Effect of thapsigargin on calcium homeostasis in Trypanosoma cruzi trypomastigotes and epimastigotes

By using the fluorescent calcium indicator fura-2, it was found that the concentration of free Ca2+ in the cytoplasm of Trypanosoma cruzi trypomastigotes incubated in the presence or absence of external calcium was maintained at very low levels (10-20 nM). When trypomastigotes were incubated in the presence of succinate and ATP and permeabilized with digitonin, they lowered the medium calcium concentration to a submicromolar level. In the presence of 1 microM FCCP the initial rate of Ca2+ sequestration by these permeabilized cells was very slow. When succinate alone was present, the initial rate of Ca2+ accumulation was slower than with ATP plus succinate, and the calcium set point was about 0.6 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. High concentrations of the tumor promoter thapsigargin slightly increased cytosolic Ca2+ in the presence of extracellular Ca2+ but had no effect on the FCCP- and oligomycin/antimycin A-insensitive Ca2+ pool. In addition, when used at those concentrations (4-20 microM), thapsigargin was shown to release Ca2+ from the mitochondria and to decrease the inner mitochondrial membrane potential of trypomastigotes and epimastigotes as measured using safranine O. Despite the presence of inositol phosphates as determined by [3H]inositol incorporation, no IP3-sensitive Ca2+ release could be detected in trypomastigotes.     

Analyzing expression of the calmodulin and ubiquitin-fusion genes of Trypanosoma cruzi using simultaneous, independent dual gene replacements

Previous work has shown that abrupt visual onsets capture attention. Possible attention. Possible mechanisms for this phenomenon include (a) a luminance-change detection system and (b) a mechanism that detects the appearance of new perceptual objects. Experiments 1 and 2 revealed that attention is captured in visual search by the appearance of a new perceptual object even when the object is equiluminant with its background and thus exhibits no luminance change when it appears. Experiment 3 showed that a highly salient luminance increment alone is not sufficient to capture attention. These findings suggest that attentional capture is mediated by a mechanism that detects the appearance of new perceptual objects.     

Trypanosoma rangeli sialidase: cloning, expression and similarity to T. cruzi trans-sialidase

Sialidases are hydrolytic enzymes present from virus to higher eukaryotes, catalyzing the removal of sialic acid from glycoconjugates. Some protozoa Trypanosomatidae secrete high levels of sialidase into the medium. We have now purified the secreted sialidase from Trypanosoma rangeli. Its N-terminal sequence reveals 100% identity with the corresponding region of the trans-sialidase from T. cruzi. Trans-sialidase, although homologous to viral and bacterial sialidases, displays a novel sialyltransferase activity and is involved in host cell invasion. Several homologous transsialidase-like genes were cloned from genomic DNA of T. rangeli, and grouped in three subfamilies. Active sialidase-encoding genes were found in one of them. The recombinant sialidase shows similar properties to those of the native enzyme, including undetectable trans-sialidase activity. Nevertheless, it has an overall identity of 68.9% with the catalytic domain of T. cruzi trans-sialidase, increasing to 86.7% admitting conservative substitutions. Only three other eukaryotic sialidases have been previously cloned, none of them showing significant homology to trans-sialidase. The isolation of a highly similar sialidase is relevant to further identify the molecular determinants allowing trans-sialidase activity. As a first approach, chimeric constructs between sialidase and trans-sialidase were generated, one of them rendering a sialidase with three times lower Km than the natural enzyme.     

The Trypanosoma cruzi ribosomal RNA-encoding gene: analysis of promoter and upstream intergenic spacer sequences

The present study was designed to investigate the influence of endothelium-derived nitric oxide on the contractile responses of isolated human omental arteries to electrical field stimulation and noradrenaline. We measured isometric tension in artery rings obtained from portions of human omentum during the course of abdominal operations (32 patients). Electrical field stimulation induced frequency-dependent contractions which were abolished by tetrodotoxin (10(-6) M) and prazosin (10(-6) M), thus indicating that this effect was due to noradrenaline released from adrenergic nerves acting on alpha 1-adrenoceptors. The increases in tension induced by electrical field stimulation were of greater magnitude in arteries denuded of endothelium. NG-Nitro-L-arginine (L-NAME, 10(-4) M) potentiated the contractile response to electrical field stimulation in artery rings with endothelium but did not influence the contractile responses of endothelium-denuded arteries. The potentiation induced by L-NAME was completely reversed by L-arginine (10(-4) M), but not by D-arginine (10(-4) M). Contractile responses to noradrenaline were similar in arteries with and without endothelium. L-NAME (10(-4) M) had no significant effect on the contractile responses to noradrenaline. Our results suggest that electrical field stimulation releases endothelium-derived nitric oxide which inhibits the contractile responses of human omental arteries. The constrictor responses to noradrenaline are not modulated by the endothelium.     

