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ABSTRACT: 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.
Memórias do Instituto Oswaldo Cruz 02/2013; 108(1):110-2. · 2.15 Impact Factor
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ABSTRACT: Giardia lamblia is a protozoan parasite that presents both trophozoite and cyst forms. In this study, the distribution of the different sugar residues and the origin of the carbohydrate components of the cyst wall were studied using transmission electron microscopy, ultrastructural cytochemistry for carbohydrate detection and immunocytochemistry. Immunofluorescence microscopy using anti-cyst wall protein 1 (CWP1) and gold- and fluorescent-conjugated lectins, such as WGA and DBA, were also used. Interestingly, a population of carbohydrate-containing vesicles, distinct from the encystation-specific vesicles (ESVs) was found in the encysting cells and was named encystation carbohydrate-positive vesicles (ECVs). The differences between the ECVs and the ESVs were: (1) they are electron-translucent, whereas ESVs are electron dense; (2) they do not react with antibodies against cyst wall proteins; (3) the contents are positive for carbohydrates, whereas ESVs display a negative reaction; and (4) they exhibit a positive labeling for DBA indicating the presence of N-acetyl-galactosamine, whereas ESVs are negative. To evaluate if ECVs could be vesicles involved in the endocytic pathway, endocytic markers were used. No co-localization of these markers with ECVs was observed. We suggest that the ECVs may represent a new structure involved in cyst wall formation.
Protist 12/2012; · 3.14 Impact Factor
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ABSTRACT: Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such infections could cause serious risks to the infected host.
Memórias do Instituto Oswaldo Cruz 09/2012; 107(6):720-7. · 2.15 Impact Factor
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ABSTRACT: 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 2.5h of co-incubation with MDCK cells. Our results suggest that the T. foetus pseudocyst might be a more aggressive form.
Protist 02/2012; 163(4):529-43. · 3.14 Impact Factor
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ABSTRACT: Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process.
Memórias do Instituto Oswaldo Cruz 09/2011; 106(6):701-4. · 2.15 Impact Factor
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ABSTRACT: Tritrichomonas mobilensis is an intestinal parasite of squirrel monkeys. There are few reports concerning the morphological aspects of this parasite. In addition, the taxonomic relationship between T. mobilensis and Tritrichomonas foetus, a serious pathogen that causes bovine and feline trichomonosis, has been questioned. For this reason, in the present study, we examined and compared both tritrichomonads with regard to their morphology, ultrastructure, endocytic activity and cytotoxicity when in the presence of host cells. Electron microscopy demonstrated consistent morphological differences between the hydrogenosomes of both parasites. Moreover, T. mobilensis and T. foetus had striking differences in their endocytic behavior. Thus, this work provides additional data that support the hypothesis that T. mobilensis is a distinct species from T. foetus.
Veterinary Parasitology 05/2011; 182(2-4):171-80. · 2.58 Impact Factor
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ABSTRACT: 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.
FEMS Microbiology Letters 02/2011; 315(1):72-8. · 2.04 Impact Factor
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ABSTRACT: Tritrichomonas foetus is a serious veterinary pathogen that causes bovine trichomonosis, a sexually transmitted disease that eventually leads to abortion and infertility. T. foetus has a simple life cycle that consists of only a trophozoitic form. During unfavorable environmental conditions, the trophozoites, which are polar and flagellated, can adopt a spherical shape and internalize their flagella. These rounded organisms are known as pseudocysts. Although it is currently assumed that T. foetus pseudocyst formation is reversible and that it represents a response to stressful conditions, there are no reports showing the presence of this form in vivo. For this reason, the aim of this study was to verify whether T. foetus pseudocysts are encountered in naturally infected bulls. Towards this goal, fresh preputial samples obtained from seven mature bulls that were naturally infected with T. foetus were analyzed using complementary techniques, such as video microscopy, fluorescence microscopy, scanning and transmission electron microscopy. The analyses revealed that approximately 55% of the parasites were in pseudocyst form in each preputial sample, whereas approximately 25% of T. foetus displayed pear-shaped bodies. Previous research demonstrated that in vitro T. foetus pseudocysts are able to divide by a budding process. Here, this division mode was observed in approximately 20% of fresh T. foetus obtained from preputial bovine samples. Thus, this study shows that in infected bulls, pseudocysts are present and occur more frequently than the pear-shaped parasites.
Veterinary Parasitology 10/2010; 175(1-2):1-8. · 2.58 Impact Factor
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ABSTRACT: Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca(2+)-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca(2+) effect in BAT mitochondria thermogenesis. We found that Ca(2+) increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca(2+) concentration needed for half-maximal activation varied between 0.08 and 0.11 microM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN. Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca(2+) strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca(2+) activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver.
