DOCSLIB.ORG

26-4125I]Iodoponasterone a Is a Potent Ecdysone and a Sensitive

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
26-4125I]Iodoponasterone a Is a Potent Ecdysone and a Sensitive Proc. Nail. Acad. Sci. USA Vol. 85, pp. 2096-2100, April 1988 Biochemistry 26-4125I]Iodoponasterone A is a potent ecdysone and a sensitive radioligand for ecdysone receptors (steroid hormone/26-iodoponasterone A/Drosophila/Kc cells) PETER CHERBAS*t, Lucy CHERBAS*, SHOEI-SHENG LEEt§, AND Koji NAKANISHIt *Department of Biology and Program in Molecular, Cellular and Developmental Biology, Indiana University, Bloomington, IN 47405; and tDepartment of Chemistry, Columbia University, New York, NY 10027 Communicated by Carroll M. Williams, December 7, 1987 ABSTRACT The effects of ecdysone, the steroid molting introduction of [3H]ponasterone A (>100 Ci/mmol; 1 Ci = hormone of arthropods, are of considerable interest both to 37 GBq) has been critical to recent progress in the study of insect physiologists and to those studying steroid-regulated ecdysone receptors (31, 36, 38). Still, this material has not gene expression. Yet progress in understanding ecdysone been widely available and, given the small amounts of receptors has been inhibited by the lack of a suitable highly biological material that often must be assayed, is still mar- radioactive hormone analog with high affinity for the receptor. ginal in its specific activity. Here we report that the synthetic Here we report that the synthetic ecdysteroid 26-iodoponaste- analog 26-iodoponasterone A (called hereafter iodoponaste- rone A is one of the most active ecdysones known, inducing rone) is still more active biologically than ponasterone A, half-maximal morphological transformation in Drosophila that it can be synthesized readily with carrier-free 125I, and Kc167 cells when present at 0.5 nM. 26-[12'I5Iodoponasterone that the radiolabeled material (2175 Ci/mmol) functions well A can be prepared at a specific activity of 2175 Ci/mmol (1 Ci as a ligand for the detection of ecdysone receptors. = 37 GBq) by reaction of the precursor 26-mesylinokosterone with carrier-free Na'25I. The radiolabeled material binds to Kc167 cell ecdysone receptors specifically and with affinity (Kd MATERIALS AND METHODS ca. 3.8 x 10-10 M). Thus, 26-['2I]iodoponasterone A ap- Cells. Kc167 cells were a gift from M. Bourouis (43). We pears to be a superior radioligand for ecdysone receptors on have adapted them to growth in M3 medium (44) supple- grounds both of affinity and of specific activity. Its ready mented with 5% fetal calf serum. availability should greatly facilitate studies of these receptors. Bioassay. Ecdysones were bioassayed by their activity in inducing morphological transformation of Kc cells (42). The Steroid hormone receptors are generally detected by the use assay procedure was that described in ref. 42 save that of high-affinity radioligands. Because such receptors are Kc167 cells were used. Ecdysteroids to be tested were present at very low concentrations (typically 1-10 ppm of dissolved at known concentrations in ethanol [assuming 6242 cellular protein), a useful ligand must exhibit not only high (ethanol) = 12,400 (45)], serially diluted, and tested in 1-ml affinity for the receptor but also unusually high radiological cell cultures. 20-Hydroxyecdysone, 26-mesylinokosterone, specific activity. Indeed, the development and use of such and iodoponasterone were checked for purity by HPLC (see radioligands has played an important role in the advance- below) and assayed in parallel at concentrations ranging ment of our knowledge of the vertebrate steroid receptors from 10 ,uM to 10 pM. Bioassays of 125I-substituted steroid (1-7). were carried out similarly. Dilutions of [1251]iodoponaste- Ecdysone,$ the steroid molting hormone of arthropods, rone were prepared (assuming a counting efficiency of 50% regulates the timing of development and metamorphosis at for 125I in solution), and cells were treated in parallel with it least in part by its effects on gene expression, effects that are and with a series of concentrations of 20-hydroxyecdysone. under study in numerous laboratories (9-29). Nevertheless, The cultures were compared qualitatively under the micro- our knowledge of the molecular biology and physiology of ecdysone receptors is still fragmentary. Ecdysone-binding scope, the iodoponasterone cultures being assigned posi- proteins thought to be ecdysone receptors have been iden- tions in the series, and then the 20-hydroxyecdysone series tified in ecdysone-responsive Drosophila tissues and cells in was scored in the usual way. culture. These receptors are present at low abundance (ca. Steroids. 