Differential Effects of Cocaine-Induced Seizures and Lethality on M1-Like Muscarinic and Dopaminergic D1- and D2-Like Binding Receptors in Mice Brain

Summary This work was designed to study the changes produced by cocaine-induced seizures and lethality on dopaminergic D₁- and D₂-like receptors, muscarinic M₁-like binding sites, as well as acetylcholinesterase activity in mice prefrontal cortex (PFC) and striatum (ST). Binding assays were performed in brain homogenates from the PFC and ST and ligands used were [³H]-N-methylscopolamine, [³H]-NMS (in the presence of carbachol), [³H]-SCH 23390 and [³H]-spiroperidol (in presence of mianserin), for muscarinic (M₁-like), D₁- and D₂-like receptors, respectively. Brain acetylcholinesterase (AChE) activity was also determined in these brain areas. Cocaine-induced SE decreased [³H]-SCH 23390 binding in both ST and PFC areas. A decrease in [³H]-NMS binding and an increase in [³H]-spiroperidol binding in PFC was also observed. Cocaine-induced lethality increased [³H]-spiroperidol binding in both areas and decreased [³H]-NMS binding only in PFC, while no difference was seen in [³H]-SCH 23390 binding. Neither SE, nor lethality altered [³H]-NMS binding in ST. AChE activity increased after SE in ST while after death the increase occurred in both PFC and ST. In conclusion, cocaine-induced SE and lethality produces differential changes in brain cholinergic and dopaminergic receptors, depending on the brain area studied suggesting an extensive and complex involvement of these with cocaine toxicity in central nervous system.     

Invasion by Limnoperna fortunei (Dunker, 1857) (Bivalvia, Mytilidae) of the Pantanal Wetland, Brazil

Limnoperna fortunei (Bivalvia, Mitylidae) was introduced into South America in 1991 in the La Plata River (Argentina). It arrived in the ballast water of ships coming from Asia, where this species is native. It was first observed in 1998 in the Paraguay River. Limnoperna was introduced into the Pantanal region as hull fouling of vessels using the Paraguay–Parana waterway. This study describes how L. fortunei came to the Pantanal region, and provides details of its occurrence, density, and impacts. From 1999 to 2002, observations and sampling on natural and artificial substrates in the Paraguay River were made. Some aspects of the spread and impacts, based on local community information, were also analyzed. On artificial substrate the density reached 523.8 individuals m⁻² and on natural substrate (rocks), up to 10,000 individuals m⁻² were found. The densities observed were quite low compared to those found in Southern Brazil, where values up to 100,000 individuals m⁻² have been recorded in the last 3 years. In the Paraguay River, the population density of L. fortunei can be negatively impacted by periodic low levels of dissolved oxygen and decreases in pH to between 5 and 6. Such conditions are frequently present during the periodic flooding or inundation of this area. Under these conditions, a high mortality of L. fortunei was recorded in March of 2002, on both natural and artificial substrates. Despite low densities, L. fortunei can colonize water cooling systems of boats, obstructing water circulation and causing motor overheating. Accumulation in water supply equipment, such as pumps and pipes has also been observed. An erratum to this article is available at .     

Interaction of Metarhizium anisopliae (Metsch.) Sorok., Beauveria bassiana (Bals.) Vuill. and the parasitoid Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) with larvae of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Chemical insecticides are broadly applied to control diamondback moth, Plutella xylostella (L.). Diamondback moth is a major pest of cruciferous worldwide, and resistance of this pest to insecticide has been often reported. Thus, this research investigated the interactions among the fungi Metarhizium anisopliae (Metsch.) Sorok., Beauveria bassiana (Bals.) Vuill., and the larval-pupal parasitoid Oomyzus sokolowskii (Kurdjumov) before and after application of the fungi on DBM larvae offered to the parasitoid. The experiment was carried out at 26+/-l degreeC, 75+/- 5% RH and 12h photophase using a completely randomized design, with eight treatments with six replications each. The isolates E9 of M. anisopliae and ESALQ 447 of B. bassiana, were used at the concentration of 10(7) conidia ml(-1). The results showed that M. anisopliae and B. bassiana reduced the parasitism of P. xylostella by O. sokolowskii. Additive effects were found on the mortality of P. xylostella with the different combinations among the fungi and parasitoid, except for the treatment B. bassiana inoculated 24h before exposition of the larvae to O. sokolowskii. The isolates were more efficacious when applied after exposition of the larvae to the parasitoid. The efficiency of O. sokolowskii was negatively influenced by the presence of the fungi, mainly when the fungi were applied 24h before diamondback's larvae were exposed to the parasitoid. The association of the fungi with the parasitoid presents potential to be tested in field. The use of these natural enemies in the integrated management of P. xylostella may economically improve the cabbage productive system, especially for organic farming.     

