DOCSLIB.ORG

Regulation of Phosphatidylinositol Turnover in Brain

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Phosphatidylinositol Turnover in Brain Proc. Nati. Acad. Sci. USA Vol. 83, pp. 3003-3007, May 1986 Neurobiology Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: Stimulatory effects of agents that enhance influx of sodium ions (inositol phosphates/pumiliotoxin/batrachotoxin/tetrodotoxin/saidtoxin) FABIAN GUSOVSKY, ELIZABETH B. HOLLINGSWORTH, AND JOHN W. DALY Laboratory of Bioorganic Chemistry, Building 4, Room 212, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Bernhard Witkop, December 2, 1985 ABSTRACT Norepinephrine and carbamoylcholine stim- might be the basis for the biochemical and pharmacological ulate accumulation of [33H]inositol phosphates from [3H]- activity of this alkaloid. The finding also suggested that inositol-labeled guinea pig cerebral cortical synaptoneuro- nervous activity involving sodium channels might have a somes through interaction with a1-adrenergic and muscarinic regulatory input to the systems involved in phosphatidylino- receptors, respectively. In addition to such agonists, a variety sitol turnover. Sodium channel activity does appear to of natural products that affect voltage-dependent sodium provide another stimulatory input to phosphatidylinositol channels can markedly stimulate accumulation of [3H]inositol systems, since a variety of agents that enhance sodium phosphates. These include (a) alkaloids that activate sodium channel function have now been shown to stimulate phos- channels, such as batrachotoxin, veratridine, and aconitine; (ii) phatidylinositol turnover in brain preparations. peptide toxins that alter activation or slow inactivation of sodium channels, such as various scorpion toxins from Leiurus, MATERIALS AND METHODS Centruroides, and Tityus species; and (ii) agents that cause repetitive fring of sodium channel-dependent action poten- Materials. [3H]Inositol (14 Ci/mmol; 1 Ci = 37 GBq) was tials, such as pyrethroids and pumiliotoxin B. Ouabain, an purchased from Amersham. Batrachotoxin was isolated from agent that will increase accumulation of internal sodium by the skin of the dart poison frog Phyllobates terribilis (15). inhibition of Na+,K+-ATPase, also stimulates formation of Pumiliotoxin B was isolated from the skin of the poison frog [3H]inositol phosphates, as does monensin, a sodium iono- Dendrobates pumilio (16). Scorpion venoms (Leiurus phore. Tetrodotoxin and saxitoxin, specific blockers ofvoltage- quinquestriatus, Tityus serrulatus), carbamoylcholine, nor- dependent sodium channels, prevent or reduce the stimulatory epinephrine, tetrodotoxin, atropine sulfate, veratridine, effects of sodium channel agents and ouabain on phosphati- monensin, and aconitine were from Sigma. AG 1-X8 (100-200 dylinositol turnover, while having lesser or no effect, respec- mesh, formate form) anion-exchange resin was from Bio- tively, on receptor-mediated or monensin-mediated stimula- Rad. Prazosin was provided by Pfizer (Groton, CT). Scorpion tion. Removal of extracellular sodium ions markedly reduces (Leiurus) toxin (an a-scorpion toxin) was kindly provided by stimulatory effects of sodium channel agents, while removal of W. Catterall (University of Washington, Seattle). Another extracellular calcium ions with EGTA blocks both receptor- scorpion (Centruroides suffusus) toxin (toxin II, /-scorpion mediated and sodium channel agent-mediated phosphatidyl- toxin) was kindly provided by F. Couraud (Institut National inositol turnover. The results provide evidence for a hitherto de la Sante et de la Recherche Mddicale, Marseille, France). unsuspected messenger role for sodium ions in excitable tissue, The pyrethroids deltamethrin and permethrin were kindly whereby neuronal activity