Participation of the Entner-Doudoroff pathway in Escherichia coli strains with an inactive phosphotransferase system (PTS- Glc+) in gluconate and glucose batch cultures

The activity of the enzymes of the central metabolic pathways has been the subject of intensive analysis; however, the Entner-Doudoroff (ED) pathway has only recently begun to attract attention. The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS- Glc+ was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed. Even though the specific growth rates between the strain carrying the edd gene knockout and its parent JM101 and PTS- Glc+ edd and its parent PTS- Glc+ were very similar, reproducible changes in the specific consumption rates and biomass yields were obtained when grown on glucose. These results support the participation of the ED pathway not only on gluconate metabolism but on other metabolic and biochemical processes in E. coli. Despite that gluconate is a non-PTS carbohydrate, the PTS- Glc+ and derived strains showed important reductions in the specific growth and gluconate consumption rates. Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS- Glc+ and PTS- Glc+ pykF mutants. Additional results obtained with the pykA pykF mutant indicate the important contribution of the pyruvate kinase enzymes to pyruvate synthesis and energy production in both carbon sources.     

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.     

Absence of VanA- and VanB-Containing Enterococci in Poultry Raised on Nonintensive Production Farms in Brazil

We examined cloacal samples from poultry raised on nonintensive production farms in Brazil for the presence of vancomycin-resistant enterococci. No VanA- or VanB-containing enterococci were identified in a total of 200 cloacal swabs. The most prevalent species were Enterococcus gallinarum (vanC1; 13.0%) and E. casseliflavus (vanC2/3; 5.5%).     

A malaria parasite-encoded vacuolar H(+)-ATPase is targeted to the host erythrocyte

The asexual development of malaria parasites inside the erythrocyte is accompanied by changes in the composition, structure, and function of the host cell membrane and cytoplasm. The parasite exports a membrane network into the host cytoplasm and several proteins that are inserted into the erythrocyte membrane, although none of these proteins has been shown to have enzymatic activity. We report here that a functional malaria parasite-encoded vacuolar (V)-H(+)-ATPase is exported to the erythrocyte and localized in membranous structures and in the plasma membrane of the infected erythrocyte. This localization was determined by separation of parasite and erythrocyte membranes and determination of enzyme marker activities and by immunofluorescence microscopy assays using antibodies against the B subunit of the malarial V-H(+)-ATPase and erythrocyte (spectrins) and parasite (merozoite surface protein 1) markers. Our results suggest that this pump has a role in the maintenance of the intracellular pH (pH(i)) of the infected erythrocyte. Our results also indicate that although the pH(i) maintained by the V-H(+)-ATPase is important for maximum uptake of small metabolites at equilibrium, it does not appear to affect transport across the erythrocyte membrane and is, therefore, not involved in the previously described phenomenon of increased permeability of infected erythrocytes that is sensitive to chloride channel inhibitors (new permeation pathway). This constitutes the first report of the presence of a functional enzyme of parasite origin in the plasma membrane of its host.     

Therapeutic treatment with scFv–PLGA nanoparticles decreases pulmonary fungal load in a murine model of paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic mycosis with lymphatic dissemination that is caused by Paracoccidioides species. Treatment of PCM consists of chemotherapeutics such as itraconazole, trimethoprim, sulfamethoxazole or amphotericin B. However, several studies are aiming to develop therapeutic alternatives for the treatment of fungal infection using new molecules as adjuvants. The single-chain variable fragments (scFv) from an antibody that mimics the main fungal component incorporated within poly(lactide-co-glycolic) acid (PLGA) nanoparticles helped treat the fungal disease. After expressing the scFv in Picchia pastoris (P. pastoris), the recombinant molecules were coupled with PLGA, and the BALB/c mice were immunized before or after infection with yeast Paracoccidioides brasiliensis (P. brasiliensis). Our results showed decreased disease progression and decreased fungal burden. Taken together, our results showed an increased of IFN-γ and IL-12 cytokine production and an increased number of macrophages and dendritic cells in the pulmonary tissue of BALB/c mice treated with a high concentration of our molecule. Our data further confirm that the scFv plays an important role in the treatment of experimental PCM.     

Src kinase activation by nitric oxide promotes resistance to anoikis in tumour cell lines

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.     

Integrating alpha,beta, and phylogenetic diversity to understand anuran fauna along environmental gradients of tropical forests in western Ecuador

The study of current distribution patterns of amphibian species in South America is of particular interest in areas such as evolutionary ecology and conservation biology. These patterns could be playing an important role in biological interactions, population size, and connectivity, and potential extinction risk in amphibians. Here, we tested the effects of spatial and environmental factors on the variation, turnover, and phylogenetic diversity of anuran amphibian species in tropical forests of western Ecuador. Data for presence/absence of 101 species of 34 genera and 10 families registered in 12 sites (nested in four biogeographic units) were obtained through fieldwork, museum collections, and literature records. We examined the influence of geographical, altitudinal, temperature, and precipitation distances on differences in anuran composition between sites. We found significant positive correlations among all of these variables with anuran distribution. The greatest alpha diversity (species richness) was found in the Equatorial Chocó biogeographic unit. Equatorial Pacific biogeographic unit could act as a transition zone between the Equatorial Chocó and Equatorial Tumbes. The western Andes (Western Cordillera biogeographic unit) was the most dissimilar and exhibited a higher species turnover rate than the other biogeographic units. Our results suggest that precipitation and elevation play a key role in maintaining the diversity of amphibian species in western Ecuador.