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Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor–ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions. Fil: Nievas, Yésica Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Cóceres, Verónica Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Midlej, Victor. Universidade Federal do Rio de Janeiro; Brasil Fil: de Souza, Wanderley. Universidade Federal do Rio de Janeiro; Brasil Fil: Benchimol, Marlene. Universidade Federal do Rio de Janeiro; Brasil Fil: Pereira Neves, Antonio. Fundación Oswaldo Cruz; Brasil Fil: Vashisht, Ajay A.. University of California at Los Angeles; Estados Unidos Fil: Wohlschlegel, James A.. University of California at Los Angeles; Estados Unidos Fil: Johnson, Patricia J.. University of California at Los Angeles; Estados Unidos Fil: de Miguel, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. Cell division has been described as a key player in controlling cell survival in other cells, including parasites but there is no information on the regulation of this process in T. foetus. The regulation of cytokinetic abscission, the final stage of cell division, is mediated by members of the ESCRT (endosomal sorting complex required for transport) machinery. VPS32 is a subunit within the ESCRTIII complex and here, we report that TfVPS32 is localized on cytoplasmic vesicles and a redistribution of the protein to the midbody is observed during the cellular division. In concordance with its localization, deletion of TfVPS32 C-terminal alpha helices (α5 helix and/or α4-5 helix) leads to abnormal T. foetus growth, an increase in the percentage of multinucleated parasites and cell cycle arrest at G2/M phase. Together, these results indicate a role of this protein in controlling normal cell division. Fil: Iriarte, Lucrecia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Midlej, Victor. Universidade Federal do Rio de Janeiro; Brasil Fil: Frontera, Lorena Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Moros Duarte, Daniel Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Barbeito, Claudio Gustavo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Ciencias Básicas. Cátedra de Histología y Embriología; Argentina Fil: de Souza, Wanderley. Universidade Federal do Rio de Janeiro; Brasil Fil: Benchimol, Marlene. Universidade Federal do Rio de Janeiro; Brasil. Universidade Federal do Rio Grande; Brasil Fil: de Miguel, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Cóceres, Verónica Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Trichomonas tenax is considered a commensal organism found under poor oral hygiene conditions. T. tenax presents morphological similarities with T. vaginalis, and there are doubts concerning whether this protist is a parasite and whether it is a genetic variant of T. vaginalis. This study aimed to investigate the capacity of T. tenax to cause mammalian cell damage and compare its cytotoxicity with that of T. vaginalis. Protozoan-host cell interaction assays were performed with Madin-Darby canine kidney, HeLa, and gum cells and 3D spheroids, which were examined by scanning electron and transmission electron microscopy. Cellular viability experiments were also performed. T. tenax attached and had different forms when interacting with mammalian cells and caused damage with time-dependent host-cell viability. We observed that T. tenax produced plasma membrane projections and phagocytosed portions of the mammalian cells. In addition, T. tenax caused membrane blebbing and apoptotic bodies in HeLa cells, thus inducing cell death. Spheroids were also used in interaction assays with T. tenax and they were damaged by these cells. This study shows that T. tenax fulfills the requisites of a parasite, causing damage to different mammalian cells and behaving similarly to T. vaginalis when in contact with target cells in vitro.

Summary In vitro studies show that cholesterol is essential to myogenesis. We have been using zebrafish to overcome the limitations of the in vitro approach and to study the sub-cellular structures and processes involved during myogenesis. We use simvastatin—a drug widely used to prevent high levels of cholesterol and cardiovascular disease—during zebrafish skeletal muscle formation. Simvastatin is an efficient inhibitor of cholesterol synthesis that has various myotoxic consequences. Here, we employed simvastatin concentrations that cause either mild or severe morphological disturbances to observe changes in the cytoskeleton (intermediate filaments and microfilaments), extracellular matrix and adhesion markers by confocal microscopy. With low-dose simvastatin treatment, laminin was almost normal, and alpha-actinin was reduced in the myofibrils. With high simvastatin doses, laminin and vinculin were reduced and appeared discontinuous along the septa, with almost no myofibrils, and small amounts of desmin accumulating close to the septa. We also analyzed sub-cellular alterations in the embryos by electron microscopy, and demonstrate changes in embryo and somite size, septa shape, and in myofibril structure. These effects could be reversed by the addition of exogenous cholesterol. These results contribute to the understanding of the mechanisms of action of simvastatin in muscle cells in particular, and in the study of myogenesis in general. (J Histochem Cytochem 63:427–437, 2015)

The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T.vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite:parasite interaction. Fil: Cóceres, Verónica Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Alonso, Andrés Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Nievas, Yésica Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Midlej, V.. Universidade Federal do Rio de Janeiro; Brasil Fil: Frontera, L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina Fil: Benchimol, M.. Universidade Federal do Rio de Janeiro; Brasil Fil: Johnson, P. J.. University of California at Los Angeles; Estados Unidos Fil: de Miguel, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina

O presente artigo apresenta os resultados de uma pesquisa que procurou evidenciar a percepção dos egressos de Cursos de Ciências Biológicas do Estado do Rio de Janeiro como indicador de avaliação institucional. O instrumento de pesquisa utilizado foi um questionário online, composto de perguntas multiopcionais e uma dissertativa. Este foi respondido por 241 egressos, sendo 83 da modalidade de ensino a distância e 158 de ensino presencial. Os resultados revelaram que os egressos do ensino a distância foram os mais satisfeitos em relação à Graduação que cursaram, considerando-a como “ótima”. A análise de conteúdo mostrou que a maior insatisfação entre os egressos do Curso presencial foi que o Curso deveria oferecer melhor preparação para o mercado de trabalho. Outros tópicos – necessidade de melhorias na infraestrutura, aumento das atividades práticas em campo, renovação e atualização da grade curricular, e qualidade dos professores – também foram comentados em maior frequência.