and the resultant influx of sodium provided by G. Brown (University of Alabama, Birming- will cause activation of phospholipase systems involved in ham). hydrolysis of phosphatidylinositols, thereby generating two Preparation of Synaptoneurosomes. Male Hartley guinea second messengers, (') the inositol phosphates, which mobilize pigs (175-220 g) were decapitated and the brains were rapidly calcium from internal stores, and (ii) the diacylglycerols, which removed. The cerebral cortex was dissected out and activate protein kinase C. synaptoneurosomes were obtained according to Hollings- worth et al. (17). Briefly, the cortex of one brain was Biogenic amines, acetylcholine analogs, and certain peptides homogenized in 7-10 vol of Krebs-Henseleit buffer in a stimulate phosphatidylinositol turnover in brain tissue glass-glass homogenizer (five strokes). When the nonffitered through interaction with specific receptors (refs. 1-10 and preparation was used, the suspension was centrifuged at this references therein). The mechanism(s) involved in receptor- point at 1000 x g for 10 min, the supernatant was decanted, mediated stimulation ofphosphatidylinositol turnover are not and the pellet was reconstituted in an appropriate volume of well understood. The products of phosphatidylinositol turn- buffer. When a filtered preparation was used, the over, namely inositol phosphates and diacylglycerides, ap- homogenate was diluted with 35 ml offresh buffer and filtered parently serve as "second messengers," with the inositol first through two layers ofnylon material (100 mesh) and then phosphates eliciting mobilization of internal calcium and the with pressure through 10-,um-pore Millipore filters (LCWP- diacylglycerides activating protein kinase C (11). An alkaloid, 047). The filtrate was centrifuged at 1000 x g for 10 min, the pumiliotoxin B, which causes repetitive firing of neurons and supernatant was decanted, and the pellet was resuspended in enhances excitation contraction coupling (12, 13), was found an appropriate volume of buffer. The synaptoneurosome to stimulate phosphatidylinositol turnover (14), suggesting preparation contains many synaptosomes with attached that generation of diacylglycerides and inositol phosphates resealed postsynaptic entities (neurosomes) (17) and is prov- ing to be a useful, readily obtained, brain preparation for a The publication costs of this article were defrayed in part by page charge variety ofbiochemical studies on cyclic AMP generation (ref. payment. This article must therefore be hereby marked "advertisement" 17 and references therein), phosphatidylinositol breakdown in accordance with 18 U.S.C. §1734 solely to indicate this fact. (9, 14), and ion fluxes (18, 19). 3003 Downloaded by guest on September 26, 2021 3004 Neurobiology: Gusovsky et al. Proc. Natl. Acad. Sci. USA 83 (1986) Phosphatidylinositol Turnover. The nonfiltered pellet from RESULTS one guinea pig brain or the combined filtered pellet from two guinea pigs was resuspended in 20 ml of Krebs-Henseleit Carbamoylcholine and norepinephrine stimulate phosphati- buffer. Similar results were obtained with nonfiltered and dylinositol turnover in [3H]inositol-labeled guinea pig cere- filtered pellets. To the suspension was added 270 ,Ci of bral cortical synaptoneurosomes (Table 1). The effects of are completely blocked by a muscarinic [3H]inositol (1 ,M), and the mixture was incubated for 60mm carbamoylcholine at 37TC with continuous gassing with 02/CO2 (95:5, vol/vol). antagonist, atropine, while effects of norepinephrine are blocked by a specific a1-adrenergic antagonist, After centrifugation at 1000 x g for 10 min and decantation completely of the supernatant, the labeled synaptoneurosomes were prazosin (data not shown). Tetrodotoxin caused a 20-35% reconstituted