Effect of exogenous galectin-1 on leukocyte migration: modulation of cytokine levels and adhesion molecules

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Distinct localization of T cell Agrin during antigen presentation - evidence for the expression of Agrin receptor(s) in antigen-presenting cells

Agrin is over-expressed by activated and autoimmune T cells, and synergizes with the T cell receptor (TCR) to augment cell activation. In the present study, we show that Agrin accumulates to distinct areas of the plasma membrane and that cell activation causes its redistribution. During antigen presentation, Agrin primarily accumulates to the periphery of the mature immunological synapse, mostly in lamellipodia-like protrusions that wrap around the antigen-presenting cell and, conversely, anti-Agrin sera induced a significant redistribution of TCR at the plasma membrane. We also provide evidence for the expression of Agrin receptors in peripheral blood monocytes, dendritic cells and a fraction of B cells. Interestingly, interferon-a treatment, which induces the expression of Agrin in T cells, also augmented Agrin binding to monocytes. Stimulation of monocytes with recombinant Agrin induced the clustering of surface receptors, including major histocompatibility complex class II, activation of intracellular signalling cascades, as well as enhanced dsRNA-induced expression of pro-inflammatory cytokines interleukin-6 and tumour necrosis factor-a. Collectively, these results confirm the location of Agrin at the immunological synapse between T cells and antigen-presenting cells and justify further characterization of its receptors in the immune system.

Mecanismos da intoxicação do fígado de rato causada pelo gossipol

O fígado desempenha uma função central no metabolismo devido à sua interposição entre o trato digestivo e a circulação geral do organismo. Ele é também o principal órgão envolvido na biotransformação de substâncias exógenas (xenobióticos), com capacidade de converter compostos hidrofóbicos em hidrossolúveis, mais facilmente eliminados pelo organismo. O gossipol é uma substância fenólica tóxica presente na semente de algodão (Gossypium sp). Com o objetivo de estudar os mecanismos envolvidos na hepatotoxicidade do gossipol avaliou-se os seus efeitos no sistema antioxidante do fígado de ratos no que diz respeito ao estresse oxidativo e aspectos histopatológicos. Foram utilizados ratos machos da linhagem Wistar, separados em dois grupos, sendo que um recebeu óleo de canola (veículo, grupo Controle) e o outro recebeu gossipol na dosagem de 40 mg/kg de peso vivo do animal por 15 dias (grupo Tratado). O tratamento com gossipol promoveu alterações na atividade sérica das enzimas marcadoras de dano hepático e um significativo estresse oxidativo caracterizado pela diminuição nos níveis da glutationa reduzida (GSH) e consequente aumento da glutationa oxidada (GSSG), incluindo, ainda, danos à membrana plasmática e de organelas demonstrados pela peroxidação lipídica. O resultado da avaliação histopatológica demonstrou degeneração dos hepatócitos.

Microscopia eletrônica de varredura do endosperma de café (Coffea arabica L.) durante o processo de secagem

A manutenção da integridade das membranas celulares, entre outros eventos, é um forte indicativo de que a qualidade do café foi preservada na pós-colheita. Objetivou-se neste trabalho, analisar o efeito de diferentes métodos de secagem na manutenção da integridade da parede celular e da membrana plasmática de café natural e café despolpado, buscando determinar as condições e o momento em que ocorrem as rupturas microscópicas. Os cafés foram submetidos a um período de pré-secagem em terreiro. Após este, uma parcela de cada tipo de café foi desidratada no terreiro e, outra, à temperatura de 40ºC e 60ºC em secadores de camada fixa, monitorando-se a temperatura e o teor de água até 11% (bu). Nesse período, grãos foram aleatoriamente amostrados e fragmentos do endosperma preparados para a microscopia eletrônica de varredura, registrando-se diversas eletromicrografias, avaliando-se as alterações na membrana plasmática da célula do endosperma dos grãos de cafés em função do teor de água e tempo de secagem. O citoplasma das células a 11% (bu) de teor de água não foi comprometido na secagem em terreiro e a 40°C; na secagem a 60°C, observou-se comprometimento nas estruturas celulares nos cafés com teor de água de 20% (bu).

Efeitos da pentoxifilina sobre a viabilidade in vitro dos espermatozóides de eqüinos, após o resfriamento a 5ºC

