RESUMEN

This communication reviews most of the important findings related to venom components isolated from scorpions and spiders, mainly by means of gene cloning and expression. Rather than revising results obtained by classical biochemical studies that report structure and function of venom components, here the emphasis is placed on cloning and identification of genes present in the venomous glands of these arachnids. Aspects related to cDNA library construction, specific or random ESTs cloning, transcriptome analysis, high-throughput screening, heterologous expression and folding are briefly discussed, showing some numbers of species and components already identified, but also shortly mentioning limitations and perspectives of research for the future in this field.

Asunto(s)

Péptidos/metabolismo , Venenos de Escorpión/genética , Venenos de Araña/genética , Animales , Clonación Molecular , Etiquetas de Secuencia Expresada/química , Etiquetas de Secuencia Expresada/metabolismo , Perfilación de la Expresión Génica , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Péptidos/genética , Proteómica , Venenos de Escorpión/química , Venenos de Araña/química

RESUMEN

Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.

Asunto(s)

Péptidos beta-Amiloides/administración & dosificación , Péptidos beta-Amiloides/inmunología , Anticuerpos Monoclonales/química , Bacteriófago M13/inmunología , Epítopos/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Anticuerpos de Cadena Única/química , Secuencia de Aminoácidos , Péptidos beta-Amiloides/genética , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Bacteriófago M13/química , Bacteriófago M13/genética , Sitios de Unión de Anticuerpos/genética , Línea Celular Tumoral , Epítopos/genética , Epítopos/metabolismo , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/genética

RESUMEN

This communication revises the state of the art concerning antivenoms against snakes, spiders and scorpions. An overview of the historical facts that preceded the therapeutic use of antibodies is mentioned. A brief list of the major protein components of these venomous animals is revised with a short discussion of what is known on the proteomic analysis of their venoms, but the emphasis is placed on the type of antivenoms available commercially, including pertinent literature and addresses of the companies that prepare these antivenoms. The final section revises and discusses current research on the field and new potential applications that are being developed geared at obtaining new therapeutic antibodies or fragments of antibodies for neutralization of toxic components of venomous animals.

Asunto(s)

Antivenenos/análisis , Proteómica/métodos , Venenos de Escorpión/toxicidad , Venenos de Serpiente/toxicidad , Venenos de Araña/toxicidad , Animales , Antivenenos/inmunología , Cromatografía Líquida de Alta Presión , Humanos , Inmunoglobulina G/química , Ratones , Proteoma , Proteínas Recombinantes/química , Venenos de Escorpión/análisis , Escorpiones , Venenos de Serpiente/análisis , Venenos de Araña/análisis , Arañas

RESUMEN

We performed a comparative analysis of the conformation of the CDR1 of the human lambdaVI variable domains JTO and WIL and the equivalent loop of the lambdaI light chains RHE and KOL, which are representative of the type I canonical structure for lambda light chains. On the basis of the differences found in the main chain conformation, as well as the identity of the residues at key positions, we showed that the L1 of some lambdaVI light chains adopts a conformation that represents a new type of canonical structure. The conformation of the L1 of those lambdaVI light chains, is primarily determined by the presence of an Arg residue at position 25. The analysis of the lambdaVI light chain sequences so far reported, showed that near 25% of those proteins have Gly at position 25 instead of Arg, which represents an allotypic variant of the lambdaVI variable locus. The presence of Gly at position 25 in the L1 of lambdaVI light chains would imply a different conformation for this loop. Additionally, the position 68 in lambdaVI light chains, which is at the top of the FR3 loop, showed such spatial orientation and variability that suggested its participation in the conformation of the antigen recognition surface in this subgroup of lambda chains.

Asunto(s)

Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Autoantígenos , Bases de Datos de Proteínas , Humanos , Sustancias Macromoleculares/química , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia

RESUMEN

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antivenenos/metabolismo , Fragmentos de Inmunoglobulinas/inmunología , Mutación/genética , Venenos de Escorpión/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos , Antivenenos/genética , Antivenenos/inmunología , Evolución Biológica , Clonación Molecular , Mapeo Epitopo , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Pruebas de Neutralización , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Péptidos/aislamiento & purificación , Especificidad por Sustrato

RESUMEN

A library of phage-displayed human single-chain Fv (scFv) antibodies was selected against the human amyloid-beta peptide (Abeta42). Two new anti-Abeta42 phage-displayed scFvs antibodies were obtained, and the sequences of their V(H) and Vkappa genes were analyzed. A synthetic peptide based on the sequence of Ig heavy chain (V(H)) complementarity-determining region (HCDR3) of the clone with the highest recognition signal was generated and determined to bind to Abeta42 in ELISA. Furthermore, we showed for the first time that an HCDR3-based peptide had neuroprotective potential against Abeta42 neurotoxicity in rat cultured hippocampal neurons. Our results suggest that not only scFvs recognizing Abeta42 but also synthetic peptides based on the V(H) CDR3 sequences of these antibodies may be novel potential candidates for small molecule-based Alzheimer's disease (AD) therapy.

