A new scenario of bioprospecting of Hymenoptera venoms through proteomic approach

Venoms represent a huge and essentially unexplored reservoir of bioactive components that may cure diseases that do not respond to currently available therapies. This review select advances reported in the literature from 2000 to the present about the new scenario of Hymenoptera venom composition. On account of new technologies in the proteomic approach, which presents high resolution and sensitivity, the combination of developments in new instruments, fragmentation methods, strategic analysis, and mass spectrometry have become indispensable tools for interrogation of protein expression, molecule interaction, and post- translational modifications. Thus, the biochemical characterization of Hymenoptera venom has become a major subject of research in the area of allergy and immunology, in which proteomics has been an excellent alternative to assist the development of more specific extracts for diagnosis and treatment of hypersensitive patients to Hymenoptera venoms.

Insects as biological models to assay spider and scorpion venom toxicity

This study was undertaken to develop an experimental protocol using insects as biological models to assay venom toxicity of the following spiders Loxosceles gaucho, Phoneutria nigriventer, Nephilengys cruentata and Tityus serrulatus scorpion. Three different insect species were bioassayed: Apis mellifera (Hymenoptera), Grillus assimilis (Orthoptera), and Diatraea saccharalis (Lepidoptera). Venoms were injected into the hemocele of insects with a microsyringe at concentrations that caused dose/weight-dependent effects; doses causing either paralysis (ED50) or death (LD50) were recorded for each venom and insect test-species. T. serrulatus and L. gaucho venoms were lethal to all tested species, while P. nigriventer venom caused paralysis and death, and N. cruentata venom caused only paralysis at the doses assayed. A comparison between the insect test species described above revealed that A. mellifera was highly sensitive to all venoms tested; even a tiny amount of N. cruentata non-lethal venom caused a change in the walking pattern leading to transient paralysis. D. saccharalis larvae were very resistant to all four venoms.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Alarm pheromones and the influence of pupal odor on the aggressiveness of Polybia paulista (Ihering) (Hymenoptera: Vespidae)

Field bioassays were used to demonstrate that aggressive behavior of Polybia paulista (Ihering) workers is elicited by alarm pheromones present in the venom reservoirs of nest defenders and that the brood care pheromone (pupal odor) produced by the young inside the nest also plays an important defensive role. Pupal odor was extracted from the surface of pupa bodies with methanol. When bioassayed alone, the pupal odor elicited onlyu attractiveness of workers towards the odor source, but no stinging attacks were observed. However, in the presence of alarm pheromones, the brood care pheromone potentiated the effect caused by the pupal odors, increasing the number of stinging attacks during an action of colony defense. Thus, the presence of pupae within the nest not only releases brood care but also enhances the aggressiveness of workers in P. paulista colonies.

Chemical communication in Apis mellifera: temporal modulation of alarm behaviors

The effect of pheromones and their chemical analogues in honeybee alarm behaviors was studied in observation boxes. Defensive behaviors, as follows: a) attraction to scent source, b) elevation of wings in "V", c) abdomen elevation d) abdomen elevation and pumping and e) first leg pair elevation had been temporarily registered when the following compounds were presented: isoamyl alcohol, octyl alcohol, benzyl alcohol, n-butyl acetate, n-octyl acetate, isopentyl acetate, benzyl acetate and 2-heptanone. The results were as follows: 1. the bees elicited some characteristic behaviors when chemical alarm messages were presented, 2. agression (stinging) was not completed with any compound tested, probably because there was not a target (visual stimulus), 3. in all situations the attraction to scent source was low, 4. all the behaviors were elicited in a temporarily different way, 5. the compounds that elicited stronger responses and a greater number of the investigated behaviors were: isopentyl acetate, 2-heptanone, octyl acetate and n-octyl alcohol. In all situations, the first behavior response (and the most intense one) was the elevation and pumping the abdomen. This suggests that the chemical message was promptly recognized and then transmitted to each worker. So, the results obtained in the present work, suggest that chemical alarm messages may be recognized by different mechanisms of neural integration.

Bioinsecticide action of the venom of africanized bees Apis mellifera Linnaeus 1758 (hymenoptera: apidae): I-the most susceptible age of diatrae saccharalis fabricius 1794 (Lepeletier: pyralidae) eggs to the venom action

The present paper aimed at testing the action of non-lyophilized venom of Africanized bees Apis mellifera through topical applications on Diatraea saccharalis egg masses. The CL50, DL50 and the most susceptible age of eggs to the venom topic application were also determined. Three-day-old eggs were the most susceptible to the venom action with CL50 equal to 8.6 mg/ml and DL50 equal to 0.173 mg/mass. The venom loses its action after being stored for 15 days.