The protist Tritrichomonas foetus (Excavata, Parabasalia) is a parasite that causes bovine and feline trichomonosis. Bovine trichomonosis is a venereal disease that leads to abortion and reproductive problems in herds. Feline trichomonosis affects domestic cats. Here, we report the genome sequence of the T. foetus K strain, isolated in Brazil.

Tritrichomonas foetus is a serious veterinary parasite that causes bovine trichomoniasis, a sexually transmitted disease that results in reproductive failure and considerable economic losses in areas that practice natural breeding. T. foetus is an extracellular parasite, which initially adheres to and infects the urogenital tract using a diverse array of surface glycoconjugates, including adhesins and extracellular matrix-binding molecules. However, the cellular mechanisms by which T. foetus colonizes mucosal surfaces and causes tissue damage are not well defined. Several studies have demonstrated the involvement of pathogen or host lipid rafts in cellular events that occur during pathogenesis, including adhesion, invasion and evasion of the immune response. In this study, we demonstrate that detergent-resistant membranes are present in the plasma membrane of T. foetus. We further demonstrate that microdomains are cholesterol-enriched and contain ganglioside GM1-like molecules. In addition, we demonstrate that lipid microdomains do not participate in T. foetus adhesion to host cells. However, the use of agents that disrupt and disorganize the plasma membrane indicated the involvement of the T. foetus lipid microdomains, in cell division and in the formation of endoflagellar forms. Our results suggest that trophozoites and endoflagellar forms present a different plasma membrane organization.

SUMMARYTritrichomonas foetusis a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi ofT. foetusand used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of theTrichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in theT. foetusdatabase, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved betweenT. foetusandT. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin fromT. foetusand (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.

The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry.Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction.The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments.Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the organisation and function of this structure in unicellular organisms.

The protozoan parasite Tritrichomonas foetus displays a pear-shaped form and a pseudocyst stage. However, little is known about the biology of the pseudocyst. The aim of this work was to assess whether pseudocysts exert cytotoxic effects during their interaction with MDCK cells (an epithelial kidney canine cell line) and compare their behavior to that of the pear-shaped parasites. Pseudocysts and pear-shaped parasites from both cultured and freshly isolated T. foetus were used. Electron microscopy revealed that the epithelial cells exhibited more signs of injury, such as depletion of microvilli, retraction from neighboring cells and swollen mitochondria with loss of electron density in the matrix, when the pseudocysts were used in interaction experiments. In addition, during the co-incubation with MDCK cells, pseudocysts exhibited a more intense amoeboid transformation than that found in pear-shaped parasites. The MTT viability assay demonstrated that the pseudocysts were more cytotoxic when in contact with host cells as compared to the flagellated pear-shaped parasites. The JC-1 viability assay revealed that pseudocysts induced a higher loss of mitochondrial membrane potential compared to pear-shaped parasites. Pseudocysts undergoing a budding process were observed after 2.5 h of co-incubation with MDCK cells. Our results suggest that the T. foetus pseudocyst might be a more aggressive form.

In this work, evidence for a critical role of Trichomonas vaginalis protein phosphatase 1 gamma (TvPP1γ) in proliferation and attachment of the parasite to the mammalian cell is provided. Firstly, proliferation and attachment of T. vaginalis parasites to HeLa cells was blocked by calyculin A (CA), a potent PP1 inhibitor. Secondly, it was demonstrated that the enzyme activity of native and recombinant TvPP1γ proteins was inhibited by CA. Thirdly, reverse genetic studies confirmed that antisense oligonucleotides targeted to PP1γ but not PP1α or β inhibited proliferation and attachment of trichomonads CA-treated parasites underwent cytoskeletal modifications, including a lack of axostyle typical labelling, suggesting that cytoskeletal phosphorylation could be regulated by a CA-sensitive phosphatase where the role of PP1γ could not be ruled out. Analysis of subcellular distribution of TvPP1γ by cell fractionation and electron microscopy demonstrated the association between TvPP1γ and the cytoskeleton. The expression of adhesins, AP120 and AP65, at the cell surface was also inhibited by CA. The concomitant inhibition of expression of adhesins and changes in the cytoskeleton in CA-treated parasites suggest a specific role for PP1γ -dependent dephosphorylation in the early stages of the host-parasite interaction. Molecular modelling of TvPP1γ showed the conservation of residues critical for maintaining proper folding into the gross structure common to PP1 proteins. Taken together, these results suggest that TvPP1γ could be considered a potential novel drug target for treatment of trichomoniasis.

The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc.

Trichomonas vaginalis is an important human parasite that causes trichomoniasis, a cosmopolitan sexually transmitted disease. Currently, the treatment of choice for T. vaginalis infections is metronidazole. The increase in metronidazole-resistant parasites and undesirable side effects of this drug make the search for alternative chemotherapeutic approaches a priority for the management of trichomoniasis. Here, the antiproliferative and ultrastructural effects of sterol biosynthesis inhibitors against T. vaginalis were investigated. It was found that 22,26-azasterol (5 μM) and 24(R,S),25-epiminolanosterol (10 μM), known inhibitors of Δ(24(25))-sterol methyltransferase, exhibited antiproliferative effects on T. vaginalis trophozoites cultured in vitro. Morphological analyses showed that azasterols induced changes in the ultrastructure of T. vaginalis. The most significant alterations were (1) membrane blebbing and disruption, (2) wrinkled cells and (3) the formation of cell clusters. In addition, autophagic vacuoles, Golgi duplication arrest, an abnormal Golgi enlargement and damaged hydrogenosomes were also observed. Nonspecific cytotoxicity assays using the cultured mammalian cell lines Madin-Darby canine kidney cells showed no effect of the azasterols on the viability and proliferation of these cells at a concentration that significantly inhibited the proliferation of T. vaginalis, indicating a selective antiparasitic action. Taken together, these results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis.