20-Hydroxyecdysone and muristerone were pur- 1-2 x 103 molecules per cell); they exhibit high-affinity chased from Simes (Milan). Ponasterone A was the purified binding to active ecdysteroids, the relative affinities being natural product (40). Inokosterone was a gift from T. Take- approximately in proportion to relative ecdysone activities moto (Tohoku University, Sendai, Japan). All were homo- (9, 30-38); and their concentration and/or activity is de- geneous by HPLC save the ponasterone A, which was pressed in ecdysone-resistant tissue culture cells (K. Wing, estimated to be ca. 85% pure. 26-Mesylinokosterone and personal communication). Similar receptors have been iden- iodoponasterone were synthesized from inokosterone. De- tified from hypodermal cells of the crayfish Orconectes (39). tails of the syntheses and physical data for these compounds Because the natural hormone, 20-hydroxyecdysone, is will be reported elsewhere (S.-S.L., K.N., and P.C., unpub- effective only at relatively high concentration (ca. 10 nM) lished data; inquiries concerning the availability of both and its receptor complex is kinetically unstable, success in compounds should be directed to S.-S.L.). studies of the receptor(s) has relied upon use of a more potent analog, the phytoecdysone ponasterone A (40, 41), tTo whom reprint requests should be addressed. which is 10-50 times as active in biological assays and which §Present address: School of Pharmacy, College of Medicine, Na- is comparably more active as a ligand (30-38, 42). Thus, the tional Taiwan University, Taipei 100, Taiwan, Republic of China. IEcdysteroid is the generic name for compounds structurally related to a-ecdysone (8). We use ecdysone as the generic name for The publication costs of this article were defrayed in part by page charge compounds with the appropriate biological activity in accordance payment. This article must therefore be hereby marked "advertisement" with common usage in the literature and in analogy with the terms in accordance with 18 U.S.C. §1734 solely to indicate this fact. estrogen or progestin (9). Downloaded by guest on September 24, 2021 20% Biochemistry: Cherbas et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2097 ["'Il]Iodoponasterone Synthesis. Radiolabeled iodopo- Cell Extracts. Whole-cell extracts have been prepared by nasterone was prepared from the mesyl intermediate as procedures slightly modified from those of ref. 36. Data are follows. The label was purchased as carrier-free Na125I (2175 shown for two extracts similarly prepared. Kc167 cells were Ci/mmol in dilute base free of reducing agents; Amersham) harvested at a density of 2 x 107 cells per ml, washed in lots of 1 or 2 mCi. It was transferred from the shipping vial extensively in saline, and sonicated in an ice-water bath in to a 100-,ul conical glass reaction vial by using dry acetone, 1.25 ml of buffer A [40 mM Hepes, pH 7.0 at 230C/1 mM which subsequently was evaporated under a stream of dry dithiothreitol/5% (vol/vol) glycerol]. The sonicate was ad- nitrogen gas. (To prepare dry solvents, reagent grade ace- justed to 0.4 M KCI by addition of 2.0 M KCI and incubated tone and acetonitrile in lots of a few milliliters were perco- for 5 min at 0C. Finally the extract was centrifuged for 50 lated through 2-ml columns of silica gel immediately before min at 20,000 rpm in a Beckman JA 20 rotor. The superna- use.) 26-Mesylinokosterone (19 nmol/mCi of 1251I) was added, tant was immediately diluted 1:9 with cold buffer A to bring and residual water was partially eliminated by repeatedly the extract to 40 mM KCI in buffer A. Extract 1 (used in the dissolving the mixture in a small volume of dry acetonitrile experiment of Fig. 3) contained 210 gg of protein per 100 ,ul; and evaporating. Finally the mixture was dissolved in dry extract 2 (experiments of Figs. 4 and 5) contained 380 ,ug of acetone (20 tkl/mCi of 1251) as reaction solvent, and the protein per 100 jLd (Bio-Rad protein assay). Extracts were reaction vessel was capped tightly and heated to 800C for 8-12 immediately frozen (liquid nitrogen) in 2-ml aliquots and hr. At the conclusion the acetone was evaporated, and the stored at - 90'C. Receptor activity remained constant under reaction mixture was dissolved in ca. 10 jul of methanol and these conditions for a period of at least 2 months. purified by HPLC. The major radioactive product was eluted Ligand Binding by Extracts. Experimental tubes contained at ca. 7 ml and cochromatographed with authentic iodopo- 0.1 volume (typically 14 ILI) of [1251]iodoponasterone in nasterone both by HPLC and by thin-layer chromatography buffer A, 0.1 volume of additional buffer A or competitor on silica gel. Typical yields have been 12-15%, based on input A, and 1 volume of extract. As the 1251 (six syntheses). Three lots of [1251]iodoponasterone were steroid dissolved in buffer used in the experiments described here. Each was checked by competitor steroid, we used muristerone (42)-the most HPLC and found to be >98% radiochemically pure; residual potent ecdysone available to us in quantity-at a final 26-mesylinokosterone was undetectable by absorbance. concentration of 4.5 ,uM. Binding reactions were carried out Iodoponasterone solutions are quite stable both radio- at room temperature for 1 hr. chemically and chemically when stored over a period of at Aliquots (10 pll) of each tube were removed and assayed to least 4 months in ethanol at - 20'C.
