A short framework-III (mini-M-2) conotoxin from the venom of a vermivorous species, Conus archon, inhibits human neuronal nicotinic acetylcholine receptors.

The venoms of Conus snails contain neuroactive peptides named conotoxins (CTXs). Some CTXs are nicotinic acetylcholine receptor (nAChRs) antagonists. nAChRs modulate the release of neurotransmitters and are implicated in several pathophysiologies. One venom peptide from Conus archon, a vermivorous species from the Mexican Pacific, was purified by RP-HPLC and its activity on human α7, α3ß2, and α7ß2 nAChRs was assessed by the two-electrode voltage clamp technique. At 36.3 µM the purified peptide (F27-1, renamed tentatively ArchIIIA) slowly reversibly inhibited the ACh-induced response of the hα7 subtype by 44.52 ± 5.83%, while it had low or no significant effect on the response of the hα3ß2 and hα7ß2 subtypes; the EC50 of the inhibiting effect was 45.7 µM on the hα7 subtype. This peptide has 15 amino acid residues and a monoisotopic mass of 1654.6 Da (CCSALCSRYHCLPCC), with three disulfide bridges and a free C-terminus. This sequence with a CC-C-C-CC arrangement (framework III) belongs to the M superfamily of conotoxins, corresponding to the mini-M´s (M-1-M-3) conotoxins; due to its size and inter-Cys spacings it is an M-2 conotoxin. This toxin is a novel mini-M conotoxin affecting ligand-gated ion channels, like the maxi-M CTX ψ-conotoxins and α-MIIIJ conotoxin (nAChRs blockers). This peptide seems to be homologous to the reg3b conotoxin (from Conus regius) with an identity of 93.3%, differing only in the third residue in the sequence, serine for threonine, both uncharged polar residues. We obtained, in silico, a probable 3D structure, which is consistent with its effect on neuronal subtypes.

A turripeptide from Polystira nobilis venom inhibits human α3ß2 and α7 nicotinic acetylcholine receptors.

Almost all marine snails within superfamily Conoidea produce venoms containing numerous neuroactive peptides. Most toxins characterized from members of this superfamily are produced by species belonging to family Conidae. These toxins (conotoxins) affect diverse membrane proteins, such as voltage- and ligand-gated ion channels, including nicotinic acetylcholine receptors (nAChRs). Family Turridae has been considerably less studied than their Conidae counterpart and, therefore, turrid toxins (turritoxins) have just been barely described. Consequently, in this work the most prominent chromatographic (RP-HPLC) fractions from the East Pacific species Polystira nobilis venom duct extract were isolated. The biological activity of six selected fractions was assayed on human (h) α7 AChRs expressed in Xenopus laevis oocytes. One of these fractions, F21, inhibited the acetylcholine-elicited response by 62 ± 12%. Therefore, this fraction was further purified and the F21-2 peptide was obtained. This peptide (at 5.6 µM) strongly and irreversibly inhibited the acetylcholine-induced response on hα7 and hα3ß2 nAChRs, by 55 ± 4 and 91 ± 1%, respectively. Electrospray mass spectrometry indicates that the average molecular mass of this toxin is 12 358.80 Da. The affinity for hα3ß2 nAChRs is high (IC50 of 566.2 nM). A partial sequence without cysteines was obtained by automated Edman degradation: WFRSFKSYYGHHGSVYRPNEPNFRSFAS…; blastp search revealed that this sequence has low similarity to some non-Cys-containing turripeptides. This is the first report of a turritoxin from a species of the American Pacific and the second description of a turripeptide inhibiting nAChRs.

Peptide pal9a from the venom of the turrid snail Polystira albida from the Gulf of Mexico: purification, characterization, and comparison with P-conotoxin-like (framework IX) conoidean peptides.

