks was suspended in 10 to 25 was suspended in distilled water (4 mg/ml) and then ml of medium), and 0.1-ml volumes of these suspen- filtered (Sartorius SM/S13400 [diameter, 47 mm; pore sions were seeded into wells of microtiter plates size, 5 ,m] followed by Sartorius SM/N11306 [diame- (Titertek plates with 96 flat-bottom wells; Flow Labo- ter, 25 mm; pore size, 0.45 pLm]) for testing. ratories). Dilutions of test suspensions (0.1 ml) were Escherichia coli enterotoxin. E. coli strains 18, B7A, added immediately or after 24 h. Sterile distilled water and H10407, which all produce both heat-labile and and similar preparations of nontoxic strains of M. heat-stable enterotoxins (8, 9, 15, 33), were supplied aeruginosa were used in negative controls. After incu- by A. S. Greeff (Institute of Pathology, Pretoria). bation at 37°C in an atmosphere of 5% C02, the cells Cultures were grown in Oxoid Nutrient Broth no. 2 for were studied for cytopathogenic effects. 48 h at 35°C without shaking and centrifuged (10,000 x Hemagglutination and hemolysis tests. Sheep, Mus- g for 30 min); then the supernatant was filtered covy duck, mouse, rat, and human (group A, RH+) through a membrane (pore size, 0.45 pm) for toxicity erythrocytes were washed three times with 0.85% testing. sodium chloride and resuspended in sodium chloride. Cell cultures. The human hepatoma cell lines PLC/ In hemagglutination tests, 1% suspensions (0.1 ml) of PRF/5 (hepatitis B surface antigen positive) (passage erythrocytes were seeded into wells of microtiter 91) and Mahlavu (hepatitis B surface antigen negative) plates (Titertek plates with 96 V-shaped wells; Flow (passage 102) (1, 29, 30) were obtained from J. J. Laboratories), and dilutions of the test suspensions Alexander, R. Saunders, and J. A. Pienaar (National (0.1 ml) were added immediately. Saline and similar Institute for Virology, Johannesburg). The Chinese preparations of nontoxic strains of M. aeruginosa hamster ovary cell line CHO-Kl (passage 14) was were used in negative controls. The plates were cov- purchased from Flow Laboratories, Inc., and the fetal ered and allowed to stand for 3 to 4 h at room rhesus monkey kidney cell line MA-104 and the human temperature. Results were recorded as ++ (no cells embryo lung cell line MRC-5 (passage 27) were pur- settling at the bottom, erythrocyte agglutination), + - chased from M. A. Bioproducts. Cultures of the Vero (partially positive: about half of the cells settling at the (African green monkey kidney) and HeLa (human bottom, the rest remaining diffused), or - - (negative: cervical carcinoma) cell lines were provided by E. M. well-defined spot of erythrocytes settled at the bottom Bey (National Institute for Virology) and J. A. Pien- of the well and clear solution at the top), as described aar. The BGM (African green monkey kidney cell elsewhere (6). In hemolysis tests, equal volumes of lines) and primary vervet kidney cells have been erythrocytes in saline (10% [vol/vol]) and test material described elsewhere (14). Hepatoma, HeLa, MA-104, suspended in saline were mixed and kept at 35°C for 60 Vero, BGM, and primary vervet kidney cells were min. Reaction mixtures were then macrohematocrit grown in Eagle minimal essential medium with Earle centrifuged in Wintrobe tubes (3,000 x g for 3 min at salts EMEM (Auto-Pow; Flow Laboratories) supple- room temperature or 2,260 x g for 30 min at 5°C) (41) mented (per milliliter) with 100 U of penicillin, 100 pg or microhematocrit centrifuged by the method de- of streptomycin, 50 ptg of neomycin, 292 pg of L- scribed previously (41) or in 1.5-ml Eppendorff tubes glutamine, and 2 mg of sodium bicarbonate. Gentamy- with a Heraeus-Christ type 00912 microhematocrit cin (50 ,ug/ml) and tylosin (8 p.g/ml) were occasionally centrifuge for 5 min at room temperature.