Load more

Recommended publications
  • Ecdysone: Structures and Functions Guy Smagghe Editor
    Ecdysone: Structures and Functions Guy Smagghe Editor Ecdysone: Structures and Functions Editor Guy Smagghe Laboratory of Agrozoology Faculty of Bioscience Engineering Ghent University Belgium ISBN 978-1-4020-9111-7 e-ISBN 978-1-4020-9112-4 Library of Congress Control Number: 2008938015 © 2009 Springer Science + Business Media B.V. No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper springer.com Preface The 16th International Ecdysone Workshop took place at Ghent University in Belgium, July 10–14, 2006 and drew some 150 attendees, many of these young students and postdoctoral associates. These young scientists had the opportunity to dis- cuss their work with many senior scientists at meals, breaks and during the several social events, and were encouraged to do so. This book resulting from the meeting is more up-to-date than might be expected since manuscripts were not delivered to the editor until 2007. The workshop itself had 54 oral presentations as well as many posters. This book, and the meeting itself, is comprised of 23 contributed chapters falling into five general categories: Fundamental Aspects of Ecdysteroid Research: The Distribution and Diversity of Ecdysteroids in Animals and Plants; Ecdysteroid Genetic Hierarchies in Insect Growth and Reproduction; Role of Cross Talk and Growth Factors in Ecdysteroid Titers and Signaling; Ecdysteroid Function Through Nuclear and Membrane Receptors; Ecdysteroids in Modern Agriculture, Medicine, Doping and Ecotoxicology.
    [Show full text]
  • Nuclear Receptor Ftz-F1 Promotes Follicle Maturation and Ovulation
    RESEARCH ARTICLE Nuclear receptor Ftz-f1 promotes follicle maturation and ovulation partly via bHLH/PAS transcription factor Sim Elizabeth M Knapp1, Wei Li1, Vijender Singh2, Jianjun Sun1,2* 1Department of Physiology & Neurobiology, University of Connecticut, Storrs, United States; 2Institute for Systems Genomics, University of Connecticut, Storrs, United States Abstract The NR5A-family nuclear receptors are highly conserved and function within the somatic follicle cells of the ovary to regulate folliculogenesis and ovulation in mammals; however, their roles in Drosophila ovaries are largely unknown. Here, we discover that Ftz-f1, one of the NR5A nuclear receptors in Drosophila, is transiently induced in follicle cells in late stages of oogenesis via ecdysteroid signaling. Genetic disruption of Ftz-f1 expression prevents follicle cell differentiation into the final maturation stage, which leads to anovulation. In addition, we demonstrate that the bHLH/PAS transcription factor Single-minded (Sim) acts as a direct target of Ftz-f1 to promote follicle cell differentiation/maturation and that Ftz-f1’s role in regulating Sim expression and follicle cell differentiation can be replaced by its mouse homolog steroidogenic factor 1 (mSF-1). Our work provides new insight into the regulation of follicle maturation in Drosophila and the conserved role of NR5A nuclear receptors in regulating folliculogenesis and ovulation. Introduction *For correspondence: [email protected] Female fertility, an essential half of the reproductive equation, requires proper follicle maturation and ovulation. The NR5A family of nuclear receptors are critical for the success of these complex Competing interests: The ovarian processes across species (Jeyasuria et al., 2004; Meinsohn et al., 2019; Mlynarczuk et al., authors declare that no 2013; Sun and Spradling, 2013; Suresh and Medhamurthy, 2012).