Trichomonas vaginalis is an important human parasite of the urogenital tract. Jasmonates are a group of small lipids that are produced in plants and function as stress hormones. Naturally occurring methyl jasmonate (MJ) has been used to treat several types of cancer cells and it is cytotoxic to protistan parasites. It has been suggested that mitochondria are the target organelles of jasmonates. Here, we tested this drug against T. vaginalis. Although metronidazole has been the drug of choice for trichomoniasis, side effects from this treatment are common, and nausea and dizziness have been reported in up to 12% of patients. In addition, there has been increased recognition of resistance to metronidazole. We demonstrate here using flow cytometry, JC-1 and scanning and transmission electron microscopy that MJ induced the cell death of T. vaginalis parasites. Our results are discussed with previous findings published by others.

Tritrichomonas foetus is an extracellular parasite of the reproductive tract in cattle. To investigate the cytopathic effects of T. foetus in deeper parts of the reproductive tract, a bovine primary oviduct epithelial cell system (BOECs) was developed. Reproductive tracts were obtained from cows and the effect of co-incubating T. foetus with BOECs was analyzed by scanning electron, transmission electron and fluorescence microscopy. Viability tests were performed using colorimetric methods, TUNEL (Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling), fluorescein diacetate, propidium iodide, JC-1 and annexin-V. The results demonstrate that: (1) the in vitro oviduct epithelium is useful for interaction experiments with T. foetus; (2) T. foetus adheres to the BOECs as single separate cells, and later on the cells aggregate as large clusters; (3) the posterior region of the cell initiates the process of adhesion and forms filopodia and digitopodia; (4) T. foetus severely damages BOECs leaving imprints in the epithelial cells, wide intercellular spaces, and large lesions in the epithelium; and (5) T. foetus provokes bovine oviduct cell death by apoptosis and secondary necrosis. Our observations indicate the possibility that T. foetus can move through the reproductive tract to the oviduct and that infertility in cows can be mediated by an attack on the oviduct cells by T. foetus.

SUMMARYThis study reports that the cytopathic effect ofTrichomonas vaginalis, an important human parasite of the urogenital tract, occurs due to mechanical stress and subsequent phagocytosis of the necrotic cells. The investigation was done using a primary culture of bovine oviduct epithelial cells (BOECs), grown either in monolayers or as floating cells. Trophozoites displaying different virulence levels were co-incubated with BOECs for times varying between 1 min and 48 h. Analyses were performed using videomicroscopy, scanning and transmission electron microscopy, colourimetric assays and cytochemistry. Injury was observed as early as 1 h after incubation, while after 12 h the host cells were severely damaged when a fresh trichomonad isolate was used. Trichomonads attack the host cells by clustering around them. Mechanical stress on the microvilli of the host cells was observed and appeared to induce plasma membrane damage and cell death. After membrane injury and lysis, fragments of the necrotic cells were ingested by trichomonads. Phagocytosis occurred by trichomonads avidly eating large portions of epithelial cells containing the nucleus and other organelles, but living or intact cells were not ingested. Necrotic fragments were rapidly digested in lysosomes, as shown by acid phosphatase and ruthenium red assays where only the BOECs were labelled. The lytic capacity of the trichomonads was more pronounced in host cell suspensions.

A hydrogenosome is a hydrogen-producing organelle, evolutionary related to mitochondria and is found in Parabasalia protozoa, certain chytrid fungi and certain ciliates. It displays similarities to and differences from mitochondria. Hydrogenosomes are spherical or slightly elongated organelles, although very elongated hydrogenosomes are also found. They measure from 200 nm to 1 microm, but under stress conditions can reach up to 2 microm. Hydrogenosomes are surrounded by two closely apposed membranes and present a granular matrix. Cardiolipin has been detected in their membranes, and frataxin, which is a conserved mitochondrial protein involved in iron metabolism, was also recently found. Hydrogenosomes have one or multiple peripheral vesicles, which incorporate calcium. The peripheral vesicle can be isolated from the hydrogenosomal matrix and can be considered as a distinct hydrogenosomal compartment. Dysfunctional hydrogenosomes can be removed by an autophagic process and further digested by lysosomes. Hydrogenosomes divide in three different ways, like mitochondria, by segmentation, partition and the heart form. They may divide at any phase of the cell cycle. Nucleoid or electron dense deposits found in hydrogenosomes can be considered artifacts or dysfunctional hydrogenosomes. The hydrogenosome does not contain a genome, although DNA has already been detected in one anaerobic ciliate. Hydrogenosomes can be considered as good drug targets since their metabolism is distinct from mitochondria.

