Mutacytin-1, a New C-Type Lectin-Like Protein from the Venezuelan Cuaima (Lachesis muta muta Linnaeus, 1766) (Serpentes: Viperidae) Snake Venom Inducing Cardiotoxicity in Developing Zebrafish (Danio rerio) Embryos.

Envenomation by the Venezuelan bushmaster snake (Lachesis muta muta) (Serpentes: Viperidae) is characterized by local and cardiac alterations. This study investigates the in vivo cardiac dysfunction, tissue destruction, and cellular processes triggered by Lachesis muta muta snake crude venom and a C-type lectin (CTL)-like toxin named Mutacytin-1 (MC-1). The 28 kDa MC-1 was obtained by molecular exclusion, ion exchange, and C-18 (checking pureness) reverse-phase chromatographies. N-terminal sequencing of the first eight amino acids (NNCPQ LLM) revealed 100% identity with Mutina (CTL-like) isolated from Lachesis stenophrys, which is a Ca2+-dependent-type galactoside-binding lectin from Bothrops jararaca and CTL BpLec from Bothrops pauloensis. The cardiotoxicity in zebrafish of MC-1 was evaluated by means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40 and 60 min post-treatment. The L. muta muta venom and MC-1 also produced heart rate/rhythm alterations, circulation modifications, and the presence of thrombus and apoptotic phenomenon with pericardial damages. Acridine orange (100 µg/mL) was used to visualize apoptosis cellular process in control and treated whole embryos. The cardiotoxic alterations happened in more than 90% of all larvae under the action of L. muta muta venom and MC-1. The findings have demonstrated the potential cardiotoxicity by L. muta muta venom, suggesting the possibility of cardiovascular damages to patients after bushmaster envenoming.

Intraspecies geographical variability in the South American tigra mariposa (Bothrops venezuelensis Sandner 1952) snake venom activities.

Bothrops venezuelensis snake venoms, from five localities in the North-Central Venezuelan regions, showed biochemical and haemostatic differences. In this study, bioactivities of B. venezuelensis venoms from different regions (Aragua state; Waraira Repano (Capital District); Baruta, La Boyera and Lagunetica (Miranda state)) were compared using both natural and synthetic substrates. The protein contents of these venoms were Lagunetica 89%, La Boyera 79%, Baruta 71%, Waraira Repano 68% and Aragua 64%. Toxic activities effects were: Intraperitoneal LD50s: Aragua-14 mg/kg; Waraira Repano-6.4 mg/kg; Baruta: 8.3 mg/kg; La Boyera-4.4 mg/kg; Lagunetica-16.2 mg/kg. The MHD results: Aragua-21.4 µg/mouse; Waraira Repano-2.5 µg/mouse; Baruta-1.2 µg/mouse; La Boyera-1.4 µg/mouse and Lagunetica-12 µg/mouse. The hide powder azure results: Aragua-1.24 U/mg; La Boyera-2.26 U/mg; Baruta-2.83 U/mg; Lagunetica-3.28 U/mg and Waraira Repano-5.77 U/mg. Esterase specific activity on BAEE results: Waraira Repano-666.66 U/mg; La Boyera-805.5 U/mg; Baruta-900.00 U/mg; Lagunetica-922.19 U/mg and Aragua-1960.67 U/mg. Casein zymography showed digestion bands in the molecular weight above 100 and at 66.2 and 21.5 kDa. Analysis of casein degradation by SDS-PAGE showed two different degradation patterns. Fibrinolytic activity (mm2/µg) on fibrin plates results: Aragua-6.07; Lagunetica-27.6; Waraira Repano-35.7; La Boyera-44.27 and Baruta-45.63. In the fibrinogenolytic assay, the five venoms completely degraded the α chain after 1 min of incubation. None of the venoms completely degraded the ß and γ chains after 24 h incubation. The research indicated that venoms of B. venezuelensis of different geographic areas in Venezuela exhibit variances in composition and component concentrations; except the Aragua venom, all of them had high proteolytic activities.