Asunto(s)

Péptidos beta-Amiloides/farmacología , Regiones Determinantes de Complementariedad/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Fragmentos de Péptidos/farmacología , Animales , Anticuerpos/análisis , Anticuerpos/metabolismo , Células Cultivadas , Regiones Determinantes de Complementariedad/análisis , Humanos , Región Variable de Inmunoglobulina/análisis , Unión Proteica/fisiología , Ratas , Ratas Wistar

RESUMEN

Most scorpion toxins are ligand peptides that recognize and bind to integral membrane proteins known as ion-channels. To date there are at least 202 distinct sequences described, obtained from 30 different species of scorpions, 27 from the family Buthidae and three from the family Scorpionidae. Toxins that recognize potassium and chloride channels are usually from 29 to 41 amino acids long, stabilized by three or four disulfide bridges, whereas those that recognize sodium channels are longer, 60 to 76 amino acid residues, compacted by four disulfide bridges. Toxins specific for calcium channels are scarcely known and have variable amino acid lengths. The entire repertoire of toxins, independently of their specificity, was analyzed together by computational programs and a phylogenetic tree was built showing two separate branches. The K(+) and Cl(-) channel specific toxins are clustered into 14 subfamilies, whereas those of Na(+) and Ca(2+) specific toxins comprise at least 12 subfamilies. There are clear similarities among them, both in terms of primary sequence and the main three-dimensional folding pattern. A dense core formed by a short alpha helix segment and several antiparallel beta-sheet stretches, maintained by disulfide pairing, seems to be a common structural feature present in all toxins. The physiological function of these peptides is manifested by a blockage of ion passage through the channels or by a modification of the gating mechanism that controls opening and closing of the ion pore.

Asunto(s)

Canales Iónicos/efectos de los fármacos , Péptidos/farmacología , Venenos de Escorpión/farmacología , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Filogenia , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Venenos de Escorpión/química , Venenos de Escorpión/genética , Homología de Secuencia de Aminoácido

RESUMEN

The primary structure of a phospholipase A2, with unique structural and functional characteristics, was determined. The large subunit has 108 amino acid residues, linked by a disulfide bridge to the small subunit, which contains 17 residues. Its gene was cloned from a cDNA library. The nucleotide sequence showed that the same RNA messenger encodes both subunits, separated only by a pentapeptide, that is processed during maturation.

Asunto(s)

Fosfolipasas A/aislamiento & purificación , Venenos de Escorpión/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Dimerización , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Fosfolipasas A/química , Fosfolipasas A/genética , Fosfolipasas A2 , Venenos de Escorpión/química , Venenos de Escorpión/genética , Homología de Secuencia de Aminoácido

RESUMEN

Na+-channel specific scorpion toxins are peptides of 60-76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three-dimensional structure has been solved by X-ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of alpha-helix plus a triple-stranded beta-sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na+-channel function, affecting either the inactivation (alpha-toxins) or the activation (beta-toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical alpha- and beta-types, but also the species specificity of their action. There are peptides that bind or affect the function of Na+-channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or 'active sites' of these peptides with Na+-channel functions. Segments containing positively charged residues in special locations, such as the five-residue turn, the turn between the second and the third beta-strands, the C-terminal residues and a segment of the N-terminal region from residues 2-11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well-folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.

