Cytoskeletal rearrangement and cell death induced by Bothrops alternatus snake venom in cultured Madin-Darby canine kidney cells.

Bothrops snake venoms cause renal damage, with renal failure being the main cause of death in humans bitten by these snakes. In this work, we investigated the cytoskeletal rearrangement and cytotoxicity caused by Bothrops alternatus venom in cultured Madin-Darby canine kidney (MDCK) cells. Incubation with venom (10 and 100 microg/mL) significantly (p <0.05) decreased the cellular uptake of neutral red dye after 1 and 3 h. Venom (100 microg/mL) also markedly decreased the transepithelial electrical resistance (RT) across MDCK monolayers. Staining with rhodamine-conjugated phalloidin revealed disarray of the cytoskeleton that involved the stress fibers at the basal cell surface and focal adhesion-associated F-actin in the cell-matrix contact region. Feulgen staining showed a significant decrease in the number of cells undergoing mitosis and an increase in the frequency of altered nuclei. Scanning electron microscopy revealed a decrease in the number of microvilli and the presence of cells with a fusiform format. Flow cytometry with annexin V and propidium iodide showed that cell death occurred by necrosis, with little apoptosis, a conclusion supported by the lack of DNA fragmentation characteristic of apoptosis. Pretreating the cells with catalase significantly attenuated the venom-induced loss of viability, indicating a possible involvement of H2O2 in the cellular damage; less protection was observed with superoxide dismutase or N omega-nitro-L-arginine methyl ester. These results indicate that Bothrops alternatus venom is cytotoxic to cultured MDCK cells, possibly via the action of reactive oxygen species. This cytotoxicity could contribute to nephrotoxicity after envenoming by this species.     

Influence of training on the response to exercise of adrenocorticotropin and growth hormone plasma concentrations in human swimmers

The aim of the present study was to evaluate the response of adrenocorticotropin ([ACTH]) and growth hormone ([GH]) concentrations to a typical aerobic swimming set during a training season. Nine top-level male endurance swimmers (age range 17–23 years) were tested during three training sessions occurring 6, 12 and 18 weeks after the beginning of the season. During each session, after a standard warm-up, the swimmers performed a training set of 15 × 200-m freestyle, with 20 s of rest between repetitions, at a predetermined individual speed. Blood samples were collected before warm-up and at the end of the training set. A few days before each session, the individual swimming velocity corresponding to the 4 mmol · l⁻¹ blood lactate concentration (v ₄) was assessed as a standard of aerobic performance. Aerobic training affected v ₄ levels, which were highest 18 weeks after the beginning of the season; at the same time, while [ACTH] response was attenuated, [GH] response was enhanced. These results could be considered as adaptations to the exercise intensity. In our training programme, these adaptations seemed to have occurred between the 12th and 18th weeks of the training season. Accepted: 21 April 1998     

Occurrence of Tuber melanosporum in relation to soil surface layer properties and soil differentiation

An intensive survey was carried out on a 12-year-old experimental truffle bed of Tuber melanosporum Vitt. located in the central Apennines. The aim of the investigation was to relate the presence and carpophore production of T. melanosporum to changes in soil structure, aeration and fertility — expressed in terms of 0.25–2.00 mm aggregate fraction, total organic carbon, DTPA-extractable Mn and host plant height — and to determine if these modifications, whenever present, could be ascribed to soil differentiation within the truffle bed. The occurrence of pianelli — i.e. areas with little herbaceous ground cover created by T. melanosporum — showed a close relationship with host plant height and aeration of soil surface layers. Where pianelli occurred, the height of symbiont trees increased and the content of reduced Mn, indicating the presence of a well-aerated soil environment, decreased. The variation of host plant height was attributable not only to the increased absorption of nutrients related to the ectomycorrhizal partnership, but also to soil differentiation. The soils of the investigated area were characterized by a relatively low slope gradient, a rigid framework of gravel and a homogeneous physico-chemical behaviour, due to the predominance of Ca among exchangeable bases. In these environmental conditions, T. melanosporum was present in the rather thick soil belonging to Typic Rendolls, whereas it was absent in the area characterized by thin Lithic Rendolls. In the latter case, the plant cover was probably too scarce to protect T. melanosporum from summer dryness, and consequently the more resistant T. aestivum species prevailed. This revised version was published online in June 2006 with corrections to the Cover Date.     

One‐Sided Test to Assess Correlation in Linear Logistic Models using Estimating Equations

A score‐type test is proposed for testing the hypothesis of independent binary random variables against positive correlation in linear logistic models with sparse data and cluster specific covariates. The test is developed for univariate and multivariate one‐sided alternatives. The main advantage of using score test is that it requires estimation of the model only under the null hypothesis, that in this case corresponds to the binomial maximum likelihood fit. The score‐type test is developed from a class of estimating equations with block‐diagonal structure in which the coefficients of the linear logistic model are estimated simultaneously with the correlation. The simplicity of the score test is illustrated in two particular examples.     

