Analysis of expressed sequence tags from Trypanosoma cruzi amastigotes

A total of 880 expressed sequence tags (EST) originated from clones randomly selected from a Trypanosoma cruzi amastigote cDNA library have been analyzed. Of these, 40% (355 ESTs) have been identified by similarity to sequences in public databases and classified according to functional categorization of their putative products. About 11% of the mRNAs expressed in amastigotes are related to the translational machinery, and a large number of them (9% of the total number of clones in the library) encode ribosomal proteins. A comparative analysis with a previous study, where clones from the same library were selected using sera from patients with Chagas disease, revealed that ribosomal proteins also represent the largest class of antigen coding genes expressed in amastigotes (54% of all immunoselected clones). However, although more than thirty classes of ribosomal proteins were identified by EST analysis, the results of the immunoscreening indicated that only a particular subset of them contains major antigenic determinants recognized by antibodies from Chagas disease patients.     

Histoplasmosis in a Brazilian center: clinical forms and laboratory tests

Histoplasmosis, caused by the dimorphic fungus Histoplasma capsulatum, is endemic in many regions of the Americas, Asia and Africa. It has a wide spectrum of clinical manifestations, from asymptomatic infection to severe disseminated disease. A retrospective study was carried out to describe the clinical forms and assess the clinical significance of the laboratory diagnostic tests of patients with histoplasmosis during the period of July 1987 to December 2003 at Instituto de Pesquisa Clínica Evandro Chagas/ FIOCRUZ, RJ, Brazil. Seventy-four patients were included. Forty-nine percent of the cases (n = 36) occurred in HIV positive patients who presented with disseminated disease. The remaining 38 cases were classified in different clinical forms. Histoplasma capsulatum was isolated from 69.5% of the clinical specimens sent to culture. Immunodiffusion and immunoblot were positive in 72.6% and 100% of the performed tests, respectively. Histopathologic findings suggestive of H. capsulatum were found in 63.2% of the performed exams. Serology had a lower proportion of positivity amongst AIDS patients, when compared with HIV negative patients (X2 = 6.65; p lower than 0.008). Statistical differences between AIDS and non-AIDS patients were not observed with culture and histopathology. The specific role of each test varies according to the clinical form. Physicians need to know the value and limitations of the available diagnostic tests, but before that, they have to think about histoplasmosis and consider this clinical entity in their differential diagnosis.     

Functional characterization of the thi1 promoter region from Arabidopsis thaliana

The Arabidopsis thaliana THI1 protein is involved in thiamine biosynthesis and is targeted to both chloroplasts and mitochondria by N-terminal control regions. To investigate thi1 expression, a series of thi1 promoter deletions were fused to the beta-glucuronidase (GUS) reporter gene. Transgenic plants were generated and expression patterns obtained under different environmental conditions. The results show that expression derived from the thi1 promoter is detected early on during development and continues throughout the plant's life cycle. High levels of GUS expression are observed in both shoots and roots during vegetative growth although, in roots, expression is restricted to the vascular system. Deletion analysis of the thi1 promoter region identified a region that is responsive to light. The smallest fragment (designated Pthi322) encompasses 306 bp and possesses all the essential signals for tissue specificity, as well as responsiveness to stress conditions such as sugar deprivation, high salinity, and hypoxia.     

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

Cytokine gene expression in Walker 256: a comparison of variants A (aggressive) and AR (regressive)

Two variants of this Walker 256 tumor have been previously reported as Walker 256 A and variant AR. The variant A has more aggressive property than variant AR and can induce systemic effects such as anorexia, sodium and water retention, followed by weight loss and death. The mechanisms involved in enhancing tumor regression and progression in this model are still incompletely understood. In the present study, serum and spleen mononuclear cells and tumor cells from animals inoculated with variants A and AR, were isolated to investigate the TGF-beta, IL-12, IFN-gamma and TNF-alpha and relationship with anemia, weight of animals, weight of spleen, volume of tumor and osmotic fragility compared with controls inoculated with Ringer Lactate. Results demonstrate that the group inoculated with variant A, compared to variant AR, shows high levels of TGF-beta gene expression in both tumor tissue and spleen cells, no expression of IFN-gamma and a progressive and higher levels of IL-12 in tumor tissue without inflammatory infiltrate visualized by optical microscopy. These results suggest that the aggressively of variant A is relate to cytokine modulation, facilitating the growth and escape of tumor cells. Furthermore, IL-12 seems to be constitutively expressed in both tumor lineage A and AR.     

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.     

Germination of Geonoma brevispatha (Arecaceae) in laboratory and its relation to the palm spatial distribution in a swamp forest

Geonoma brevispatha is a clonal palm species commonly found as tussocks in swamp and riparian forests in SE Brazil, occurring in the very transition between flooded pits/channels and dry mounds. Aiming to verify whether the germination responses could explain the spatial pattern of the palm tussocks in swamp forests, we investigated the germination in relation to the main environmental factors to which the seeds are subjected in their natural habitat. The seeds are recalcitrant since germination percentage is reduced with loss of moisture content. Germination did not differ on moist filter paper, semi-immersion (water up to half of the seed diameter) or complete immersion (water up to double of the seed diameter). Seeds did not germinate when stored in a closed bottle with water (that is, under low oxygen concentration), but remained viable for up to 8 weeks under this condition. The capacity to germinate in water with minimum oxygen availability may be considered ecologically advantageous in a swamp habitat. On the other hand, the capacity to germinate in moist filter paper indicates that, in natural conditions, the seed can germinate, provided there is a critical minimum of water availability. We concluded that the germination responses per se are not enough to explain the palm tussocks spatial pattern, but they are very well tuned with the conditions of the transitional zone between channels/pits (ever flooded micro-sites) and mounds (well-drained micro-sites), in which the species occurs in the swamp forest.     

Is the expression of kinin B(1) receptor related to intrahepatic vascular response?

Bradykinin elicits an intrahepatic vascular response (IHVR) mediated by the constitutive B(2) receptor (B(2)R). The biological effects of kinins may also be mediated by the inducible B(1) receptor (B(1)R). AIM: To verify if the hepatic B(1)R expression modulates IHVR to kinins. METHOD: We evaluated the ability of bradykinin and B(1)R agonists to elicit an IHVR in normal rats and in those submitted to acute or chronic inflammatory stimuli, fibrosis, cirrhosis, or hepatic regeneration. RESULTS: Bradykinin-induced IHVR was similar in all groups. B(1)R agonists did not elicit in any of them either a hypertensive or a hypotensive response. B(1) receptor induction was observed in all experimental groups (Western blot), except for the acute inflammatory group. CONCLUSION: B(1)R hepatic expression did not modulate IHVR to kinins.     

Functional Characterization of TbMCP5, a Conserved and Essential ADP/ATP Carrier Present in the Mitochondrion of the Human Pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u⁻ revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.