Asunto(s)

Neurotoxinas/química , Péptidos/química , Venenos de Escorpión/química , Bloqueadores de los Canales de Sodio , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Neurotoxinas/genética , Filogenia , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia

RESUMEN

The antibody BCF2 generated against the mammal-specific toxin Cn2 of the scorpion Centuroides noxius Hoffmann neutralizes the effect of both the toxin and the venom. We cloned and sequenced the genes coding for the Fv fragment of BCF2. A three-dimensional (3D) model of the Fv fragment was generated using a knowledge-based approach. Furthermore, a 3D model of the complex Cn2-BCF2 was built using the nuclear magnetic resonance (NMR) structure of Cn2 and experimental results on a putative epitope region around the N and C termini. The initial complex conformations were submitted to a new refinement procedure of rigid-body energy minimization combined with flexible-side-chain molecular dynamics. The final complex, selected after an extensive evaluation, uses the loop 7-11 as the central part of the epitope. The generated complex allows the following conclusions: 1) the neutralizing capacity of BCF2 toward the venom of C. noxius might rather be caused by the high venom concentration and toxicity of Cn2 than by a broad specificity, 2) the region involved in the binding of Cn2 to the Na(+) channel, should overlap with the employed epitope region, and 3) contact residues SerL91, AsnL92, LeuH50, AspH56, TyrH95, and TyrH98 of BCF2 are candidates for mutations to broaden its specificity. Proteins 1999;37:130-143.

Asunto(s)

Simulación por Computador , Fragmentos de Inmunoglobulinas/inmunología , Modelos Moleculares , Neurotoxinas/inmunología , Conformación Proteica , Venenos de Escorpión/inmunología , Algoritmos , Secuencia de Aminoácidos , Animales , Reacciones Antígeno-Anticuerpo , Secuencia de Bases , Epítopos/química , Epítopos/inmunología , Fragmentos de Inmunoglobulinas/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Neurotoxinas/antagonistas & inhibidores , Venenos de Escorpión/antagonistas & inhibidores , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Potassium-channel-blocking scorpion toxins (alpha-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other alpha-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of alpha-K-toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and Kv1.1 K+ channels with moderate affinities, with Kd values of around 0.7 microM and 4.1 microM (Shaker) and 0.5 microM and 1.0 microM (Kv1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.

Asunto(s)

Bloqueadores de los Canales de Potasio , Venenos de Escorpión/química , Venenos de Escorpión/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Clonación Molecular , ADN Complementario , Radioisótopos de Yodo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Venenos de Escorpión/genética , Venenos de Escorpión/metabolismo , Homología de Secuencia de Aminoácido , Sinaptosomas/metabolismo

RESUMEN

Scorpion venoms contain a variety of low mol. wt peptides toxic to different organisms. These peptides have been intensively studied because they represent excellent models for investigating structure-function relationships and they are also fine probes for studying ionic channel functions. This review deals with the biological and chemical aspects of toxic peptides that affect Na+ or K+ channels and the cloning of the cDNAs and genes encoding the main alpha and beta neurotoxins present in the venom of the three most dangerous species of Brazilian scorpion, Tityus bahiensis, Tityus stigmurus and Tityus serrulatus, and the Venezuelan scorpion Tityus discrepans. At least 16 different peptides specific for Na+ channels and five affecting K+ channels were isolated and characterized from the venom of these scorpions. The isolation of cDNAs and genes encoding four distinct toxins has permitted the elucidation of their nucleotide sequences as well as their genomic organization. Venoms and isolated toxins from scorpions of the genus Tityus were shown to enhance the secretory activity of the pancreas. Antisera obtained against venom of T. serrulatus show cross-reactivity with other species of the Brazilian scorpions.

Asunto(s)

Genes de Insecto , Venenos de Escorpión/aislamiento & purificación , Escorpiones/genética , Toxinas Biológicas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Canales de Sodio/efectos de los fármacos

RESUMEN

We present an in-depth analysis of the structural and functional properties of Imperatoxin I (IpTxi), an approximately 15-kDa protein from the venom of the scorpion Pandinus imperator that inhibits Ca2+ release channel/ryanodine receptor (RyR) activity (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12185-12189). A cDNA library was prepared from the venomous glands of this scorpion and used to clone the gene encoding IpTxi. From a single continuous messenger RNA, the information coding for the toxin is translated into two mature polypeptide subunits after elimination of a basic pentapeptide. The IpTxi dimer consists of a large subunit (104-amino acid residues) with phospholipase A2 (PLA2) activity covalently linked by a disulfide bond to a smaller (27 amino acid residues), structurally unrelated subunit. Thus, IpTxi is a heterodimeric protein with lipolytic action, a property that is only shared with beta-bungarotoxins, a group of neurotoxins from snake venoms. The enzymatic subunit of IpTxi is highly homologous to PLA2 from bee (Apis mellifera) and lizard (Heloderma horridum) venoms. The small subunit has no significant similarity to any other known peptide, including members of the Kunitz protease inhibitors superfamily that target the lipolytic effect of beta-bungarotoxins. A synthetic peptide with amino acid sequence identical to that of the small subunit failed to inhibit RyR. On the other hand, treatment of IpTxi with p-bromophenacylbromide, a specific inhibitor of PLA2 activity, greatly reduced the capacity of IpTxi to inhibit RyRs. These results suggested that a lipid product of PLA2 activity, more than a direct IpTxi-RyR interaction, was responsible for RyR inhibition.

