Bothrops moojeni venom kills Leishmania spp. with hydrogen peroxide generated by its L-amino acid oxidase

Leishmaniasis is an endemic tropical disease in South America, with few therapeutic approaches. Snake venoms are complex protein mixtures with biological actions that could be used as tools for drug development. Here we show that Bothrops moojeni crude venom presented a killing effect in vitro against Leishmania spp. promastigotes, but not with amastigotes, as determined by a viability assay using the mitochondrial oxidative function. Purification of active fractions from crude venom was performed by molecular exclusion and ion exchange chromatography. Anti-Leishmania and l-amino acid oxidase (L-AAO, EC.1.4.3.2.) activities co-eluted in the same fractions. The molecular weight of the active enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa by SDS--PAGE, with a 4.8 isoelectric point. Using substrate subtraction and catalase for scavenging, the action of L-AAO was demonstrated to be hydrogen-peroxide-dependent.     

Effect of heme iron valence state on the conformation of cytochrome c and its association with membrane interfaces. A CD and EPR investigation

Recently cytochrome c has been mentioned as an important mediator in the events of cellular oxidative stress and apoptosis. To investigate the influence of charged interfaces on the conformation of cytochrome c, the CD and magnetic circular dichroic behavior of ferric and ferrous cytochrome c in homogeneous medium and in phosphatidylcholine/phosphatidylethanolamine/cardiolipin and dicetylphosphate liposomes was studied in the 300-600 and 200-320 nm wavelength region. EPR spectra demonstrate that the association of cytochrome c with membranes promotes alterations of the crystal field symmetry and spin state of the heme Fe(3+). The studies also include the effect of P(i), NaCl, and CaCl(2). Magnetic circular dichroism and CD results show that the interaction of both ferrous and ferric cytochrome c with charged interfaces promotes conformational changes in the alpha-helix content, tertiary structure, and heme iron spin state. Moreover, the association of cytochrome c with different liposomes is sensitive to the heme iron valence state. The more effective association with membranes occurs with ferrous cytochrome c. Dicetylphosphate liposomes, as a negatively charged membrane model, promoted a more pronounced conformational modification in the cytochrome c structure. A decrease in the lipid/protein association is detected in the presence of increasing amounts of CaCl(2), NaCl, and P(i), in response to the increase of the ionic strength.     

Impaired pulmonary host defense in mice lacking expression of the CXC chemokine lungkine

Lungkine (CXCL15) is a novel CXC chemokine that is highly expressed in the adult mouse lung. To determine the biologic function of Lungkine, we generated Lungkine null mice by targeted gene disruption. These mice did not differ from wild-type mice in their hematocrits or in the relative number of cells in leukocyte populations of peripheral blood or other tissues, including lung and bone marrow. However, Lungkine null mice were more susceptible to Klebsiella pneumonia infection, with a decreased survival and increased lung bacterial burden compared with infected wild-type mice. Histologic analysis of the lung and assessment of leukocytes in the bronchioalveolar lavage revealed that neutrophil numbers were normal in the lung parenchyma, but reduced in the airspace. The production of other neutrophil chemoattractants in the Lungkine null mice did not differ from that in wild-type mice, and neutrophil migration into other tissues was normal. Taken together, these findings demonstrate that Lungkine is an important mediator of neutrophil migration from the lung parenchyma into the airspace.     

Evaluation of the effect of 90Sr beta-radiation on human blood cells by chromosome aberration and single cell gel electrophoresis (comet assay) analysis

Among various environmental genotoxins, ionizing radiation has received special attention because of its mutagenic, carcinogenic and teratogenic potential. In this context and considering the scarcity of literature data, the objective of the present study was to evaluate the effect of 90Sr beta-radiation on human cells. Blood cells from five healthy donors were irradiated in vitro with doses of 0.2-5.0Gy from a 90Sr source (0.2Gy/min) and processed for chromosome aberration analysis and for comet assay. The cytogenetic results showed that the most frequently found aberration types were acentric fragments, double minutes and dicentrics. The alpha and beta coefficients of the linear-quadratic model, that best fitted the data obtained, showed that 90Sr beta-radiation was less efficient in inducing chromosome aberrations than other types of low linear energy transfer (LET) radiation such as 3H beta-particles, 60Co gamma-rays, 137Cs and 192Ir and X-rays. Apparently, 90Sr beta-radiation in the dose range investigated had no effect on the modal chromosome number of irradiated cells or on cell cycle kinetics. Concerning the comet assay, there was an increase in DNA migration as a function of radiation dose as evaluated by an image analysis system (tail moment) or by visual classification (DNA damage). The dose-response relation adequately fitted the non-linear regression model. In contrast to the cytogenetic data, 90Sr beta-radiation induced more DNA damage than 60Co gamma-radiation when the material was analyzed immediately after exposures. A possible influence of selective death of cells damaged by radiation was suggested.     