    [Show full text]
  • Characterization of the Novel Role of Ninab Orthologs from Bombyx Mori and Tribolium Castaneum T
    Insect Biochemistry and Molecular Biology 109 (2019) 106–115 Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb Characterization of the novel role of NinaB orthologs from Bombyx mori and Tribolium castaneum T Chunli Chaia, Xin Xua, Weizhong Sunb, Fang Zhanga, Chuan Yea, Guangshu Dinga, Jiantao Lia, ∗ Guoxuan Zhonga,c, Wei Xiaod, Binbin Liue, Johannes von Lintigf, Cheng Lua, a State Key Laboratory of Silkworm Genome Biology, Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of Biotechnology, Southwest University, Chongqing 400715, China b College of Animal Science and Technology, Southwest University, Chongqing 400715, China c Life Sciences Institute and the Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou 310058, China d College of Plant Protection, Southwest University, Chongqing 400715, China e Sericulture Research Institute, Sichuan Academy of Agricultural Science, Chengdu 610066, China f Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106, USA ABSTRACT Carotenoids can be enzymatically converted to apocarotenoids by carotenoid cleavage dioxygenases. Insect genomes encode only one member of this ancestral enzyme family. We cloned and characterized the ninaB genes from the silk worm (Bombyx mori) and the flour beetle (Tribolium castaneum). We expressed BmNinaB and TcNinaB in E. coli and analyzed their biochemical properties. Both enzymes catalyzed a conversion of carotenoids into cis-retinoids. The enzymes catalyzed a combined trans to cis isomerization at the C11, C12 double bond and oxidative cleavage reaction at the C15, C15′ bond of the carotenoid carbon backbone. Analyses of the spatial and temporal expression patterns revealed that ninaB genes were differentially expressed during the beetle and moth life cycles with high expression in reproductive organs.
    [Show full text]
  • Molecular Biology of Bhlh PAS Genes Involved in Dipteran Juvenile Hormone Signaling
    Molecular Biology of bHLH PAS Genes Involved in Dipteran Juvenile Hormone Signaling Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Aaron A. Baumann. B.S. Graduate Program in Entomology The Ohio State University 2010 Dissertation Committee: Thomas G. Wilson, Advisor David Denlinger H. Lisle Gibbs Amanda Simcox Copyright by Aaron A. Baumann 2010 Abstract Methoprene tolerant (Met), a member of the bHLH-PAS family of transcriptional regulators, has been implicated in juvenile hormone (JH) signaling in Drosophila melanogaster. Met mutants are resistant to the toxic and morphogenetic defects of exogenous JH application. A paralogous gene in D. melanogaster, germ cell expressed (gce), forms JH-sensitive heterodimers with MET, but a function for gce has not been reported. DmMet orthologs from three mosquito species are characterized and, based on sequence analysis and intron position, are shown to have higher sequence identity to Dmgce than to DmMet. An evolutionary scheme for the origin of Met from a gce-like ancestor gene in lower Diptera is proposed. RNAi-driven underexpression of Met in the Yellow Fever mosquito, Aedes aegypti, results in the concomitant reduction of putative JH-inducible genes, suggesting involvement in JH signaling. The viability of D. melanogaster Met mutants is thought to result from functional redundancy conferred by gce. Therefore, genetic manipulation of gce expression was used to probe the function of this gene. Overexpression of gce was shown to alleviate preadult, but not adult Met phenotypes. RNAi-driven underexpression of gce resulted in ii preadult lethality in both Met+ and Met mutant backgrounds.
