Biomedical subjects

Marlene Benchimol

Publications and source records attributed to Marlene Benchimol.

At least 37 records · Page 2Linked to original sources

PubMed2004

The median body of Giardia lamblia: an ultrastructural study.

Giardia lamblia is an intestinal parasite of several mammals. The most striking feature of Giardia is the presence of a complex and unique cytoskeleton, and among its components the median body (MB) is the least defined microtubular structure. In the present study, we used a technique that allowed the removal of the plasma membrane and observation of cytoskeletal structures by both routine scanning electron microscopy (SEM) and field emission high resolution SEM. This technique permitted new observations such as details and insights of the median bodies, not previously described or controversial in the literature. Light microscopy after Panotic staining, immunofluorescence microscopy using several antibodies, and thin sections were also used to better characterized the Giardia MB. The new observations concerning the median bodies were : (1) they are not one or two structures, but varied in number, shape and position ; (2) they were found in mitotic and interphasic trophozoites, in disagreement with previous works ; (3) they were present in about 80 % of the cells, and not in 50 % of the cells, as previously described ; (4) they could be connected either to the plasma membrane, to the adhesive disc, and caudal flagella, and thus they are not completely free in the cells, as published before ; (5) they can protrude the cell surface ; (6) their microtubules react with several anti-tubulin and -beta giardin antibodies. These observations add new data on the scarce literature and to this largely understudied cell structure.

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PubMed2004

Molecular phylogeny of Trichomonadidae family inferred from ITS-1, 5.8S rRNA and ITS-2 sequences.

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.

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PubMed2004

Visualization of the funis of Giardia lamblia by high-resolution field emission scanning electron microscopy--new insights.

Giardia lamblia is a multiflagellar parasite and one of the earliest diverging eukaryotic cells. It possesses a cytoskeleton made of several microtubular structures-an adhesive disc, four pairs of flagella, median body, and funis. This protozoan displays different types of movements, including a lateral and dorso-ventral dislocation of its posterior region, which has not been completely elucidated. In the present study, high-resolution field emission scanning electron microscopy was used to analyze the funis structure of G. lamblia trophozoites. It was shown that the funis is made of short arrays of microtubules emanating from the axonemes of the caudal flagella, which are anchored to dense rods that run parallel to the posterior-lateral flagella. After emergence of the posterior-lateral flagella, funis microtubules are anchored to the epiplasm, a fibrous layer that underlies the portion of membrane that presents tail contractility. Based on these observations a model for the tail flexion of G. lamblia is proposed.

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PubMed2004

Trichomonads under Microscopy.

Trichomonads are flagellate protists, and among them Trichomonas vaginalis and Tritrichomonas foetus are the most studied because they are parasites of the urogenital tract of humans and cattle, respectively. Microscopy provides new insights into the cell biology and morphology of these parasites, and thus allows better understanding of the main aspects of their physiology. Here, we review the ultrastructure of T. foetus and T. vaginalis, stressing the participation of the axostyle in the process of cell division and showing that the pseudocyst may be a new form in the trichomonad cell cycle and not simply a degenerative form. Other organelles, such as the Golgi and hydrogenosomes, are also reviewed. The virus present in trichomonads is discussed.

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PubMed2004

Mitosis in Giardia lamblia: multiple modes of cytokinesis.

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.

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PubMed2003

The effect of drugs on cell structure of Tritrichomonas foetus.

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.

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PubMed2003

Ultrastructural study of a tetratrichomonad species isolated from prepucial smegma of virgin bulls.

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.

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PubMed2003

Ca(2+) sequestering in the early-branching amitochondriate protozoan Tritrichomonas foetus: an important role of the Golgi complex and its Ca(2+)-ATPase.

