Species diversity, novel interactions and absence of well-supported host-guided phylogenetic groupings of Neotropical Alternaria isolates causing foliar lesions in Solanaceae.

AIM: To report the characterization of 120 Alternaria isolates inducing early blight-like foliar lesions in nine species of five Solanaceae genera collected across all macrogeographical Brazilian regions. MATERIAL AND RESULTS: Phylogenetic relationships were assessed via analyses of the Alternaria alternata allergenic protein-coding, glyceraldehyde-3-phosphate dehydrogenase and the calmodulin gene sequences. Most of the tomato isolates were placed into the Alternaria linariae cluster, whereas most of the potato isolates were grouped with Alternaria grandis. Novel host-pathogen interactions were also reported. Seventeen isolates were selected for morphometrical characterization, and a subsample of 13 isolates was employed in pathogenicity assays on tomato, potato, eggplant, scarlet eggplant, Capsicum annuum, Datura stramonium, Physalis angulata and Nicotiana tabacum. Eleven isolates were able to induce foliar lesions in tomatoes but none in C. annuum. Potato was susceptible to a subgroup of isolates but displayed a subset of isolate-specific interactions. Morphological traits were in overall agreement with molecular and host range data. CONCLUSION: Alternaria linariae and A. grandis were confirmed as the major causal agents of tomato and potato early blight, respectively. However other Alternaria species are also involved with early blight in solanaceous hosts in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity and host-specific patterns of the Alternaria isolates from Solanaceae may have practical implications in establishing effective early blight genetic resistance and cultural management strategies especially for tomato and potato crops.

NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia.

Changes in high-energy phosphate metabolites (ATP and phosphocreatine) were monitored, in real time, by 31P-nuclear magnetic resonance in primary cell cultures of neurons and astrocytes during periods of hypoxia, ischemia and hypoglycemia, and also during the recovery periods following the re-establishment of standard conditions. Cells were immobilized in basement membrane gel threads and perfused with oxygen-depleted medium (oxygen concentration below 30 microM), to create hypoxic conditions, or with aerobic medium (oxygen concentration approximately 460 microM) containing different concentrations of glucose (hypoglycemia). Ischemic conditions were imposed by stopping perfusion for different periods of time (15 min to 2 h). The experimental set-up enabled the acquisition of 31P-spectra with high signal-to-noise ratio within 10-20 min for both cell types. The effect of hypoxia on glucose metabolism was assessed by 13C-NMR using [1-13C]glucose as substrate. The levels of ATP and PCr in astrocytes were unaffected during hypoxia (up to 2 h), but decreased notably under ischemia. In neurons, hypoxic periods caused a sharp drop of the ATP and PCr levels, and considerable damage to the capacity of neurons to replenish the ATP and PCr pools upon returning to normoxic conditions. However, neurons were remarkably less sensitive to ischemic conditions, the ATP and PCr pools being restored quickly, even after 2 h under challenging conditions. The data show that neurons were more resistant to ischemia than astrocytes, and suggest that the capacity to sustain the pools of ATP and PCr was part of the neuronal protective strategy.

Proteolytic activity in infected and noninfected insect cells: degradation of HIV-1 Pr55gag particles.

In this work the proteolytic activity in the supernatant and inside insect cells in culture was evaluated for different multiplicities of infection (MOI) and times of infection (TOI). Several methods to detect proteolytic activity in insect cells were tested and that using fluorescein thiocyanite-casein as a substrate was chosen. It was observed that infection caused not only a reduction in the concentration of proteases by decreasing their synthesis but also an inhibition of the intracellular proteolytic activity by increasing the intracellular ATP level (measured by in vivo nuclear magnetic resonance, NMR). The maximum proteolytic activity in the supernatant was observed at 72 hpi except when the cells were infected in the late exponential growth phase or with very low MOI, yielding a nonsynchronous infection. The proteolytic degradation of Pr55gag particles was studied during culture and after harvest. In this particular case it was concluded that the supernatant should be stored at low temperature or quickly purified, since the degradation after 24 h is only 3% at 4 degrees C while at 27 degrees C this value rises to 23%. There is a complex relationship between MOI, TOI, proteolytic activity, and product titer and quality. Thus, the optimal conditions for each case will be a compromise between the final product titer, the desired product quality, and operational issues like process time and capacity, requiring proper integration between bioreaction and downstream processing.

