Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris

Sarafotoxins are peptides isolated from the Atractaspisw snake venom. with strong constrictor effect on cardiac and smooth muscle. They are structurally and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer, in the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris. and secreted to the culture medium, Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred. Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium. Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5 X 10(-10) M and 5 x 10(-11) M of sarafotoxin b, respectively. The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Morphological modifications induced by an artificial diet on the hypopharyngeal glands of Apis mellifera (Hymenoptera, apidae) during their degenerative process

The morphological aspects of the hypopharyngeal glands were analyzed in worker bees of Apis mellifera of 15 and 30 days of age. The individuals were kept in a room with controlled temperature at 32degreesC where they received water and either a protein or a high energy food. Nurse and foraging workers were used as a control for the experiment. The morphological results showed that the different diets modified the cell death characteristics and intensified its occurrence. Both diets caused precocious glandular degeneration. However, this anticipation of cell death was more pronounced in the glandular tissue of the workers who received the high energy diet when compared to the glands of the bees fed with the protein meal.The degenerative signs observed were an intense cytoplasmic vacuolization, with a loss of cytoplasm and of the cell boundaries, dilation or condensation of the cells and nuclei, and nuclear fragmentation. At the end of the degenerative process, we observed the extrusion of nuclei and, finally, the dissolution of the glands. The hypopharyngeal glands' remains were found in the haemolymph.

Ultrastructural studies of the mandibular glands of the minima, media and soldier ants of Atta sexdens rubropilosa (Forel 1908)

The mandibular glands of Hymenoptera are structures associated with the mandibles and constitute part of the salivary glands system. Histological studies in workers of Atta sexdens rubropilosa revealed that this gland contains two portions: a secretory and a storage portion or reservoir. Both portions are connected by means of canaliculi. The object of the present work was the study of the ultratructure of the mandibular glands of minima, media and soldier ant of A. s. rubropilosa by TEM techniques. The glands, in the three castes studied, possess a reservoir, constituted by a simple pavementous epithelium surrounded by the cuticular intima and the secretory portion is constituted by cells of rounded shape. The secretory cells, mainly of minima and soldier, were rich in smooth endoplasmic reticulum. The media worker and soldier presented a large number of mitochondria, of varying shape. Well-developed Golgi complexes were also present in the soldiers. The secretory cells in minima, media and soldier were provided with collecting intracellular canaliculi, which were linked to the reservoir through the extracellular portion. The cytoplasm of the canaliculi-forming cell was poor in organelles. In the individuals of the three castes of A. s. rubropilosa, the presence of lipid secretion granules suggested, beyond the other functions, also a possible pheromonal action. The different roles executed by the different insect castes are directly dependent on the glandular products and, consequently, on the secretory cellular characteristics. (c) 2005 Elsevier Ltd. All rights reserved.

Class III glands in the abdomen of Meliponini

Class III tegumentar glands were studied in workers, as well as in queens and males when available, of 56 Meliponini species. The presence and development of these glands varies widely among and within species. However, the queen typically has more glands than do workers, and males rarely have any. Gland development in workers was evaluated by counting and determining the size of cells in histological sections. Laying queens were found to have more active gland cells than did virgins. Cell numbers and cell ultrastructure differed among glands similarly located in workers, queens and males. Cell size and ultrastructure also varied from tergite to tergite. In conclusion, since it is likely that most of them produce pheromones, the wide variability in these glands suggests that they are important to social interaction.

Comparative study of mandibular glands of Melipona bicolor queens obtained from polygynic and monogynic colonies

The aim of the present study was analyze, by histological and morphometrical studies, mandibular glands of Melipona bicolor queens collected from monogynic and polygynic colonies and compare their level of development. The results showed that the glands of physogastric queens from monogynic colony present a higher level of activity in relation to the queens of polygynic colonies; this is explained by the fact that just a unique queen controls the monogynic colony. In the polygynic colonies, the queens may divide such control to each other.

Lipid distribution in salivary glands of larvae and adult bees (Hymenoptera : Apidae)

Cytochemical studies were carried out to establish lipid distribution in the salivary glands of larvae and adult bees, using the imidazole buffer technique. In the duct cells of the larval salivary gland, the reaction was positive in the epicuticle and negative in the glandular lumen. The absence of smooth endoplasmic reticulum and the presence of lipids in the intercellular space suggest that lipids absorbed from the haemolymph could be used in the constitution of the epicuticle, after having been conveyed through the epithelium. In adult workers (new-emerged, nurse and forager workers), the head salivary glands presented a positive reaction in the secretion in glandular lumen, identifying its lipidic nature.

A morpho-histological and ultrastructural study of the mandibular glands of Atta sexdens worker ants (Hymenoptera : Formicidae)

We describe here the morphology, histology and ultrastructure of the mandibular glands of Atta sexdens (Hymenoptera: Formicidae) worker ants. The mandibular glands consist of a reservoir with an excretory duct and a secretory portion linked to the reservoir trough canaliculi. These glands have the same morphology in the three castes studied, differing only in size, with smaller glands observed in minima workers and larger glands in soldiers.