in 11-14 ml of fresh buffer containing 10 mM inhibition of these receptor-mediated responses (Table 1). LiCl so that 320-,l aliquots would contain approximately 1 Saxitoxin (0.5 ,M) in one experiment caused 40% inhibition mg of protein. Aliquots were transferred to polypropylene of the carbamoylcholine response and a 9% inhibition of the tubes (Falcon 2063) and, after 10 min at room temperature, norepinephtine response. Such inhibitions suggest that re- agents in buffer or buffer alone (20,ul) was added. The tubes ceptor-mediated stimulation of phosphatidylinositol break- were then gassed briefly with 02/CO2 (95:5), capped, and down in brain preparations involves to some extent influx of placed in a water bath at 37°C. Unless stated otherwise sodium ions through voltage-dependent sodium channels. incubations were for 90 min. A variety of agents that are known to affect the state or The analysis of [3H]inositol metabolites was as described function of voltage-dependent sodium channels enhance by Berridge et al. (1, 20). In brain preparations, the major accumulation of [3H]inositol phosphate in synaptoneuro- product ofphosphatidylinositol breakdown in the presence of somes (Table 1). The time courses for responses to a receptor lithium ions is inositol phosphate, with inositol bis- and agonist, carbamnoylcholine, to scorpion (Leiurus) venom, and trisphosphates being very minor (data not shown; see also to pumiliotoxin B are shown in Fig. 1. The concentration refs. 1-10). In the present study, total accumulation of dependency of the effect of purified scorpion (Leiurus) toxin [3H]inositol phosphates in inositol-labeled synaptoneuro- on phosphatidylinositol breakdown is shown in Fig. 2. The somes has been used to monitor phosphatidylinositol turn- concentration of 2 nM for half-maximal effect is commensu- over. Briefly, the tubes were centrifuged for 5 min at 1000 x rate with the potency of scorpion toxin in enhancing sodium g and the supernatant was discarded. The pellets were fluxes and allosterically
Load more

Recommended publications
  • Pertussis Toxin
    Pertussis Toxin Publication Number MAN0004270 Revision Date 09 May 2011 Catalog Number: PHZ1174 Quantity: 50 μg Lot Number: See product label. Appearance: Lyophilized solid. Origin: Bordetella pertussis. Purity: >99%. This preparation migrates as five distinct bands when analyzed by SDS–Urea PAGE. The five bands correspond to one A protomer subunit, designated S1 (Mr=26.2 kDa), and four B oligomer subunits, designated S2- S5 (Mr’s= 21.9, 21.9, 12.1, and 10.9 kDa, respectively). Summary: Islet-activating protein Pertussis toxin consists of an A protomer subunit (S1) which possesses both NAD+ glycohydrolase and ADP–ribosyltransferase activities, and B oligomer subunits (S2, S3, S4, and S5) which are responsible for attachment of the native toxin to eukaryotic cell surfaces. Pertussis toxin uncouples G proteins from receptors by ADP ribosylating a cysteine residue near the carboxyl terminus of the α subunit. Biological Activity: The lowest concentration which produces a clustered growth pattern with CHO cells is 0.03 ng/mL. The adenylate cyclase activity of this preparation is 20.1 picomoles/minute/μg in the presence of 1 μg calmodulin. Reconstitution Reconstitute the contents of this vial with 500 μL sterile, distilled water. The composition of the solution will be Recommendation: 50 μg Pertussis toxin, 10 mM sodium phosphate, pH 7.0, 50 mM sodium chloride. Because Pertussis toxin is relatively insoluble, the resulting suspension should be made uniform by gentle mixing prior to withdrawing aliquots. It is important to note that this suspension should not be sterile-filtered. This preparation is not activated. For use with intact cells or extracts, activation is not necessary.