Load more

Recommended publications
  • Letter from the Desk of David Challinor December 1992 We Often
    Letter from the Desk of David Challinor December 1992 We often identify poisonous animals as snakes even though no more than a quarter of these reptiles are considered venomous. Snakes have a particular problem that is ameliorated by venom. With nothing to hold its food while eating, a snake can only grab its prey with its open mouth and swallow it whole. Their jaws can unhinge which allows snakes to swallow prey larger in diameter than their own body. Clearly the inside of a snake's mouth and the tract to its stomach must be slippery enough for the prey animal to slide down whole, and saliva provides this lubricant. When food first enters our mouths and we begin to chew, saliva and the enzymes it contains immediately start to break down the material for ease of swallowing. We are seldom aware of our saliva unless our mouths become dry, which triggers us to drink. When confronted with a chocolate sundae or other favorite dessert, humans salivate. The very image of such "mouth­ watering" food and the anticipation of tasting it causes a reaction in our mouths which prepares us for a delightful experience. Humans are not the only animals that salivate to prepare for eating, and this fluid has achieved some remarkable adaptations in other creatures. scientists believe that snake venom evolved from saliva. Why it became toxic in certain snake species and not in others is unknown, but the ability to produce venom helps snakes capture their prey. A mere glancing bite from a poisonous snake is often adequate to immobilize its quarry.
    [Show full text]
  • Role of the Inflammasome in Defense Against Venoms
    Role of the inflammasome in defense against venoms Noah W. Palm and Ruslan Medzhitov1 Department of Immunobiology, and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520 Contributed by Ruslan Medzhitov, December 11, 2012 (sent for review November 14, 2012) Venoms consist of a complex mixture of toxic components that are Large, multiprotein complexes responsible for the activation used by a variety of animal species for defense and predation. of caspase-1, termed inflammasomes, are activated in response Envenomation of mammalian species leads to an acute inflamma- to various infectious and noninfectious stimuli (14). The activa- tory response and can lead to the development of IgE-dependent tion of inflammasomes culminates in the autocatalytic cleavage venom allergy. However, the mechanisms by which the innate and activation of the proenzyme caspase-1 and the subsequent – immune system detects envenomation and initiates inflammatory caspase-1 dependent cleavage and noncanonical (endoplasmic- – fl and allergic responses to venoms remain largely unknown. Here reticulum and Golgi-independent) secretion of the proin am- matory cytokines IL-1β and IL-18, which lack leader sequences. we show that bee venom is detected by the NOD-like receptor fl family, pyrin domain-containing 3 inflammasome and can trigger In addition, activation of caspase-1 leads to a proin ammatory cell death termed pyroptosis. The NLRP3 inflammasome con- activation of caspase-1 and the subsequent processing and uncon- “ ” ventional secretion of the leaderless proinflammatory cytokine sists of the sensor protein NLRP3, the adaptor apoptosis-as- sociated speck-like protein (ASC) and caspase-1. Damage to IL-1β in macrophages.
