Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has been associated with cognitive, behavioral and motor dysfunctions, seizures and coma. The underlying mechanisms of CM are incompletely understood. Glutamate and other metabolites such as lactate have been implicated in its pathogenesis. In the present study, we investigated the involvement of glutamate in the behavioral symptoms of CM. Seventeen female C57BL/6 mice (20-25 g) aged 6-8 weeks were infected with P. berghei ANKA by the intraperitoneal route using a standardized inoculation of 10(6) parasitized red blood cells suspended in 0.2 mL PBS. Control animals (N = 17) received the same volume of PBS. Behavioral and neurological symptoms were analyzed by the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. Glutamate release was measured in the cerebral cortex and cerebrospinal fluid of infected and control mice by fluorimetric assay. All functional categories of the SHIRPA battery were significantly altered in the infected mice at 6 days post-infection (dpi) (P ≤ 0.05). In parallel to CM symptoms, we found a significant increase in glutamate levels in the cerebral cortex (mean ± SEM; control: 11.62 ± 0.90 nmol/mg protein; infected at 3 dpi: 10.36 ± 1.17 nmol/mg protein; infected at 6 dpi: 26.65 ± 0.73 nmol/mg protein; with EGTA, control: 5.60 ± 1.92 nmol/mg protein; infected at 3 dpi: 6.24 ± 1.87 nmol/mg protein; infected at 6 dpi: 14.14 ± 0.84 nmol/mg protein) and in the cerebrospinal fluid (control: 128 ± 51.23 pmol/mg protein; infected: 301.4 ± 22.52 pmol/mg protein) of infected mice (P ≤ 0.05). These findings suggest a role of glutamate in the central nervous system dysfunction found in CM.

RESUMO O objetivo do trabalho foi avaliar a capacidade de voo, o parasitismo e a emergência de linhagens de Trichogramma pretiosum Riley, criadas em laboratório por 54 gerações sobre Sitotroga cerealella (Olivier) e oriundas de quatro municípios do Mato Grosso. O delineamento estatístico foi o inteiramente casualizado com quatro tratamentos (linhagens) e sete repetições (cada uma com 30 ovos inviáveis de S. cerealella). Avaliou-se a porcentagem de insetos voadores, caminhadores, não-voadores, parasitismo e emergência. A porcentagem de parasitoides voadores de Rondonópolis (97,7) foi superior às de Primavera do Leste (91,7), Jaciara (85,9) e Pedra Preta (94) pelo teste de ScottKnott (P < 0,05). A porcentagem de caminhadores não diferiu nas linhagens de Rondonópolis (0,6) e Pedra Preta (2,6). Para os parasitoides não voadores não se detectaram diferenças nas linhagens de Primavera do Leste (3,6%), Jaciara (3,1%), Rondonópolis (1,7%) e Pedra Preta (3,7%). A taxa de parasitismo foi semelhante entre as linhagens e variou de 69,2% (Pedra Preta) a 81,9% (Rondonópolis). A emergência foi superior a 80% em todas as linhagens e também não diferiu entre elas. Após 54 gerações as quatro linhagens estão em ótimas condições de atividade de voo, parasitismo e emergência, para serem utilizadas em programas de controle biológico.

ABSTRACT The objective of this study was to evaluate the flight capacity, parasitism and emergence of Trichogramma pretiosum Riley strains, collected from 4 localities of Mato Grosso State, Brazil, reared in the laboratory for 54 generations, and fed on Sitotroga cerealella (Olivier) eggs. A completely randomized statistical design was adopted, with 4 treatments (strains) and 7 replicates (30 unviable eggs of S. cerealella). The percentage of flying, running and nonflying insects, parasitism and emergence rate were evaluated. Percentage of flying parasitoids from the Rondonópolis strain (97.7) was higher than for the Primavera do Leste (91.7), Jaciara (85.9) and Pedra Preta (94.0) strains, according to the Scott-Knott test (P < 0.05). The percentage of running insects did not differ between the Rondonópolis (0.6) and Black Stone strains (2.6). For the nonflying parasitoids, differences were not detected in the Primavera do Leste (3.6%), Jaciara (3.1%), Rondonópolis (1.7%) or Pedra Preta strains (3.7%). The parasitism rate was similar among the strains and varied from 69.2% (Pedra Preta strain) to 81.9% (Rondonópolis strain). The emergence rate did not differ among the strains and was superior to 80% in all the strains. After 54 generations the 4 strains are in adequate flight activity, parasitism and emergence conditions to be used in biological control programs.