Total membrane vesicles isolated from Tritrichomonas foetus showed an ATP-dependent Ca(2+) uptake, which was not sensitive to 10 microM protonophore FCCP but was blocked by orthovanadate, the inhibitor of P-type ATPases (I(50)=130 microM), and by the Ca(2+)/H(+) exchanger, A-23187. The Ca(2+) uptake was prevented also by thapsigargin, an inhibitor of the SERCA Ca(2+)-ATPases. The sensitivity of the Ca(2+) uptake by the protozoan membrane vesicles to thapsigargin was similar to that of Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. Fractionation of the total membrane vesicles in sucrose density gradient revealed a considerable peak of Ca(2+) transport activity that co-migrated with the Golgi marker guanosine diphosphatase (GDPase). Electron microscopy confirmed that membrane fractions of the peak were enriched with the Golgi membranes. The Golgi Ca(2+)-ATPase contributed to the Ca(2+) uptake by all membrane vesicles 80-85%. We conclude that: (i) the Golgi and/or Golgi-like vesicles form the main Ca(2+) store compartment in T. foetus; (ii) Ca(2+) ATPase is responsible for the Ca(2+) sequestering in this protozoan, while Ca(2+)/H(+) antiporter is not involved in the process; (iii) the Golgi pump of this ancient eukaryotic microorganism appears to be similar to the enzymes of the SERCA family by its sensitivity to thapsigargin.

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PubMed2003

Alternative programs of cell death in developing retinal tissue.

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.

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PubMed2003

Hydrogen peroxide induces caspase activation and programmed cell death in the amitochondrial Tritrichomonas foetus.

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.

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PubMed2003

Fungal hydrogenosomes contain mitochondrial heat-shock proteins.

At least three groups of anaerobic eukaryotes lack mitochondria and instead contain hydrogenosomes, peculiar organelles that make energy and excrete hydrogen. Published data indicate that ciliate and trichomonad hydrogenosomes share common ancestry with mitochondria, but the evolutionary origins of fungal hydrogenosomes have been controversial. We have now isolated full-length genes for heat shock proteins 60 and 70 from the anaerobic fungus Neocallimastix patriciarum, which phylogenetic analyses reveal share common ancestry with mitochondrial orthologues. In aerobic organisms these proteins function in mitochondrial import and protein folding. Homologous antibodies demonstrated the localization of both proteins to fungal hydrogenosomes. Moreover, both sequences contain amino-terminal extensions that in heterologous targeting experiments were shown to be necessary and sufficient to locate both proteins and green fluorescent protein to the mitochondria of mammalian cells. This finding, that fungal hydrogenosomes use mitochondrial targeting signals to import two proteins of mitochondrial ancestry that play key roles in aerobic mitochondria, provides further strong evidence that the fungal organelle is also of mitochondrial ancestry. The extraordinary capacity of eukaryotes to repeatedly evolve hydrogen-producing organelles apparently reflects a general ability to modify the biochemistry of the mitochondrial compartment.

Amino Acid Sequence↗

PubMed2003

Hydrogenosome behavior during the cell cycle in Tritrichomonas foetus.

The hydrogenosome is an unusual organelle found in several trichomonad species and other protists living in oxygen poor or anoxic environments. The hydrogenosome behavior in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle, is reported here. The hydrogenosomes were followed by light and transmission electron microscopy during the whole cell cycle. Videomicroscopy, immunofluorescence microscopy, and immunocytochemistry were also used. It is shown that the hydrogenosomes divide at any phase of the cell cycle and that the organellar division is not synchronized. During the interphase the hydrogenosomes are distributed mainly along the axostyle and costa, and at the beginning of mitosis migrate to around the nucleus. Three forms of hydrogenosome division were seen: (1). segmentation, where elongated hydrogenosomes are further separated by external membranous profiles; (2). partition, where rounded hydrogenosomes, in a bulky form, are further separated by a membranous internal septum and, (3). a new dividing form: heart-shaped hydrogenosomes, which gradually present a membrane invagination leading to the organelle division. The hydrogenosomes divide at any phase of the cell cycle. A necklace of intramembranous particles delimiting the outer hydrogenosomal membrane in the region of organelle division was observed by freeze-etching. Similarities between hydrogenosomes and mitochondria behavior during the cell cycle are discussed.