Hemostatic and toxinological diversities in venom of Micrurus tener tener, Micrurus fulvius fulvius and Micrurus isozonus coral snakes.

The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A2s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD50s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD50 of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD50s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bß chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders.

Characterization of toxins from the broad-banded water snake Helicops angulatus (Linnaeus, 1758): isolation of a cysteine-rich secretory protein, Helicopsin.

Helicops angulatus (broad-banded water snake) according to recent proposals is presently cited in the family Dipsadidae, subfamily Xenodontinae, forming the tribe Hydropsini along with the genera Hydrops and Pseudoeryx. The current work characterizes the proteolytic and neurotoxic activities of H. angulatus crude toxins from salivary excretion (SE) and describes the isolation and identification of a cysteine-rich secretory protein (CRISP) called helicopsin. The SE lethal dose (LD50) was 5.3 mg/kg; however, the SE did not contain hemorrhagic activity. Helicopsin was purified using activity-guided, Superose 12 10/300 GL molecular exclusion, Mono Q10 ion exchange, and Protein Pak 60 molecular exclusion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a highly purified band of approximately 20 kDa. The minimal lethal dose for helicopsin was 0.4 mg/kg. Liquid chromatography mass spectrometry (LC-MS/MS) analysis identified 2 unique peptides MEWYPEAAANAER and YTQIVWYK, representing a protein sequence (deleted homology) belonging to cysteine-rich secretory proteins, which are conserved in snake venoms (CRISPs). CRISPs are a large family of cysteine-rich secretory proteins found in various organisms and participate in diverse biological processes. Helicopsin exhibited robust neurotoxic activity as evidenced by immediate death (~8 min) due to respiratory paralysis in NIH mice. These observations for helicopsin purified from H. angulatus provide further evidence of the extensive distribution of highly potent neurotoxins in the Colubroidea superfamily of snakes than previously described.

Intensa reacción alérgica en paciente mordido por la hormiga negra (Odontomachus bauri) / Intense allergic reaction in a patient stung by the black ant (Odontomachus bauri)

INTRODUCCIÓN: en países del área tropical y subtropical las picaduras de hormiga eran generalmente benignas, autolimitadas; sin embargo, en la actualidad se describe un mayor número de casos, que se desarrollan con cuadros alérgicos graves que pueden evolucionar hasta el choque anafiláctico o urticarias severas. MÉTODOS: un adulto joven con una reacción alérgica moderada para una picadura de hormiga es revisado y comentado en cuanto a su importancia biomédica. Se trata del segundo accidente descrito en la literatura causado por la hormiga Odontomachus bauri, lo cual acentúa su importancia epidemiológica futura. Se hace una descripción de los signos y síntomas clínicos de una reacción alérgica, así como de algunos aspectos biológicos de la hormiga. El paciente fue tratado con succinato sódico de hidrocortisona (100 mg/stat) y el antihistamínico sistémico loratadina. CONCLUSIONES: las reacciones alérgicas a consecuencia de reacciones de picadura de himenóptera se están haciendo un problema creciente en muchos países, es importante que las autoridades médicas se hagan conscientes de sus manifestaciones dermatológicas y sistémicas, que aquejan a algunos de los pacientes picados o mordidos por hormigas.

INTRODUCTION: ant stings in nations located in the tropical and subtropical regions were generally benign and not frequent; however, at present a higher number of cases develop serious allergic reactions that may evolve into anaphylactic shock or severe urticaria. METHODS: review of the case of a young man suffering moderate allergic reaction to ant stings and comments on the biomedical importance of this event. This is the second accident described in literature and associated with Odontomachus bauri ant, which stresses its epidemiological weight in the future. The clinical signs and symptoms of an allergic reaction together with some biological aspects of the ant were described. The patient was treated with hydrocortisone sodium succynate (100mg/stat) and systemic antihistaminic drug Loratadina. CONCLUSIONS: allergic reactions as a result of hymenoptera stings is becoming a growing problem in many countries; therefore it is essential that the medical authorities be aware of the dermatological and systemic manifestations affecting some patients stung by ants.