O objetivo deste trabalho foi avaliar, in vitro, o efeito da adição de 1 mg pentoxifilina por mL de amostras seminais diluídas em meio Kenney, e resfriadas a 5º C, de garanhões normospérmicos, sobre os seguintes parâmetros: taxas de motilidade total e motilidade progressiva, velocidade espermática ao longo da trajetória real, índice retilíneo, taxa de linearidade, velocidade progressiva, taxa de viabilidade e taxa de integridade da membrana plasmática. Foram avaliados 20 ejaculados de quatro garanhões, com o auxílio do Hamilton Thorn Research (Animal Version 12.0 L, EUA), em câmara de Makler. A taxa de viabilidade foi realizada por coloração de eosina e microscopia de contraste e a taxa de integridade de membrana, por coloração com fluorocromos, iodeto de propídio e diacetato de carboxifluoresceina e microscopia de fluorescência. As análises foram realizadas aos 30, 60, e 120 minutos de incubação a 37º C, após 12, 24 e 48 horas de resfriamento a 5ºC. A pentoxifilina incrementou significativamente, em relação ao grupo controle, os parâmetros espermáticos relacionados à motilidade dos espermatozóides e de integridade da membrana plasmática, durante todo o período de incubação, da seguinte forma: taxa de motilidade total em 8,2%; motilidade progressiva em 4,7%; velocidade real em 23,1 mm/s; taxa de espermatozóides com velocidade rápida em 7,4%; taxa de viabilidade em 4,7%; e integridade da membrana plasmática em 3,5 % do tratado. Quanto aos parâmetros índice de retilinidade e linearidade, não foi observada diferença significativa entre os grupos. Os resultados obtidos nas condições deste experimento indicam que a adição de pentoxifilina ao ejaculado resfriado de garanhões, até 48 horas após o resfriamento, pode ser benéfica à qualidade dos espermatozóides para uso na inseminação artificial.

Functional ultrastructure of the midgut of the fire ant Solenopsis saevissima Forel 1904 (Formicidae : Myrmicinae)

Solenopsis saevissima has a midgut composed of columnar, regenerative, and goblet cells. The midgut epithelium was covered by a basal lamina. Outside the basal lamina, layers of inner oblique, circular, and outer longitudinal muscles were present. Columnar cells showed a basal plasma membrane containing numerous folds, mitochondria, and the nucleus. Rough endoplasmic reticulum, Golgi bodies, membrane bounded vacuoles, and spherocrystals were found in this region. The apical plasma membrane was constituted by microvilli, which were above a region rich in mitochondria. Regenerative cells were found in groups lying by the basal lamina. Goblet cells were associated with an ion-transporting mechanism between the haemolymph and the midgut epithelium. These cells were lying by the midgut lumen and large microvilli were evident, but the cytoplasmic features were similar to the columnar cells.

Secretion of salivary glands of the Brazilian termite Serritermes serrifer Hagen&Bates (Isoptera: Serritermitidae)

The salivary glands of termites are composed of several secretory acini connected by ducts. These glands, in the Brazilian termite Serritermes serrifer, were examined through the electron microscope. The ultrastructure of worker salivary acinus revealed central ductule cells and four different types of cells. Cells of type I contain an abundance of electron-lucid vacuoles of various sizes which fuse to form enormous vacuolar structures that fill up most of the cell. Cells of type II are narrow cells in which the secretion is contained in small clear vacuoles of approximately equal diameter. Both of these cellular types have numerous Golgi bodies and rough endoplasmic reticulum. Type III or parietal cells have an apical plasma membrane deeply infolded and lined by microvilli. This type of cell is located in the acinar periphery and occurs in pairs. Cells of type IV are completely filled with electrondense secretion. The secretory granules can be small in some cells or large and similar to fingerprints in others. This is the first report of the occurrence of these spiral or concentric rings of dense material in the salivary gland of Isoptera.

Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression

Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation, This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis, For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus tie, able to recognize intact ANX-A1) or the whole protein tie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority (similar to 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to similar to6%), concomitant with an increase in total amount of the protein; only similar to 25% of the total protein was now recognized by the antibody raised against the N-terminus tie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes, In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.

The N terminus of the human alpha(1D)-adrenergic receptor prevents cell surface expression

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A) NT-D, alpha(1B) NT-D, alpha(1D) NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)- or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

Ultrastructure of the Lyonet's glands in larvae of Diatraea saccharalis Fabricius (Lepidoptera : Pyralidae)

The Lyonet's gland is found in Lepidoptera larvae, close to the excretory duct of the silk gland. The role played by this gland is still uncertain. This work aims to describe the ultrastructure of the Lyonet's bland in Diatraea saccharalis larvae, offering suggestions regarding its possible function. The insects were reared under laboratory-controlled conditions. The glands were conventionally prepared for transmission (TEM) and scanning (SEM) electron microscopy. SEM showed that Lyonet's glands are paired small structures located in the ventral side of the head. They are composed by clustered long cells resembling leaves. Under TEM observations, each cell is surrounded by a thin basal lamina and contains large stellate nucleus. The cytoplasm presents large and empty canaliculi with small microvilli. The basal plasma membrane forms numerous infoldings where numerous and well-developed mitochondria are concentrated. The cytoplasmic membrane system is poorly developed. Our ultrastructural results suggest that the Lyonet's gland in D. saccharalis larvae may be involved in the uptake of small molecules from the hemolymph no morphological evidences of macromolecules synthesis and secretion were noticed. The detection of nerve fibers in the gland suggest a neural control for the glandular cell function.

