Isolation and characterization of five novel disulfide-poor conopeptides from Conus marmoreus venom

Background: Conopeptides from cone snail venom have aroused great interest related to the discovery of novel bioactive candidates, due to their excellent prospects for the treatment of various health problems such as pain, addiction, psychosis and epilepsy. In order to explore novel biopeptides, we investigated the structure and function of five novel conopeptides isolated from the venom of Conus marmoreus from South China Sea. Methods: C. marmoreus crude venom was prepared, fractionated and purified by HPLC system. The primary sequences of the five novel disulfide-poor conopeptides Mr-1 to Mr-5 were identified by comprehensive analysis of de novo MALDI-TOF tandem mass spectrometry and Edman degradation data. In order to investigate their function, these five conopeptides were synthesized by Fmoc-SPPS chemistry, and their biological effects at several heterologous rat nicotinic acetylcholine receptor (nAChR) subtypes (α1β1δε, α3β2, α3β4, α4β2) were determined by electrophysiological technique. Results: Five novel disulfide-poor conopeptides were identified and named as follows: Mr-1 (DWEYHAHPKPNSFWT), Mr-2 (YPTRAYPSNKFG), Mr-3 (NVIQAPAQSVAPP NTST), Mr-4 [KENVLNKLKSK(L/I)] and Mr-5 [NAVAAAN(L/I)PG(L/I)V]. None of them contains a disulfide bond. The sequences of conopeptides Mr-2 to Mr-5 do not belong to any category of the known disulfide-poor conopeptides. No significant activity against the above nAChR subtypes were observed for the five conopeptides at 100 µM. Conclusion: We purified and structurally characterized five novel disulfide-poor conopeptides from C. marmoreus crude venom and first investigated their nAChR inhibitory effects. This work expanded our knowledge on the structure and function of disulfide-poor conopeptides from this cone snail venom.(AU)

Proteomic analysis of Red Sea Conus taeniatus venom reveals potential biological applications

Diverse and unique bioactive neurotoxins known as conopeptides or conotoxins are produced by venomous marine cone snails. Currently, these small and stable molecules are of great importance as research tools and platforms for discovering new drugs and therapeutics. Therefore, the characterization of Conus venom is of great significance, especially for poorly studied species. Methods: In this study, we used bioanalytical techniques to determine the venom profile and emphasize the functional composition of conopeptides in Conus taeniatus, a neglected worm-hunting cone snail. Results: The proteomic analysis revealed that 84.0% of the venom proteins were between 500 and 4,000 Da, and 16.0% were > 4,000 Da. In C. taeniatus venom, 234 peptide fragments were identified and classified as conotoxin precursors or non-conotoxin proteins. In this process, 153 conotoxin precursors were identified and matched to 23 conotoxin precursors and hormone superfamilies. Notably, the four conotoxin superfamilies T (22.87%), O1 (17.65%), M (13.1%) and O2 (9.8%) were the most abundant peptides in C. taeniatus venom, accounting for 63.40% of the total conotoxin diversity. On the other hand, 48 non-conotoxin proteins were identified in the venom of C. taeniatus. Moreover, several possibly biologically active peptide matches were identified, and putative applications of the peptides were assigned. Conclusion: Our study showed that the composition of the C. taeniatus-derived proteome is comparable to that of other Conus species and contains an effective mix of toxins, ionic channel inhibitors and antimicrobials. Additionally, it provides a guidepost for identifying novel conopeptides from the venom of C. taeniatus and discovering conopeptides of potential pharmaceutical importance.(AU)

The inhibitory effect of a non-yessotoxin-producing dinoflagellate, Lingulodinium polyedrum (Stein) Dodge, towards Vibrio vulnificus and Staphylococcus aureus