A novel peptide, pal9a, was purified from the venom duct extract of the turrid snail, Polystira albida (superfamily Conoidea, family Turridae), collected in the Gulf of Mexico. Its primary structure was determined by automated Edman degradation and confirmed by mass spectrometry. Turritoxin pal9a contains 34 amino acid residues, including 6 Cys residues arranged in the pattern C-C-C-C-C-C (framework IX, where "-" represents one or more non-Cys amino acids), which characterizes the P-conotoxins. Peptide pal9a is the first P-conotoxin-like turritoxin characterized from a member of family Turridae of the Western Atlantic. The primary structure of turritoxin pal9a, NVCDGDACPDGVCRSGCTCDFNVAQRKDTCFYPQ-nh(2) (-nh(2), amidated C-terminus; calculated monoisotopic mass, 3679.48Da; experimental monoisotopic mass, 3678.84Da), shows variable degrees of low sequence similarity with framework IX-toxins from turrid (three species of Lophiotoma, and four species of Gemmula), terebrid (Hastula hectica), and Conus species of the Indo-Pacific (C. textile, C. gloriamaris, C. amadis, and C. litteratus) and of the Western Atlantic (C. regius). During the comparison of peptide pal9a with the other framework IX-toxins known to date, we realized that, in general, these peptides are hydrophilic, acidic compounds that have not been found in the fish-hunting Conus species studied thus far; we also found support for the notion that they may belong to several distinct gene superfamilies, even those from the same species. Given the broad distribution of framework IX-toxins within superfamily Conoidea, it will be interesting to identify the still-unknown molecular targets of P-conotoxins, P-conotoxin-like turritoxins, and P-conotoxin-like augertoxins.

Structural characterization of five post-translationally modified isomorphs of a novel putative delta-conotoxin from the vermivorous snail Conus delessertii from the Mexican Caribbean Sea.

A novel peptide, de7b, was isolated from the venom of Conus delessertii, a worm-hunting species collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and confirmed by mass spectrometry: it contains 28 amino acids, including six Cys residues. Peptide de7b is the second, O-conotoxin-like peptide isolated from the venom of this species, and it exists in different post-translationally modified isomorphs, some of which contain gamma-carboxy-glutamate (gamma) and/or 4-hydroxy-proline (O) at positions 4, 7, and/or 14. Its primary structure is DCI(P/O)GG(E/gamma)NCDVFR(O/P)YRCCSGYCILLLCA, with molecular masses varying from 3078.6 to 3154.6Da, depending on the number and kind of modified amino acid residues. Peptide de7b shows significant sequence identity with several O-conotoxins purified and biologically characterized from molluscivorous and piscivorous cone snails of the Indo-Pacific region, the tropical Atlantic and Eastern Pacific Oceans, especially with the delta-conotoxins but also with the omega-conotoxins from molluscivorous species, which suggests that it might affect voltage-gated Na(+) or Ca(2+)channels. Peptide de7b has 32% sequence identity with putative gamma-conotoxin de7a, previously characterized from the same species; both peptides contain the same number of amino acid residues and of non-Cys residues between the pairs of consecutive Cys residues. However, these peptides have charge differences at seven positions within the N-terminal half indicating that they might have distinct molecular targets that remain to be identified.

αD-Conotoxins in Species of the Eastern Pacific: The Case of Conus princeps from Mexico.

Conus snails produce venoms containing numerous peptides such as the α-conotoxins (α-CTXs), which are well-known nicotinic acetylcholine receptor (nAChR) antagonists. Thirty-eight chromatographic fractions from Conus princeps venom extract were isolated by RP-HPLC. The biological activities of 37 fractions (0.07 µg/µL) were assayed by two-electrode voltage clamp on human α7 nAChRs expressed in Xenopus laevis oocytes. Fractions F7 and F16 notably inhibited the response elicited by acetylcholine by 52.7 ± 15.2% and 59.6 ± 2.5%, respectively. Fraction F7 was purified, and an active peptide (F7-3) was isolated. Using a combination of Edman degradation, mass spectrometry, and RNASeq, we determined the sequence of peptide F7-3: AVKKTCIRSTOGSNWGRCCLTKMCHTLCCARSDCTCVYRSGKGHGCSCTS, with one hydroxyproline (O) and a free C-terminus. The average mass of this peptide, 10,735.54 Da, indicates that it is a homodimer of identical subunits, with 10 disulfide bonds in total. This peptide is clearly similar to αD-CTXs from species of the Indo-Pacific. Therefore, we called it αD-PiXXA. This toxin slowly and reversibly inhibited the ACh-induced response of the hα7 nAChR subtype, with an IC50 of 6.2 µM, and it does not affect the hα3ß2 subtype at 6.5 µM.

Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice.

As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92Da; calculated monoisotopic mass 2884.20Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63Da; calculated monoisotopic mass 3308.34Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.

Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks.

A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPM gammaDPLgammaIIRI-nh2, (gamma, gamma-carboxyglutamate; -nh2, amidated C-terminus; calculated monoisotopic mass, 1468.72Da; experimental monoisotopic mass, 1468.70Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.

An O-conotoxin from the vermivorous Conus spurius active on mice and mollusks.

Here, we report the purification, amino acid sequence and a preliminary biological characterization of a peptide, sr7a, from the venom of Conus spurius, a vermivorous species collected in the Yucatan Channel, Mexico. The peptide consists of 32 amino acid residues (CLQFGSTCFLGDDDICCSGECFYSGGTFGICS&; &, amidated C-terminus) and contains six cysteines arranged in the pattern (C-C-CC-C-C) that characterizes the O-superfamily of conotoxins. This superfamily includes several pharmacological families (omega-, kappa-, muO-, delta- and gamma-conotoxins) that target Ca(2+), K(+), Na(+) and pacemaker voltage-gated ion channels. Compared with other O-conotoxins that were purified from venoms, this peptide displays sequence similarity with omega-SVIA (from Conus striatus), delta-TxVIA/B (from Conus textile), omega-CVID (from Conus catus) and kappa-PVIIA (from Conus purpurascens). At a dose of 250 pmol, peptide sr7a elicited hyperactivity when injected intracranially into mice and produced paralysis when injected into the pedal muscle of freshwater snails, Pomacea paludosa, but it had no apparent effect after intramuscular injection into the limpet Patella opea or the freshwater fish Lebistes reticulatus.

I-conotoxins in vermivorous species of the West Atlantic: peptide sr11a from Conus spurius.

Peptide sr11a was purified from the venom of Conus spurius, a vermivorous cone snail collected in the Yucatan Channel, in the Western Atlantic. Its primary structure was determined by automatic Edman degradation after reduction and alkylation. Its molecular mass, as determined by MALDI-TOF mass spectrometry (average mass 3650.77 Da), confirmed the chemical data (calculated average mass, 3651.13 Da). The sequence of peptide sr11a (CRTEGMSCgamma gamma NQQCCWRSCCRGECEAPCRFGP&; gamma, gamma-carboxy-Glu; &, amidated C-terminus) shows eight Cys residues arranged in the pattern that defines the I-superfamily of conotoxins. Peptide sr11a contains two gamma-carboxy-Glu residues, a post-translational modification that has been found in other I-conotoxins from species that live in the West Pacific: r11e from the piscivorous Conus radiatus, and kappa-BtX from the vermivorous Conus betulinus. Peptide sr11a is the eighth I-conotoxin isolated from a Conus venom and the first I-conotoxin from a species from the Western Atlantic. Peptide sr11a produced stiffening of body, limbs and tail when injected intracranially into mice.