Probing DMPG vesicle surface with a cationic aqueous soluble spin label

A small, highly aqueous soluble, deuterated, cationic spin label, 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (dCAT1), was used to directly monitor the negatively charged DMPG vesicle surface in order to test a recent suggestion (Riske et al., Chem. Phys. Lipids, 89 (1997) 31-44) that alterations in the surface potential accompanied apparent phase transitions observed by light scattering. The temperature dependence of the label partition between the lipid surface and the aqueous medium indicated an increase in the surface potential at the gel to liquid-crystal transition, supporting the previous suggestion. Results at the phase transition occurring at a higher temperature were less definitive. Although some change in the dCAT1 ESR spectra was observed, the interpretation of the phenomena is still rather unclear. DMPG surface potentials were estimated from the dCAT1 partition ratios (surface label moles/total label moles), using a simple two-sites model, where the electrostatic potential is zero everywhere but at the vesicle surface, and the interaction between the spin label and the membrane surface is chiefly electrostatic. The Gouy-Chapman-Stern model predicts surface potentials similar to those observed, although the measured decrease in the surface potential with ionic strength is somewhat steeper than that predicted by the model.     

CXC chemokine receptor-2 ligands are necessary components of neutrophil-mediated host defense in invasive pulmonary aspergillosis

Invasive pulmonary aspergillosis is a devastating complication of immunosuppression, which occurs in association with neutrophil dysfunction or deficiency. ELR+ CXC chemokines are a subfamily of chemokines that play a critical role in neutrophil chemotaxis and activation both in vitro and in vivo. We hypothesized that interaction of these ligands with CXC chemokine receptor-2 (CXCR2), their sole murine receptor, is a major component of neutrophil-dependent pulmonary host defense against Aspergillus fumigatus. In immunocompetent animals, neutrophils were recruited to the lung in response to intratracheally administered A. fumigatus conidia. In a model of transient in vivo depletion of neutrophils, animals developed invasive pulmonary aspergillosis, associated with delayed influx of neutrophils into the lung. In both normal and neutrophil-depleted animals, the ELR+ CXC chemokines MIP-2 and KC were induced in response to intratracheal administration of conidia. Ab-mediated neutralization of the common ELR+ CXC chemokine receptor, CXCR2, resulted in development of invasive disease indistinguishable from the disease in neutrophil-depleted animals, while control animals were highly resistant to the development of infection. CXCR2 neutralization was associated with reduced lung neutrophil influx and resulted in a marked increase in mortality compared with controls. In contrast, animals with constitutive lung-specific transgenic expression of KC were resistant to the organism, with reduced mortality and lower lung burden of fungus. We conclude that CXCR2 ligands are essential mediators of host defense against A. fumigatus, and may be important targets in devising future therapeutic strategies in this disease.     

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.     

Diagnostic of Biomphalaria snails and Schistosoma mansoni: DNA obtained from traces of shell organic materials

Freshwater snails belonging to the genus Biomphalaria act as intermediate hosts for the parasite trematode Schistosoma mansoni in Africa and in the neotropical region. Identification of such molluscs is carried out based on morphological characters and the presence of cercariae is verified through squeezing snails between two glass slides or by exposing them to artificial light. However, sometimes, the material collected includes molluscs with decomposed bodies or, yet, only empty shells, which precludes their identification and S. mansoni detection. Due to these difficulties, we have developed a methodology in which DNA may be extracted from traces of organic material from inside shells in order to identify molluscs through polymerase chain reaction and restriction fragment length polymorphism and to detect S. mansoni into these snails, by using low stringency polymerase chain reaction. Species-specific profiles obtained from B. glabrata, B. straminea, and B. tenagophila snails and their shells, maintained in laboratory for ten years, showed the same profiles. S. mansoni profiles showed to be present in shell specimens as far as the eighth week after being removed from aquarium.     

Zinc nutritional status and its relationships with hyperinsulinemia in obese children and adolescents

A perturbation of zinc metabolism has been noted in subjects with obesity. The present work intends to investigate whether the zinc nutritional status is associated with hyperinsulinemia in obesity. A study was carried out in a group of obese children and adolescents (n=23) and compared to a control group (n=21), both between 7 and 14 yr of age. Software analyzed diet information from 3-d food records. Body composition was evaluated by body mass index (BMI), bioelectrical impedance, and skinfold measurements. Zinc nutritional status was evaluated by Zn determination in plasma, erythrocyte, and 24-h urine, by atomic absorption spectrophotometry (λ=213.9 nm). Insulin was measured by radioimmunoassay (Linco Res). Diets consumed by both groups had marginal concentrations of zinc. Zinc concentrations in plasma and erythrocytes were significantly lower in the obese group. Urinary zinc excretion and serum insulin were significantly higher in the same group, although the insulinemia and the parameters of zinc nutritional status were not significantly correlated. As a result, considering that zinc is part of the synthesis and secretion of this hormone, an assessment is necessary of the possible participation of the oligoelement in the mechanisms of insulin resistance, commonly present in obese patients.     

DNA double-strand breaks, recombination and synapsis: the timing of meiosis differs in grasshoppers and flies

The temporal and functional relationships between DNA events of meiotic recombination and synaptonemal complex formation are a matter of discussion within the meiotic field. To analyse this subject in grasshoppers, organisms that have been considered as models for meiotic studies for many years, we have studied the localization of phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks (DSBs), in combination with localization of cohesin SMC3 and recombinase Rad51. We show that the loss of gamma-H2AX staining is spatially and temporally linked to synapsis, and that in grasshoppers the initiation of recombination, produced as a consequence of DSB formation, precedes synapsis. This result supports the idea that grasshoppers display a pairing pathway that is not present in other insects such as Drosophila melanogaster, but is similar to those reported in yeast, mouse and Arabidopsis. In addition, we have observed the presence of gamma-H2AX in the X chromosome from zygotene to late pachytene, indicating that the function of H2AX phosphorylation during grasshopper spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA DSBs.