High-performance gel filtration patterns of venoms from three species of wasps of the genus Polybia (Hymenoptera, vespidae)

The profiles of high-performance gel filtration of venoms from Polybia paulista, Polybia ignobilis and Polybia occidentalis showed 13 peaks distributed among the three species. These profiles presented similarities that permitted the chromatographic characterization of the genus Polybia and differences that permitted the identification of each species studies. Thus, the comparative analysis of chromatographic profiles of high-performance gel filtration of venoms may be used as an auxiliary tool in taxonomic studies of Polybia wasps.

Molecular exclusion chromatography of crude venom as an auxiliary tool to identify hybrid honeybee populations

A comparison among the profiles of molecular exclusion chromatography in Sephadex G 100 column of venoms from Apis mellifera adansonii and Africanized honeybees revealed unique peaks which might be used to identify these populations. The venoms from hybrid populations resulting from the reciprocal mating of Apis mellifera adansonii and Africanized honeybees presented unique peaks, probably resulting from a synergistic effect between the parental genomes. The occurrence of characteristic peaks in venoms of hybrid populations might be used to identify these populations as well as to distinguish them from their parents.

The effect of a polydnavirus of Apanteles Galleriae on the hemolymph proteins of host Galleria Mellonela

The presence of a polydnavirus in the ovary of the microhymennoptera Apanteles galleriae was detected. This wasp parasites the larvae of the wax moth Galleria mellonella. To study the possible viral effects on the total protein of G. mellonella, hemolymph extracts of the ovary calyxes of parasitized A. galleriae were injected into 6th and 7th instar cells of G. mellonella larvae. Protein profiles on polyacrilamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) of host cell extracts showed a suppression of several protein bands when infection occurred in the 6th instar. However, in the 7th instar, parasitism caused an amplification in the synthesis of most proteins with some changes in the electrophoretic profile.

Regulatory mechanisms of blood lactate production during exercise in man

During cycloergometric exercise at progressively increasing loads, blood lactate concentration increased about 12-fold. Pyruvate concentration decreased initially(for loads of 50-75 W), increased with loads of 75 to 125 W and then decreased again until the end of exercise. the malate concentration increased abruptly between 50 and 75 W, followed by a slow decline; citrate increased about nine-fold as the exercise load was increased to 125 W and then fell sharply. Thus, the production of lactate during low-intensity exercise seems to occur by the "mass-action effect" caused by enhanced glycolysis, whereas with moderate loads the glycolysis rate is very much reduced and most of the lactate production seems to involve the action of the malate-aspartate shuttle. For high-intensity exercise, both mechanisms appear to participate in lactate production

Helados de elaboracion artesanal y semiindustrial. Incidencia de Staphylococcus aureus y enterotoxigenicidad. / Craft or semi-industrial ice cream. Occurence of Staphylococous aureus and enterogenicity

En 7 heladerias de elaboracion artesanal y semiindustrial se retiraron 67 muestras de helados a base de crema de leche y de agua. El 43,3% de las muestras fueron positivas para la investigacion de Staphylococcus aureus en 1 g, el 88% de las mismas presentaron menos de 10 St. aureus por g, 10,5% entre 10 y 100 y 1,5% entre 101 y 1.000.A 29 cepas se les determino la enterotoxigenicidad, la respuesta a diversas pruebas bioquimicas y la sensibilidad a antibioticos. El 21% de las cepas resultaron enterotoxigenicas (4 produjeron toxina A, 1 toxina B y 1 toxinas A y D). No se hallo diferencia en las pruebas bioquimicas realizadas (produccion de catalasa, coagulasas, termonucleasas, fermentacion de glucosa y manitol, hidrolisis de gelatina y caseina, produccion de pigmentos y hemolisinas) entre las cepas toxigenicas y no toxigenicas. Todas las cepas presentaron una amplia sensibilidad frente a los antibioticos. Como resultado del presente estudio se propone incorporar al articulo 1075 del Codigo Alimentario Argentino el limite de 100 Staphylococcus aureus por g en helados a base de crema y de agua