Giardia lamblia is a protozoan parasite with many characteristics common among eukaryotic cells, but lacking other features found in most eukaryotes. Cardiolipin is a phospholipid located exclusively in energy transducing membranes and it was identified in mitochondria, bacteria, hydrogenosomes and chloroplasts. In eukaryotes, cardiolipin is the only lipid that is synthesized in the mitochondria. Biochemical procedures (TLC, HPLC) and fluorescent tools (NAO) were applied in order to search for cardiolipin in G. lamblia. In addition, BLAST searches were used to find homologs of enzymes that participate in the cardiolipin synthesis. Cardiolipin synthase was searched in the Giardia genome, using Saccharomyces cerevisiae and Mycoplasma penetrans sequences as bait. However, a good match to G. lamblia related proteins was not found. Here we show that mitosomes of G. lamblia apparently do not contain cardiolipin, which raises the discussion for its endosymbiotic origin and for the previous proposal that Giardia mitosomes are modified mitochondria.

The hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. The hydrogenosome of Tritrichomonas foetus, a cattle parasite, is a spherical organelle that presents a peripheral vesicle the origin and behavior of which is poorly known. In this article it is reported an ultrastructural and microanalytical study using energy dispersive X-ray analysis, 3D reconstruction and cytochemistry of the hydrogenosome peripheral vesicle and then compare the results with the endoplasmic reticulum and the nuclear envelope of T. foetus. Similarities between the hydrogenosome peripheral vesicle and the ER are presented. This study included: (1) the detection of ER enzymes by cytochemistry, such as glucose-6-phosphatase, IDPase, acid phosphatase and Ca(2+) -ATPase; (2) elemental composition by X-ray microanalysis and the mapping of calcium, phosphorus and oxygen in both ER and hydrogenosome peripheral vesicle; (3) freeze-fracture; (4) TEM of routine and cryofixed cells by high-pressure freezing and freeze-substitution; (5) 3D reconstruction, (6) monoclonal antibody anti-trichomonads ER; and (6) other cytochemical techniques that detects ER, such as the ZIO and lectins. We found a similar composition of the tested enzymes and other elements present in the ER when compared with the hydrogenosome's peripheral vesicle. It was concluded that, like mitochondria, hydrogenosome presents relationships with the ER, especially the peripheral vesicle.

Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional glycophosphoprotein, secreted by different cell types, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated in this work by immunofluorescence and immunoelectron microscopy approaches the localization of an OPN-like protein in dense granules of extracellular T. gondii tachyzoites. Western blotting and RT-PCR confirmed this protein expression by the parasites. Our results also showed, after macrophage invasion, an intense positive labeling for OPN-like protein at the sub-apical portion of tachyzoites, the site of dense granules secretion, and the localization of this protein at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we speculate that this protein participates during the parasite interaction process with host cells and parasitophorous vacuole formation.

Tritrichomonas foetus and Trichomonas vaginalis are extracellular parasites of the urogenital tract of cattle and humans, respectively. They cause infertility and abortion, but there is no documented information on the susceptibility of bovine sperm cells to this cattle parasite. The aim of this present work was to study the effects provoked by T. foetus and T. vaginalis when in interaction with bovine and human sperm cells. The bovine and human spermatozoa were obtained from uninfected bulls and men, respectively, and were exposed to living trichomonads over different periods of time. Light microscopy, video microscopy, scanning, and transmission electron microscopy first revealed a tropism, then a close proximity followed by a tight adhesion between these two different cells. A decrease in the spermatozoa motility was observed as well intense semen agglutination. The adhesion between trichomonads to the sperm cell occurred either by the flagella or sperm head. Motile parasites were observed during the next 12 h, whereas sperm cells in contact with the parasites rapidly became immotile. The parasites were able to maintain the sperm cells attached to their cell surface, followed by phagocytosis. This process began with a tight membrane-membrane adhesion and the incorporation of the sperm cell within an intracellular vacuole. Afterwards, the sperm cell was gradually digested in lysosomes. Many trichomonads were injured and/or died on making contact with the spermatozoa possibly due to necrosis. Results from this study demonstrated that both T. foetus and T. vaginalis interact with sperm cells provoking damage and death of these reproductive cells. Differences in the behavior of both trichomonads were evident, showing that T. vaginalis was much more virulent than T. foetus. The possible role of trichomonads in reproductive failure is discussed.

We examined the participation of MAPK and PKA in the Golgi complex disassembly caused by light-activated Calphostin C in HT-29 cells. When these cells were incubated with Calphostin C, fragmentation and dispersal of the Golgi complex was observed as assessed by immunofluorescence microscopy. Electron microscopy analysis showed that clusters of vesicles and large tubule-vesicular membrane structures, resembling the Golgi remnants present in mitotic cells, substituted the Golgi stacks. In addition, Calphostin C treatment caused inhibition of the endocytic route. We confirmed that the Golgi disassembly was not due to PKC inhibition, and suggested, based on the use of specific inhibitors, that other kinases are involved. It was shown that pretreatment with PD98059 and H-89, both inhibitors of MAPK and PKA, respectively, prior to incubation with Calphostin C, caused blockade of the Golgi disassembly, as well as the inhibition of the endocytic pathway caused by this drug. This finding supports the existence of a novel mechanism by which MAPK and PKA may regulate the Golgi breakdown caused by Calphostin C in HT-29 cells.

The cholesterol synthesis inhibitor simvastatin, which is used to treat cardiovascular diseases, has severe collateral effects. We decided to comprehensively study the effects of simvastatin in zebrafish development and in myogenesis, because zebrafish has been used as a model to human diseases, due to its handling easiness, the optical clarity of its embryos, and the availability of physiological and structural methodologies. Furthermore, muscle is an important target of the drug. We used several simvastatin concentrations at different zebrafish developmental stages and studied survival rate, morphology, and physiology of the embryos. Our results show that high levels of simvastatin induce structural damage whereas low doses induce minor structural changes, impaired movements, and reduced heart beating. Morphological alterations include changes in embryo and somite size and septa shape. Physiological changes include movement reduction and slower heartbeat. These effects could be reversed by the addition of exogenous cholesterol. Moreover, we quantified the total cell number during zebrafish development and demonstrated a large reduction in cell number after statin treatment. Since we could classify the alterations induced by simvastatin in three distinct phenotypes, we speculate that simvastatin acts through more than one mechanism and could affect both cell replication and/or cell death and muscle function. Our data can contribute to the understanding of the molecular and cellular basis of the mechanisms of action of simvastatin.

Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca2+ concentration from 0.05 up to 0.70 μm. It is not known how the rise of Ca2+ concentration activates BAT thermogenesis. In this report we compared the effects of Ca2+ in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca2+-ATPase bound to the inner membrane of BAT mitochondria. A Ca2+-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca2+ enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca2+ concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 μm. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca2+-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca2+ did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca2+-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca2+ concentrations similar to those noted in the cell during adrenergic stimulation.

Giardia lamblia, a flagellated protist, is the parasite most commonly found in the intestinal tract of humans and other mammals causing a disease known as giardiasis. This parasite presents several cytoskeletal structures whose major components are microtubules, namely: the ventral adhesive disk, eight flagella axonemes, the median body, and funis. However, the cytoskeletal filamentous structures are poorly understood, and therefore, less studied. In the present work, we used actin-interacting drugs such as cytochalasin B and D to investigate their effects on Giardia ultrastructure. Axenically grown G. lamblia trophozoites were treated with these drugs and analyzed by fluorescence microscopy and scanning and transmission electron microscopy. It was observed that trophozoites became completely misshapen, detached from the glass surface, and failed to complete cell division. The main alterations observed included: (1) disk fragmentation, (2) presence of large vacuoles, (3) alterations in flagella number and flagella internalization, (4) blocked cytokinesis but not the karyokinesis, and (5) presence of membrane undulations and blebs. These findings are discussed.

Tritrichomonas foetus is an amitochondriate parasite that possesses hydrogenosomes, unusual anerobic energy-producing organelles. In these organisms the "mitochondrial cell death machinery" is supposed to be absent, and the mechanisms that lead to cell demise remain to be elucidated. The presence of a cell death program in trichomonads has already been reported, suggesting the existence of a caspase-like execution pathway in such organisms. Here we demonstrate the alterations provoked by the fungicide griseofulvin and raise the possibility that other cell death pathways may exist in T. foetus. Dramatic changes in trichomonads morphology are presented after griseofulvin treatment, such as intense plasma membrane and nuclear envelope blebbing, nucleus fragmentation, and an abnormal number of oversized vacuoles. One important finding was the exposition of phosphatidylserine (PS) in the outer leaflet of the plasma membrane in cells after drug treatment, and also the presence of a high amount of misshapen flagella and tubulin precipitates as vacuolar contents, suggesting an autophagic process of abnormal cellular elements. Interestingly, immunoreactivity for activated caspase-3 was not detected during griseofulvin treatment, a finding distinct from the observed when this cell was treated with H(2)O(2). The possibility of the existence of different pathways to cell death in trichomonads is discussed.

Tritrichomonas foetus, a parasitic protozoon of the urogenital tract in cattle, presents a poorly known cytoskeleton, formed by rootlets and proteinaceous structures, many of which have not yet been characterized. Studies on its skeletal organization sheds light on the evolution of the matrix system, characteristic of higher eukaryotes. The skeletal matrix system of T. foetus in interphasic and dividing cells were studied using whole mount cell procedures observed either in field emission scanning electron microscopy (FESEM) or in transmission electron microscope (TEM) after the cell-sandwich technique, where the plasma membrane was mechanically removed. Three-dimensional-like images of the cell matrix were attained revealing a network of filaments that has not been described previously. Freeze-etching and cytochemistry using acridine orange for TEM, were also used. Membrane-skeleton interactions were examined in the hydrogenosomes, on the nuclear envelope at mitosis and interphase, and in the overall matrix filling of the cytoplasm and nucleoplasm. It was demonstrated that this eukaryote has a complex skeletal matrix other than just the rigid cytoskeletal structures. Our analysis indicated that the nucleus has a defined position, and fibrils perform an anchoring system for the nucleus. The possibility of a mechanism for nuclei fidelity migration during mitosis is discussed.

Tritrichomonas foetus is a parasitic protist that infects the urogenital tract of cattle causing bovine trichomonosis. Virus-like particles (VLPs) in protozoa have been reported in several parasites including Trichomonas vaginalis , a human flagellate, but viruses were never described in T. foetus so far. Herein we show for the first time the presence of VLPs in T. foetus after several drug treatments. They were detected by electron microscopy and were confirmed by immunofluorescence microscopy using antibodies anti-virus proteins. These VLPs were always observed in clusters of variable size. Their preferential locations were at the cell periphery, close to the axostyle, and interestingly in some cases, inside the nucleus. Their appearance occurred when the parasites were under drug-treatments, such as cytoskeleton-affecting drugs (colchicine, vinblastine, taxol, nocodazole, and griseofulvin) or drugs inducing cell death, such as lactacystin and H 2 O 2 . We propose that cytoskeleton participates in trichomonads of the process of virus release or maturation. These virus particles were not described previously probably because they were either in low amount or in a latent state.