Trypanosoma cruzi: nitrogenous-base-containing phosphatides in trypomastigote forms--isolation and chemical analysis

Increasingly clinicians attempt to base decisions regarding patient management on the results of clinical studies in addition to expert opinion and their own practical experience. In this article, the author reviews the published studies available to assist clinicians to make evidence-based decisions in three topics related to small volume red blood cell (RBC) transfusions for preterm infants; namely, studies examining the effects of RBC transfusions on possible symptoms of anemia such as tachypnea, apnea or other cardiorespiratory irregularities, studies investigating the collection and transfusion of umbilical cord blood and finally studies addressing the duration of storage and use of additive solutions for RBCs for transfusion to neonates. Based on the review of these studies, guidelines for small volume RBC transfusions in preterm infants are suggested.     

Localization of peroxidase activity in Trypanosoma cruzi microbodies

Electron microscopic observation of Trypanosoma cruzi epimastigotes reveals the presence of microbody-like structures (microperoxisomes) in which 3,3'-diaminobenzidine (DAB) is peroxidized to electron-opaque material. The role of peroxidase in DAB peroxidation is supported by the enzyme demonstration in disrupted epimastigotes and the microbody-containing cell fractions.     

Trypanosoma cruzi meningoencephalitis in AIDS mimicking cerebral metastases: case report

BACKGROUND: It has been suggested that citrate salts might enhance aluminum (Al) absorption from a normal diet, posing a threat of Al toxicity even in subjects with normal renal function. We have recently reported that in normal subjects and patients with moderate renal failure, short-term treatment with tricalcium dicitrate (Ca3Cit2) does not significantly change urinary and serum Al levels. However, we have not assessed total body Al stores in patients on long-term citrate treatment. OBJECTIVE: The objective of this study was to ascertain body content of Al non-invasively using the increment in serum and urinary Al following the intravenous administration of deferoxamine (DFO) in patients with kidney stones and osteoporotic women undergoing long-term treatment with potassium citrate (K3Cit) or Ca3Cit2, respectively. METHODS: Ten patients with calcium nephrolithiasis and five with osteoporosis who were maintained on potassium citrate (40 mEq/day or more) or calcium citrate 800 mg calcium/day (40 mEq citrate) for 2 to 8 years, respectively, and 16 normal volunteers without a history of regular aluminum-containing antacid use participated in the study. All participants completed the 8 days of study, during which they were maintained on their regular home diet. Urinary Al excretion was measured during a two-day baseline before (Days 5, 6) and for 1 day (Day 7) immediately following a single intravenous dose of DFO (40 mg/kg). Blood for Al was obtained before DFO administration, and at 2, 5 and 24 hours following the start of the infusion. RESULTS: The median 24-hour urinary Al excretion (microgram/day) at baseline versus post-DFO value was 15.9 vs. 44.4 in the normal subjects and 13.3 vs. 35.7 in the patients. These values were all within normal limits and did not change significantly following DFO infusion (p = 0.003 and p = 0.0001, respectively). The median change of 17.1 micrograms/day in urinary Al in the normal subjects was not significantly different from the 18.7 micrograms/day change measured in the patient group (p = 0.30). Similarly, no change in the mean serum Al was detected at any time following the DFO infusion, either in the patient or control group (patients 4.1 to 4.3 ng/ml, controls 7.4 to 4.6 ng/ml). CONCLUSION: The results suggest that abnormal total body retention of Al does not occur during long-term citrate treatment in patients with functioning kidneys.     

Trypanosoma rangeli and trypanosoma cruzi: cross-reaction among their immunogenic components

A pilot presented with a 2-month history of dysgeusia following treatment for sinusitis. This was accompanied by a 30-lb weight loss due to the abnormal taste of most foods. An extensive evaluation failed to demonstrate a cause for the malady. The clinical course and diagnostic evaluation were consistent with a diagnosis of idiopathic dysgeusia. The clinical presentation, evaluation, and diagnosis of a pilot with dysgeusia are discussed.     

