Low and high-dose intradermal infection with Leishmania major and Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensis with low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 103 or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 103 parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensis displayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensis in the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.     

Genetic similarity among male bees of Euglossa truncata Rebêlo & Moure (Hymenoptera: Apidae) revealed by RAPD markers

The genetic RAPD (random amplified polymorphic DNA) markers have been used successfully in taxonomical studies of several groups of organisms. In the present study these molecular markers were used to analyze the genetic similarity among eighteen males of Euglossa truncata Rebêlo & Moure exhibiting variations in two morphological characters (colour of the antennal scape and metaepisternal hairs) which are frequently used to identify this species of euglossine bee. The twelve primers used in the RAPD analysis amplified 127 loci, of which 40 (31.5%) were polymorphic, showing some variation among the individuals. The coefficients of genetic similarity among the individuals ranged from 0.79 to 0.95, indicating a rather high genetic similarity among the 18 male bees studied. No RAPD band was specific to any morphological character analyzed. The results indicate that all bees analyzed belong to the same species. The high genetic similarity among the eighteen euglossine males studied indicates that the variations observed in the morphological characters are not in disagreement with the identification of this species of Euglossina and these characters can vary among males of E. truncata.     

CFD-aided design of a dynamic filter for mammalian cell separation

In the present work, a rotating disk filter was designed for mammalian cell separation with the aim of avoiding both cell damage and membrane fouling. Different geometric and operational variables of the rotating disk filter were studied using computational fluid dynamics (CFD) by varying rotor radius, rotor angle, membrane-rotor distance, and angular velocity. The combinations of these variables followed a statistical design, so that an analysis of the CFD results provided correlations describing the average shear stress on the membrane surface and the maximum shear stress in the whole module as a function of the variables studied. Based on these correlations, and on the shear resistance levels of Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cell lines, which were investigated using a cone-and-plate viscosimeter, it was possible to determine the geometry and angular velocity that would minimize both cell damage and membrane fouling. After construction, the filter was tested in filtration experiments at increasing permeate fluxes. Cell viability remained >90% for the duration of the experiments (2.5 h), and no indication of fouling was observed. It was shown that the designed dynamic filter is able to effectively avoid both cell damage and membrane fouling, and thus can be used for mammalian cell harvesting and perfusion.     

Diversity of Bacillus thuringiensis strains from Latin America with insecticidal activity against different mosquito species

The characterization of selected Bacillus thuringiensis strains isolated from different Latin America countries is presented. Characterization was based on their insecticidal activity against Aedes aegypti, Culex quinquefasciatus, and Anopheles albimanus larvae, scanning electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and plasmid profiles as well as PCR analysis using novel general and specific primers for cry and cyt genes encoding proteins active against mosquitoes (cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17, cry19, cry24, cry25, cry27, cry29, cry30, cry32, cry39, and cry40). Strains LBIT315, LBIT348, and IB604 showed threefold higher mosquitocidal activity against A. aegypti and C. quinquefasciatus larvae than B. thuringiensis subsp. israelensis and displayed high similarities with the B. thuringiensis subsp. israelensis used in this study with regard to protein and plasmid profiles and the presence of cry genes. Strain 147-8906 has activity against A. aegypti similar to that of B. thuringiensis subsp. israelensis but has different protein and plasmid profiles. This strain, harboring cry11, cry30, cyt1, and cyt2 genes, could be relevant for future resistance management interventions. Finally, the PCR screening strategy presented here led us to identify a putative novel cry11B gene.     

Pyrethroid-Acarophenax lacunatus interaction in suppressing the beetle Rhyzopertha dominica on stored wheat

Any biocontrol agent used against stored grain insects will have to interact with insecticides used as grain protectants due to their heavy use especially in tropical areas. Therefore, the interaction between the pyrethroid insecticides bifenthrin and deltamethrin and Acarophenax lacunatus (Cross & Krantz) (Prostigmata: Acarophenacidae), an egg parasite of the stored cereal pest Rhyzopertha dominica(F.) (Coleoptera: Bostrichidae), was assessed in a range of mite densities and insecticide doses for each compound. Despite the mite species presence on all insecticide doses, the lowest instantaneous rate of increase of A.lacunatus was recorded with the highest doses of insecticides. The presence of the biological control agent at all doses of both insecticides suggests its tolerance to these compounds. A. lacunatusshowed higher parasitism rate with bifenthrin than with deltamethrin indicating a higher selectivity of the first compound to the biocontrol agent. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of the prosegment of Fasciola hepatica cathepsin L1 in folding of the catalytic domain

The N-terminal propeptides of cysteine proteinases play regulatory roles in the folding and stability of their catalytic domains, as well as being potent and highly specific inhibitors of their parental mature enzymes. Cysteine proteinases play a major role in the biology of the parasitic trematode Fasciola hepatica; in particular, this organism secretes significant amounts of cathepsin L enzymes. The isolated propeptide of F. hepatica cathepsin L1 functioned as a chaperone for the mature enzyme in renaturation experiments. A double point mutation (N701/F721) within the GxNxFxD motif of the propeptide affected its conformation and markedly decreased its affinity for the mature enzyme. When this mutation was introduced into preprocathepsin L1 expressed in yeast, the secretion of active enzyme dropped dramatically. However, significant enzyme activity was recovered from the culture supernatants after denaturation and renaturation in the presence of native propeptide. Thus, the variant prosegment gave rise to an enzyme with altered conformation, which could be refolded to the active form with the assistance of the native propeptide.     

Repressor Mutant Forms of the Azospirillum brasilense NtrC Protein

The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source. Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site. The phenotypes of these strains are discussed in relation to their genotypes.     

Sesquiterpenes and a phenylpropanoid from Cordia trichotoma

Two new secondary metabolites, the phenylpropanoid 3-(2',4',5'-trimethoxyphenyl)propanoic acid (1) and the sesquiterpene (+)-1beta,4beta,6alpha-trihydroxyeudesmane (2) were isolated from the heartwood of Cordia trichotoma Vell., along with the known sesquiterpenes (-)-1beta,4beta,7alpha-trihydroxyeudesmane (3) and (+)-1beta,4beta,11-trihydroxyoppositane (4). Their structures were elucidated by means of spectroscopic data interpretation, mainly 1D and 2D NMR and mass spectrometry.     

Development of a Bacillus sphaericus tablet formulation and its evaluation as a larvicide in the biological control of Culex quinquefasciatus

This study aimed to analyze the final fermentation culture of Bacillus sphaericus 2362, standardize it and develop an active tablet formulation for use in urban mosquito breeding sites. It was performed in three phases: analysis and standardization of a B. sphaericus fermented culture; physical, chemical, and biological analysis of the active powder (solubility, residual humidity, particle size, resting angle, flowing off time, compacted density, and biological activity against Culex quinquefasciatus larvae); and the development of fast-disintegrating tablets. Five formulations with differing compositions were developed and a UV protector was added to the selected formulation. The formulation products with or without UV protector, as well as the active powder caused 100% larval mortality from 1 day to 2 months after a single treatment under simulated field conditions. These results show that the UV protector does not affect the initial larvicide activity of B. sphaericus, nor its persistence over a period of two months.