ResumenEl aumento de la resistencia bacteriana a los antibióticos ha causado preocupación a nivel mundial, por lo que se ha promovido la búsqueda de nuevos compuestos. Debido a su abundancia y diversidad, el fitoplancton marino constituye una importante fuente potencial de tales compuestos. La investigación sobre dinoflagelados ha llevado al descubrimiento de inhibidores de crecimiento bacteriano. El dinoflagelado marino Lingulodinium polyedrum causa proliferaciones algales en diferentes regiones del mundo, incluyendo México, y también se sabe que regula el crecimiento de otras especies en las aguas costeras. En este trabajo, se investiga la taxonomía de este dinoflagelado y se caracteriza la capacidad de sus extractos para inhibir el crecimiento de dos bacterias de importancia médica (Vibrio vulnificus y Staphylococcus aureus) en placas de cultivo de agar. La caracterización taxonómica se realizó por PCR y amplificación del gen de ITS, y se confirmó que la especie aislada en la costa del Pacífico de México fue L. polyedrum. Para demostrar el efecto inhibidor de los extractos de L. polyedrum, los cultivos se cosecharon por centrifugación. Los pellets de tres abundancias celulares se extrajeron con agua, metanol, hexano y cloroformo. Los experimentos en V. vulnificus mostraron una inhibición alta del crecimiento para los cuatro extractos, variando entre 77 y 98 %. Sorprendentemente, la inhibición del crecimiento fue menor cuando los extractos se originaron a partir de una mayor abundancia de células L. polyedrum, varía de 0 a 34 %. Para S. aureus, la inhibición del crecimiento también fue alta, pero no estadísticamente diferente para todos los extractos y abundancias de células, con un rango de 62 hasta 99 %. Esto resultados son prometedores para futuras aplicaciones farmacológicas. La cepa mexicana de L. polyedrum no produjo yesotoxinas detectables.

AbstractThe increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins. Rev. Biol. Trop. 64 (2): 805-816. Epub 2016 June 01.

In the picture: disulfide-poor conopeptides, a class of pharmacologically interesting compounds

During evolution, nature has embraced different strategies for species to survive. One strategy, applied by predators as diverse as snakes, scorpions, sea anemones and cone snails, is using venom to immobilize or kill a prey. This venom offers a unique and extensive source of chemical diversity as it is driven by the evolutionary pressure to improve prey capture and/or to protect their species. Cone snail venom is an example of the remarkable diversity in pharmacologically active small peptides that venoms can consist of. These venom peptides, called conopeptides, are classified into two main groups based on the number of cysteine residues, namely disulfide-rich and disulfide-poor conopeptides. Since disulfide-poor conotoxins are minor components of this venom cocktail, the number of identified peptides and the characterization of these peptides is far outclassed by its cysteine-rich equivalents. This review provides an overview of 12 families of disulfide-poor peptides identified to date as well as the state of affairs.(AU)

Infiltrated plaques resulting from an injury caused by the common octopus (Octopus vulgaris): a case report

Several species of octopus are considered venomous due to toxins present in the glands connected to their beak, which may be associated with hunt and kill of prey. Herein, we report an accident involving a common octopus (Octopus vulgaris) that injured an instructor during a practical biology lesson and provoked an inflamed infiltrated plaque on the hand of the victim. The lesion was present for about three weeks and was treated with cold compresses and anti-inflammatory drugs. It was healed ten days after leaving a hyperchromic macule at the bite site. The probable cause of the severe inflammation was the digestive enzymes of the glands and not the neurotoxins of the venom.

Conus vexillum venom induces oxidative stress in Ehrlich's ascites carcinoma cells: an insight into the mechanism of induction