In situ compositional analysis of acidocalcisomes in Trypanosoma cruzi

We measured the elemental content of different compartments in Trypanosoma cruzi epimastigotes using quick freezing, ultracryomicrotomy, and electron probe microanalysis. Vacuoles identified by high electron density contained (in units of mmol/kg dry weight +/- S.E.) large amounts of phosphorus (1390 +/- 13), magnesium (646 +/- 19), calcium (171 +/- 5), sodium (161 +/- 18), and zinc (148 +/- 6). No other compartment had appreciable calcium or zinc content. Iron (128 +/- 16 mmol/kg) was detected only in vacuoles distinct from the electron-dense vacuoles and other organelles. Incubation of cells for 70 min in culture medium in the presence of ionomycin plus nigericin led to a very significant 3- or 2-fold increase in potassium in the electron-dense vacuoles and the iron-rich vacuoles, respectively, with no significant change in the other elements investigated. This indicated the acidic nature of the vacuoles and demonstrated that the electron-dense vacuoles correspond to what were described previously as acidocalcisomes, i.e. acidic compartments rich in Ca2+. The acidocalcisomes were investigated by separation of epimastigote fractions on Percoll gradients in combination with Triton WR-1339 treatment. This detergent caused a rapid vacuolation; these vacuoles were shown by electron microscopy to be largely transparent, with a diffuse matrix. Percoll gradient fractionation demonstrated decreases in the density of various organelle markers in detergent-treated cells compared with controls. Large decreases in the density of the acidocalcisome and the mitochondrion were seen, as well as smaller decreases in the density of the other markers. Conventional electron microscopy of epimastigotes loaded with gold-labeled transferrin indicated that the endosomal system was separate from vacuoles that probably corresponded to the calcium-containing organelles detected by electron probe microanalysis. The combined results provide evidence that acidocalcisomes are organelles different from lysosomes or other organelles previously described in these parasites.     

Induction of resistance to azole drugs in Trypanosoma cruzi

Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a frequently fatal illness affecting the heart and gastrointestinal systems. An estimated 16 million to 18 million people in Latin America and 50,000 to 100,000 people in the United States are infected with this pathogen. Treatment options for T. cruzi infections are suboptimal due to the toxicities and limited effectiveness of the available drugs. Azole antimicrobial agents have been discovered to have antitrypanosomal activity by inhibition of ergosterol synthesis. The triazole itraconazole was recently shown to produce a parasitologic cure rate of 53% in chronically infected patients (W. Apt et al., Am. J. Trop. Med. Hyg. 59:133-138, 1998), a result which may lead to more use of this family of drugs for the treatment of T. cruzi infections. In the experiments reported on here, resistance to azoles was induced in vitro by serial passage of mammalian-stage parasites in the presence of fluconazole for 4 months. These parasites were cross resistant to the other azoles, ketoconazole, miconazole, and itraconazole. They remained susceptible to benznidazole and amphotericin B. The azole-resistant phenotype was stable for more than 2 months of in vitro serial passage without fluconazole. In addition, the parasites resisted treatment in mice receiving ketoconazole. The rapid development of azole resistance in T. cruzi in vitro suggests that resistance to azole drugs has the potential to occur in patients and may pose an impediment to the progress being made in the treatment of T. cruzi infection.     

The activities of four anticancer alkyllysophospholipids against Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei

The in-vitro activities of four anticancer alkyllysophospholipids, ET-18-OCH3 (edelfosine), hexadecylphosphocholine (miltefosine), ilmofosine and SRI 62-834, were determined with ED50 values for Leishmania donovani and Trypanosoma cruzi amastigotes between 0.2 and 5.0 microM and for Trypanosoma brucei trypomastigotes between 7.0 and 50.8 microM. In BALB/c mice miltefosine and ilmofosine were the most active compounds with ED50 values of 2.9 and 14.5 mg/kg x 5 doses against L. donovani and a suppressive effect on T. cruzi infections at 30 mg/kg x 5 doses.     

Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi

The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat. This is particularly important in the case of Ca2+, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value. Ca2+ entry across the plasma membrane is a widely recognized mechanism for Ca2+ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites. Here we show that Trypanosoma cruzi amastigotes possess a higher Ca2+ content than the extracellular stages of the parasite. This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced. The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively. The tca1 gene suppresses the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H+-ATPase to the plasma membrane and to intracellular vacuoles of T. cruzi. These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D. A. Scott, R. Docampo, J. A. Dvorak, S. Shi, and R. D. Leapman, J. Biol. Chem. 272:28020-28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes). Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites and underscore the ability of intracellular parasites to adapt to the hostile environment of their hosts.     