Cardiolipin in hydrogenosomes: evidence of symbiotic origin

Hydrogenosomes are found in organisms that lack typical mitochondria. Cardiolipin is a phospholipid located exclusively in bacterial membranes and the inner membrane of mitochondria. Here we show, by cell fractionation, thin-layer chromatography, high-pressure liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry that hydrogenosomes of Tritrichomonas foetus, a cattle vaginal parasite, contain cardiolipin, which is strong evidence for its endosymbiotic origin.     

The PbMDJ1 gene belongs to a conserved MDJ1/LON locus in thermodimorphic pathogenic fungi and encodes a heat shock protein that localizes to both the mitochondria and cell wall of Paracoccidioides brasiliensis

J-domain (DnaJ) proteins, of the Hsp40 family, are essential cofactors of their cognate Hsp70 chaperones, besides acting as independent chaperones. In the present study, we have demonstrated the presence of Mdj1, a mitochondrial DnaJ member, not only in the mitochondria, where it is apparently sorted, but also in the cell wall of Paracoccidioides brasiliensis, a thermodimorphic pathogenic fungus. The molecule (PbMdj1) was localized to fungal yeast cells using both confocal and electron microscopy and also flow cytometry. The anti-recombinant PbMdj1 antibodies used in the reactions specifically recognized a single 55-kDa mitochondrial and cell wall (alkaline beta-mercaptoethanol extract) component, compatible with the predicted size of the protein devoid of its matrix peptide-targeting signal. Labeling was abundant throughout the cell wall and especially in the budding regions; however, anti-PbMdj1 did not affect fungal growth in the concentrations tested in vitro, possibly due to the poor access of the antibodies to their target in growing cells. Labeled mitochondria stood preferentially close to the plasma membrane, and gold particles were detected in the thin space between them, toward the cell surface. We show that Mdj1 and the mitochondrial proteinase Lon homologues are heat shock proteins in P. brasiliensis and that their gene organizations are conserved among thermodimorphic fungi and Aspergillus, where the genes are adjacent and have a common 5' region. This is the first time a DnaJ member has been observed on the cell surface, where its function is speculative.     

Peroxisomal fatty acid beta-oxidation is not essential for virulence of Candida albicans

Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans. Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid beta-oxidation pathway. Here, we asked whether fatty acid beta-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5Delta/pex5Delta mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2Delta/fox2Delta mutant, which lacks the second enzyme of the beta-oxidation pathway. Both mutant strains had strongly reduced beta-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2Delta/fox2Delta mutant, and not the pex5Delta/pex5Delta mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2Delta/fox2Delta mutant was comparable to that of the icl1Delta/icl1Delta mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid beta-oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2Delta/fox2Delta mutant is largely due to a dysfunctional glyoxylate cycle.     

Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A

Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150-250 ng per 10(9) CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.     

Analysis of Genetic Diversity of Fusarium oxysporum f. sp. phaseoli Isolates, Pathogenic and Non-pathogenic to Common Bean (Phaseolus vulgaris L.)

Twenty isolates of Fusarium oxysporum from Brazil, pathogenic and non‐pathogenic to common bean, were analysed using random amplified polymorphic DNA (RAPDs) to study the genetic diversity. RAPD analysis using 23 oligonucleotides resulted in the amplification of 229 polymorphic and 7 monomorphic DNA fragments ranging from 234 to 2590 bp. High genetic variability was observed among the isolates, with the distances varying between 8% and 76% among pathogenic, 2% and 63% among the non‐pathogenic and 45% and 76% between pathogenic and non‐pathogenic isolates. The analysis of genetic distance data showed that the pathogenic isolates tended to group in one group and the non‐pathogenic in another. The genetic distance values of 30% among the pathogenic isolates in cluster A are compatible with the genetic distance values observed within the physiological races, but the distance values among the pathogenic isolates in clusters B and G are not compatible with the distance values observed within the race. Although our results are preliminary, it was not possible to exclude the existence of more than one race of this fungus in Brazil.