[Intense allergic reaction in a patient stung by the black ant (Odontomachus bauri)]. / Intensa reacción alérgica en paciente mordido por la hormiga negra (Odontomachus bauri).

INTRODUCTION: ant stings in nations located in the tropical and subtropical regions were generally benign and not frequent; however, at present a higher number of cases develop serious allergic reactions that may evolve into anaphylactic shock or severe urticaria. METHODS: review of the case of a young man suffering moderate allergic reaction to ant stings and comments on the biomedical importance of this event. This is the second accident described in literature and associated with Odontomachus bauri ant, which stresses its epidemiological weight in the future. The clinical signs and symptoms of an allergic reaction together with some biological aspects of the ant were described. The patient was treated with hydrocortisone sodium succynate (100 mg/stat) and systemic antihistaminic drug Loratadina. CONCLUSIONS: allergic reactions as a result of hymenoptera stings is becoming a growing problem in many countries; therefore it is essential that the medical authorities be aware of the dermatological and systemic manifestations affecting some patients stung by ants.

Use of hen egg derived immunoglobulin against scolopendra (Scolopendra gigantea) venom

Las picaduras de escolopendra (Scolopendra gigantea) en seres humanos y animales domésticos representan un accidente agudo y muy doloroso. La necrosis y otros daños ocasionados por este veneno pueden ser prevenidos, si se inyecta un antiveneno. Este estudio propone producir anticuerpos policlonales en gallinas hiper-inmunizadas contra el veneno de escolopendra (Scolopendra gigantea Linneaus 1758). Un grupo de gallinas fue inyectado subcutánea e intramuscularmente con diluciones de venenos, de acuerdo con tres rutinas diferentes. Los huevos fueron recogidos diariamente y los anticuerpos en la yema fueron purificados con un método modificado de polietilen-glicol y cloroformo. Los niveles de anticuerpos en yema fueron calculados con prueba de precipitación de gel de agar y pruebas de protección (ED50). Los huevos cosechados 15 días post-inyección tenían los títulos más altos de anticuerpos. Después de seis meses, los anticuerpos liofilizados y guardados a 5°C mantenían su actividad. Ratones envenenados, inyectados posteriormente con anticuerpos purificados, tuvieron un 100% de supervivencia al compararse con los controles. La limpieza, la eficacia, y la sencillez de producir los antivenenos en gallina, y la incapacidad de estos anticuerpos (IgY) para fijarse al complemento humano, formulan una alternativa interesante a otros antivenenos producidos en mamíferos. Este estudio puntualiza que los anticuerpos de huevo de gallina pueden ser provechosos como un instrumento terapéutico para tratar escolopendrismo en seres humanos y animales domésticos. Además, abre un campo terapéutico para la fabricación de otros antivenenos contra el espectro amplio de las toxinas y como probable herramienta de diagnóstico.

A scolopendra sting in humans and domestic animals is an acute and highly painful accident. The present study was an attempt to raise specific hyper-immune polyclonal antibodies against scolopendra centipede (Scolopendra gigantea Linneaus 1758) venom. A group of hens were injected with venoms subcutaneously and intramuscularly according to three different routines. This protocol was found to be effective for hyperimmunization. Eggs were gathered daily and antibodies were purified from yolk with a polyethylene-glycol and chloroform modified method. Titers of antibodies in yolk were estimated with an agar gel precipitation test, and a serum protection (ED50) test. Eggs harvested at 15 days post-injection had maximum antibody titers. After six months, antibodies lyophilized and stored at 5°C still maintained their activity. Envenomed mice were injected with purified antibodies, which induced 100% recovery as compared to those not treated with the antibodies. The cleanliness, effectiveness, and simplicity of producing antibodies against scolopendra venom in avian egg yolk, and their incapability to attach human complement, formulates an interesting option to equine and other mammalian antivenoms. This study infers that avian egg yolk antibodies may be useful as a therapeutic tool in treating scolopendrism in humans and domestic animals. It also opens a field for the production of other antivenoms against the wide spectrum of toxins as well as the use of these antibodies as a diagnostic tool.