Effects of light-curing time on the cytotoxicity of a restorative resin composite applied to an immortalized odontoblast-cell line

This in vitro study evaluated the cytotoxic effects of a restorative resin composite applied to an immortalized odontoblast-cell line (MDPC-23). Seventy-two round resin discs (2-mm thick and 4 mm in diameter) were light-cured for 20 or 40 seconds and rinsed, or not, with PBS and culture medium. The resin discs were divided into four experimental groups: Group 1: Z-100/20 seconds; Group 2: Z-100/20 seconds/rinsed; Group 3: Z100/40 seconds; Group 4: Z-100/40 seconds/rinsed. Circular filter paper was used as a control material (Group 5). The round resin discs and filter papers were placed in the bottom of wells of four 24-well dishes (18 wells for each experimental and control group). MDPC-23 cells (30,000 cells/cm(2)) were plated in the wells and allowed to incubate for 72 hours. The zone of inhibition around the resin discs was measured under inverted light microscopy; the MTT assay was carried out for mitochondrial respiration and cell morphology was measured under SEM. The scores obtained from inhibition zone and MTT assay were analyzed with the Kruskal-Wallis followed by Dunnett tests. In Groups 1, 2, 3 and 4, the thickness of the inhibition zone was 1,593 +/- 12.82 mum, 403 +/- 15.49 mum, 1,516 +/- 9.81 mum and 313 +/- 13.56 mum, respectively. There was statistically significant difference among the experimental and control groups at the 0.05 level of significance. The MTT assay demonstrated that the resin discs of the experimental groups 1, 2, 3 and 4 reduced the cell metabolism by 83%, 40.1%, 75.5% and 24.5%. Only between the Groups 2 and 4 was there no statistically significant difference for mitochondrial respiration. Close to the resin discs, the MDPC-23 cells exhibited rounded shapes, with only a few cellular processes keeping the cells attached to the substrate or, even disruption of plasma membrane. Adjacent to the inhibition zone, the cultured cells exhibited multiple fine cellular processes on the cytoplasmic membrane organized in epithelioid nodules, similar to the morphology observed to the control group. Based on the results, the authors may conclude that the Z-100 resin composite light cured for 20 seconds was more cytopathic to MDPC-23 cells than Z-100 light cured for 40 seconds. The cytotoxic effects of the resin discs decreased after rinsing them with PBS and culture medium. This was confirmed by MTT assay and upon evaluation of the inhibition zone, which was narrower following rinsing of the resin discs.

Bull semen varibles after experimental exposure with Bovine Herpesvirus type 5

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Secretion of salivary glands of the Brazilian termite Serritermes serrifer Hagen & Bates (Isoptera : Serritermitidae)

The salivary glands of termites are composed of several secretory acini connected by ducts. These glands, in the Brazilian termite Serritermes serrifer, were examined through the electron microscope. The ultrastructure of worker salivary acinus revealed central ductule cells and four different types of cells. Cells of type I contain an abundance of electron-lucid vacuoles of various sizes which fuse to form enormous vacuolar structures that fill up most of the cell. Cells of type II are narrow cells in which the secretion is contained in small clear vacuoles of approximately equal diameter. Both of these cellular types have numerous Golgi bodies and rough endoplasmic reticulum. Type III or parietal cells have an apical plasma membrane deeply infolded and lined by microvilli. This type of cell is located in the acinar periphery and occurs in pairs. Cells of type IV are completely filled with electrondense secretion. The secretory granules can be small in some cells or large and similar to fingerprints in others. This is the first report of the occurrence of these spiral or concentric rings of dense material in the salivary gland of Isoptera.

Ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermiogenesis

The spermiogenesis and the spermatozoon ultrastructure of Sorubim lima were studied. Our observations showed that early spermatids are round-shaped cells, have spherical nucleus with diffuse chromatin, small quantity of mitochondria and large amount of vesicles in the cytoplasm. During the differentiation process in the nucleus, chromatin compacts in a progressive and homogeneous way, and the flagellum is formed. In the cytoplasm the vesicles, that have double membranes, aggregate and fuse on the plasma membrane. The spermatozoa of 5. lima have no acrosome and show spherical nucleus with homogeneous and highly compacted chromatin, intermediary piece with mitochondria and double wall vesicles contiguous to the plasma membrane, as well as a flagellum formed by a basic axoneme (9 + 2).

Papel das Yops secretadas por Yersinia sobre a resposta imune do hospedeiro

The genus Yersinia contains three species pathogenic to humans: Y. pestis, Y. enterocolitica e Y. pseudotuberculosis. The pathogenicity of Yersinia is linked to the presence of a 70-kb virulence plasmid (pYV) that is common to the three species and codifies a type III secretion system and a set of virulence proteins, including those known as Yersinia outer proteins (Yops), that are exported by this system when the bacteria encounter host cells. Two Yops translocators (YopB and YopD) are inserted into the host plasma membrane and transport six effectors (YopO, YopH, YopM, YopJ and YopT) across the membrane into the cytosol of the host cell. The Yops effectors interfere with multiple signaling pathways of the infected cell, affecting both the innate and adaptive immune responses. This article focuses on the role of Yops in the modulation of the host immune response.