    [Show full text]
  • Venom Week 2012 4Th International Scientific Symposium on All Things Venomous
    17th World Congress of the International Society on Toxinology Animal, Plant and Microbial Toxins & Venom Week 2012 4th International Scientific Symposium on All Things Venomous Honolulu, Hawaii, USA, July 8 – 13, 2012 1 Table of Contents Section Page Introduction 01 Scientific Organizing Committee 02 Local Organizing Committee / Sponsors / Co-Chairs 02 Welcome Messages 04 Governor’s Proclamation 08 Meeting Program 10 Sunday 13 Monday 15 Tuesday 20 Wednesday 26 Thursday 30 Friday 36 Poster Session I 41 Poster Session II 47 Supplemental program material 54 Additional Abstracts (#298 – #344) 61 International Society on Thrombosis & Haemostasis 99 2 Introduction Welcome to the 17th World Congress of the International Society on Toxinology (IST), held jointly with Venom Week 2012, 4th International Scientific Symposium on All Things Venomous, in Honolulu, Hawaii, USA, July 8 – 13, 2012. This is a supplement to the special issue of Toxicon. It contains the abstracts that were submitted too late for inclusion there, as well as a complete program agenda of the meeting, as well as other materials. At the time of this printing, we had 344 scientific abstracts scheduled for presentation and over 300 attendees from all over the planet. The World Congress of IST is held every three years, most recently in Recife, Brazil in March 2009. The IST World Congress is the primary international meeting bringing together scientists and physicians from around the world to discuss the most recent advances in the structure and function of natural toxins occurring in venomous animals, plants, or microorganisms, in medical, public health, and policy approaches to prevent or treat envenomations, and in the development of new toxin-derived drugs.
    [Show full text]
  • The Effect of Agkistrodon Contortrix and Crotalus Horridus Venom Toxicity on Strike Locations with Live Prey
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Honors Theses, University of Nebraska-Lincoln Honors Program 5-2021 The Effect of Agkistrodon Contortrix and Crotalus Horridus Venom Toxicity on Strike Locations with Live Prey. Chase Giese University of Nebraska-Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/honorstheses Part of the Animal Experimentation and Research Commons, Higher Education Commons, and the Zoology Commons Giese, Chase, "The Effect of Agkistrodon Contortrix and Crotalus Horridus Venom Toxicity on Strike Locations with Live Prey." (2021). Honors Theses, University of Nebraska-Lincoln. 350. https://digitalcommons.unl.edu/honorstheses/350 This Thesis is brought to you for free and open access by the Honors Program at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Honors Theses, University of Nebraska-Lincoln by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. THE EFFECT OF AGKISTRODON COTORTRIX AND CROTALUS HORRIDUS VENOM TOXICITY ON STRIKE LOCATIONS WITH LIVE PREY by Chase Giese AN UNDERGRADUATE THESIS Presented to the Faculty of The Environmental Studies Program at the University of Nebraska-Lincoln In Partial Fulfillment of Requirements For the Degree of Bachelor of Science Major: Fisheries and Wildlife With the Emphasis of: Zoo Animal Care Under the Supervision of Dennis Ferraro Lincoln, Nebraska May 2021 1 Abstract THE EFFECT OF AGKISTRODON COTORTRIX AND CROTALUS HORRIDUS VENOM TOXICITY ON STRIKE LOCATIONS WITH LIVE PREY Chase Giese, B.S. University of Nebraska, 2021 Advisor: Dennis Ferraro This paper aims to uncover if there is a significant difference in the strike location of snake species that have different values of LD50% venom.
    [Show full text]
  • Snake Venom in Relation to Haemolysis, Bacteriolysis, and Texoicity
    SNAKE VENOI~ IN RELATION TO H2EMOLYSIS, BACTERIOLYSIS, AND TOXICITY. BY SIMON FLEXNER, M.D., AND HIDEYO NOGUCHI, M.D. (From the -Pathological Laboratory of the University of Pennsylvania.) CONTENTS. PA.GE. INTRODUCTION. GENERAL CONSIDERATIONS CONCERNING H~MOLYSIS AND BAC- TERIOLYSIS .......................................................... 277 VENOM-.AGGLUTINATIO~ .................................................... 283 VENOM-H-~EMOLYSIS ........................................................ 284 Defibrinated blood .................................................. 285 Effect of heat upon hmmolytic power of venoms ........................ 286 Effect of venoms upon ~vashcd blood-corpuscles ........................ 286 Combined action of venom and ricin. Relation of agglutination and h~emolysis ........................................................ 289 VENOM-LEUCOLYSIS ......................................... ~ .............. 289 Are the h~emolysins (erythrolysins) identical with leucolysins ? .......... 291 VENoM-ToxXcXTY .......................................................... 291 Relation of neurotoxie to h~emolytic principle ......................... 292 EFFECTS OF VENOM UPON BACTERICIDAL PROPERTIES OF BLOOD SERUM ...... 294 Serum venomized in vivo ............................................. 294 Blood mixed with venom in vitro ...................................... 295 The mechanism of the action of venom upon serum .................... 298 EFFECTS OF ANTIVENIN ON H~EMOLYSIS AND BACTERIOLYSIS ................. 300 INTRODUCTION.
