RESUMEN
We described the phylogenetic affiliation, development in cultures and ultrastructural features of a trypanosome of Leptodacylus chaquensis from the Pantanal biome of Brazil. In the inferred phylogeny, this trypanosome nested into the Anura clade of the basal Aquatic clade of Trypanosoma, but was separate from all known species within this clade. This finding enabled us to describe it as Trypanosoma herthameyeri n. sp., which also infects other Leptodacylus species from the Pantanal and Caatinga biomes. Trypanosoma herthameyeri multiplies as small rounded forms clumped together and evolving into multiple-fission forms and rosettes of epimastigotes released as long forms with long flagella; scarce trypomastigotes and glove-like forms are common in stationary-phase cultures. For the first time, a trypanosome from an amphibian was observed by field emission scanning electron microscopy, revealing a cytostome opening, well-developed flagellar lamella, and many grooves in pumpkin-like forms. Transmission electron microscopy showed highly developed Golgi complexes, relaxed catenation of KDNA, and a rich set of spongiome tubules in a regular parallel arrangement to the flagellar pocket as confirmed by electron tomography. Considering the basal position in the phylogenetic tree, developmental and ultrastructural data of T. herthameyeri are valuable for evolutionary studies of trypanosome architecture and cell biology.
Asunto(s)
Anuros/parasitología , Filogenia , Trypanosoma/clasificación , Trypanosoma/ultraestructura , Tripanosomiasis/veterinaria , Animales , Anuros/sangre , Biodiversidad , Brasil , Clasificación , ADN Protozoario/genética , Ecología , Ecosistema , Tomografía con Microscopio Electrónico/métodos , Flagelos/ultraestructura , Aparato de Golgi/ultraestructura , Especificidad del Huésped , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión/métodos , Trypanosoma/crecimiento & desarrollo , Trypanosoma/aislamiento & purificación , Tripanosomiasis/sangre , Tripanosomiasis/diagnóstico , Tripanosomiasis/parasitologíaRESUMEN
Three-dimensional electron microscopy tools have revolutionized our understanding of cell structure and molecular complexes in biology. Here, we describe methods for studying flagellar ultrastructure and biogenesis in two unicellular parasites-Trypanosoma brucei and Leishmania mexicana. We describe methods for the preparation of these parasites for scanning electron microscopy cellular electron tomography, and serial block face scanning electron microscopy (SBFSEM). These parasites have a highly ordered cell shape and form, with a defined positioning of internal cytoskeletal structures and organelles. We show how knowledge of these can be used to dissect cell cycles in both parasites and identify the old flagellum from the new in T. brucei. Finally, we demonstrate the use of SBFSEM three-dimensional models for analysis of individual whole cells, demonstrating the excellent potential this technique has for future studies of mutant cell lines.
Asunto(s)
Movimiento Celular/fisiología , Flagelos/ultraestructura , Leishmania mexicana/fisiología , Trypanosoma brucei brucei/fisiología , Animales , Ciclo Celular/genética , ADN Protozoario/genética , Tomografía con Microscopio Electrónico/métodos , Flagelos/fisiología , Imagenología Tridimensional/métodos , Leishmania mexicana/genética , Leishmania mexicana/crecimiento & desarrollo , Microscopía Electrónica de Rastreo/métodos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Moscas Tse-Tse/parasitologíaRESUMEN
The cytostome-cytopharynx complex is the main site of endocytosis of Trypanosoma cruzi epimastigotes. Little is known about the detailed morphology of this remarkable structure. We used serial electron tomography and focused-ion-beam scanning electron microscopy to reconstruct the entire complex, including the surrounding cytoskeleton and vesicles. Focusing on cells that had taken up gold-labeled tracers, we produced three-dimensional snapshots of the process of endocytosis. The cytostome cytoskeleton was composed of two microtubule sets--a triplet that started underneath the cytostome membrane, and a quartet that originated underneath the flagellar-pocket membrane and followed the preoral ridge before reaching the cytopharynx. The two sets accompanying the cytopharynx formed a 'gutter' and left a microtubule-free side, where vesicles were found to be associated. Cargo was unevenly distributed along the lumen of the cytopharynx, forming clusters. The cytopharynx was slightly longer during the G2 phase of the cell cycle, although it did not reach the postnuclear region owing to a bend in its path. Therefore, the cytopharynx is a dynamic structure, undergoing remodeling that is likely associated with endocytic activity and the preparation for cell division.