Asunto(s)

Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Proteínas Musculares/fisiología , Venenos de Escorpión/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Cromatografía por Intercambio Iónico , Clonación Molecular , ADN Complementario , Biblioteca de Genes , Cinética , Membrana Dobles de Lípidos , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas Musculares/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Fosfolipasas A/química , Fosfolipasas A/aislamiento & purificación , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/metabolismo , Venenos de Escorpión/biosíntesis , Venenos de Escorpión/aislamiento & purificación , Escorpiones , Homología de Secuencia de Aminoácido , Porcinos

RESUMEN

A novel crustacean-specific toxin, Cn5, containing 66 amino acid residues was isolated from the venom of the scorpion Centruroides noxius Hoffmann. It is stabilized by four disulfide bridges, formed between Cys12-Cys65, Cys16-Cys41, Cys25-Cys46 and Cys29-Cys48. Toxicity tests revealed that Cn5 is a toxin that affects arthropods but not mammals. However, at high concentrations, Cn5 does displace the mammal-specific toxin Cn2 from rat brain synaptosomes. The concentration of Cn5 that produces half-maximal inhibition (IC50) was estimated to be 100 microM. Sequence comparison of Cn5 with toxin Cn2, a mammal-specific toxin from the same scorpion, showed the presence of two sequence stretches, at positions 30 to 38 and 49 to 58, where the majority of the differences are concentrated. On the three-dimensional structure of Cn5 it is demonstrated that these two sequence stretches form a continuous surface region near the site thought to bind to the sodium channel. We assume that this region might be implicated in determining species specificity.

Asunto(s)

Crustáceos/efectos de los fármacos , Mamíferos , Venenos de Escorpión/química , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/toxicidad , Secuencia de Aminoácidos , Animales , Astacoidea , Encéfalo/metabolismo , Disulfuros , Relación Dosis-Respuesta a Droga , Dosificación Letal Mediana , Ratones , Ratones Endogámicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Ratas , Venenos de Escorpión/metabolismo , Venenos de Escorpión/farmacología , Escorpiones/química , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo

RESUMEN

Scorpion toxins acting on sodium channels differ in their specificity. Toxic peptides specific towards mammals and arthropods (insects and/or crustaceans) have been described. Because of the similar three-dimensional fold of these peptides, the molecular base of their specificity is thought to reside in certain differences at the level of amino acid residues especially within or near the binding site of the toxin to the particular ion channel. The cDNA, amino acid sequence and biological activity of an insect-specific toxin, Cn10, from the scorpion Centruroides noxius Hoffmann is reported. The electrostatic potential surface around a three-dimensional model of Cn10 was calculated. It revealed that residues Tyr4, Lys13, Ile18, Leu19, Gly20, Lys43, Leu44, Thr57, Tyr58, Pro59, Thr64 and Cys65, situated at the side of the toxin proposed in the literature to bind to the sodium channel, constitute a positive surface region. Therefore, they may form the site that binds to the channel. Cn10 was included in a comparative analysis of two groups of natural variants, highly similar peptides of the genus Centruroides with specificities towards mammals or arthropods. A number of surface-accessible residues, consistently different between the two groups and situated near the putative binding site, may be of importance for the specificity of the analyzed toxins.