    [Show full text]
  • Biological Toxins Fact Sheet
    Work with FACT SHEET Biological Toxins The University of Utah Institutional Biosafety Committee (IBC) reviews registrations for work with, possession of, use of, and transfer of acute biological toxins (mammalian LD50 <100 µg/kg body weight) or toxins that fall under the Federal Select Agent Guidelines, as well as the organisms, both natural and recombinant, which produce these toxins Toxins Requiring IBC Registration Laboratory Practices Guidelines for working with biological toxins can be found The following toxins require registration with the IBC. The list in Appendix I of the Biosafety in Microbiological and is not comprehensive. Any toxin with an LD50 greater than 100 µg/kg body weight, or on the select agent list requires Biomedical Laboratories registration. Principal investigators should confirm whether or (http://www.cdc.gov/biosafety/publications/bmbl5/i not the toxins they propose to work with require IBC ndex.htm). These are summarized below. registration by contacting the OEHS Biosafety Officer at [email protected] or 801-581-6590. Routine operations with dilute toxin solutions are Abrin conducted using Biosafety Level 2 (BSL2) practices and Aflatoxin these must be detailed in the IBC protocol and will be Bacillus anthracis edema factor verified during the inspection by OEHS staff prior to IBC Bacillus anthracis lethal toxin Botulinum neurotoxins approval. BSL2 Inspection checklists can be found here Brevetoxin (http://oehs.utah.edu/research-safety/biosafety/ Cholera toxin biosafety-laboratory-audits). All personnel working with Clostridium difficile toxin biological toxins or accessing a toxin laboratory must be Clostridium perfringens toxins Conotoxins trained in the theory and practice of the toxins to be used, Dendrotoxin (DTX) with special emphasis on the nature of the hazards Diacetoxyscirpenol (DAS) associated with laboratory operations and should be Diphtheria toxin familiar with the signs and symptoms of toxin exposure.
    [Show full text]
  • Pertussis Toxin
    PLEASE POST THIS PAGE IN AREAS WHERE PERTUSSIS TOXIN IS USED IN RESEARCH LABORATORIES UNIVERSITY OF CALIFORNIA, SAN FRANCISCO ENVIRONMENT, HEALTH AND SAFETY/BIOSAFETY PERTUSSIS TOXIN EXPOSURE/INJURY RESPONSE PROTOCOL Organism or Agent: Pertussis Toxin Exposure Risk; Multiple Endocrine/Metabolic Effects Exposure Hotline Pager: 415/353-7842 (353-STIC) (Available 24 hours) Office of Environment, Health & Safety: 415/476-1300 (Available during work hours) 415/476-1414 or 9-911 (In case of emergency, available 24 hours) EH&S Biosafety Officer 415/514-2824 EH&S Public Health Officer: 415/514-3531 UCSF Occupational Health Services: 415/885-7580 (Available during work hours) California Poison Control: 800/222-1222 SFDPH Emergency Number: 415/554-2830 CDC Emergency Operations: 770/488-7100 _________________________________________________________________________ PROTOCOL SUMMARY In the event of an accidental exposure or injury, the protocol is as follows: 1. Modes of Exposure: a. Skin puncture or injection b. Ingestion c. Contact with mucous membranes (eyes, nose, mouth) d. Contact with non-intact skin e. Exposure to aerosols f. Respiratory exposure from inhalation of toxin 2. First Aid: a. Skin Exposure, immediately go to the sink and thoroughly wash the skin with soap and water. If working with pertussis, decontaminate any exposed skin with an antiseptic scrub solution. b. Skin Wound, immediately go to the sink and thoroughly wash the wound with soap and water and pat dry. c. Splash to Eye(s), Nose or Mouth, immediately flush the area with running water for at least 5- 10 minutes. d. Splash Affecting Garments, remove garments that may have become soiled or contaminated and place them in a double red plastic bag.
    [Show full text]
  • Recent Advances in Research on Widow Spider Venoms and Toxins
    Review Recent Advances in Research on Widow Spider Venoms and Toxins Shuai Yan and Xianchun Wang * Received: 2 August 2015; Accepted: 16 November 2015; Published: 27 November 2015 Academic Editors: Richard J. Lewis and Glenn F. King Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081, China; [email protected] * Correspondence: [email protected]; Tel.: +86-731-8887-2556 Abstract: Widow spiders have received much attention due to the frequently reported human and animal injures caused by them. Elucidation of the molecular composition and action mechanism of the venoms and toxins has vast implications in the treatment of latrodectism and in the neurobiology and pharmaceutical research. In recent years, the studies of the widow spider venoms and the venom toxins, particularly the α-latrotoxin, have achieved many new advances; however, the mechanism of action of the venom toxins has not been completely clear. The widow spider is different from many other venomous animals in that it has toxic components not only in the venom glands but also in other parts of the adult spider body, newborn spiderlings, and even the eggs. More recently, the molecular basis for the toxicity outside the venom glands has been systematically investigated, with four proteinaceous toxic components being purified and preliminarily characterized, which has expanded our understanding of the widow spider toxins. This review presents a glance at the recent advances in the study on the venoms and toxins from the Latrodectus species. Keywords: widow spider; venom; toxin; latrotoxin; latroeggtoxin; advance 1. Introduction Latrodectus spp.