Asunto(s)
Trypanosoma cruzi/ultraestructura , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Tomografía con Microscopio Electrónico/métodos , Endocitosis , Microtúbulos/metabolismo , Trypanosoma cruzi/metabolismoRESUMEN
The perinuclear theca (PT) is a cytoskeletal structure that surrounds the mammal sperm nucleus which must be disrupted once the sperm has penetrated the oocyte to permit normal chromatin decondensation and formation of male pronucleus. F-actin is a thermo sensitive protein found in the equatorial segment which is involved in the stability of PT. It has been reported that cryopreservation induces alterations in nuclear decondensation of spermatozoa, which have been interpreted as an over condensation. The aims of the present study were identified the presence of changes in sperm sPT integrity of frozen-thawed boar spermatozoa and its effect in sperm nuclei decondensation; and whether changes in the actin cytoskeleton are involved using an in vitro model to test probably differences in a chemical decondensation (DTT/heparin) between fresh (FS) and frozen-thawed (TS) spermatozoa. Results showed an increase on sPT damage in TS (P<0.001), and significant changes in sperm chromatin nuclear decondensation (P<0.05). In same way differences on the swelling degree was found assessed by measures in equatorial region of head sperm (P<0.05). Evaluation with rodamine-labeled actin (0.2µM) showed two different patterns with differences in percentages before and after cryopreservation (P<0.001). F-actin stabilization constrained the equatorial segment of FS while this was not observed in TS. The data showed that the presence of early changes in sPT integrity and changes in the F-actin localization on TS may suggest the participation in F-actin in decondensation process and probably that the disruption of actin-PT interaction during freezing-thawing process could have far-reaching consequences for the subsequent fertility of spermatozoa.
Asunto(s)
Actinas/metabolismo , Cromatina/ultraestructura , Criopreservación/métodos , Congelación/efectos adversos , Preservación de Semen/efectos adversos , Cabeza del Espermatozoide/ultraestructura , Sus scrofa , Animales , Citoesqueleto/fisiología , Tomografía con Microscopio Electrónico/métodos , Masculino , Semen/citología , Semen/fisiología , Preservación de Semen/métodos , Preservación de Semen/veterinaria , Cabeza del Espermatozoide/fisiología , Motilidad Espermática/fisiología , Espermatozoides/fisiología , Espermatozoides/ultraestructuraRESUMEN
We describe the interactions of two benzimidazole derivatives, astemizole (AST) and lansoprazole (LNS), with anomalous aggregates of tau protein (neurofibrillary tangles). Interestingly, these compounds, with important medical applications in the treatment of allergies and gastrointestinal disorders respectively, specifically bind to aggregated variants of tau protein and to paired helical filaments isolated from brains of Alzheimer's disease (AD) patients. These ligands appear to be a powerful tool to tag brain-isolated tau-aggregates and heparin-induced polymers of recombinant tau. The interactions of AST and LNS with tau aggregates were assessed by classical radioligand assays, surface plasmon resonance, and bioinformatic approaches. The affinity of AST and LNS for tau aggregates was comparatively higher than that for amyloid-beta polymers according to our data. This is relevant since senile plaques are also abundant but are not pathognomonic in AD patients. Immunochemical studies on paired helical filaments from brains of AD patients and surface plasmon resonance studies confirm these findings. The capacity of these drugs to penetrate the blood-brain barrier was evaluated: i) in vitro by parallel artificial membrane permeability assay followed by experimental Log P determinations; and ii) in vivo by pharmacokinetic studies comparing distribution profiles in blood and brain of mice using HPLC/UV. Importantly, our studies indicate that the brain/blood concentration ratios for these compounds were suitable for their use as PET radiotracers. Since neurofibrillary tangles are positively correlated with cognitive impairment, we concluded that LNS and AST have a great potential in PET neuroimaing for in vivo early detection of AD and in reducing the formation of neurofibrillary tangles.
Asunto(s)
2-Piridinilmetilsulfinilbencimidazoles , Enfermedad de Alzheimer/diagnóstico , Astemizol , Inhibidores Enzimáticos , Antagonistas de los Receptores Histamínicos H1 no Sedantes , Proteínas tau/metabolismo , 2-Piridinilmetilsulfinilbencimidazoles/química , 2-Piridinilmetilsulfinilbencimidazoles/farmacocinética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Astemizol/química , Astemizol/farmacocinética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Cromatografía Líquida de Alta Presión , Biología Computacional , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Técnicas Electroquímicas , Tomografía con Microscopio Electrónico/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Antagonistas de los Receptores Histamínicos H1 no Sedantes/química , Antagonistas de los Receptores Histamínicos H1 no Sedantes/farmacocinética , Humanos , Lansoprazol , Modelos Moleculares , Ovillos Neurofibrilares/efectos de los fármacos , Ovillos Neurofibrilares/patología , Ovillos Neurofibrilares/ultraestructura , Ensayo de Unión Radioligante , Resonancia por Plasmón de Superficie , Tritio/farmacocinética , Proteínas tau/ultraestructuraRESUMEN
Synaptic dysfunction has been associated with neuronal cell death following hypoxia. The lack of knowledge on the mechanisms underlying this dysfunction prompted us to investigate the morphological changes in the postsynaptic densities (PSDs) induced by hypoxia. The results presented here demonstrate that PSDs of the rat neostriatum are highly modified and ubiquitinated 6 months after induction of hypoxia in a model of perinatal asphyxia. Using both two dimensional (2D) and three dimensional (3D) electron microscopic analyses of synapses stained with ethanolic phosphotungstic acid (E-PTA), we observed an increment of PSD thickness dependent on the duration and severity of the hypoxic insult. The PSDs showed clear signs of damage and intense staining for ubiquitin. These morphological and molecular changes were effectively blocked by hypothermia treatment, one of the most effective strategies for hypoxia-induced brain injury available today. Our data suggest that synaptic dysfunction following hypoxia may be caused by long-term misfolding and aggregation of proteins in the PSD.