Animals↗

PubMed2003

Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis.

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 immuno-cytochemical 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.

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PubMed2003

Pseudocysts in trichomonads--new insights.

Tritrichomonas foetus and Trichomonas vaginalis, parasitic protists of the urogenital tract, display a trophozoite and a pseudocyst stage. The ultrastructure of the trophozoite was compared with the pseudocyst form. The latter appears under unfavorable environmental conditions when the flagella are internalized, and a true cell wall is not formed. Although some authors consider this form as a degenerate stage, the cell behaves as a resistant form. Pseudocysts were found in natural culture conditions and also under induction by hydroxyurea or cycles of cooling and warming cultures. They were studied by light and scanning and transmission electron microscopy, using immunofluorescence and videomicroscopy. This report presents evidence that the trichomonad pseudocysts appear under stress conditions and that they are competent to divide. Pseudocysts differ from trophozoites in that: (1) the flagella are located in endocytic vacuoles and remain beating; (2) the axostyle and the costa are not depolymerized but present a curved shape; (3) the axostyle does not exhibit staining with antitubulin antibodies when the mitotic spindle is observed; (4) the mitotic process occurs within pseudocysts but differs from that described for trophozoites; (5) a nuclear canal is formed connecting the two spindle poles; and (6) the process is reversible if the cells are transferred to fresh medium.

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PubMed2002

Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate.

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.

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PubMed2002

Tritrichomonas foetus: induced division synchrony by hydroxyurea.

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.

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PubMed2002

A proton pumping pyrophosphatase in the Golgi apparatus and plasma membrane vesicles of Trypanosoma cruzi.

The proton pumping pyrophosphatase (H(+)-PPase) is an enzyme that has been identified in membranes of plant vacuoles, in the Golgi complex of plants and Chlamydomonas reinhardtii, and more recently in acidocalcisomes of different trypanosomatids and apicomplexan parasites. Immunofluorescence and immunoelectron microscopy studies using antibodies against the plant enzyme also suggested a plasma membrane localization in different stages of Trypanosoma cruzi. In this report we provide immunogold electron microscopy evidence of the presence of the H(+)-PPase in the Golgi complex and plasma membrane of epimastigotes of T. cruzi. Pyrophosphate promoted acidification of plasma membrane vesicles as determined using acridine orange. This activity was stimulated by K(+) ions, inhibited by the pyrophosphate analogs imidodiphosphate (IDP) and aminomethylenediphosphonate (AMDP) by KF, NaF and DCCD, and it had different responses to ions and inhibitors as compared with the activity present in acidocalcisomes. Surface localization of the H(+)-PPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against H(+)-PPase. Taken together, these results are consistent with the presence of a functional H(+)-PPase in the plasma membrane of these parasites.

Animals↗

PubMed2002

Ultrastructural localization of glycoconjugates in Tritrichomonas foetus.

We have probed Tritrichomonas foetus, a protozoan parasite of the urogenital tract of cattle, with gold-labeled and fluorescent lectins, for the localization of glycoconjugates, at the cell surface and in internal cell compartments. The following lectins were used: Con A, PNA, WGA, BSI, BSII, HPA, WFA, SBA, LFA, HPA, and LCA. Carbohydrates were also localized using the Thiéry's technique. Carbohydrate residues were observed in the Golgi and vesicles with all lectins used. However, the labeling pattern varied among the cisternae. A high heterogeneity of cell labeling was detected in the same preparation. The nucleus and the nuclear envelope were labeled with Con A, WGA, BSI, and UEA I, suggesting the presence of D-Man, and/or D-glucose, L-fucose, and GlcNAc. Lysosomes were labeled with Con A and intensely labeled with HPA. The intensity of labeling of the plasma and flagellar membranes varied according to the lectin used.

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