    [Show full text]
  • STATE of the ART ASSESSMENT of ENDOCRINE DISRUPTERS Final Report
    STATE OF THE ART ASSESSMENT OF ENDOCRINE DISRUPTERS Final Report Project Contract Number 070307/2009/550687/SER/D3 Authors: Andreas Kortenkamp, Olwenn Martin, Michael Faust, Richard Evans, Rebecca McKinlay, Frances Orton and Erika Rosivatz 23.12.2011 TABLE OF CONTENTS TABLE OF CONTENTS 0 EXECUTIVE SUMMARY ......................................................................................................................... 7 1 INTRODUCTION .................................................................................................................................... 9 1.1 TERMS OF REFERENCE, SCOPE OF THE REPORT ........................................................................... 9 1.2 STRUCTURE OF THE REPORT ....................................................................................................... 11 2 DEFINITION OF ENDOCRINE DISRUPTING CHEMICALS ...................................................................... 13 2.1 THE ENDOCRINE SYSTEM ............................................................................................................ 13 2.2 ADVERSITY ................................................................................................................................... 15 2.2.1 DEFINITION........................................................................................................................... 15 2.2.2 ASSAY REQUIREMENTS ........................................................................................................ 16 2.2.3 ECOTOXICOLOGICAL EFFECTS .............................................................................................
    [Show full text]
  • Compared Activity of Agonist Molecules Towards Ecdysone
    PESTICIDES / SCIENTIFIC COMMUNICATION DOI: 10.1590/1808-1657000312019 Compared activity of agonist molecules towards ecdysone receptor in insect cell-based screening system Comparação da atividade de moléculas agonistas em relação ao receptor de ecdisona em um sistema de triagem baseado em linhagens celulares de insetos Ciro Pedro Guidotti Pinto1,2* , Letícia Neutzling Rickes2 , Moisés João Zotti2 , Anderson Dionei Grutzmacher2 ABSTRACT: The ecdysone receptor, naturally activated by ste- RESUMO: O receptor de ecdisona, naturalmente ativado por hor- roidal hormones, is a key protein for molting and reproduction mônios esteroidais, é uma proteína-chave nos processos de muda e processes of insects. Artificial activation of such receptor by specific reprodução de insetos. A ativação artificial desse receptor por meio de pesticides induces an anomalous process of ecdysis, causing death pesticidas específicos induz um processo de ecdise anômala, levando o of insects by desiccation and starvation. In this paper, we establi- inseto à morte por dessecação e inanição. Neste trabalho, foi estabele- shed a protocol for screening agonistic molecules towards ecdysone cido um protocolo para a triagem de moléculas agonistas em relação ao receptor of insect cells line S2 (Diptera) and Sf9 (Lepidoptera), receptor de ecdisona nas linhagens celulares responsivas S2 (Diptera) e transfected with the reporter plasmid ere.b.act.luc. Therefore, we Sf9 (Lepidoptera), transfectadas com o plasmídeo repórter ere.b.act.luc. set dose-response curves with the ecdysteroid 20-hydroxyecdysone, Para tanto, curvas de dose-resposta foram estabelecidas com o ecdiste- the phytoecdysteroid ponasterone-A, and tebufenozide, a pesticide roide 20-hidroxiecdisona, o fitoecdisteroide ponasterona-A e tebufeno- belonging to the class of diacylhydrazines.
    [Show full text]
  • Binding Proteins for an Ecdysone Metabolite in the Crustacean Hepatopancreas (Molting Hormone/Steroid/Sucrose Gradient Oentrifugation) THOMAS A
    Proc. Nat. Acad. Sci. USA Vol. 69, No. 4, pp. 812-815, April 1972 Binding Proteins for an Ecdysone Metabolite in the Crustacean Hepatopancreas (molting hormone/steroid/sucrose gradient oentrifugation) THOMAS A. GORELL*, LAWRENCE I. GILBERTt, AND JOHN B. SIDDALL Department of Biological Sciences, Northwestern University, Evanston, Illinois 60201; and Zoecon Research Corporation, Palo Alto, California 94304 Communicated by William S. Johnson, January 20, 1972 ABSTRACT When crustacean hepatopancreas is incu- and incubated at 250 in 3 ml of saline containing [8H]ecdysone bated in the presence of a-[3Hlecdysone of high specific (see figure legends for quantities). After incubation, the tissue activity and is then homogenized and centrifuged, a peak of protein-radioactivity is recovered after gel filtration of was rinsed twice in cold 0.1 M phosphate buffer (pH 7.3) and the 105,000 X g supernatant. Analysis of this peak by su- homogenized in buffer in a ground glass homogenizer at 4°. crose gradient centrifugation revealed the presence of two After centrifugation of the homogenate at 105,000 X g for 1.5 complexes of protein and labeled material (-11.5 S and hr at 40, the supernatant was concentrated with lyphogel and 6.35 S). The same results were obtained in vivo. On stand- for ing at low ionic strength, the lighter component disap- aliquots of the concentrated supernatant were assayed peared, suggesting that the heavier component is an aggre- protein (13) and radioactivity (Packard Tri-carb scintillation gate of the lighter one. Chemical analysis of radioactive ma- spectrometer, model 3375) in ethyl alcohol-toluene [2,5 di- terial in the complex revealed that it is not a- or j3-ecdysone phenyloxazole (PPO), 5 g/l; 1,4-bis-2-(4-methyl-5-phenoxa- nor any previously described metabolite of the ecdysones.
