Brazilian contribution for a better knowledge on the biology of Toxoplasma gondii

Historically, scientists in Brazil has significantly contributed to the biology, cultivation and structural organization of the pathogenic protozoan Toxoplasma gondiiand its interaction with host cells, starting with the description of the protozoan by Splendore in 1908. The intracellular and extracellular corpuscoli observed in rabbits, corresponded to what we now as tachyzoites. Later on, a pioneering method to grow T. gondii in tissue cultures was developed by Guimarães and Meyer, 1942. They also observed for the first time T. gondii by transmission electron microscopy and made the initial description of the cytoskeleton of T. gondii by observing negatively stained cells. In the 1980's, the relation of the cytoskeleton with the sub-pellicular microtubules was reveled by freeze-fracture. More recently, several Brazilian groups have analyzed in detail basic aspects of the early interaction of the protozoan with the host cell, such as the role of protein phosphorylation, transfer of host cell surface components to the protozoan and genesis and organization of the parasitophorous vacuole. Tachyzoites strategically inhibit nitric oxide production during active invasion of activated macrophages. In vitro studies on the sexual cycle of T. gondii using primary cultures of cat enterocytes and the egress from host cells are being carried out. Perspectives are that the contribution of Brazilian science to the knowledge on T. gondii biology will continue to flourish in years to come.

Indirect effect of neem oil on Podisus nigrispinus (Hemiptera, Pentatomidae): biology and predatory capacity

This study evaluated the effects on the development and predatory capacity of Podisus nigrispinus fed on Spodoptera frugiperda that have ingested different concentrations of neem oil. The predatory capacity of Podisus nigrispinus was assessed, separating nymphs (fourth instar) and adults (males and females). The treatments consisted of S. frugiperda larvae reared in neem oil aqueous solutions (0.077, 0.359 and 0.599%), deltamethrin EC 25 (0.100%) and control arranged in a completely randomized design, with ten replicates. Insects were offered three larval densities (one, three and six), in the third or fourth instars. The predated larvae were examined at 24 and 48 hours after the beginning of the experiment. Biological parameters of Podisus nigrispinus were evaluated in groups of ten second-instar nymphs transferred to pots, in five replicates. Insects were offered 2-6 third and/or fourth-instar larvae reared in the same neem oil concentrations in a completely randomized design. The following parameters were evaluated: duration of each nymph stage (days), nymph mortality (%), weight of fifth-instar nymphs (mg), sex ratio, weight of males and females (mg) and longevity of unfed adults (days). The predatory capacity of nymphs and adults of Podisus nigrispinus was influenced by the neem oil at the concentrations of 0.359% and 0.599% in the highest density. The concentration of 0.359% lengthened the nymphal stage and the concentration of 0.599% reduced the weight of males.

New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

Production of TNF-a by primary cultures of human keratinocytes challenged with Loxosceles gaucho venom

Primary cultures of human keratinocytes were challenged with increasing doses from 10 ng/mL to 2 mg/mL of Loxosceles gaucho venom, responsible for dermonecrotic lesion in humans. TNF-a was investigated by bioassay and ELISA in the supernatant of the cultures challenged with 100 ng/mL, 500 ng/mL, 1 and 2 mg/mL of venom. TNF-a was detected by bioassay in the supernatant of cultures challenged with 100 ng/mL, after 6 h. The cytokine was detected by ELISA in the supernatant of the cells challenged with doses of l mg/mL, after 6 and 12 h. The results point out the capacity of this venom to activate the keratinocytes in primary cultures to produce TNF-a. The production of cytokines could contribute to the local inflammatory process in patients bitten by Loxosceles sp.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.