Trichomonas vaginalis secretes putrescine that is readily detected in vaginal secretions. We wanted to examine the effect of decreased putrescine synthesis by inhibition of ornithine decarboxylase (ODC) on T. vaginalis . One reason is because inhibition of Tritrichomonas foetus ODC results in growth arrest, destruction of hydrogenosomes, and decreased amounts of hydrogenosomal enzymes. Treatment of T. vaginalis T016 with ≥20 mM 1,4-diamino-2-butanone (DAB) to inhibit ODC resulted in growth arrest, which was reversed by addition of exogenous putrescine. No similar reversal of growth arrest was achieved with the polyamines spermine or spermidine or with iron. Electron microscopic examination of control versus DAB-treated trichomonads did not reveal any adverse effects on the number and integrity of hydrogenosomes. Further, the adhesins AP65, AP51, and AP33 mediating binding to immortalized vaginal epithelial cells (VECs) share identity to enzymes of the hydrogenosome organelle, and there was no difference in amounts of adhesins between control versus DAB-treated T. vaginalis parasites. Likewise, similar patterns and extent of fluorescence were evident for the prominent AP65 adhesin. Surprisingly, DAB treatment increased by 4- to 20-fold above untreated trichomonads handled identically the level of adherence mediated by adhesins. Interestingly, the enhanced attachment to VECs was reversed by exogenous putrescine added to DAB-treated trichomonads. Equally noteworthy was that DAB-treated T. vaginalis with enhanced adherence did not possess the previously reported ability to kill host cells in a contact-dependent fashion mediated by cysteine proteinases, and total cysteine proteinase activity patterns were identical between control and DAB-treated trichomonads. Overall, these data suggest that polyamine metabolism and secreted putrescine are linked to host cell adherence and cytotoxicity.

Giardia lamblia is the most commonly detected parasite in the intestinal tract of humans and other mammals causing giardiasis. Giardia presents several cytoskeletal structures with microtubules as major components such as the ventral adhesive disk, eight flagella axonemes, the median body and funis. Many drugs have already been tested as antigiardial agents, such as albendazole and mebendazole, which act by specifically inhibiting tubulin polymerization and hence microtubule assembly. In the present work, we used the microtubule inhibitors nocodazole and colchicine in order to investigate their direct and indirect effects on Giardia ultrastructure and attachment to the glass surface, respectively. Axenically grown G. lamblia trophozoites were treated with nocodazole or colchicine for different time intervals and analyzed by light and electron microscopy. It was observed that trophozoites became completely misshapen, detached from the glass surface and failed to complete cell division. The main alterations observed included disc fragmentation, presence of large vacuoles, and appearance of electrondense deposits made of tubulin. The cytokinesis was blocked, but not the karyokinesis, and membrane blebs were observed. These findings show that Giardia behavior and cytoskeleton are clearly affected by the commonly used microtubule targetting agents colchicine and nozodazole.

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

Giardia Lamblia is a flagellar parasite possessing the unusual morphology of bearing two nuclei. New morphological observations on trophozoites and encysting Giardia nuclei using routine transmission electron microscopy, freeze fracture and cytochemistry are presented. Nuclear pores of both nuclei in the same cells were assessed on freeze-fracture replicas from different cell cycle phases, and compared. These techniques showed that (1) both nuclei in the same cell are distinct in nuclear pore number and distribution; (2) nuclear pore complexes are frequently clustered in nuclear envelope domains; (3) dividing nuclei display very few nuclear pores; (4) few ribosomes are found on the outer nuclear envelope of the trophozoite form; (5) nuclear membranes present spots of closely apposed membranes, which are different from the typical diaphragm nuclear pore complexes; (6) in addition to the nuclear pores, membrane blebs are also present in the nuclear envelope; (7) encysting cells show intranuclear inclusions, morphologically similar to the ESV (encystation-specific vesicles) and to the ER membranes, which may be the result of nuclear envelope folding. It is proposed that the two nuclei in Giardia are dissimilar in morphology and activity.

The Trichomonads have been the subject of several molecular studies that reported some discrepancies both at the lower and higher taxonomic levels. The purpose of this study was to make an extensive phylogenetic analysis of the Trichomonadidae using ITS-1/5.8S/ITS-2 sequences, to better understand its phylogeny and the usefulness of this marker. ITS-1/5.8S/ITS-2 sequences of 36 strains from 14 species belonging to Trichomonadidae and Monocercomonadidae were analysed, in which 20 were newly determined. Maximum likelihood, maximum parsimony, neighbour joining, and Bayesian phylogenetic methods were employed in order to reconstruct and compare the evolutionary history of this group. Tetratrichomonas gallinarum and four strains of Tetratrichomonas sp. isolated from bull genital organs were found closely related, confirming the classification of the latter, probably as a new species. The monophyly of Tritrichomonadinae and Trichomonadinae subfamilies were corroborated, with the exclusion of Trichomitus batrachorum from the latter since it grouped consistently with Hypotrichomonas acosta. Tritrichomonas foetus, Tritrichomonas suis and potentially also Tritrichomonas mobilensis seemed to correspond to the same species. Monocercomonas sp. and Ditrichomonas honigbergii emerged as independent lineages, with their phylogenetic positions undetermined. Neither Trichomonadidae nor Monocercomonadidae were supported as monophyletic groups. The ITS-1/5.8S/ITS-2 seems to be a reliable locus for phylogenetic studies in the Trichomonadida, mainly at lower taxonomic levels, and at least up to the family level.

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. It secretes an extracellular cyst wall using a poorly understood process. An encystation-specific secretory vesicle (ESV) was previously described containing cyst wall proteins. The process of release of these vesicles has been suggested to occur after fragmentation of large ESV in small secretory vesicles, followed by exocytosis, but it was not demonstrated. The release of the ESV was studied by transmission electron microscopy. It was observed: (1) the moment of vesicle release; (2) that a large vesicle is exocytosed and does not fragment into small vesicles; (3) membrane fusion is distinct from traditional exocytosis since it is incomplete; (4) the occurrence of membrane fragmentation and that those membranes reseal to form ghosts; (5) these membrane ghosts may be endocytosed, adhered to flagellar surface or/and form empty vesicles in the extracellular medium.

