Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic ␣-linked galactopyranosyl (␣-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 ؋ g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing ␣-Gal epitopes reacted strongly with anti-␣-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal ␣-Gal), but only faintly with natural anti-␣-Gal. Reactivity was inhibited after treatment with ␣-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying ␣-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-␣-Gal in ELISA only when not digested with ␣-galactosidase, while reactivity with glycoproteins was reduced after ␤-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and ␣-galactosyl epitopes.

Trypanosoma cruzi is the protozoan that causes Chagas disease. It divides in the insect vector gut or in the cytosol of an infected mammalian cell. T. cruzi has one mitochondrion, one Golgi complex, one flagellum, and one cytostome. Here, we provide threedimensional (3D) models of this protozoan based on images obtained from serial sections on electron microscopy at different stages of the cell cycle. Ultrathin serial sections were obtained from Epon TM embedded parasites, photographed in a transmission electron microscope, and 3D models were generated using Reconstruct and Blender 3D modeling softwares. The localization and distribution of organelles was evaluated and attributed to specific morphological patterns and deduced by distribution of specific markers by immunofluorescence analysis. The new features found in the 3D reconstructions are (1) the electron-dense chromatin is interconnected leaving an internal space for a centrally located nucleolus; (2) The kinetoplast is accommodated within a separated branch of the tubular and single mitochondrion; (3) The disk shaped kinetoplast, which is the mitochondrial DNA, duplicates from the interior in G2 phase; (4) The mitochondrion faces the external membrane and shrinks to accommodate an enlarged number of cytosolic vesicles from G1 to G2; (5) The cytostome progress from the parasite surface toward the posterior end contouring the kinetoplast and nucleus and retracts during cell cycle. These new observations might help understanding how organelles are formed and distributed in early divergent eukaryotic cells and provides a useful method to understand the organelle distribution in small eukaryotic cells. ' International Society for Advancement of Cytometry Key termsTrypanosoma cruzi; electron microscopy; 3D-reconstruction, nucleus; nucleolus; mitochondrion; kinetoplast; flagellum; cell cycle TRYPANOSOMA cruzi is the protozoan that causes Chagas' disease. It divides in the insect vector gut or in the cytosol of an infected mammalian cell. Although its ultrastructure has been extensively described (1-5), little is known about how it changes during the cell division cycle. Several techniques have been recently developed to observe the ultrastructure at the three-dimensional (3D) levels, particularly electrontomography, which provide detailed information about specific organelles (6). Reconstruction of entire cells requires extensive imaging and computer processing. In contrast, 3D reconstruction using serial sections is a very powerful method to visualize the morphological aspects of entire cells, which would allow observing the variable distribution of diverse organelles in different situations (7,8).We have recently described how epimastigotes, the parasite stage that divides in the insect gut, progress through the cell division cycle (9). When the nuclear DNA is replicated, basal bodies are found duplicated and a new flagellum starts to be synthesized. At this moment, recognized as the G2 stage of the cell cycle, a series of morphological alterations ...

Protein dystrophin is a component of the dystrophin-associated protein complex, which links the contractile machinery to the plasma membrane and to the extracellular matrix. Its absence leads to a condition known as Duchenne muscular dystrophy (DMD), a disease characterized by progressive skeletal muscle degeneration, motor disability, and early death. In mdx mice, the most common DMD animal model, loss of muscle cells is observed, but the overall disease alterations are less intense than in DMD patients. Alterations in gastrointestinal tissues from DMD patients and mdx mice are not yet completely understood. Thus, we investigated the possible relationships between morphological (light and electron microscopy) and contractile function (by recording the isometric contractile response) with alterations in Ca²⁺ handling in the ileum of mdx mice. We evidenced a 27% reduction in the ileal muscular layer thickness, a partial damage to the mucosal layer, and a partial damage to mitochondria of the intestinal myocytes. Functionally, the ileum from mdx presented an enhanced responsiveness during stretch, a mild impairment in both the electromechanical and pharmacomechanical signaling associated with altered calcium influx-induced contraction, with no alterations in the sarcoplasmic reticulum Ca²⁺ storage (maintenance of the caffeine and thapsigargin-induced contraction) compared with control animals. Thus, it is evidenced that the protein dystrophin plays an important role in the preservation of both the microstructure and ultrastructure of mice intestine, while exerting a minor but important role concerning the intestinal contractile responsiveness and calcium handling.

Choline - now recognized as an essential nutrient - is the most common polar group found in the outer leaflet of the plasma membrane bilayer. Brain ischemia-reperfusion causes lipid peroxidation triggering multiple cell death pathways involving necrosis and apoptosis. Membrane breakdown is, therefore, a major pathophysiologic event in brain ischemia. The ability to achieve membrane repair is a critical step for survival of ischemic neurons following reperfusion injury. The availability of choline is a rate-limiting factor in phospholipid synthesis and, therefore, may be important for timely membrane repair and cell survival. This work aimed at verifying the effects of 7-day oral administration with different doses of choline on survival of CA1 hippocampal neurons following transient global forebrain ischemia in rats. The administration of 400 mg/kg/day divided into two daily doses for 7 consecutive days significantly improved CA1 pyramidal cell survival, indicating that the local availability of this essential nutrient may limit postischemic neuronal survival.