Background: It is estimated that venoms of marine cone snails (genus Conus) contain more than 100,000 different small peptides with a wide range of pharmacological and biological actions. Some of these peptides were developed into potential therapeutic agents and as molecular tools to understand biological functions of nervous and cardiovascular systems. In this study we examined the cytotoxic and anticancer properties of the marine vermivorous cone snail Conus vexillum (collected from Hurgada and Sharm El-Shaikh, Red Sea, Egypt) and suggest the possible mechanisms involved. The in vitro cytotoxic effects of Conus venom were assessed against Ehrlich's ascites carcinoma (EAC) cells. Results: Conus venom treatment resulted in concentration-dependent cytotoxicity as indicated by a lactate dehydrogenase leakage assay. Apoptotic effects were measured in vivo by measuring levels of reactive oxygen species and oxidative defense agents in albino mice injected with EAC cells. Conus venom (1.25 mg/kg) induced a significant increase (p < 0.05) in several oxidative stress biomarkers (lipid peroxidation, protein carbonyl content and reactive nitrogen intermediates) of EAC cells after 3, 6, 9 and 12 hours of venom injection. Conus venom significantly reduced (p < 0.05) the activities of oxidative defense enzymes (catalase and superoxide dismutase) as well as the total antioxidant capacity of EAC cells, as evidenced by lowered levels of reduced glutathione. Conclusions: These results demonstrate the cytotoxic potential of C. vexillum venom by inducing oxidative stress mediated mechanisms in tumor cells and suggest that the venom contains novel molecules with potential anticancer activity.(AU)

Conus vexillum venom induces oxidative stress in Ehrlich's ascites carcinoma cells: an insight into the mechanism of induction

It is estimated that venoms of marine cone snails (genus Conus) contain more than 100,000 different small peptides with a wide range of pharmacological and biological actions. Some of these peptides were developed into potential therapeutic agents and as molecular tools to understand biological functions of nervous and cardiovascular systems. In this study we examined the cytotoxic and anticancer properties of the marine vermivorous cone snail Conus vexillum (collected from Hurgada and Sharm El-Shaikh, Red Sea, Egypt) and suggest the possible mechanisms involved. The in vitro cytotoxic effects of Conus venom were assessed against Ehrlich’s ascites carcinoma (EAC) cells. Results Conus venom treatment resulted in concentration-dependent cytotoxicity as indicated by a lactate dehydrogenase leakage assay. Apoptotic effects were measured in vivo by measuring levels of reactive oxygen species and oxidative defense agents in albino mice injected with EAC cells. Conus venom (1.25 mg/kg) induced a significant increase ( p  < 0.05) in several oxidative stress biomarkers (lipid peroxidation, protein carbonyl content and reactive nitrogen intermediates) of EAC cells after 3, 6, 9 and 12 hours of venom injection. Conus venom significantly reduced ( p  < 0.05) the activities of oxidative defense enzymes (catalase and superoxide dismutase) as well as the total antioxidant capacity of EAC cells, as evidenced by lowered levels of reduced glutathione.Conclusions These results demonstrate the cytotoxic potential of C. vexillum venom by inducing oxidative stress mediated mechanisms in tumor cells and suggest that the venom contains novel.

Estudio histológico del tubo digestivo y aparato venenoso de Gemmula periscelida (Gastropoda: Turridae) / Histologic study of digestive tract and venom apparatus of Gemmula periscelida (Gastropoda: Turridae)

En el presente trabajo se describe anatómica e histológicamente el tubo digestivo y aparato venenoso de Gemmula periscelida (Gastropoda: Turridae) en ejemplares colectados al Noroeste de la Plataforma Continental Yucateca. Se determinó que el tipo de epitelio que reviste a cada una de las zonas del tubo digestivo (probóscide, esófago anterior, medio y posterior, estómago, glándula digestiva e intestino) y al aparato venenoso, es diferente a lo reportado para otros túrridos; por lo que se infiere el posible mecanismo de alimentación para esta especie.

In this paper we realized anatomical and histologically description of the digestive tract and venom apparatus of Gemmula periscelida (Gastropoda: Turridae) specimens collected northwest of the Yucatan Shelf. Results of analysis show that there are differences in the type of epithelium coating each of the areas of the digestive tract (proboscis, anterior, middle and posterior esophagus, stomach, digestive gland and intestine) and of a venom apparatus with respect to that reported for other turrid snails. This suggests the possible feeding mechanism for this species.

Report of a human accident caused by Conus regius (Gastropoda, Conidae) / Relato de um acidente em ser humano causado por Conus regius (Gastropoda, Conidae)

Conus regius is a venomous mollusc in the Conidae family, which includes species responsible for severe or even fatal accidents affecting human beings. This is the first report on a clinical case involving this species. It consisted a puncture in the right hand of a diver who presented paresthesia and movement difficulty in the whole limb. The manifestations disappeared after around twelve hours, without sequelae.