PLoS ONE 01/2010; 5(3):e9439. · 4.09 Impact Factor
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ABSTRACT: Giardia lamblia is a protozoan that parasitizes the small intestine of vertebrates. It is a cause of intestinal infection and diarrhea and infects millions of people worldwide. This protozoan presents many characteristics common to eukaryotic cells but it lacks organelles found in most eukaryotes (e.g., peroxisomes, typical Golgi complex and mitochondria). Also it presents mitosomes, a relic organelle that appears to be a mitochondrial remnant. Cell death in Giardia was induced by the drug beta-Lapachone and by starvation. Giardia behavior was followed by scanning, transmission and fluorescence microscopy, quantification of cell metabolism using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide), changes in lipid rafts, using DiIC(16) and cholera toxin. Cell shrinkage, chromatin condensation, membrane blebbing and vacuolization provided ultrastructural evidence of apoptosis, whereas the myelinic figures in large vacuoles and LC-3 staining suggested an autophagic process. Lipids rafts were altered by drug treatment and co-localized with regions containing membrane blebbing. The treatment with beta-Lap induced encystation. A search for sequence similarities in databases and protein alignments was carried out. Although Giardia is an amitochondrial organism, it presented some autophagic-like cell death characteristics and several, but not all, apoptotic characteristics, induced by beta-Lapachone and starvation.
Parasitology International 09/2009; 58(4):424-37. · 2.13 Impact Factor
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ABSTRACT: Tritrichomonas foetus is a flagellated protozoan parasite that causes trichomoniasis, a major sexually transmitted disease in cattle. T. foetus presents a simple life cycle, exhibiting only the trophozoitic form. However, under unfavorable growth conditions, the trophozoites, which are polar and flagellated, can round up and internalize their flagella forming pseudocysts. In this form no cyst wall surrounds the cell and it also displays a distinct mitosis when compared with the trophozoite form. In pseudocyst mitosis, the cell proceeds with duplication of cytoskeletal and mastigont structures; nuclear division occurs but without the corresponding cytoplasm division. Thus, giant multinucleated cells which present many mastigont structures are formed (approximately 62% of the population). These polymastigont/multinucleated cells are maintained when the cells are under stress conditions. When environmental conditions become favorable, the flagella are externalized and new flagellated trophozoites one by one, gradually bud from the multinucleated cell. Thus, in order to better understand the pseudocyst mitosis, the polymastigont formation and the generation of new cells by this budding process, video microscopy and other complementary techniques, such as immunofluorescence and transmission electron microscopy were used.
Protist 08/2009; 160(4):536-51. · 3.14 Impact Factor
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ABSTRACT: Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. Different stages in the Giardia cyst formation include distinctive changes in the trophozoite shape and polarization, from the characteristic flattened dorsal-ventral axis found in motile trophozoites to a rounded appearance and also the appearance of a "tail-like" appendage in later stages of cyst formation. In addition, the flagella disappear and the cyst is oval or rounded and immotile. Since we found no clear information describing how the cells change shape and how the flagella disappear, we applied videomicroscopy, scanning and transmission electron microscopy to follow the gradual modifications that occur in the trophozoite, including alterations in the cell shape, the manner of flagella internalization and changes in disc behavior. Based on the data presented here, it was possible to construct a temporal sequence of changes during Giardia encystation. In this article we show how the membrane growth of the flange contributes to cell shape changes during encystment. In addition, an operculum and flagella internalization is shown. There is a video as a supplement showing these modifications. In other procedure, the plasma membrane was removed and the disc was seen by high resolution scanning electron microscopy where the modifications of the disc spiral can be followed.
Parasitology International 01/2009; 58(1):72-80. · 2.13 Impact Factor
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ABSTRACT: 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.
Experimental Parasitology 08/2008; 120(3):215-20. · 2.12 Impact Factor
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ABSTRACT: 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.
Parasitology Research 04/2008; 102(4):597-604. · 2.15 Impact Factor
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ABSTRACT: In this work it is shown that Trichomonas vaginalis remains viable and infective in swimming pool water samples for several hours. After survival, trichomonad cytotoxicity was tested on primary cultures of epithelial cells. It demonstrates that the some trichomonad strains are able to survive in water pools and survival time is dependent on the degree of strain infectivity and also if it is a long term cultured or fresh isolate.
Experimental Parasitology 04/2008; 118(3):438-41. · 2.12 Impact Factor
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Marlene Benchimol
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ABSTRACT: 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.