Asunto(s)

Estructura Secundaria de Proteína , Venenos de Escorpión/química , Venenos de Escorpión/toxicidad , Secuencia de Aminoácidos , Animales , Astacoidea , Sitios de Unión , Clonación Molecular , ADN Complementario , Gryllidae , Canales Iónicos/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/toxicidad , Venenos de Escorpión/biosíntesis , Escorpiones , Homología de Secuencia de Aminoácido , Electricidad Estática

RESUMEN

BCF2 is a murine hybridoma cell line that produces a neutralizing antibody against toxin 2 (Cn2) from the scorpion Centruroides noxius Hoffmann of Mexico. In this communication we report the preparation and use of the BCF2 antibody and its antigen binding fragments (Fab) in experiments aiming at obtaining protection of experimental albino mice (strain CD1) challenged with purified toxin Cn2, as well as, with whole soluble venom from C. noxius. The monoclonal antibody BCF2 in amounts of 1 mg neutralizes 28 LD50 of soluble venom of C. noxius, whereas the Fab fragments of BCF2 (1 mg) are capable of neutralizing 43 LD50 dose of the same venom. To reach the same level of neutralization, with the commercially available horse antiserum [F(ab')2], we need to use about ninefold more material.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Venenos de Escorpión/inmunología , Animales , Dosificación Letal Mediana , Ratones , Pruebas de Neutralización

RESUMEN

Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed.

Asunto(s)

Genes , Péptidos/química , Péptidos/genética , Venenos de Escorpión/química , Venenos de Escorpión/genética , Escorpiones/genética , Secuencia de Aminoácidos , Animales , Anticuerpos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Inmunoquímica , Ratones , Datos de Secuencia Molecular , Conejos , Venenos de Escorpión/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la Especie

RESUMEN

By means of PCR and using synthetic oligonucleotides designed from the reported cDNA, we amplified the gene that codes for toxin IV-5 from the Brazilian scorpion Tityus serrulatus. The analysis of the nucleotide sequence shows that the amplified genomic region is composed of 659 base pairs (bp) comprising two exons (28 and 284 bp) and an intron of 347 bp interrupting the region that encodes the signal peptide of the precursor toxin. Based on these findings a model for the structural organization of scorpion toxin genes is proposed.

Asunto(s)

Clonación Molecular/métodos , Venenos de Escorpión/genética , Escorpiones/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , ADN/aislamiento & purificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Venenos de Escorpión/química

RESUMEN

Using a cDNA library prepared from venomous glands of the Mexican scorpion Centruroides noxius Hoffmann the genes that encode toxins 1 and 2 were identified, cloned and sequenced. In view of the proposed mechanism for processing the mature peptides coded by these two genes, the corresponding peptide-toxins were sequenced de novo. Mass spectrometric and 1H-NMR analyses of the C-terminal peptide produced by enzymatic digestion of both toxins indicated that the last residue is serine-amide. Sequence comparison revealed that these two genes have a similarity of 56% and 80% at the amino acid and nucleotide levels, respectively. Small corrections to the published primary structures were introduced: Cn toxin 1 has an extra serine residue at position 65 and the residue in position 60 is a proline, while the amino acids at positions 34 and 35 of Cn 2 are, respectively, tyrosine and glycine. Sequence comparison of toxins from the genus Centruroides suggests the presence of at least three classes of distinct peptides in these venoms.

Asunto(s)

Clonación Molecular , ADN Complementario/química , Neurotoxinas/genética , Venenos de Escorpión/química , Canales de Sodio/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Venenos de Escorpión/genética , Homología de Secuencia de Aminoácido , Canales de Sodio/metabolismo

RESUMEN

1. An experimental model system was developed in the rabbit to study the transport of iron and the erythropoietic response to chronic bloodletting. 2. This model presents certain novel features as the capacity to measure total red blood cell production and total hemoglobin production in a daily basis. 3. Daily red blood cell production and output of hemoglobin are directly proportional to the volume of blood extracted. The limits of the erythropoietic response were determined. 4. When only minimal bloodletting was performed (3-6 ml blood/kg body weight) a normocytic response was obtained; with the removal of larger volumes of blood, a switch to macrocytic anemia was observed. Cats and guinea pigs responded in a similar fashion. 5. After induction of macrocytic anemia, the diameter of erythroid precursor cells in the bone marrow of the long bones increased and their numbers increased 11.5-fold.

Asunto(s)

Anemia/fisiopatología , Modelos Animales de Enfermedad , Eritropoyesis , Hierro/metabolismo , Conejos/fisiología , Anemia/sangre , Anemia/etiología , Animales , Médula Ósea/patología , Gatos/sangre , Gatos/fisiología , Recuento de Células , Enfermedad Crónica , Envejecimiento Eritrocítico , Recuento de Eritrocitos , Células Precursoras Eritroides/patología , Cobayas/sangre , Cobayas/fisiología , Hemoglobinas/análisis , Hemorragia/complicaciones , Conejos/sangre , Reproducibilidad de los Resultados , Reticulocitos , Especificidad de la Especie