    [Show full text]
  • Human Peptides -Defensin-1 and -5 Inhibit Pertussis Toxin
    toxins Article Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin Carolin Kling 1, Arto T. Pulliainen 2, Holger Barth 1 and Katharina Ernst 1,* 1 Institute of Pharmacology and Toxicology, Ulm University Medical Center, 89081 Ulm, Germany; [email protected] (C.K.); [email protected] (H.B.) 2 Institute of Biomedicine, Research Unit for Infection and Immunity, University of Turku, FI-20520 Turku, Finland; arto.pulliainen@utu.fi * Correspondence: [email protected] Abstract: Bordetella pertussis causes the severe childhood disease whooping cough, by releasing several toxins, including pertussis toxin (PT) as a major virulence factor. PT is an AB5-type toxin, and consists of the enzymatic A-subunit PTS1 and five B-subunits, which facilitate binding to cells and transport of PTS1 into the cytosol. PTS1 ADP-ribosylates α-subunits of inhibitory G-proteins (Gαi) in the cytosol, which leads to disturbed cAMP signaling. Since PT is crucial for causing severe courses of disease, our aim is to identify new inhibitors against PT, to provide starting points for novel therapeutic approaches. Here, we investigated the effect of human antimicrobial peptides of the defensin family on PT. We demonstrated that PTS1 enzyme activity in vitro was inhibited by α-defensin-1 and -5, but not β-defensin-1. The amount of ADP-ribosylated Gαi was significantly reduced in PT-treated cells, in the presence of α-defensin-1 and -5. Moreover, both α-defensins decreased PT-mediated effects on cAMP signaling in the living cell-based interference in the Gαi- mediated signal transduction (iGIST) assay.
    [Show full text]
  • Α-Neurexins Together Withα2δ-1 Auxiliary Subunits Regulate Ca
    The Journal of Neuroscience, September 19, 2018 • 38(38):8277–8294 • 8277 Cellular/Molecular ␣-Neurexins Together with ␣2␦-1 Auxiliary Subunits 2ϩ Regulate Ca Influx through Cav2.1 Channels X Johannes Brockhaus,1* Miriam Schreitmu¨ller,1* Daniele Repetto,1 Oliver Klatt,1,2 XCarsten Reissner,1 X Keith Elmslie,3 Martin Heine,2 and XMarkus Missler1,4 1Institute of Anatomy and Molecular Neurobiology, Westfa¨lische Wilhelms-University, 48149 Mu¨nster, Germany, 2Molecular Physiology Group, Leibniz- Institute of Neurobiology, 39118 Magdeburg, Germany, 3Department of Pharmacology, AT Still University of Health Sciences, Kirksville, Missouri 63501, and 4Cluster of Excellence EXC 1003, Cells in Motion, 48149 Mu¨nster, Germany Action potential-evoked neurotransmitter release is impaired in knock-out neurons lacking synaptic cell-adhesion molecules ␣-neurexins (␣Nrxns), the extracellularly longer variants of the three vertebrate Nrxn genes. Ca 2ϩ influx through presynaptic high- ␣ ␦ voltage gated calcium channels like the ubiquitous P/Q-type (CaV2.1) triggers release of fusion-ready vesicles at many boutons. 2 Auxiliary subunits regulate trafficking and kinetic properties of CaV2.1 pore-forming subunits but it has remained unclear if this involves ␣Nrxns. Using live cell imaging with Ca 2ϩ indicators, we report here that the total presynaptic Ca 2ϩ influx in primary hippocampal ␣ neurons of Nrxn triple knock-out mice of both sexes is reduced and involved lower CaV2.1-mediated transients. This defect is accom- ␣ ␦ panied by lower vesicle release, reduced synaptic abundance of CaV2.1 pore-forming subunits, and elevated surface mobility of 2 -1 on axons. Overexpression of Nrxn1␣ in ␣Nrxn triple knock-out neurons is sufficient to restore normal presynaptic Ca 2ϩ influx and synaptic vesicle release.