Ultrastructural study of the salivary glands of the sugarcane spittlebug Mahanarva fimbriolata (Stal, 1854) (Euhemiptera : Cercopidae)

Spittlebugs are insects that suck sap from plants and regurgitate saliva containing toxic enzymes into the leaves. As a consequence, the conductive channels are blocked resulting in dry leaves, thus giving a burned aspect to the plantation. This work performed ultrastructural analyses of the salivary glands of the sugarcane spittlebug Mahanarva fimbriolata, since these organs produce the enzymes that are injected into the plants, thus being responsible for the economic losses in the production of sugarcane. Three kinds of secretory cells are found in principal gland, forming the lobules I-IV. The main differences among these cells relate to size, morphology and electron density of the secretory vesicles. The accessory glands contain different secretory vesicles to those in the principal gland. Muscular cells are found around the entire gland. The different secretory vesicles found in both principal and accessory glands indicate that the gland produces different substances or that the secretion in the interior of cells passes through a maturation process. (c) 2005 Elsevier Ltd. All rights reserved.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

Changes in the size of cephalic salivary glands of Apis mellifera and Scaptotrigona postica (Hymenoptera: Apidae) queens and workers in different life phases

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cephalic salivary glands of two species of advanced eusocial bees (Hymenoptera: Apidae): morphology and secretion

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ocorrência e morfologia de glândulas tegumentares no abdome de algumas abelhas (Hymenoptera: Apidae): um estudo comparado

No presente trabalho foi estudada a ocorrência, distribuição e morfologia de glândulas tegumentares do abdome em Exomalopsis auropilosa Spinola, Centris fuscata Lepeletier, Epicharis flava Friese e Xylocopa (Neoxylocopa) suspecta Moure & Camargo. Os resultados mostraram a ocorrência de glândulas epiteliais (classe I) e unicelulares (classe III) com distribuição dorsal (tergais) e ventral (esternais) sem que se pudesse caracterizar um padrão relacionável com a posição filogenética ou grau de sociabilidade. No entanto, verificou-se uma tendência para maior número de glândulas em espécies com algum grau de sociabilidade como E. auropilosa e X. suspecta.

Análise morfológica da glândula de veneno de Apis mellifera L. (Hymenoptera: Apidae) em populações de Mato Grosso do Sul

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ocorrência, morfologia e ultra-estrutura da glândula de Dufour de Scaptotrigona postica Latreille (Hymenoptera: Apidae)

A glândula de Dufour é uma glândula acessória do aparelho reprodutivo feminino das abelhas. Nas abelhas neotropicais sem ferrão, tem sido pouca estudada sob todos os aspectos: morfológico, ontogenético e bioquímico. Na tentativa de colaborar com o conhecimento dessa glândula em abelhas sem ferrão, foi realizado um estudo da sua ocorrência, morfologia e desenvolvimento em Scaptotrigona postica Latreille. Os resultados mostraram que ela se encontra ausente nas operárias, como ocorre em muitas outras espécies desse grupo. Nas rainhas, as células glandulares parecem mais ativas nas virgens, possuindo uma desenvolvida rede de retículo endoplasmático liso tubular, grânulos de secreção e polirribossomos dispersos no citoplasma, além de apresentarem núcleos maiores do que os das células glandulares das fisogástricas. Nas rainhas fisogástricas há dois tipos de células glandulares, ambas aparentemente inativas sinteticamente. As glândulas das rainhas fisogástricas são claramente capazes de captar substâncias da hemolinfa, provavelmente lipídios, que não penetram nas células, mas passam pelos espaços intercelulares e, através da cutícula, chegam diretamente à luz da glândula. A bem desenvolvida dupla camada de lâmina basal ao redor da glândula pode atuar no processo de captação de substâncias da hemolinfa. A secreção, e conseqüentemente sua função, pode ser diferente nas duas classes de rainhas.

Dufour glands in the hymenopterans (Apidae, Formicidae, Vespidae): a review

Anexas ao aparelho do ferrão dos himenópteros aculeados encontram-se as glândulas de veneno e as de Dufour. A glândula de veneno é originada das glândulas associadas ao ovopositor dos himenópteros ancestrais não aculeados, já a glândula de Dufour é menos derivada, homóloga das glândulas colateriais dos outros insetos, sendo encontrada em todas as fêmeas dos himenópteros. Nestes insetos sua função é, em grande parte, desconhecida, mas, em formigas, parece estar envolvida com a comunicação e a defesa e, nas abelhas não sociais, com a construção e a proteção do ninho. Nas vespas pode estar relacionada ao reconhecimento parental. Foram observadas diferenças morfológicas e na composição química da secreção da glândula de Dufour entre as espécies, bem como na mesma espécie, entre as castas dos himenópteros sociais e entre indivíduos da mesma casta desempenhando diferentes funções ou pertencentes a ninhos diferentes. Portanto, nos himenópteros, sua função original de produzir substâncias para proteger os ovos ou favorecer a ovoposição parece ter sido substituída ou complementada com a função de produzir semioquímicos com função na comunicação.