    [Show full text]
  • Snake and Spider Toxins Induce a Rapid Recovery of Function of Botulinum Neurotoxin Paralysed Neuromuscular Junction
    Article Snake and Spider Toxins Induce a Rapid Recovery of Function of Botulinum Neurotoxin Paralysed Neuromuscular Junction Elisa Duregotti 1, Giulia Zanetti 1, Michele Scorzeto 1, Aram Megighian 1, Cesare Montecucco 1,2, Marco Pirazzini 1,* and Michela Rigoni 1,* Received: 23 October 2015; Accepted: 30 November 2015; Published: 8 December 2015 Academic Editor: Wolfgang Wüster 1 Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35131 Padova, Italy; [email protected] (E.D.); [email protected] (G.Z.); [email protected] (M.S.); [email protected] (A.M.); [email protected] (C.M.) 2 Institute for Neuroscience, National Research Council, Via U. Bassi 58/B, 35131 Padova, Italy * Correspondence: [email protected] (M.P.); [email protected] (M.R.); Tel.: +39-049-827-6057 (M.P.); +39-049-827-6077 (M.R.); Fax: +39-049-827-6049 (M.P. & M.R.) Abstract: Botulinum neurotoxins (BoNTs) and some animal neurotoxins (β-Bungarotoxin, β-Btx, from elapid snakes and α-Latrotoxin, α-Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis.
    [Show full text]
  • Snake Venom Detection Kit (SVDK)
    SVDK Template In non-urgent situations, serum or plasma may also be used. Other samples such as lymphatic fluid, tissue fluid or extracts may 8. Reading Colour Reactions be used. • Place the test strip on the template provided over page and observe each well continuously over the next 10 minutes whilst the colour develops. Any test sample used in the SVDK must be mixed with Yellow Sample Diluent (YSD-yellow lid), prior to introduction into the The first well to show visible colour, not including the positive control well, is assay. Samples mixed with YSD should be clearly labelled with the patient’s identity and the type of sample used. The volume of diagnostic of the snake’s venom immunotype – see interpretation below. YSD in each sample vial is sufficient to allow retesting of the sample or referral to a reference laboratory for further investigation. Well 1 Tiger Snake Immunotype Snake Venom Detection Kit (SVDK) Note: Strict adherence to the 10 minute observation period after addition of Tiger Snake Antivenom Indicated Detection and Identification of Snake Venom SAMPLE PREPARATION the Chromogen and Peroxide Solutions is essential. Slow development of 1. Prepare the Test Sample. colour in one or more wells after 10 minutes should not be interpreted ENZYME IMMUNOASSAY METHOD • Any test sample used in the SVDK must be mixed with Yellow Sample Diluent (YSD-yellow lid), prior to introduction as positive detection of snake venom. Well 2 Brown Snake Immunotype into the assay. INTERPRETATION OF RESULTS Brown Snake Antivenom Indicated Note: There is enough YSD in one vial to perform two snake venom detection tests.