Envenomation by neotropical opisthoglyphous colubrid Thamnodynastes cf. pallidus Linne, 1758 (Serpentes:Colubridae) in Venezuela.

This is a case report of a "non-venomous" snake bite in a herpetologist observed at the Sciences Faculty of the Universidad de los Andes (Mérida, Venezuela). The patient was bitten on the middle finger of the left hand, and shows signs of pronounced local manifestations of envenomation such as bleeding from the tooth imprint, swelling and warmth. He was treated with local care, analgesics, and steroids. He was dismissed from the hospital and observed at home during five days with marked improvement of envenomation. The snake was brought to the medical consult and identified as a Thamnodynastes cf. pallidus specimen. This report represents the first T. pallidus accident described in a human.

Envenomation by neotropical Opisthoglyphous colubrid Thamnodynastes cf. pallidus Linné, 1758 (Serpentes: Colubridae) in Venezuela

Se reporta un caso de una mordedura de serpiente "no venenosa", en un herpetólogo observado en la Facultad de Ciencias de la Universidad de los Andes (Mérida, Venezuela). El paciente fue mordido en el dedo medio de la mano izquierda, mostrando pronunciados signos locales de sangramiento por la impronta ocasionada por los dientes de la serpiente, edema y calor local. El paciente fue tratado con cuidados locales, analgésicos y esteroides. Fue dado de alta del hospital y observado en el hogar durante 5 días, con marcada mejoría del envenenamiento. La serpiente fue traída a la consulta médica e identificada como un espécimen de Thamnodynastes cf. pallidus. Este es el primer caso humano descrito, ocasionado por un T. pallidus.

Proteolytic, hemorrhagic, and neurotoxic activities caused by Leptodeira annulata ashmeadii (Serpentes: Colubridae) Duvernoy's gland secretion.

OBJECTIVE: The main goal was to explore the different toxin properties (proteolytic, hemorrhagic, and neurotoxic) of Leptodeira annulata ashmeadii Duvernoy's gland secretion (DGS). METHODS: To separate and characterize the different proteins present in L. annulata ashmeadii DGS, 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was run. To partially purify the proteolytic activity, Mono Q2 column ion-exchange chromatography was used. Hemorrhagic activity was analyzed in chicken embryos and mice (skin and peritoneum). Neurotoxic disorders were analyzed in mice inoculated with L. annulata ashmeadii DGS. RESULTS: The approximate relative mass of 21 protein bands was determined using the Multi-Analyst PC version 1.1 (Bio-Rad) program. L. annulata ashmeadii DGS proved to have proteolytic, hemorrhagic, and neurotoxic activities. CONCLUSIONS: Given the properties of the secretions of its Duvernoy's gland, L. annulata ashmeadii should be added to the list of venomous colubrids.

On the unsual hemorrhagic and necrotic activities caused by the rattlesnake (Crotalus durissus cumanensis) in a Venezuelan patient.

The hemorrhagic, necrotic and edematous effects observed in a 23-year-old patient from Lagunetica, Los Teques, state of Miranda, Venezuela, that was bitten by a common Venezuelan rattlesnake (Crotalus durissus cumanensi), were described. The patient was treated with polyvalente serum, antibiotics and autograft. This finding allows to suggest that the poison of some Venezuelan common rattlesnakes has a systemic effect on the skeletal muscle and on capillaries that generate edema, hemorragic phenomena and necrosis.