Many publications describe methods for peroxidase purification from plant material. When the goal was to obtain a high purity enzyme every purification method included an affinity chromatography step using Concanavalin A as the ligand. However, the adsorption step was carried out under quite different conditions with regard to pH, ionic strength and metal cation content in the binding buffer, thus leading to a rather confuse situation. To establish the best conditions for purification of peroxidases from horseradish root (HRP) and soybean hull (SBP), equilibrium adsorption isotherms were fitted to the Langmuir-type model, where ionic strength, pH and cation concentration were chosen as the variables. For SBP, our results showed that Kd rounded 10-6 M in all cases (pH 5.0 - 7.0, 1 and 5 mM Mn2+ / Ca2+, 0 - 0.75 M NaCl). For HRP, Kd ranged between 10-5 M and 10-6 M depending on the parameters. Under optimised binding conditions, 84.3% SBP was recovered after elution carried out with 0.74 M a -methyl-D-mannopyranoside and 0.37 M NaCl. For HRP, the recovery was lower (75%) and 0.36M a -methyl-D-mannopyranoside was necessary for the elution step.

A process of directed evolution applied to obtain a library of mutants of horseradish peroxidase (HRP) enzyme is described. We have introduced slight variations into the original DNA shuffling protocol. A DNA template was prepared by PCR amplification and digested with Dnase I during 1 hour. Dnase I products were concentrated by precipitation with isopropanol. Gel electrophoresis showed fragments of the desired size range (20-600 pb) without a full-length template remaining in the reaction mixture. A high concentration of fragments was crucial for performing PCR without primers. In this case, a template concentration of 32.5 ng/mu l was appropriate. Amplification of recombinant genes in a standard PCR reaction (template dilution 1:100) produced a smear with a low yield for the full-length sequence. A single product of the correct size was obtained by PCR with nested primers separated from the previously used primers by 40 pb. In our laboratory, native HRP has been functionally expressed in a baculovirus expression vector system. The purpose is to develop the screening of the first generation of random mutants in this system. To facilitate detection of those clones that have high peroxidase activity, we developed a rapid method: after five days postinfection agarose plates with six wells were covered with DAB (3,3´-diaminobenzidine) and H2O2. The appearance of brown-black stain allows visualization of up to 100 active clones/well in only 1 min.

Com o propósito de determinar a relação filogenética entre a cana-de-açúcar e membros da subtribo Saccharinae, a região gênica nuclear ITS1-5,8S-ITS2 (ITS: espaçador interno transcrito; 5,8S: DNA ribossomal 5.8S), com alta taxa evolutiva, foi identificada no banco de dados do projeto genoma "Sugarcane Expressed Sequence Tag" (SUCEST). Uma análise através do método de parcimônia, utilizando esta região e seqüências homólogas de 23 Andropogoneae retiradas da base de dados GenBank, indicou que a cana-de-açúcar é o grupo-irmão de Saccharum sinense. No entanto, devido à pequena quantidade de caracteres informativos para parcimônia e à homoplasia presentes na região ITS1-5,8S-ITS2, não foi possível determinar com segurança a relação filogenética entre a cana-de-açúcar e alguns dos demais membros da tribo Saccharine. Como alternativa para esta baixa resolução, dezessete regiões gênicas nucleares, cloroplasmáticas ou mitocondriais foram selecionadas a partir do banco de dados SUCEST com o objetivo de encontrar marcadores mais apropriados para a reconstrução da filogenia da cana-de-açúcar. Entre elas, aquelas correspondentes à alfa-tubulina, rpl16, e rpoC2 apresentaram baixa incidência de polimorfismo e taxas de evolução equivalentes ou mesmo maiores do que a observada para a região ITS1-5,8S-ITS2. Estes marcadores são propostos como preferenciais para estudos filogenéticos da subtribo Saccharinae.

To determine the phylogenetic relationship between sugarcane cultivars and other members of the Saccharinae subtribe, we identified the fast evolving ITS1-5.8S-ITS2 (ITS = internal transcribed spacer; 5.8S = 5.8S ribosomal DNA) region of the sugarcane genome in the Sugarcane Expressed Sequence Tag (SUCEST) genome project database. Parsimony analysis utilizing this region and homologs belonging to the 23 closely related Andropogoneae currently deposited in the GenBank database has shown sugarcane as the sister group of Saccharum sinense. However, because there are few parsimony-informative characters and high homoplasy in the ITS1-5.8S-ITS2 region we were not able to determine with confidence the phylogenetic relationship between sugarcane and some of the remaining members of Saccharine subtribe. To find alternatives for the phylogenetic reconstruction of sugarcane evolutionary history, we selected 17 markers (nuclear, chloroplastic or mitochondrial) from the SUCEST database of which apha-tubulin, ribosomal protein L16 (rpl16) and DNA-directed RNA polymerase beta chain (rpoC2) were found to have a low incidence of polymorphism and comparable, or even faster, rates of evolution than the ITS1-5.8S-ITS2 region. We suggest that these markers should be considered as preferential choices for phylogenetic studies of Saccharinae subtribe.