    [Show full text]
  • Genetical and Physiological Mechanisms of Face Fly, Musca Autumnalis Degeer, Diapause Yonggyun Kim Iowa State University
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1993 Genetical and physiological mechanisms of face fly, Musca autumnalis DeGeer, diapause Yonggyun Kim Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons, Entomology Commons, and the Genetics Commons Recommended Citation Kim, Yonggyun, "Genetical and physiological mechanisms of face fly, Musca autumnalis DeGeer, diapause " (1993). Retrospective Theses and Dissertations. 10245. https://lib.dr.iastate.edu/rtd/10245 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps.
    [Show full text]
  • Epidermis As the Source of Ecdysone in an Argasid Tick (Ecdysteroids/Tissue Culture/Integument/Ornithodors Parkeri) X
    Proc. Natl. Acad. Sci. USA Vol. 88, pp. 3744-3747, May 1991 Developmental Biology Epidermis as the source of ecdysone in an argasid tick (ecdysteroids/tissue culture/integument/Ornithodors parkeri) X. X. ZHU, J. H. OLIVER, JR.*, AND E. M. DOTSON Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro, GA 30460-8056 Communicated by Wendell Roelofs, February 7, 1991 (received for review November 19, 1990) ABSTRACT Various tissues excised from nymphs of the MATERIALS AND METHODS tick Ornithodoros parkeri at the time of epicuticle deposition were incubated in vitro. The medium from the incubation of Ticks (0. parkeri) were reared at 270C and 85% relative salivary glands, coxal glands, synganglion, testis, midgut, and humidity by standard techniques (10). RIA of ecdysteroids fat body associated with tracheal trunk showed little or no was carried out following established methods (11, 12). For each assay, a standard curve was prepared using ecdysone ecdysteroid immunoreactivity. Only medium from incubated England Nuclear). integument contained ecdysteroids. The following evidence (Sigma) and [23,24-3H]ecdysone (New The antiserum used was a generous gift from T. Ohtaki the source of ecdysone: (i) indicated that epidermal cells are (Kanazawa, Japan) and has a 2.5-fold higher sensitivity for when dorsal and/or ventral integuments were incubated sep- ecdysone than for 20-hydroxyecdysone when their 50% arately, both produced ecdysteroid immunoreactive material cross-reaction was compared. during the course of incubation. As compared with the ecdy- Ecdysteroid titer was highest during epicuticle deposition steroid content in the integument before incubation, the in third instar nymphal 0. parkeri (unpublished data), and amount of ecdysteroids produced after a 24-h incubation thus nymphs at this stage were used in all our experiments.
    [Show full text]
  • And Type the TITLE of YOUR WORK in All Caps
    THE EFFECTS OF AZADIRACHTIN, METHOXYFENOZIDE, AND TEBUFENOZIDE ON THE YELLOW FEVER MOSQUITO, AEDES AEGYPTI by DANIEL JACK USRY (Under the Direction of Mark R. Brown) ABSTRACT Mosquito control is important to control the spread of pathogens which are transmitted by the bites of infected mosquitoes. To this end, research has focused on insecticides that are insect specific and have relatively low impact on the environment. Here I report on the plant botanical azadirachtin and the ecdysone agonists methoxyfenozide and tebufenozide. Overall, my results suggest that azadirachtin and tebufenozide are not suitable for use as insecticides, even though they cause mortality at high doses. Methoxyfenozide kills mosquitoes at high doses and reduces the number of eggs oviposited by females. Additionally, methoxyfenozide is able to stimulate egg production in non-bloodfed females. However, it is not able to cause eggs to develop all the way to oviposition. INDEX WORDS: Aedes aegypti, Azadirachtin, Methoxyfenozide, Tebufenozide THE EFFECTS OF AZADIRACHTIN, METHOXYFENOZIDE, AND TEBUFENOZIDE ON THE YELLOW FEVER MOSQUITO, AEDES AEGYPTI by DANIEL JACK USRY B.S., Georgia Southern University, 2009 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2012 © 2012 Daniel Jack Usry All Rights Reserved THE EFFECTS OF AZADIRACHTIN, METHOXYFENOZIDE, AND TEBUFENOZIDE ON THE YELLOW FEVER MOSQUITO, AEDES AEGYPTI by DANIEL JACK USRY Major Professor: Mark R. Brown Committee: Michael R. Strand Donald E. Champagne Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2012 DEDICATION I dedicate this to my family.
