Cloning, synthesis and target identification of a novel α-conotoxin Lt1.1 / 军事医学

Objective To discover novel conopeptides which are the antagonists of neuronal nicotinic acetylcholine receptors (nAChRs) in order to contribute to the development of novel analgesic drugs and neuropharmacological probes.Methods Based on the conserved untranslated region and intron of A-superfamily conotoxins,a novel α-conotoxin Lt1.1 was cloned from Conus litteratus.The peptide-resin was synthesized using the solid-phased method and was cleaved.The resulting linear peptide was oxidized by air to give the product containing disulfide bridges.The folding product was finally purified by HPLC.The disulfide bond connectivity was determined using the two-step oxidative folding methods.The cRNA of rat nAChRs was expressed on the membrane of Xenopus oocyte.Membrane currents were recorded using the two electrode voltage-clamp technique.Results A novel α-conotoxin designated as Lt1.1(GCCSHPACNVNNPDIC-NH2) was cloned and its disulfide connectivity was C1-C3,C2-C4.Lt1.1 selectively inhibited the α3β2 and α3β4 nAChRs with an IC50 of 166.76 and 190.00 nmol/L,respectively.Conclusion Lt1.1 is a novel 4/7 α-conotoxin that selectively targets α3β2 and α3β4 nAChRs.

Preparation of conotoxin GI antiserum and determination of neutralization activity / 军事医学

Objective To determine whether the antiserum produced by immunizing mice with conotoxin GI coupled with bovine serum albumin (BSA) could neutralize GI conotoxin.Methods The GI-BSA was prepared by glutaraldehyde-coupled method,and the mice were immunized with the GI-BSA to produce antiserum.The antibody neutralization assay was used to test the detoxication of the antiserum.Results The SDS-PAGE protein electrophoresis showed that the coupling reaction of GI hapten with BSA was successful.The two distinct protein bands of GI-BSA were more than 120×103.Each mouse was immunized four times with 99 μg every two weeks.After the fourth immunization,the serum neutralization titer was more than 1:64 000.After the intraperitoneal injection of the mixture of 100 or 200 μl of the antiserum and different doses of GI,75% of the mice survived in the group with 100 μl of the antiserum and 1× LD50 GI(16.3 μg/kg).The same percentage of mice also survived in the group of with 200 μl of serum and 25.8 μg/kg of GI.Conclusion The antiserum produced by immunizing mice with GI-BSA exhibits significant detoxication activity to conotoxin GI.

Progress in toxicology and pharmacology of conotoxins / 中国药理学与毒理学杂志

Conotoxins are secreted by Conus snail,which are mainly composed of 12-40 amino acid residues and several disulfide bridges. Their diversities in sequences,disulfide bond connections and modified amino acids are different from those of peptide toxins and proteins secreted by terrestrial animals,and their targets include sodium,potassium,calcium ion channels and membrane receptors. Conotoxins have been categorized into more than 20 superfamilies,such as A,M,O,P,S,T,I,V, Y,J,D,C and L,which are characterized by consensus signal sequences and cysteine framework. According to pharmacological functions,these superfamilies are further classified into several pharma?cological families,such as α,μ,ω,κ,δ,ψ,σ,ρ,γ,conopressin and conantokins. This review briefly introduced the classifications and diversities of conotoxins,and summarized the progresses in the toxi?cology and pharmacology of conotoxins targeting calcium,sodium ion channel,nicotinic acetylcholine receptor and N-methyl-D-aspartic acid receptor. Some of the above conotoxins are highly poisonous,some have been developed as drugs or drug candidates,or have become powerful research tools for neuro?pharmacology. We hope this review will contribute to researches of toxins and related neuropharmacology.