Incidencia de litiasis vesicular asintomática en pacientes con complicaciones biliares / Incidence of asymptomatic gallstones in patients with biliary complications / Incidência de cálculos biliares asintomáticos em pacientes com complicações biliares

Introducción: Las complicaciones de la litiasis biliar (LB) son una causa importante de morbilidad en nuestro país y en el mundo entero y generan elevados costos en salud. Objetivo: El objetivo de este trabajo fue determinar, que pacientes con una complicación de su patología litiásica de la vía biliar (colecistitis, colangitis aguda, pancreatitis aguda), fueron previamente asintomáticos, resultando dicha complicación el debut de la enfermedad. Lugar: Sanatorio Asociación Española de Socorros Mutuos, Montevideo Uruguay. Diseño: Estudio observacional descriptivo, retrospectivo, análisis de historias clínicas. Materiales y Métodos: Se analizaron 234 casos clínicos. Se constató en este grupo de pacientes, características epidemiológicas, metodología diagnóstica, tratamientos recibidos y complicaciones. Resultados: Del total de pacientes (n=234), 109 (46.6%) tenían una litiasis vesicular asintomática (LVA) y la complicación biliar, fue el debut de su enfermedad. La colecistitis aguda fue la complicación más frecuente (68%), en segundo lugar, la colangitis aguda (22%) y en tercer lugar la pancreatitis aguda (10%). La edad promedio de presentación de la enfermedad fue los 59 años. Conclusiones: Casi la mitad de los pacientes (46.6%) que presentaron una complicación de su litiasis biliar eran asintomáticos. Este sería un argumento importante para indicar la colecistectomía laparoscópica con un criterio profiláctico en pacientes con una LVA.

Introduction : Complications of gallstones are an important cause of morbidity in our country and throughout the world and generate high health costs. Objective: The objective of this study was to determine which patients with a complication of their bile duct stone pathology (cholecystitis, acute cholangitis, acute pancreatitis) were previously asymptomatic, and this complication resulted in the onset of the disease. Place: Sanatorium Asociación Española de Socorros Mutuos, Montevideo Uruguay. Design: Descriptive and retrospective observational study with an analysis of medical records. Materials and Methods: 234 clinical cases were analyzed. Epidemiological characteristics, diagnostic methodology, treatments received, and complications were assessed in this group of patients. Results: Of the total number of patients (n=234), 109 (46.6%) had an asymptomatic gallbladder lithiasis and the biliary complication was the debut of their disease. Acute cholecystitis was the most frequent complication (68%), followed by acute cholangitis (22%) and third by acute pancreatitis (10%). The average age of presentation of the disease was 59 years. Conclusions: Almost half of the patients (46.6%) who presented a complication of their gallstones were asymptomatic. This would be an important argument to indicate laparoscopic cholecystectomy with a prophylactic criterion in patients with asymptomatic gallbladder lithiasis.

Introdução: As complicações dos cálculos biliares são uma importante causa de morbidade em nosso país e em todo o mundo e geram altos custos de saúde. Objetivo: O objetivo deste estudo foi determinar quais pacientes com uma complicação de sua patologia de cálculo do ducto biliar (colecistite, colangite aguda, pancreatite aguda) eram previamente assintomáticos, e essa complicação resultou no aparecimento da doença. Local: Sanatório Asociación Española de Socorros Mutuos, Montevidéu - Uruguai. Desenho: Estudo observacional descritivo, retrospectivo, análise de histórias clínicas. Materiais e Métodos: Foram analisados ​​234 casos clínicos. Características epidemiológicas, metodologia diagnóstica, tratamentos recebidos e complicações foram avaliadas neste grupo de pacientes. Resultados: Do total de pacientes (n=234), 109 (46,6%) apresentavam litíase vesicular assintomática e a complicação biliar foi o início da doença. A colecistite aguda foi a complicação mais frequente (68%), seguida da colangite aguda (22%) e a terceira da pancreatite aguda (10%). A idade média de apresentação da doença foi de 59 anos. Conclusões: Quase metade dos pacientes (46,6%) que apresentaram complicação de seus cálculos biliares eram assintomáticos. Esse seria um argumento importante para indicar a colecistectomia laparoscópica com critério profilático em pacientes com litíase vesicular assintomática.

Amino acid sequence and biological activity of a gamma-conotoxin-like peptide from the worm-hunting snail Conus austini.