    [Show full text]
  • Release of Pertussis Toxin and Its Interaction with Outer-Membrane Antigens
    Journal of General Microbiology (1987), 133, 2427-2435. Printed in Great Britain 2427 Release of Pertussis Toxin and Its Interaction With Outer-membrane Antigens ByV. Y. PERERA, A. C. WARDLAW AND J. H. FREER* Department of Microbiology, University of Glasgow, Alexander Stone Building, Garscube Estate, Bearsden, Glasgow G61 IQH, UK (Received 16 December 1986 ;revised 22 April 1987) The absence of subunit S3 in cell-associated pertussis toxin (PT) from a mutant of Bordetella pertussis which failed to produce cell-free toxin suggested that this subunit was involved in the release of PT into the culture medium. The addition of methylated P-cyclodextrin (MCD) to the culture medium caused a small but consistent increase in the release of lipopolysaccharide (LPS) by four wild-type strains of B. pertussis. Since previous studies have shown that MCD also enhances the levels of PT in culture supernates, it seemed probable that the increased shedding of outer-membrane vesicles (OMV) may explain the increased levels of both cell-free PT and LPS. Release of PT was inhibited in media buffered with HEPES but was unaffected in Tris/HCl buffer. This suggested that in addition to shedding of the outer membrane, increased permeability and greater destabilization of the outer membrane, as caused by Tris/HCl buffer, may be important in the release of PT. Our data do not support the idea that PT is packaged into OMV because only an insignificant proportion (0.01 %) of the total cell-free PT was associated with LPS. The association of PT with small micelles derived from outer-membrane amphiphiles may be more important since the LPS content of PT purified from culture supernates (containing no large OMV) was nearly 18% by weight.
    [Show full text]
  • Mitochondrial Alarmins Released by Degenerating Motor Axon Terminals
    Mitochondrial alarmins released by degenerating PNAS PLUS motor axon terminals activate perisynaptic Schwann cells Elisa Duregottia, Samuele Negroa, Michele Scorzetoa, Irene Zornettaa, Bryan C. Dickinsonb,c,1, Christopher J. Changb,c, Cesare Montecuccoa,d,2, and Michela Rigonia,2 aDepartment of Biomedical Sciences, University of Padua, Padua 35131, Italy; bDepartment of Chemistry and Molecular and Cell Biology and cHoward Hughes Medical Institute, University of California, Berkeley, CA 94720; and dItalian National Research Council Institute of Neuroscience, Padua 35131, Italy Edited by Thomas C. Südhof, Stanford University School of Medicine, Stanford, CA, and approved December 22, 2014 (received for review September 5, 2014) An acute and highly reproducible motor axon terminal degeneration of nerve terminal degeneration (21). Indeed, these neurotoxins followed by complete regeneration is induced by some animal cause activation of the calcium-activated calpains that contribute to presynaptic neurotoxins, representing an appropriate and controlled cytoskeleton fragmentation (22). system to dissect the molecular mechanisms underlying degeneration Although clearly documented (4, 5, 20), the regeneration of and regeneration of peripheral nerve terminals. We have previously the motor axon terminals after presynaptic neurotoxins injection shown that nerve terminals exposed to spider or snake presynaptic is poorly known in its cellular and molecular aspects. Available neurotoxins degenerate as a result of calcium overload and mito- evidence indicates that, in general, regeneration of mechan- chondrial failure. Here we show that toxin-treated primary neurons ically damaged motor neuron terminals relies on all three cel- release signaling molecules derived from mitochondria: hydrogen lular components of the neuromuscular junction (NMJ): the peroxide, mitochondrial DNA, and cytochrome c.