    [Show full text]
  • Download Product Insert (PDF)
    PRODUCT INFORMATION 20-hydroxy Ecdysone Item No. 16145 CAS Registry No.: 5289-74-7 HO OH Formal Name: (5β)-2β,3β,14,20,22R,25- hexahydroxy-cholest-7-en-6-one H Synonyms: Ecdysterone, Isoinokosterone MF: C27H44O7 OH FW: 480.6 HO Purity: ≥98% H OH Stability: ≥2 years at -20°C Supplied as: A crystalline solid HO H O UV/Vis.: λmax: 243 nm Laboratory Procedures For long term storage, we suggest that 20-hydroxy ecdysone be stored as supplied at -20°C. It should be stable for at least two years. 20-hydroxy Ecdysone is supplied as a crystalline solid. A stock solution may be made by dissolving the 20-hydroxy ecdysone in the solvent of choice. 20-Hydroxy Ecdysone is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide (DMF), which should be purged with an inert gas. The solubility of 20-hydroxy ecdysone in ethanol is approximately 25 mg/ml and approximately 30 mg/ml in DMSO and DMF. Further dilutions of the stock solution into aqueous buffers or isotonic saline should be made prior to performing biological experiments. Ensure that the residual amount of organic solvent is insignificant, since organic solvents may have physiological effects at low concentrations. Organic solvent-free aqueous solutions of 20-hydroxy ecdysone can be prepared by directly dissolving the crystalline solid in aqueous buffers. The solubility of 20-hydroxy ecdysone in PBS, pH 7.2, is approximately 10 mg/ml. We do not recommend storing the aqueous solution for more than one day. Description 20-Hydroxy Ecdysone is an ecdysteroid hormone produced in arthropod
    [Show full text]
  • Toxicity and Mode of Action of Steroid and Terpenoid Secondary Plant Metabolites Against Economically Important Pest Insects in Agriculture
    Faculty of Bioscience Engineering Academic year 2011-2012 TOXICITY AND MODE OF ACTION OF STEROID AND TERPENOID SECONDARY PLANT METABOLITES AGAINST ECONOMICALLY IMPORTANT PEST INSECTS IN AGRICULTURE Lic. Ellen DE GEYTER Thesis submitted in fulfilment of the requirements for the degree of Doctor (Ph.D.) in Applied Biological Sciences Promotors: Prof. dr. ir. Guy Smagghe Department of Crop Protection Faculty of Bioscience Engineering Ghent University Prof. dr. Danny Geelen Department of Plant Production Faculty of Bioscience Engineering Ghent University Dean: Prof. dr. ir. Guido Van Huylenbroeck Rector: Prof. dr. Paul Van Cauwenberge Dutch title: Toxiciteit en werkingswijze van steroïde en terpenoïde secundaire plantmetabolieten bij economisch belangrijke schadelijke insecten in de landbouw Cite as: De Geyter, E., 2012. Toxicity and mode of action of steroid and terpenoid secondary plant metabolites against economically important pest insects in agriculture. PhD dissertation, Faculty of Bioscience Engineering, Ghent University, Ghent. ISBN: 978-90-5989-536-2 The author and promotors give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. This research was supported by a fellowship of the Special Research Fund of Ghent University (BOF-UGent). EXAMINATION COMMITTEE Promotors: Prof. dr. ir. Guy Smagghe Department of Crop Protection Faculty of Bioscience Engineering Ghent University Prof. dr. Danny Geelen Department of Plant Production Faculty of Bioscience Engineering Ghent University Members: Prof. dr. ir. Stefaan De Smet (chairman) Department of Animal Production Faculty of Bioscience Engineering Ghent University Prof.
    [Show full text]