Optimization of the production of virus-like particles in insect cells.

In this work the maximal operational hydrodynamic conditions (agitation and aeration rate) that cause no adverse effect in Sf-9 cells growth in SF900II serum-free medium were determined. Shear stresses higher than 1 N m-2 and aeration rates higher than 0.04 vvm affect cell growth and when these conditions increase to 1.5 N m-2 and 0.11 vvm, cell growth is completely inhibited with significant cell morphology changes and a strong decrease in viability. Although the pO2 did not show a significant effect upon cell growth in the range from 10 to 50%, cell infection and specific productivity were dramatically affected. The production was optimal at a pO2 of 25% with decreases higher than 50% being observed when the pO2 decreased to 10 or increased to 50%. The maximum product quality, i.e., the percentage of product in the form of high molecular weight particles, is not coincident with maximum product titer. Although the highest Pr55gag particle titer was obtained at 96 hours post infection (hpi) and at pO2 of 25%, the best product quality (defined by gel filtration chromatography and Western immunoblot) was obtained at 48 hpi, independently of the pO2 used. The effect of overcritical conditions upon productivity was also studied. As obtained for cell growth, cell infection is affected by shear stresses above 1 N m-2 and by aeration rates higher than 0.04 vvm, with decreases in Pr55gag particle titer higher than 70%, even when the overcritical values are still far from the limit at which cell death occurs. The results obtained and the optimization strategy used allowed the maximization of the oxygen supply without damaging the cells, with important consequences on the scale-up of a production process involving this insect cell/baculovirus expression system.

Revascularization of the ischaemic hand using in situ veins.

Chronic severe ischaemia of the upper limb is rare, due to the low incidence of arterial disease and to the presence of an adequate pre-existent collateral network. However, sometimes it occasionally occurs, dictating the need for revascularization. Since 1990, the authors have revascularized five upper extremities with severe ischaemic changes, employing the cephalic and/or basilic veins, used in situ. The aetiology of the arterial disease was autoimmune in one case and late sequelae of trauma in the others. Of the in situ venous bypasses performed, three were brachial-radial, and two sequential grafts (one axillary-radial-ulnar and one axillary-brachial-radial-ulnar). One concomitant sympathectomy was performed. All patients had excellent early and late results which leads the authors to recommend such revascularization of the forearm and hand, especially in the presence of severe ischaemia.

Biochemical correlate of depression in children.

The degree of depression in 88 abandoned children was analysed through a depression rating scale adapted to prepubertal children. The items were grouped into three dimensions: sociological-relational, psychological and biological. In 46 children from this sample it was dosed plasmatic cortisol and the urinary excretion of catecholamine, VMA, HVA and 5-HIAA. When analysing the principal components, the sociological and psychological dimensions were the most important ones in the sample, followed by age and catecholamine variables. The group of male depressed children presented a higher level of catecholamine urinary excretion and a lower peak of plasmatic cortisol than the non-depressed group. The variable age, in both sexes, was correlated with the biochemical variable catecholamine. Biochemical alterations are present in depressed children but it is difficult to show a correlation of dependence between them and the phenomenological aspects of depression.

Biochemical correlate of depression in children

O grau de depressäo em 88 crianças abandonadas foi analisado por escala de avaliaçäo de depressäo adaptada para crianças na pré-puberdade. Os itens da escala foram agrupados em três dimensöes: sociológico-relacional, psicológica e biológica. Em 46 crianças desta amostra foram dosados o cortisol plasmático e a excreçäo urinária de catecolomina, VMA, HVA e 5-HIAA. Pela análise dos principais componentes, mostraram-se mais importantes, na amostra, as dimensöes sociológica e psicológica, seguidas dos componentes idade e catecolomina. O grupo de crianças do sexo masculino com depressäo apresentava maior nível de excreçäo urinária de catecolomina e menor pico de cortisol plasmático que o grupo sem depressäo. A variável idade, em ambos os sexos, correlacionava-se à variável catecolomina. Alteraçöes bioquímicas estäo presentes em crianças com depressäo, mas é difícil demonstrar correlaçäo de dependência entre elas e aspectos fenomenológicos da depressäo