    [Show full text]
  • Rope Parasite” the Rope Parasite Parasites: Nearly Every AuSC Child I Ever Treated Proved to Carry a Significant Parasite Burden
    Au#sm: 2015 Dietrich Klinghardt MD, PhD Infec4ons and Infestaons Chronic Infecons, Infesta#ons and ASD Infec4ons affect us in 3 ways: 1. Immune reac,on against the microbes or their metabolic products Treatment: low dose immunotherapy (LDI, LDA, EPD) 2. Effects of their secreted endo- and exotoxins and metabolic waste Treatment: colon hydrotherapy, sauna, intes4nal binders (Enterosgel, MicroSilica, chlorella, zeolite), detoxificaon with herbs and medical drugs, ac4vaon of detox pathways by solving underlying blocKages (methylaon, etc.) 3. Compe,,on for our micronutrients Treatment: decrease microbial load, consider vitamin/mineral protocol Lyme, Toxins and Epigene#cs • In 2000 I examined 10 au4s4c children with no Known history of Lyme disease (age 3-10), with the IgeneX Western Blot test – aer successful treatment. 5 children were IgM posi4ve, 3 children IgG, 2 children were negave. That is 80% of the children had clinical Lyme disease, none the history of a 4cK bite! • Why is it taking so long for au4sm-literate prac44oners to embrace the fact, that many au4s4c children have contracted Lyme or several co-infec4ons in the womb from an oVen asymptomac mother? Why not become Lyme literate also? • Infec4ons can be treated without the use of an4bio4cs, using liposomal ozonated essen4al oils, herbs, ozone, Rife devices, PEMF, colloidal silver, regular s.c injecons of artesunate, the Klinghardt co-infec4on cocKtail and more. • Symptomac infec4ons and infestaons are almost always the result of a high body burden of glyphosate, mercury and aluminum - against the bacKdrop of epigene4c injuries (epimutaons) suffered in the womb or from our ancestors( trauma, vaccine adjuvants, worK place related lead, aluminum, herbicides etc., electromagne4c radiaon exposures etc.) • Most symptoms are caused by a confused upregulated immune system (molecular mimicry) Toxins from a toxic environment enter our system through damaged boundaries and membranes (gut barrier, blood brain barrier, damaged endothelium, etc.).
    [Show full text]
  • Α-LATROTOXIN and ITS RECEPTORS: Neurexins
    P1: FQP April 4, 2001 18:17 Annual Reviews AR121-30 Annu. Rev. Neurosci. 2001. 24:933–62 Copyright c 2001 by Annual Reviews. All rights reserved -LATROTOXIN AND ITS RECEPTORS: Neurexins and CIRL/Latrophilins ThomasCSudhof¨ Howard Hughes Medical Institute, Center for Basic Neuroscience, and the Department of Molecular Genetics, The University of Texas Southwestern Medical Center at Dallas, Texas 75390-9111, e-mail: [email protected] Key Words neurotransmitter release, synaptic vesicles, exocytosis, membrane fusion, synaptic cell adhesion ■ Abstract -Latrotoxin, a potent neurotoxin from black widow spider venom, triggers synaptic vesicle exocytosis from presynaptic nerve terminals. -Latrotoxin is a large protein toxin (120 kDa) that contains 22 ankyrin repeats. In stimulating exocytosis, -latrotoxin binds to two distinct families of neuronal cell-surface receptors, neurexins and CLs (Cirl/latrophilins), which probably have a physiological function in synaptic cell adhesion. Binding of -latrotoxin to these receptors does not in itself trigger exocytosis but serves to recruit the toxin to the synapse. Receptor-bound -latrotoxin then inserts into the presynaptic plasma membrane to stimulate exocytosis by two dis- tinct transmitter-specific mechanisms. Exocytosis of classical neurotransmitters (glu- tamate, GABA, acetylcholine) is induced in a calcium-independent manner by a direct intracellular action of -latrotoxin, while exocytosis of catecholamines requires extra- cellular calcium. Elucidation of precisely how -latrotoxin works is likely to provide major insight into how synaptic vesicle exocytosis is regulated, and how the release machineries of classical and catecholaminergic neurotransmitters differ. by SCELC Trial on 09/09/11. For personal use only. INTRODUCTION Annu. Rev. Neurosci. 2001.24:933-962.