Conus regius é um molusco venenoso da família Conidae, que inclui espécies responsáveis por acidentes graves ou mesmo fatais em humanos. Os autores relatam pela primeira vez um caso clínico envolvendo a espécie, que inclui uma punctura na mão direita de um mergulhador submarino, que apresentou parestesias e dificuldade de movimentação do membro todo. O quadro desapareceu em cerca de doze horas, sem seqüelas.

Neurotoxinas de invertebrados como alternativas terapéuticas y herramientas en investigación básica / Neurotoxins from invertebrates as alternative therapeutic agents and tools in basic research

Los venenos que producen los animales son una mezcla compleja de proteínas, péptidos, enzimas y trazas deelementos no proteicos tales como carbohidratos y sales, cuya finalidad es inmovilizar la presa y comenzar a digerirla. Las toxinas son sustancias aisladas de venenos, con una o varias acciones específicas sobre las víctimas. Entre estos compuestos, son numerosos los que tienen acción sobre receptores específicos ubicados en el sistema nervioso central y/o periférico, mientras que otros ejercen sus efectos actuando sobre otras proteínas. Desde el descubrimiento en 1971, del péptido que dio origen al Captopril, y teniendo en cuenta que muchas toxinas son útiles como herramientas para el estudio de procesos fisiológicos, se comenzó a mirar los venenos de animales como fuentes ricas en compuestos bioactivos y a pensar en su uso potencial como agentes terapéuticos. Así pues, en la actualidad disponemos de diferentes medicamentos y herramientas diagnósticas o de investigación básica derivados de toxinas. Esta revisión, basada en publicaciones realizadas en los últimos 10 años, busca proporcionar una visión actual del uso de algunas de estas moléculas como herramientas en diferentes campos de la biomedicina y la farmacia, y en su aplicación como nuevas alternativas terapéuticas o como modelos en el diseño de las mismas.

Pharmacological characterization of venoms obtained from Mexican toxoglossate gastropods on isolated guinea pig ileum

The protein-containing extracts prepared from the venom ducts of Conus austini, Conus spurius and Polystira albida caused a concentration-dependent inhibition of spontaneous contractions in guinea pig ileum. The most potent extract was obtained from P. albida venom ducts (IC50 equal 0.11 more or less 0.02 microg protein/mL). The three extracts produced a moderate inhibition of contractions elicited by acetylcholine (ACh 1 microM), suggesting the presence of anticholinergic compounds. The contractile response elicited by nicotine (10 microM) was significantly reduced by the extracts prepared from the ducts of C. austini and P. albida, which indicates that the venom produced by these species contains toxins that target neuronal nicotinic receptors. All three extracts significantly inhibited contractions evoked by histamine (0.5 miM), particularly those from C. spurius and P. albida. These findings reveal the presence of antihistaminergic compounds not previously described in any conoidean venom. Finally, we found that only the extract prepared from C. spurius ducts decreased KCl (60 mM)-induced contractions, indicating that the venom of this snail contains compounds that block voltage-dependent Ca2 more or Na more channels.

Cutaneous secretion from the giant African snail, Achatina fulica, as a source of Antileishmanial compounds

Leishmaniasis is a severe protozoan parasite disease, causing clinical symptoms from a single cutaneo usulceration to a progressive and fatal disease. The treatment is based on toxic chemotherapeutic compounds, being pentavalent antimonials the first line drugs. Natural products from Brazilian fauna, especially animal venoms and cutaneous secretions, are a rich source of novel chemical molecules which could beused as drug prototypes for the development of new therapeutics. This study describes for the first time the effective anti leishmanial activity of crude cutaneous secretion from the giant African snail, Achatinafulica, and its mammalian cells citotoxi city and also its potential mode of action against Leishmania promastigotes. The crude secretion showed an Effective Concentration (EC50) of 98.37 µg/mL againstL.(L.) chagasi promastigotes. By means of enzymatic assays, a L-amino acid oxidase (L-AAO) activity was detected in crude secretion, and the hydrogen peroxide produced by this enzyme revealed to be one of the compounds responsible for the antileishmanial effect. The use of catalase for H2O2 scavenging in Leishmania cultures incubated with crude secretion abolished 54% of parasite death.