A novel 31-residue toxin, named as7a, was isolated and characterized from the venom of Conus austini, a vermivorous cone snail collected in the western Gulf of Mexico. The complete amino acid sequence, TCKQKGEGCSLDVgammaCCSSSCKPGGPLFDFDC, was determined by automatic Edman sequencing after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and the sequence motif -gammaCCS-, which has only been found in the gamma-conotoxin family. The molecular mass of the native peptide was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which confirmed the chemical analyses and suggested a free C-terminus. The purified peptide elicited toxic effects in the freshwater snail Pomacea paludosa after intramuscular injection, but it had no effect when injected intracerebrally into mice. The structural similarity of peptide as7a to other gamma-conotoxins suggests that modulation of pacemaker channels could be responsible for its biological activity.

Putative gamma-conotoxins in vermivorous cone snails: the case of Conus delessertii.

Peptide de7a was purified from the venom of Conus delessertii, a vermivorous cone snail collected in the Yucatan Channel, Mexico. Its amino acid sequence was determined by automatic Edman degradation after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and several post-translationally modified residues. The determination of its molecular mass by means of laser desorption ionization time-of-flight mass spectrometry (average mass, 3170.0 Da) confirmed the chemical data and suggested amidation of the C-terminus. The primary structure (ACKOKNNLCAITgammaMAgammaCCSGFCLIYRCS*; O, hydroxyproline; gamma, gamma-carboxyglutamate; *, amidated C-terminus; calculated average mass, 3169.66 Da) of de7a contains a motif (gammaCCS) that has previously only been found in two other toxins, both from molluscivorous cone snails: TxVIIA from Conus textile and gamma-PnVIIA from Conus pennaceus. These toxins cause depolarization and increased firing of action potentials in molluscan neuronal systems, and toxin gamma-PnVIIA has been shown to act as an agonist of neuronal pacemaker cation currents. The similarities to toxins TxVIIA and gamma-PnVIIA suggest that peptide de7a might also affect voltage-gated nonspecific cation pacemaker channels.

Diversity of A-conotoxins of three worm-hunting cone snails (Conus brunneus, Conus nux, and Conus princeps) from the Mexican Pacific coast.

Conus marine snails (∼500 species) are tropical predators that use venoms mainly to capture prey and defend themselves from predators. The principal components of these venoms are peptides that are known as "conotoxins" and generally comprise 7-40 amino acid residues, including 0-5 disulfide bridges and distinct posttranslational modifications. The most common molecular targets of conotoxins are voltage- and ligand-gated ion channels, G protein-coupled receptors, and neurotransmitter transporters, to which they bind, typically, with high affinity and specificity. Due to these properties, several conotoxins have become molecular probes, medicines, and leads for drug design. Conotoxins have been classified into genetic superfamilies based on the signal sequence of their precursors, and into pharmacological families according to their molecular targets. The objective of this work was to identify and analyze partial cDNAs encoding conotoxin precursors belonging to the A superfamily from Conus brunneus, Conus nux, and Conus princeps. These are vermivorous species of the Mexican Pacific coast from which only one A-conotoxin, and few O- and I2-conotoxins have been reported. Employing RT-PCR, we identified 30 distinct precursors that contain 13 different predicted mature toxins. With the exception of two groups of four highly similar peptides, these toxins are diverse at both the sequence and the physicochemical levels, and they belong to the 4/3, 4/4, 4/5, 4/6, and 4/7 structural subfamilies. These toxins are predicted to target diverse nicotinic acetylcholine receptor (nAChR) subtypes: nx1d, muscle; pi1a-pi1d, α3ß2, α7, and/or α9α10; br1a, muscle, α3ß4, and/or α4ß2; and nx1a-nx1c/pi1g and pi1h, α3ß2, α3ß4, α9ß10, and/or α7.

Purification and structural characterization of lectins from the cnidarian Bunodeopsis antilliensis [corrected].