    [Show full text]
  • Bioactive Mimetics of Conotoxins and Other Venom Peptides
    Toxins 2015, 7, 4175-4198; doi:10.3390/toxins7104175 OPEN ACCESS toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Review Bioactive Mimetics of Conotoxins and other Venom Peptides Peter J. Duggan 1,2,* and Kellie L. Tuck 3,* 1 CSIRO Manufacturing, Bag 10, Clayton South, VIC 3169, Australia 2 School of Chemical and Physical Sciences, Flinders University, Adelaide, SA 5042, Australia 3 School of Chemistry, Monash University, Clayton, VIC 3800, Australia * Authors to whom correspondence should be addressed; E-Mails: [email protected] (P.J.D.); [email protected] (K.L.T.); Tel.: +61-3-9545-2560 (P.J.D.); +61-3-9905-4510 (K.L.T.); Fax: +61-3-9905-4597 (K.L.T.). Academic Editors: Macdonald Christie and Luis M. Botana Received: 2 September 2015 / Accepted: 8 October 2015 / Published: 16 October 2015 Abstract: Ziconotide (Prialt®), a synthetic version of the peptide ω-conotoxin MVIIA found in the venom of a fish-hunting marine cone snail Conus magnus, is one of very few drugs effective in the treatment of intractable chronic pain. However, its intrathecal mode of delivery and narrow therapeutic window cause complications for patients. This review will summarize progress in the development of small molecule, non-peptidic mimics of Conotoxins and a small number of other venom peptides. This will include a description of how some of the initially designed mimics have been modified to improve their drug-like properties. Keywords: venom peptides; toxins; conotoxins; peptidomimetics; N-type calcium channel; Cav2.2 1. Introduction A wide range of species from the animal kingdom produce venom for use in capturing prey or for self-defense.
    [Show full text]
  • (Oxyuranus) and Brown Snakes (Pseudonaja) Differ in Composition of Toxins Involved in Mammal Poisoning
    bioRxiv preprint doi: https://doi.org/10.1101/378141; this version posted July 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Venoms of related mammal-eating species of taipans (Oxyuranus) and brown snakes (Pseudonaja) differ in composition of toxins involved in mammal poisoning Jure Skejic1,2,3*, David L. Steer4, Nathan Dunstan5, Wayne C. Hodgson2 1 Department of Biochemistry and Molecular Biology, BIO21 Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia 2 Monash Venom Group, Department of Pharmacology, Monash University, 9 Ancora Imparo Way, Clayton, Victoria 3800, Australia 3 Laboratory of Evolutionary Genetics, Division of Molecular Biology, Ruder Boskovic Institute, Bijenicka 54, 10000 Zagreb, Croatia 4 Monash Biomedical Proteomics Facility, Monash University, 23 Innovation Walk, Clayton, Victoria 3800, Australia 5 Venom Supplies Pty Ltd., Stonewell road, Tanunda, South Australia 5352, Australia * Correspondence: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/378141; this version posted July 26, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Background Differences in venom composition among related snake lineages have often been attributed primarily to diet. Australian elapids belonging to taipans (Oxyuranus) and brown snakes (Pseudonaja) include a few specialist predators as well as generalists that have broader dietary niches and represent a suitable model system to investigate this assumption. Here, shotgun high-resolution mass spectrometry (Q Exactive Orbitrap) was used to compare venom proteome composition of several related mammal-eating species of taipans and brown snakes.