A Leishmaniose é uma doença parasitária grave que causa desde uma única ulceração cutânea até uma doença progressiva e fatal. O tratamento é baseado em agentes quimioterápicos tóxicos, sendo indicados como fármacos de primeira escolha os antimoniais pentavalentes. Os produtos naturais derivados da fauna brasileira, especialmente venenos e secreções cutâneas de anfíbios, são fontes ricas de novas moléculas químicas, as quais podem ser utilizadas como protótipos farmacêuticos no desenvolvimento de novos fármacos. No presente trabalho, é feita a descrição inédita sobre a efetiva atividade anti-Leishmania da secreção cutânea do caramujo gigante Africano, Achatina fulica, bem como a sua citotoxicidade em células de mamíferos e seu possível mecanismo de ação contra os promastig.

Venomous mollusks: the risks of human accidents by conus snails (gastropoda: conidae) in Brazil

Mollusks of the genus Conus present a venomous apparatus composed of radulae, a chitin structure linked to glands, which injects potent neurotoxic peptides, causing serious human envenomation and even death, associated with the blockage of certain receptors and muscular paralysis. No reported envenomation has occurred in Brazil, but certain populations are at risk of accidents.

Os moluscos do gênero Conus apresentam um aparato venenoso composto de uma rádula quitinosa ligada a glândulas de peçonha, causando envenenamentos humanos graves e mesmo óbitos pela ação neurotóxica indutora do bloqueio de vários receptores e paralisia muscular. Não há casos descritos de envenenamento no país, mas determinadas populações correm risco de acidentes.

The red tide event in El Salvador, August 2001-January 2002

A red tide event occurred in El Salvador from August 2001 to January 2002. National health authorities usually measured toxin levels in Ostrea iridescens, however other species were analyzed during this microalgae bloom: Anadara similis, Anadara tuberculosa and Modiolus sp. El Salvador authorities consider 400 mouse units/100 g the highest value that is safe for human health. During this period toxin levels in O. iridescens and Modiolus sp. increased from values under 400 to 3 977 and 15 468 mouse units/100 g, respectively. Persistent and higher levels were recorded in oyster and mussel banks on the west part of the country. The Ministry of Health and Social Assistance treated 41 slight to moderate intoxications associated to bivalve mollusks consumption

Hubo una marea roja en El Salvador desde agosto de 2001 hasta enero de 2002. Las autoridades de salud usualmente medían niveles de toxinas en Ostrea iridescens, pero durante esta proliferación de microalgas se estudió también Anadara similis, Anadara tuberculosa y Modiolus sp. En El Salvador se consideran 400 unidades ratón/ 100 g como el límite sobre el cual el consumo de moluscos representa riesgo para la salud humana. Durante este período se detectó que los niveles de toxinas en O. iridescens y Modiolus sp. variaron desde niveles menores a 400 hasta 3 977 y 15 468 unidades ratón/100 g, respectivamente. Los niveles más altos y persistentes se registraron en los bancos de ostras y mejillones de la zona oeste del país. El Ministerio de Salud y Asistencia Social atendio 41 intoxicaciones de leves a moderadas atribuidas al consumo de moluscos bivalvos

Simultaneous presence of Paralytic and Diarrheic Shellfish Poisoning toxins in Mytilus chilensis samples collected in the Chiloe Island, Austral Chilean Fjords