Vitamin D overload and experimental Trypanosoma cruzi infection: parasitological and histopathological aspects

1. Six groups of 45-day-old, 23.0 +/- 1.7 g, female Balb/c mice were inoculated intraperitoneally with 63, 252, 440, 630, 2520 or 6300 I.U. of vitamin D for 6 days. A seventh group was inoculated with saline. Each group consisted of 30 animals. 2. All animals inoculated with the doses of 2520 and 6300 and 70% of mice which received 630 I.U. of vitamin D died 21 days after the first administration of the vitamin. The LD50 was 630 I.U. 3. The survivors were divided into two groups inoculated intraperitoneally with 5000 trypomastigotes of either Y or CL strain of Trypanosoma cruzi. 4. Based on the survival index on day 73 after infection, Vitamin D gave statistically significant protection (P < 0.01) for mice inoculated with doses of 63 or 430 I.U. of Y or CL strains, respectively. 5. On histopathological examination, inflammatory reaction and cellular and tissue parasitism were less intense in animals which received higher doses of vitamin D. 6. It is concluded that an overload of vitamin D had a protective effect against CL and Y strains of Trypanosoma cruzi infection in Balb/c mice.     

A pteridine reductase gene ptr1 contiguous to a P-glycoprotein confers resistance to antifolates in Trypanosoma cruzi

We have isolated the pteridine reductase-1 gene (ptr1), from Trypanosoma cruzi (Y strain), located contiguous to the Trypanosoma cruzi P-glycoprotein-2 (tcpgp2). The gene encodes a member of the family of short-chain dehydrogenases, enzymes that are involved in several oxidoreduction reactions. One member of the family, pteridine reductase-1 (PTR1) has been previously described in Leishmania as being involved in antifolate resistance. The ptr1 gene from T. cruzi presents an 828 bp open reading frame, coding for a 276 amino acid protein with a predicted molecular mass of 30 kDa. The deduced amino acid sequence exhibited a remarkable homology with the ptr1 genes of Leishmania major and Leishmania tarentolae. Southern blot analysis using a specific probe indicated that T. cruzi PTR1 is encoded by a single copy gene located in two chromosomes of about 0.9 and 1.2 Mb. Western blot analysis using a polyclonal antiserum against recombinant PTR1 revealed that the protein is only expressed in the epimastigote forms of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. Purified recombinant PTR1 exhibits a NADPH-dependent pteridine reductase activity comparable with those described in Leishmania. Gene transfection experiments using the pTEX expression vector show that, under the conditions tested, T. cruzi PTR1 is involved in resistance to the methotrexate, aminopterin and trimethoprim antifolates.     

Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lacked markers for mitochondria, lysosomes, glycosomes, cytosol, and plasma membrane. Antiserum raised against specific sequences of the plant V-H+-PPase cross-reacted with a T. cruzi protein, which was also detectable in the dense Percoll fraction. The organelles in this fraction appeared by electron microscopy to consist mainly of acidocalcisomes (acidic calcium storage organelles). This identification was confirmed by x-ray microanalysis. Immunofluorescence and immunoelectron microscopy indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyrophosphate was able to drive calcium uptake in permeabilized T. cruzi. This uptake depended upon a proton gradient and was reversed by a specific V-H+-PPase inhibitor. Our results imply that the phylogenetic distribution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.     

Structural variation in the glycoinositolphospholipids of different strains of Trypanosoma cruzi

The structures of the glycoinositolphospholipids (GIPLs) from five strains of the protozoan parasite Trypanosoma cruzi have been determined. Two series of structures were identified, all but one containing the same Man4(AEP)GlcN-Ins-PO4 core. Series 1 oligosaccharides are substituted at the third mannose distal to inositol (Man 3) by ethanolamine-phosphate or 2-aminoethylphosphonic acid, as are some glycosyl-phosphatidylinositol-protein anchors of T. cruzi. The core can be further substituted by terminal (1-3)-linked beta-galactofuranose units. In contrast, Series 2 oligosaccharides do not have additional phosphorus-containing groups attached to Man 3, the latter being substituted instead by a single side chain unit of beta-galactofuranose. Series 1 oligosaccharides are present in all strains (G, G-645, Tulahuen CL, and Y) whereas Series 2 structures are present mainly in CL and Y strains. The lipid moiety in the GIPLs from the G, G-645 and Tulahuen strains is predominantly ceramide, as reported for the Y strain, whilst that from the CL strain is a mixture of ceramide and alkylacylglycerol species. The lipid moiety of the GIPLs, and probably also the phosphoinositol-oligosaccharide structures may play an important immunomodulatory role in infection by T. cruzi.