Tritrichomonas foetus is a parasitic protist of the urogenital tract of cattle. It presents the trophozoite stage, a motile elongated form that constitutes most of the cells in a normal population, and a pseudocyst stage, an immotile rounded form that appears under unfavourable environmental conditions. In the present report pseudocysts were studied in natural conditions and after induction by chemicals or cycles of cooling and warming of cultures. The capacity of T. foetus to adhere to vaginal epithelial cells (VECs) was compared for both trophozoite and pseudocyst forms. By the use of video-enhanced-contrast microscopy, scanning and transmission electron microscopy, and immunofluorescence microscopy techniques, we present evidence that: (1) T. foetus easily internalizes the flagella and forms pseudocysts under several unfavourable conditions; (2) T. foetus in both pseudocyst and trophozoite forms is able to adhere to VECs; (3) the adhesion rate is higher for pseudocysts than for trophozoites; (4) the adhesin Tf190 is expressed in both forms during interaction; (5) the adhesion process of pseudocysts seems to occur in a contact-dependent manner. Thus, we propose that the pseudocyst stage is not a degenerative form, but a functional life form that is able to interact with and firmly adhere to VECs.

Mitosis in Giardia is poorly understood. Until today, it is still controversial whether Giardia divides with a mirror-image symmetry (ventral-ventral or dorsal-dorsal) or in a dorsal-ventral mode. Here, we report the different modes by which cytokinesis takes place in Giardia lamblia. To determine how Giardia divides, video microscopy, scanning electron microscopy, semi-thick sections and freeze-fracture replicas were analyzed by transmission electron microscopy. Between 12 and 15% of the cells cultivated for 24-48 h were found in the process of division. Three types of cytokinesis were found: (1) ventral-ventral, where the discs face each other; (2) dorsal-dorsal, where the discs are in opposite directions; and (3) ventral-dorsal. Giardia divides with mirror-image symmetry either in ventral-ventral or dorsal-dorsal modes. During ventral-ventral type of division, Giardia becomes detached and swims freely in the culture medium, whereas, in the other modes of division, the cells can be found either adhered or swimming.

The effects of the microtubule affecting drugs taxol, nocodazole and colchicine on the cell cycle and ultrastructure of Tritrichomonas foetus, a protist parasite of cattle, were studied. Alterations in the cytoskeleton, motility and organellar ultrastructure were followed using anti-tubulin antibodies and fluorescence microscopy, scanning- and transmission-electron microscopy. Flow cytometry was also used to analyze the effect of the drugs on the cell cycle. T. foetus was treated with 10 microM taxol, 15 microM nocodazole or 1.5 mM colchicine for 12 h. The first effect observed was pseudocyst formation and alterations in cell motility. The cell cycle was affected and the cells have blocked cytokinesis, but not karyokinesis. The behavior of Golgi, hydrogenosomes and vacuoles was analyzed. The following effects were seen following drug treatments: (1) cell motility was altered and flagella internalized; (2) microtubules of the pelta-axostyle complex were not depolymerized and the axostyle assumed a curved form; (3) hydrogenosomes were of abnormal size and shape; (4) cells became multinucleate; (5) the division process was blocked in cytokinesis; (6) autophagic vacuoles containing a large amount of microtubules were seen; (7) axoneme organization was altered; (8) zoids were formed; (9) signs of cell death, such as membrane blebbing, were observed.

We present observations on an unusual tetratrichomonad species isolated from preputial smegma of virgin bulls. Ultrastructural studies were performed using scanning and electron microscopy techniques. This protozoan presents four anterior flagella of unequal length and a recurrent one forming the undulating membrane. It shows one anterior nucleus, a Golgi complex, an axostyle, and a costa. The hydrogenosomes are rather elongated, seen in groups, and presenting different electron densities. Vacuoles of different sizes containing bacteria and material in process of digestion were frequently found. PCR was also used in order to compare the species herein described with other trichomonad species. The amplification products were seen only with primers TFR1 and TFR2 (specific to trichomonads), but not with TFR3 and TFR4 (specific to Tritrichomonas foetus), suggesting that although collected from the genital tract of the bull, this protist was not T. foetus. We propose that the appearance of these tetratrichomonads were probably due to the sodomy practiced among bulls. Concomitant contamination of preputial cavity with feces could explain the presence of the opportunistic organism. The observations presented here show the importance of the correct diagnostic when investigating samples obtained from the urogenital tract of cattle. We also suggest that this flagellate belongs to the species Tetratrichomonas buttreyi.

We examined cell death in developing retinal tissue, following inhibition of protein synthesis, which kills undifferentiated post-mitotic cells. Ultrastructural features were found of both apoptosis and autophagy. Only approximately half of the degenerating cells were either terminal dUTP nick-end labeling (TUNEL)-positive or reacted with antibodies specific for activated caspases-3 or -9. Bongkrekic acid completely inhibited any appearance of cell death, whereas inhibitors of autophagy, caspases-9 or -3, prevented only TUNEL-positive cell death. Interestingly, inhibition of caspase-6 blocked TUNEL-negative cell death. Simultaneous inhibition of caspases-9 and -6 prevented cell death almost completely, but degeneration dependent on autophagy/caspase-9 still occurred under inhibition of both caspases-3 and -6. Thus, inhibition of protein synthesis induces in the developing retina various post-translational, mitochondria-dependent pathways of cell death. Autophagy precedes sequential activation of caspases-9 and -3, and DNA fragmentation, whereas, in parallel, caspase-6 leads to a TUNEL-negative form of cell death. Additional mechanisms of cell death may be engaged upon selective caspase inhibition.