The study shown here provides the first indisputable evidence that shellfish can be contaminated with Paralytic Shellfish Poisoning (PSP) and Diarrheic Shellfish Poisoning (DSP) toxins during the summer season in the Southern Chilean fjords. Quantitative analysis of the simultaneous presence of PSP and DSP toxins in Mytilus chilensis samples collected in the Chiloe Island are shown. The High Performance Liquid Chromatography (HPLC) analysis with pre-column derivatization method for DSP toxins and the post-column derivatization methods for PSP toxins, both with fluorescent on-line detections, showed that both type of toxins were concentrated by the filter bivalve Mytilus chilensis in amounts above the international safe limits. The phytoplankton analysis showed the presence of both Alexandrium catenella and Dinophysis acuta in the water column. The data shows stratification of the toxic dinoflagellates in the water column, since the lowest amount of both DSP and PSP toxins were measured in the superficial and deeper levels of the water column. Moreover, the highest toxicities of both types of toxins were shown by the shellfish samples collected at a depth of 6 meters with 190 nanograms of DTX-1 / gram of digestive gland and 709.8 mg of PSP toxins / 100 grams of mussel meat.

Veneno diarreico de moluscos y derivados del benzopireno en bivalvos ahumados / Diarrheal poison in molluscs and derivates of benzopyrene in smoked bivalves

Los florecimientos algales nocivos (FANs), corresponden al fenómeno comúnmente denominado marea roja. Esta se encuentra constituida por 5 tipos de venenos diferentes: veneno paralizante de moluscos (VPM); veneno diarreico de moluscos (VDM); veneno amnésico de moluscos (VAM); veneno neuro-tóxico de moluscos (VNM) y toxinas producidas por cianobacterias, cada una de las cuales presenta una distribución a nivel mundial. En Chile, sólo han sido identificados los venenos VPM y VDM, los que se encuentran endémicamente desde la X a XII regiones. Su identificación a nivel nacional, se realiza a través del método denominado bioensayo en ratón, el cual presenta una sensibilidad adecuada para la detección de VPM, no así para la detección de VDM, debido a las cualidades químicas que presentan estas toxinas, produciéndose cada año un número no estimado de personas intoxicadas. Dadas las características socioeconómicas del sur de Chile, la mayoría de los bivalvos son expendidos bajo condiciones de ahumado, de manera de aumentar su preservación para el consumo por parte de la población local. Esto ocasiona la fijación de hidrocarburos aromáticos policíclicos (HAPs) en los bivalvos de manera extrema. Considerando que los HAPs son iniciadores y el VDM promotor del proceso carcinogénico, sumándose a ello la importancia de este tipo de alimento en la dieta diaria de la población, en este trabajo se identificaron y cuantificaron las concentraciones HAP y VDM presentes en bivalvos de la X Región. Los resultados, muestran que los HAPs y VDM presentes en los bivalvos sobrepasan la norma internacional 10 µg/kg y 200 ng/g de hepatopáncreas, respectivamente. Siendo el HAP más relevante el benzopireno (71,62 ± 18,40 µg/kg en cholgas) y la toxina VDM identificada DTX-3 (217,33 ± 14,76 ng/g de hepatopáncreas en cholgas). Estos resultados ponen en evidencia la necesidad de realizar estudios epidemiológicos en la zona para determinar si existe una asociación entre la exposición a estos compuestos y la incidencia de cáncer gástrico de la población local.

Envenenamiento por contacto con caracol Conus / Poisoning by contact with Conus snail

De los caracoles, la familia Conidae o en forma de cono son los únicos que se sabe que pueden producir picaduras venenosas mortales. Se localizan en todos los mares tropicales del mundo en sustratos arenosos y rocosos. Es importante tener precaución al manipular cualquier caracol en forma de cono. No existe un antídoto conocido, el tratamiento médico es sintomático. Se muestra el manejo médico conocido que ha tenido éxito en evitar un desenlace fatal

Just a diarrhea? Evidence of diarrhetic shellfish poisoning in Santa Catarina, Brazil

The occurrence of phycotoxins from the diarrhetic shellfish poisoning group has been detected in mussels from Santa Catarina by two independent assays. This finding can help to elucidade several official and unofficial reported cases of human intoxication in the region. The issue becomes very important once the mussel culture activity is growing rapidly in the state which is already the largest mussel producer in the Country.