    [Show full text]
  • Safe Handling of Acutely Toxic Chemicals Safe Handling of Acutely
    Safe Handling of Acutely Toxic Chemicals , Mutagens, Teratogens and Reproductive Toxins October 12, 2011 BSBy Sco ttBthlltt Batcheller R&D Manager Milwaukee WI Hazards Classes for Chemicals Flammables • Risk of ignition in air when in contact with common energy sources Corrosives • Generally destructive to materials and tissues Energetic and Reactive Materials • Sudden release of destructive energy possible (e.g. fire, heat, pressure) Toxic Substances • Interaction with cells and organs may lead to tissue damage • EfftEffects are t tilltypically not general ltllti to all tissues, bttbut target tdted to specifi c ones • Examples: – Cancers – Organ diseases – Inflammation, skin rashes – Debilitation from long-term Poison Acute Cancer, health or accumulation with delayed (ingestion) risk reproductive risk emergence 2 Toxic Substances Are All Around Us Pollutants Natural toxins • Cigare tte smo ke • V(kidbt)Venoms (snakes, spiders, bees, etc.) • Automotive exhaust • Poison ivy Common Chemicals • Botulinum toxin • Pesticides • Ricin • Fluorescent lights (mercury) • Radon gas • Asbestos insulation • Arsenic and heavy metals • BPA ((pBisphenol A used in some in ground water plastics) 3 Application at UNL Chemicals in Chemistry Labs Toxin-producing Microorganisms • Chloro form • FiFungi • Formaldehyde • Staphylococcus species • Acetonitrile • Shiga-toxin from E. coli • Benzene Select Agent Toxins (see register) • Sodium azide • Botulinum neurotoxins • Osmium/arsenic/cadmium salts • T-2 toxin Chemicals in Biology Labs • Tetrodotoxin • Phenol
    [Show full text]
  • Cortactin: Coupling Membrane Dynamics to Cortical Actin Assembly
    Oncogene (2001) 20, 6418 ± 6434 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Cortactin: coupling membrane dynamics to cortical actin assembly Scott A Weed*,1 and J Thomas Parsons2 1Department of Craniofacial Biology, University of Colorado Health Sciences Center, Denver, Colorado, CO 80262, USA; 2Department of Microbiology, Health Sciences Center, University of Virginia, Charlottesville, Virginia, VA 22903, USA Exposure of cells to a variety of external signals causes consists of a highly organized meshwork of ®lamentous rapid changes in plasma membrane morphology. Plasma (F-) actin closely associated with the overlying plasma membrane dynamics, including membrane rue and membrane (Small et al., 1995). In motile cells, F-actin microspike formation, fusion or ®ssion of intracellular underlies two main types of protrusive membranes; vesicles, and the spatial organization of transmembrane lamellipodia, which contain short ®laments of F-actin proteins, is directly controlled by the dynamic reorgani- linked into orthogonal arrays and ®lopodia, comprised zation of the underlying actin cytoskeleton. Two of bundled, crosslinked actin ®laments (Rinnerthaler et members of the Rho family of small GTPases, Cdc42 al., 1988). Work within the last decade has demon- and Rac, have been well established as mediators of strated that Rac and Cdc42, two members of the Rho extracellular signaling events that impact cortical actin family of small GTPases, govern lamellipodia and organization. Actin-based signaling through Cdc42 and ®lopodia formation (Hall, 1998). Cdc42 and Rac play a Rac ultimately results in activation of the actin-related pivotal role in the transmission of extracellular signals protein (Arp) 2/3 complex, which promotes the forma- that induce cortical cytoskeletal rearrangement (Zig- tion of branched actin networks.
    [Show full text]