Purification and characterization of two different lectins from the Mexican anemone Bunodeopsis antilliensis are reported. These two lectins named Bunodeopsis antilliensis agglutinin-A (BAA-A) and -B (BAA-B) presented the following characteristics: BAA-A was resolved as a component, with haemagglutinating activity for human blood type A (N-acetylgalactosamine-galactose-fucose), with a molecular weight of 28,900 obtained by means of mass spectrometry, showed an isoelectric point of 5.04 with a higher carbohydrate specificity for N-acetylgalactosamine (GalNAc). The analysis of the N-terminal revealed it is related to phosphoesterase and GTP binding protein. BAA-B mainly active with human blood type B (galactose-galactose-fucose) was resolved into three fractions (BAA-B1-3). Their molecular weight were: BAA-B(1) 39,350, BAA-B(2) 28,300 and BAA-B(3) 17,550. The estimated isoelectric points were 8.05, 4.66 and 6.60, respectively. Only fraction 3 exhibited haemagglutinating activity with a higher carbohydrate specificity for galactose and mannose. The analysis of the N-terminal pointed out it is related with phospholipase A(2). We suggest these lectins could be related to a feeding strategy[corrected].

A novel arrangement of Cys residues in a paralytic peptide of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting snail from the Gulf of Mexico.

The present study details the purification, the amino acid sequence determination, and a preliminary characterization of the biological effects in mice of a new conotoxin from the venom of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting cone snail collected in the western Gulf of Mexico (Mexico). The 23-amino acid peptide, called as25a, is characterized by the sequence pattern CX1CX2CX8CX1CCX5, which is, for conotoxins, a new arrangement of six cysteines (framework XXV) that form three disulfide bridges. The primary structure (CKCPSCNFNDVTENCKCCIFRQP*; *, amidated C-terminus; calculated monoisotopic mass, 2644.09Da) was established by automated Edman degradation after reduction and alkylation, and MALDI-TOF and ESI mass spectrometry (monoisotopic mass, 2644.12/2644.08Da). Upon intracranial injection in mice, the purified peptide provokes paralysis of the hind limbs and death with a dose of 240 pmol (~0.635 µg, ~24.9 ng/g). In addition, a post-translational variant of this peptide (as25b) was identified and determined to contain two hydroxyproline residues. These peptides may represent a novel conotoxin gene superfamily.

Arrhythmogenic effect of a crude extract from sea anemone Condylactis gigantea: possible involvement of rErg1 channels.

Sea anemones possess a number of peptide toxins that target ion channels which provide powerful tools to study the molecular basis of diverse signaling pathways. It is also acknowledged that currents through Erg1 K(+) channels in cardiac myocytes are important for electrical stability of the heart and alterations in its activity has been linked to the onset of a potentially life-threatening heart condition named long QT syndrome type 2. Here, we report that a crude extract from sea anemone Condylactis gigantea significantly increases the QT interval and has arrhythmogenic effects in the rat heart. Furthermore, a bioassay-guided purification procedure allowed the isolation of a chromatographic fraction containing a major component with a molecular mass of 4478 Da from the crude extract, which causes a significant inhibition of whole-cell patch-clamp currents through recombinant Erg1 channels, responsible of the rapid delayed rectifying current crucial for electrical activity in the heart. Further studies could provide relevant information on the molecular mechanism of C. gigantea peptide toxins which represent promising tools in studying the physiology of diverse ion channels.

Identification, by molecular cloning, of a novel type of I2-superfamily conotoxin precursor and two novel I2-conotoxins from the worm-hunter snail Conus spurius from the Gulf of México.

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, México. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I(2)-conotoxins and three of I(2)-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I(2)) or 23- residue signal peptide (I(2)-like), a 31-residue "pro" region (I(2)-like), and a 32-residue mature toxin region (I(2) and I(2)-like). In addition, all the precursors have a 13-residue "post" region which contains a gamma-carboxylation recognition sequence that directs the gamma-carboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a "post" region has been found in precursors of I-conotoxins that also contain a "pro" region. The "post" peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that gamma-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I(2)-like-conotoxins of C. spurius are more related to I(2)-conotoxins than to I(1)- and I(3)-conotoxins from other species, and that they might represent a new subgroup of the I(2)-superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes.