Balancing Selection for Pathogen Resistance Reveals an Intercontinental Signature of Red Queen Coevolution

The link between long-term host–parasite coevolution and genetic diversity is key to understanding genetic epidemiology and the evolution of resistance. The model of Red Queen host–parasite coevolution posits that high genetic diversity is maintained when rare host resistance variants have a selective advantage, which is believed to be the mechanistic basis for the extraordinarily high levels of diversity at disease-related genes such as the major histocompatibility complex in jawed vertebrates and R-genes in plants. The parasites that drive long-term coevolution are, however, often elusive. Here we present evidence for long-term balancing selection at the phenotypic (variation in resistance) and genomic (resistance locus) level in a particular host–parasite system: the planktonic crustacean Daphnia magna and the bacterium Pasteuria ramosa. The host shows widespread polymorphisms for pathogen resistance regardless of geographic distance, even though there is a clear genome-wide pattern of isolation by distance at other sites. In the genomic region of a previously identified resistance supergene, we observed consistent molecular signals of balancing selection, including higher genetic diversity, older coalescence times, and lower differentiation between populations, which set this region apart from the rest of the genome. We propose that specific long-term coevolution by negative-frequency-dependent selection drives this elevated diversity at the host''s resistance loci on an intercontinental scale and provide an example of a direct link between the host’s resistance to a virulent pathogen and the large-scale diversity of its underlying genes.     

Potential predation of the exotic Amblyseius swirskii on Euseius concordis (Acari: Phytoseiidae), a predatory mite commonly found in Brazil

Intra/interspecific predation by A. swirskii.

Both Benin and Netherlands populations of Amblyseius swirskii engaged in cannibalism, and in predation on Euseius concordis larvae. Provision of pollen decreased cannibalism considerably, but had a smaller effect on predation on E. concordis. Potential effect of the eventual introduction of A. swirskii for control of Bemisia tabaci in Brazil is discussed.     

Biochemical Characterization of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. I1.2 Secretome Reveals the Presence of Multienzymatic Complexes Containing Cellulases and Accessory Enzymes

Two general strategies have been proposed for microbial cellulose degradation: filamentous fungi and aerobic bacteria secrete uncomplexed cellulases, while some anaerobic bacteria produce a cell-associated and large extracellular multienzymatic complex called cellulosomes. By using a combination of 1D-blue native (BN)-PAGE, 2D-BN/SDS-PAGE, zymography, and LC-MS/MS methods, we demonstrate here that Streptomyces sp. I1.2, an aerobic bacterium associated with the land snail Achatina fulica, is able to degrade both crystalline cellulose and sugarcane bagasse through the production of cellulolytic multienzymatic complexes containing different combinations of cellobiohydrolases, endo-glucanases, xylanases, lytic polysaccharide monooxygenases (LPMOs), and peptidases. The assembly and subunit composition of these complexes is specifically affected by the carbon source, while the multienzymatic complexes produced after growth in crystalline cellulose are composed mainly by one cellobiohydrolase and chitinase, in which the complexes produced in response to sugarcane bagasse are more heterogeneous and contain cellobiohydrolases, endo-glucanases, pectate lyases, one LPMO, β-1,3-glucanases, and one xylanase. Our results suggest that Streptomyces sp. I1.2 displays an alternative mechanism for deconstruction of cellulose that depends upon a noncellulosomic association of catalytic subunits into high molecular weight complexes in order to achieve higher catalytic efficiencies.     

Population dynamics of Aceria guerreronis Keifer (Acari: Eriophyidae) and associated predators on coconut fruits in Northeastern Brazil

Aceria guerreronis Keifer can cause severe damage to coconuts in several countries around the world. Rare studies have been conducted to determine the predatory mites associated with A. guerreronis in Brazil. The study evaluated the prevalence of A. guerreronis and associated predators on the bracts and on the surface of the fruits underneath the bracts, for 12 months, on coconut palms grown along the coast of the States of Alagoas, Paraíba and Pernambuco, Northeastern Brazil. Mites of 10 families were found, but by far the most abundant species was A. guerreronis, corresponding to 99.5% of the mites collected. The prevailing species amongst the predators were the Phytoseiidae mites Neoseiulus baraki (Athias-Henriot) and Neoseiulus paspalivorus (De Leon). These are flat mites that have short limbs, characteristics that allow them to invade the main habitat occupied by A. guerreronis. Other predators were found, but in low numbers, due mainly to their difficulty in reaching the fruit areas most inhabited by A. guerreronis. However, these mites could prey on that pest when it leaves its preferred habitat to disperse. No significant correlations were observed between the levels of abiotic factors and the population levels of A. guerreronis or of the phytoseiids associated with it. This probably occurred due to the simultaneous and conflicting effects of those factors on the mites in the field, or to significant differences between the climatic factors measured in the environment and those prevailing in the habitat occupied by A. guerreronis.     

Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is a thiol-rich metallopeptidase ubiquitously distributed in mammalian tissues and involved in oligopeptide metabolism both within and outside cells. Fifteen Cys residues are present in the rat EP24.15 protein, seven are solvent accessible, and two are found inside the catalytic site cleft; no intraprotein disulfide is described. In the present investigation, we show that mammalian immunoprecipitated EP24.15 is S-glutathionylated. In vitro EP24.15 S-glutathionylation was demonstrated by the incubation of bacterial recombinant EP24.15 with oxidized glutathione concentration as low as 10 microM. The in vitro S-glutathionylation of EP24.15 was responsible for its oxidative oligomerization to dimer and trimer complexes. EP24.15 immunoprecipitated from cells submitted to oxidative challenge showed increased trimeric forms and decreased S-glutathionylation compared to immunoprecipitated protein from control cells. Our present data also show that EP24.15 maximal enzymatic activity is maintained by partial S-glutathionylation, a mechanism that apparently regulates the protein oligomerization. Present results raise the possibility of an unconventional property of protein S-glutathionylation, inducing oligomerization by interprotein thiol-disulfide exchange.