Tissue and Cell 03/2008; 40(1):61-74. · 1.04 Impact Factor
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Marlene Benchimol
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ABSTRACT: Hydrogenosomes are spherical or slightly elongated organelles found in non-mitochondrial organisms. In Trichomonas hydrogenosomes measure between 200 to 500 nm, but under drug treatment they can reach 2 microm. Like mitochondria hydrogenosomes: (1) are surrounded by two closely apposed membranes and present a granular matrix: (2) divide in three different ways: segmentation, partition and the heart form; (3) they may divide at any phase of the cell cycle; (4) produce ATP; (5) participate in the metabolism of pyruvate formed during glycolysis; (6) are the site of molecular hydrogen formation; (7) present a relationship with the endoplasmic reticulum; (8) incorporate calcium; (9) import proteins post-translationally; (10) present cardiolipin. However, there are differences, such as: (1) absence of genetic material, at least in trichomonas; (2) lack a respiratory chain and cytochromes; (3) absence of the F(0)-F(1) ATPase; (4) absence of the tricarboxylic acid cycle; (5) lack of oxidative phosphorylation; (6) presence of peripheral vesicles. Hydrogenosomes are considered an excellent drug target since their metabolic pathway is distinct from those found in mitochondria and thus medicines directed to these organelles will probably not affect the host-cell. The main drug used against trichomonads is metronidazole, although other drugs such as beta-Lapachone, colchicine, Taxol, nocodazole, griseofulvin, cytochalasins, hydroxyurea, among others, have been used in trichomonad studies, showing: (1) flagella internalization forming pseudocyst; (2) dysfunctional hydrogenosomes; (3) hydrogenosomes with abnormal sizes and shapes and with an electron dense deposit called nucleoid; (4) intense autophagy in which hydrogenosomes are removed and further digested in lysosomes.
Current pharmaceutical design 02/2008; 14(9):872-81. · 4.41 Impact Factor
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ABSTRACT: The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. The coexistence of different sexually transmitted diseases in the same individual is very common, such as vaginal infections by T. vaginalis in association with Mycoplasma fermentans or Mycoplasma hominis. However, the consequences and behavior of mycoplasma during trichomonad infections are virtually unknown. This study was undertaken to elucidate whether mycoplasmas enter and leave trichomonad cells and if so how. M. hominis was analyzed in different trichomonad isolates and the process of internalization and the pathway within the parasite was studied. Parasites naturally and experimentally infected with mycoplasmas were used and transmission electron microscopy, cytochemistry and PCR analyses were performed. The results show that: (1) M. hominis enters T. vaginalis cells by endocytosis; (2) some mycoplasmas use a terminal polar tip as anchor to the trichomonad plasma membrane; (3) some trichomonad isolates are able to digest mycoplasmas, mainly when the trichomonads are experimentally infected; (4) some fresh virulent isolates are able to maintain mycoplasmas as cohabitants in the cell's interior; (5) some mycoplasmas are able to escape from the vacuole to the trichomonad cytosol, and trichomonad plasma membrane budding suggested that mycoplasmas could leave the parasite cell.
Archives of Microbiology 02/2008; 189(1):7-18. · 1.43 Impact Factor
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ABSTRACT: Dengue virus infection affects millions of people all over the world. Although the clinical manifestations of dengue virus-induced diseases are known, the physiopathological mechanisms involved in deteriorating cellular function are not yet understood. In this study we evaluated for the first time the associations between dengue virus-induced cell death and mitochondrial function in HepG2, a human hepatoma cell line. Dengue virus infection promoted changes in mitochondrial bioenergetics, such as an increase in cellular respiration and a decrease in DeltaPsim. These alterations culminated in a 20% decrease in ATP content and a 15% decrease in the energy charge of virus-infected cells. Additionally, virus-infected cells showed several ultrastructural alterations, including mitochondria swelling and other morphological changes typical of the apoptotic process. The alterations in mitochondrial physiology and energy homeostasis preceded cell death. These results indicate that HepG2 cells infected with dengue virus are under metabolic stress and that mitochondrial dysfunction and alterations in cellular ATP balance may be related to the pathogenesis of dengue virus infection.
Biochimica et Biophysica Acta 11/2007; 1772(10):1158-66. · 4.66 Impact Factor
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ABSTRACT: Fine structural studies of the hydrogenosomes of Tritrichomonas foetus using an improved fixative reveal that they are enclosed by two closely apposed 6 nm membranes, which separate at some regions forming a large intramembranous vacuole where Ca++-binding sites are located. Fixation of the cells in a glutaraldehyde solution containing 5 mM CaCl2 and postfixation in an osmium tetroxide-potassium ferrocyanide solution led to the appearance of a reaction product associated with certain regions of the membrane of the hydrogenosomes and in the cisternae of the endoplasmic reticulum, in the recurrent flagellum, and in the plasma membrane. Treatment of ultrathin sections with EGTA removed the reaction product. These results, in association with others previously described, indicate the existence of several similarities between the hydrogenosomes and the mitochondria.
Journal of Eukaryotic Microbiology 04/2007; 30(2):422 - 425. · 2.66 Impact Factor