Modificaciones en la plasticidad hipocampal a corto plazo por efecto de un péptido diseñado y sintetizado con base en la estructura de una conotoxina / Changes in Short Term Hippocampal Plasticity caused by a Peptide Designed and Synthesized based on the Structure of a Conotoxine

Introducción: El diseño de instrumentos o moléculas inspiradas en la naturaleza hapermitido avanzar en diferentes áreas de la ciencia y tecnología. Objetivo: Evaluarcambios plásticos a corto plazo desencadenados por la administración intraperitonealde un péptido agonista del receptor de glutamato NMDA. Materiales y métodos:Se utilizaron ratas macho divididas en grupo péptido (a quienes se administróperitonealmente el péptido) y grupo control (a quienes se administró peritonealmenteel vehículo). Por técnicas de estereotaxia se localizaron electrodos de estimulacióny registro en las áreas hipocampales CA3 y CA1 respectivamente, con el ánimo deobtener los potenciales de campo y analizar cambios en su pendiente y amplitud porla aplicación de pares de estímulos, antes y después de la administración del péptido.Resultados: Aunque con la administración del péptido no hay cambios en variablessistémicas como la temperatura o la frecuencia cardiaca, sí se pueden modificar lospotenciales de campo, específicamente cuando el intervalo entre pares de estímuloses más corto (40 a 80 ms). Conclusión: El péptido evaluado en el presente trabajotiene efectos en la facilitación por pulsos pareados tanto en la amplitud como en lapendiente de potenciales de campo en el hipocampo de ratas, pero en los intervalosentre estímulos más cortos...

Introduction: Designing bio-inspired devicesor molecules has been a longstanding methodsupporting multiple advances in science and technology.Objective: To test the potential effectof intraperitoneal administration of the bio-inspiredpeptide on short-term plasticity. Materialsand methods: Male rats in two groups (peptidetreated and control) were used for registrationof hippocampal field potentials from CA1, afterpaired stimuli in CA3 contra lateral area. Results:Amplitude and slope of field potentials’,both before and after peptide administration,showed no changes in systemic factors (temperatureand heart rate), but statistical significancein short term plasticity in small inter stimuli interval(40 to 80 ms). Conclusion: The current researchshows effects on paired pulse facilitationcaused by peptide treatment, specifically on interstimuli interval shorter...

PROGRESS IN THE INVESTIGATION ON CONOTOXINS / 中国海洋药物

This review largely deals with the peptide toxins elaborated by marine cone snails of the genus Conus . Each species of Conus contains in its venom 50 to 200 different peptides directed at different macromolecular targts. These include competitive antagonists of postsynaptic nicotinic receptors (a-conotoxins), blockers selective for Na+ channels in skeletal muscle (u- conotoxins), blockers of presynaptic of antagonists of postsynaptic Ca2+ channels (w-conotoxins), activators of Na+ channels (s-conotoxins), blockers of K+ channels (k-conotoxins), blockers of nicotinic receptor channels (u-conotoxins) and antagonists of NMDA receptors (cono-sleeper).The small size of the peptides (13 to 30 residues is typical) has facilitated synthesis of many of them. A very attractive feature is the highly cross-linked conserved 2 to 3 disulfide bonds which make conotoxins conformationally rigid, some of conotoxins, however, are stabilized by r-carboxyglutamates. The Structure-Activity Relationships of conotoxins and a brief perspective have been reviewed in the paper.

Effect of intrathecal injection of ?-conopeptide SO3 on inducible nitric oxide synthase expression in spinal cord and chronically constricted sciatic nerve in rat / 解放军医学杂志

Objective To evaluate the effect of intrathecal administration of ?-conopeptide SO3 on inducible nitric oxide synthase(iNOS)expression of spinal cord and the ligated sciatic nerve in a rat model with chronic constriction injury(CCI).Methods 40 male SD rats were randomly divided into 4 groups of 10 animals each.Rats in the N group served as controls;in group C 4 loose ligatures were placed around the right sciatic nerve for 14 days;in group CN,normal saline 1?l/h was injected intrathecally slowly for 7 days seven days after the ligation;in group CS,?-conopeptide SO3 30ng/h was administered intrathecally slowly for 7 days seven days after the ligature.Local expression of iNOS was assayed in samples taken from injured nerves(between and distal to the CCI site)and the spinal cord using Western blotting analysis,with GAPDH as an internal reference.Results A 130 kDa band,corresponding to iNOS protein was detected in the middle and distal sections of the injured nerves and the spinal cord.The iNOS immunoreactivity was inhibited by continuous intrathecal injection of ?-conopeptide SO3.There was no difference in the expression of iNOS between group CN and group C.Conclusion The expression of iNOS in the spinal cord and injured nerves of CCI rats was enhanced.Intrathecal injection of ?-conopeptide SO3 can inhibit the expression of iNOS.The data suggested that N-type calcium channel blocker took part in the expression of iNOS.