    [Show full text]
  • Venom Toxins: Plausible Evolution from Digestive Enzymes1
    AMER. ZOOL., 23:427-430 (1983) Venom Toxins: Plausible Evolution from Digestive Enzymes1 ELAZAR KOCHVA,2 ORA NAKAR, AND MICHAEL OVADIA Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel SYNOPSIS Some hydrolytic enzymes are common to the pancreas, the mammalian salivary glands and the snake venom glands. Phospholipase A, which is found in elapid and viperid venoms and in the mammalian pancreas, shows 29 common amino acid residues out of Downloaded from https://academic.oup.com/icb/article/23/2/427/302344 by guest on 23 September 2021 118-125 positions. Presynaptic neurotoxins and other venom toxins are usually composed of 2-3 units or subumts, one of which is a phospholipase. The Vipera palaestmae two- component toxin retains its lethality when the enzyme is replaced by heterologous venom phospholipases, but not by the pig pancreatic enzyme. This toxin is neutralized by a factor found in the blood serum of snakes, which binds to the phospholipase and inhibits its activity. The blood serum of snakes also neutralizes hemorrhagins and inhibits the protease activity of the venom. It is hypothesized that the developing venom glands first produced enzymes that were already secreted by the pancreas and against which inhibitors were present in the blood. These inhibitors facilitated the evolution of enzyme-based toxins by neutralizing any damaging substances that might have escaped from the venom glands. It has been suggested that enzymes pre- certain elapid venom enzymes (Table 1). ceded toxins during the evolution of the Phospholipases from Viperidae venoms are venom glands in snakes (Strydom, 1977; the most divergent not only in certain areas Gans, 1978) possibly aiding in the digestion of their sequence, but also in the presence of prey and preventing its putrefaction of a seven residue COOH-terminal exten- (Thomas and Pough, 1979; Pough and sion (Heinrikson et al., 1977).
    [Show full text]
  • Snake Presynaptic Neurotoxins with Phospholipase A2 Activity Induce Punctate Swellings of Neurites and Exocytosis of Synaptic Vesicles
    Research Article 3561 Snake presynaptic neurotoxins with phospholipase A2 activity induce punctate swellings of neurites and exocytosis of synaptic vesicles Michela Rigoni1, Giampietro Schiavo2, Anne E. Weston2, Paola Caccin1, Federica Allegrini1, Maria Pennuto3, Flavia Valtorta3, Cesare Montecucco1 and Ornella Rossetto1,* 1Department of Biomedical Sciences and CNR Institute of Neuroscience, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy 2Molecular NeuroPathoBiology Laboratory, Cancer Research UK, London Research Institute, 61 Lincoln’s Inn Fields, London, WC2A 3PX, UK 3Department of Neuroscience, S. Raffaele Scientific Institute and ‘Vita-Salute’ University, Via Olgettina 58, 20132 Milan, Italy *Author for correspondence (e-mail: [email protected]) Accepted 11 March 2004 Journal of Cell Science 117, 3561-3570 Published by The Company of Biologists 2004 doi:10.1242/jcs.01218 Summary The mechanisms of action of four snake presynaptic observed in neurons, but not in astrocytes. These findings phospholipase A2 neurotoxins were investigated in indicate that these snake presynaptic neurotoxins act with cultured neurons isolated from various parts of the rat by same mechanism and that the observed phenotype brain. Strikingly, physiological concentrations of notexin, results from the fusion of synaptic vesicles with the plasma β-bungarotoxin, taipoxin or textilotoxin induced a dose- membrane not balanced by an adequate membrane dependent formation of discrete bulges at various sites of retrieval. These changes closely resemble those occurring neuronal projections. Neuronal bulging was paralleled by at neuromuscular junctions of intoxicated animals and the redistribution of the two synaptic vesicle markers fully qualify these primary neuronal cultures as pertinent synaptophysin I (SypI) and vesicle-attached membrane models for studying the molecular mode of action of these protein 2 (VAMP2) to the bulges, and by the exposure of neurotoxins.
    [Show full text]
  • In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: the Window of Opportunity
    toxins Article In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: The Window of Opportunity Umesha Madhushani 1, Geoffrey K. Isbister 2 , Theo Tasoulis 2, Wayne C. Hodgson 3 and Anjana Silva 1,3,* 1 Department of Parasitology, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, Mihintale 50300, Sri Lanka; [email protected] 2 Clinical Toxicology Research Group, University of Newcastle, Callaghan 2308, Australia; geoff[email protected] (G.K.I.); [email protected] (T.T.) 3 Monash Venom Group, Department of Pharmacology, Biomedical Discovery Institute, Monash University, Clayton 3800, Australia; [email protected] * Correspondence: [email protected] Received: 15 October 2020; Accepted: 30 October 2020; Published: 31 October 2020 Abstract: Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL.
    [Show full text]