Peptide sr11a from Conus spurius is a novel peptide blocker for Kv1 potassium channels.

More than a hundred conotoxins are known today and from them, only seven conopeptides have been identified to target voltage-gated potassium channels (Kv). Conotoxin sr11a belongs to the I(2)-superfamily which is characterized by four disulfide bridges and provokes muscle stiffness when injected intracranially in mice. The aim of this work was to test the biological activity of sr11a on recombinant voltage-gated Kv1 potassium channels expressed in Xenopus laevis oocytes. Peptide sr11a was purified by high-performance liquid chromatography from the venom of the vermivorous Conus spurius. We found that peptide sr11a inhibits the delayed rectifiers Kv1.2 and Kv1.6 but had not effect on the slowly inactivating Kv1.3 channel. The functional dyad composed of a basic Lys and a hydrophobic amino acid residue is a crucial structural element, regarding the binding properties and blocking activities of more than a hundred K(+) channel toxins. Peptide sr11a does not contain Lys residues and then, it lacks the functional dyad. Molecular modeling of peptide sr11a reveals the presence of exposed basic residues of Arg and suggests that Arg17 and Arg29 are important on its biological activity.

Identification, by RT-PCR, of four novel T-1-superfamily conotoxins from the vermivorous snail Conus spurius from the Gulf of Mexico.

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, Mexico. From it, PCR products were generated, sequenced, and predicted to encode eight distinct precursors of T-1-conotoxins. These precursors contain five different mature toxins, of which four are novel and one (sr5a) has been previously purified and characterized from the venom of this species. Three of the novel toxins are very similar to sr5a: two have one amino acid substitution at position 8, whereas the other is predicted to have one additional residue at the C-terminus; the fourth toxin has five amino acid substitutions and is predicted to have two additional residues at the C-terminus. In general, the precursors include a 22-residue signal peptide, a 24-residue "pro" region, and a 13- to 16-residue mature toxin region; however, the C-termini of two mature toxin regions are predicted to be altered by post-translational processing. Three precursors lack, in the same positions, 15 amino acid residues included in the "pre" (one residue) and "pro" (14 residues) regions, which suggests the existence of an exon encoding the last signal peptide residue and the first 14 residues of the "pro" region. Phylogenetic analysis indicates that the T-1-conotoxin precursors and mature toxins of C. spurius are more similar to certain precursors and toxins from molluscivorous Conus species than to any precursors and toxins from vermivorous cones. The results reported here will be useful for synthesizing the novel toxins in order to identify their molecular targets.

Biochemical and pharmacological characterization of toxins obtained from the fire coral Millepora complanata.

Millepora complanata is a normal resident of coral reefs in the Mexican Caribbean. In this study, we describe for the first time the vasoconstrictor, phospholipase A2 (PLA2), and hemolytic activities elicited by a crude extract obtained from M. complanata. This extract caused a concentration-dependent contraction of isolated rat aortic rings (EC50=22.4+/-1.1 microg protein/mL). This effect was endothelium independent and significantly reduced in the absence of extracellular Ca2+ and when the intracellular Ca2+ stores were depleted. In addition, the crude extract obtained from M. complanata showed PLA2 activity (7.231+/-0.092 mmol min(-1) mg(-1)) and hemolysis of rat erythrocytes (HU50=1.64+/-1.04 mug protein/mL). The hemolysis increased in the presence of Ca2+ and decreased in the presence of cholesterol. Furthermore, this hemolysis was significantly reduced after incubation with an inhibitor of PLA2 enzymes. The hemolytic and vasoconstrictor effects were abolished after incubating the extract under denaturing conditions. Reverse phase chromatography of the M. complanata extract afforded 19 fractions (F1 to F19). F4 induced hemolysis and contained mainly a protein of 30 kDa, probably a PLA2 enzyme, while F8 and F11, containing mainly proteins of 15 and 20 kDa respectively, produced vasoconstrictor effects mediated by different mechanisms of action.