Tritrichomonas foetus is an amitochondrial parasite protist which lacks typical eukaryote organelles such as mitochondria and peroxisomes, but possesses the hydrogenosome, a double-membrane-bound organelle that produces ATP. The cell death of amitochondrial organisms is poorly studied. In the present work, the cytotoxic effects of hydrogen peroxide on T. foetus and its participation on cell death were analyzed. We took advantage of several microscopy techniques, including videomicroscopy, light microscopy immunocytochemistry for detection of caspase activation, and scanning and transmission electron microscopy. We report here that in T. foetus: (1) H(2)O(2) leads to loss of motility and induces cell death, (2) the dying cells exhibit some characteristics similar to those found during the death of other organisms, and (3) a caspase-like protein seems to be activated during the death process. Thus, we propose that, although T. foetus does not present mitochondria nor any known pathways of cell death, it is likely that it bears mechanisms of cell demise. T. foetus exhibits morphological and physiological alterations in response to H(2)O(2) treatment. The hydrogenosome, a unique organelle which is supposed to share a common ancestral origin with mitochondria and has an important role in oxidative responses in trichomonads, is a candidate for participating in this event.

The proteins AP65, AP51, AP33 and AP23 synthesized by Trichomonas vaginalis organisms in high iron play a role in adherence. Multigene families encode enzymes of the hydrogenosome organelles, which have identity to adhesins. This fact raises questions regarding the compartmentalization of the proteins outside the organelle and about the interactions of adhesins with host cells. Data here demonstrate the presence of the proteins outside the organelle under high-iron conditions. Fluorescence and immunocytochemical experiments show that high-iron-grown organisms coexpressed adhesins on the surface and intracellularly in contrast with low-iron parasites. Furthermore, the AP65 epitopes seen by rabbit anti-AP65 serum that blocks adherence and detects surface proteins were identified, and a mAb reacting to those epitopes recognized the trichomonal surface. Two-dimensional electrophoresis and immunoblot of adhesins from surface-labelled parasites provided evidence that all members of the multigene family were co-ordinately expressed and placed on the trichomonal surface. Similar two-dimensional analysis of proteins from purified hydrogenosomes obtained from iodinated trichomonads confirmed the specific surface labelling of proteins. Contact of trichomonads with vaginal epithelial cells increased the amount of surface-expressed adhesins. Moreover, we found a direct relationship between the levels of adherence and amount of adhesins bound to immortalized vaginal and ureter epithelial cells, further reinforcing specific associations. Finally, trichomonads of MR100, a drug-resistant isolate absent in hydrogenosome proteins and adhesins, were non-adherent. Overall, the results confirm an important role for iron and contact in the surface expression of adhesins of T. vaginalis organisms.

Some isolates of Trichomonas vaginalis, the number one, non-viral sexually transmitted disease agent, are infected with one or several distinct double stranded (ds)-RNA virus. Immune rabbit anti-capsid serum (IRS) reacted with the capsid protein of purified dsRNA virus of a subset of the virus-infected T. vaginalis isolates. A monoclonal antibody (mAb) that recognized the capsid protein reactive with the IRS was generated. Analysis of the virus capsid protein of virus-infected isolates by probing nitrocellulose blots with mAb revealed diversity among immunoreactivity and in the size of the reactive capsid protein. Despite difficulties in visualizing virus within parasites by cross-section electron microscopy, gold-conjugated mAb readily labeled the cytoplasm of virus-positive trichomonads. Finally and importantly, isolates infecting patients attending an STD clinic, 75% of which were virus-positive isolates, had capsid protein of the same size detected by mAb present in all dsRNA viruses.

In the present study it was demonstrated the existence of a new set of membrane-bounded vesicles in Giardia lamblia. They were found in dividing and non-dividing trophozoites studied by routine transmission electron microscopy, freeze-fracture and Thiéry's technique. Encysting cells were not studied. These vesicles appear different to the previously reported components of the Giardia endomembranous system, such as the endoplasmic reticulum (ER), lysosome-like peripheral vesicles (PV), and the encystation-specific vesicles (ESV) that appear during trophozoite differentiation into cysts. They measure 100-150 nm in diameter, and thus are smaller than the peripheral vesicles, and the encystation-specific vesicles (ESV). They were found in clusters, scattered throughout the cytoplasm, but preferentially located close to the nuclei, axonemes, median bodies, and ER profiles. These internal vesicles are roughly spherical, and their contents present different electron densities and are more electrondense than those of the peripheral vesicles. They appeared to be budding from the outer nuclear membrane envelope. These cytoplasmic vesicles were found only in cells with very good fixation. Only few cells in the same preparation exhibited these vesicles.

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.

Treatment of cultures of Tritrichomonas foetus with 4 mM hydroxyurea (HU), a known DNA synthesis inhibitor, induced pseudocyst formation and caused a mitotic burst. An hour after drug release there was a characteristic, synchronous burst of cell division. T. foetus culture was arrested in the G2/M phase. The synchrony index varied from 66% to 69%. The synchrony was maintained for several cell cycles, even in thawed cultures which had been frozen for storage in liquid nitrogen. The synchronized cells were analyzed by light and scanning electron microscopy, as well by flow cytometry.