Possible role of ionic gradients in the apical growth of <Emphasis Type="Italic">Neurospora crassa</Emphasis>

The effects of the Ca²⁺/H⁺ exchanger A23187 and the K⁺/H⁺ exchanger nigericin on the growth of Neurospora crassa were analyzed. Both ionophores had the same effects on the fungus. They both inhibited growth in liquid media, apical extension being more affected than protein synthesis. A sudden challenge to either ionophore on solid media rapidly stopped hyphal extension. Additionally, both ionophores induced profuse mycelium branching and upward hyphal growth. Hyphae growing on nigericin-containing media also burst at the apex. Both ionophores caused a rapid inhibition in the apically-occurring synthesis of structural wall polysaccharides, but they did not affect mitochondrial energy conservation. With the use of DiBAC, a membrane-potential sensitive fluorophore, it was excluded that their effects were due to depletion of the plasma membrane potential. Considering that both ionophores exchange H⁺ for different metallic ions, we concluded that their effect was due to dissipation of a proton gradient, which is directly or indirectly involved in the apical growth of the fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

In vitro response to Candida albicans in cultures of whole human blood from young and aged donors

Invasive infections with opportunistic fungi, such as Candida albicans, have become an increasing problem in aged adults in recent years. This work investigates the influence of human ageing on C. albicans recognition by toll-like receptors (TLRs), essential components of the innate immune system, using a cohort of 96 young (15-42 years) and aged (>70 years) human volunteers. No significant differences between aged and young donors were observed on (1) cell surface TLR2, TLR6 and TLR4 expression on lymphocytes, monocytes and granulocytes, (2) production of cytokines [IL-8, IL-1beta, IL-6, IL-10, tumour necrosis factor (TNF)-alpha and IL-12p70] and prostaglandin E(2) (PGE(2)) by whole human blood in response to C. albicans and (3) fungicidal activity of whole blood. A statistically significant higher titre of natural anti-C. albicans antibodies was found in plasma of volunteers between 80 and 95 years old when compared with other age groups, probably as a consequence of the increased levels of serum Ig that has been described in elderly subjects. Therefore, the results indicate that the increased susceptibility to C. albicans infections in the elderly is not a consequence of defects in TLRs expression or signalling, nor of an impaired fungicidal activity of blood.     

<Emphasis Type="Italic">RNase</Emphasis>-Based Gametophytic Self-Incompatibility Evolution: Questioning the Hypothesis of Multiple Independent Recruitments of the <Emphasis Type="Italic">S</Emphasis>-Pollen Gene

Multiple independent recruitments of the S-pollen component (always an F-box gene) during RNase-based gametophytic self-incompatibility evolution have recently been suggested. Therefore, different mechanisms could be used to achieve the rejection of incompatible pollen in different plant families. This hypothesis is, however, mainly based on the interpretation of phylogenetic analyses, using a small number of divergent nucleotide sequences. In this work we show, based on a large collection of F-box S-like sequences, that the inferred relationship of F-box S-pollen and F-box S-like sequences is dependent on the sequence alignment software and phylogenetic method used. Thus, at present, it is not possible to address the phylogenetic relationship of F-box S-pollen and S-like sequences from different plant families. In Petunia and Malus/Pyrus the putative S-pollen gene(s) show(s) variability patterns different than expected for an S-pollen gene, raising the question of false identification. Here we show that in Petunia, the unexpected features of the putative S-pollen gene are not incompatible with this gene’s being the S-pollen gene. On the other hand, it is very unlikely that the Pyrus SFBB-gamma gene is involved in specificity determination. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Supra-additive stimulation of adenylate cyclase activity by prostaglandin E2 andD-Ala2-met-enkephalinamide in the guinea-pig superior cervical ganglion: Role of Mg2+ ions

Agonists modulation of Mg²⁺-dependent adenylate cyclase activity has been studied in guinea-pig superior cervical ganglion crude membrane preparations. In the absence of receptors ligands, Mg²⁺ stimulates the enzyme in a concentration-dependent manner. The dose-activation curve shows heterogeneity and two components with higher and lower apparent affinity states, are extrapolated. In the presence ofD-Ala²-met-enkephalinamide only one component is present and the apparent affinity of the ganglionic adenylate cyclase system for the divalent cation as well as Vmax are inhibited. On the contrary, prostaglandin E₂ increases affinity and Vmax values of the lower and, to a lesser extent, of the higher Km component. When the two drugs are tested in combination, not only the inhibitory effect of the opiate is overcome, but a large increase of the apparent affinities and Vmax values for both components is obtained, suggesting the involvement of the Mg²⁺-regulated subunits of the adenylate cyclase system in the supra-additive stimulation mechanism of the enzyme.     

Absence of Gup1p in Saccharomyces cerevisiae results in defective cell wall composition, assembly, stability and morphology

Saccharomyces cerevisiae Gup1p and its homologue Gup2p, members of the superfamily of membrane-bound O-acyl transferases, were previously associated with glycerol-mediated salt-stress recovery and glycerol symporter activity. Several other phenotypes suggested Gup1p involvement in processes connected with cell structure organization and biogenesis. The gup1Delta mutant is also thermosensitive and exhibits an altered plasma membrane lipid composition. The present work shows that the thermosensitivity is independent of glycerol production and retention. Furthermore, the mutant grows poorly on salt, ethanol and weak carboxylic acids, suggestive of a malfunctioning membrane potential. Additionally, gup1Delta is sensitive to cell wall-perturbing agents, such as Calcofluor white, Zymolyase, lyticase and sodium dodecyl sulphate and exhibits a sedimentation/aggregation phenotype. Quantitative analysis of cell wall components yielded increased contents of chitin and beta-1,3-glucans and lower amounts of mannoproteins. Consistently, scanning electron microscopy showed a strikingly rough surface morphology of the mutant cells. These results suggest that the gup1Delta is affected in cell wall assembly and stability, although the Slt2p/MAP kinase from the PKC pathway was phosphorylated during hypo-osmotic shock to a normal extent. Results emphasize the pleiotropic nature of gup1Delta, and are consistent with a role of Gulp1p in connection with several pathways for cell maintenance and construction/remodelling.     

Intron-mediated gusA expression in tritordeum and wheat resulting from particle bombardment

The promoterless maize ubiquitin first exon and intron fragment can drive gusA expression in immature tritordeum inflorescences and immature wheat scutella. In fluorescence assays, this fragment induces gusA expression in tritordeum inflorescences to 50 times higher than background. The activity of the complete promoter, exon and intron cassette was up to 20000-fold higher than background but the maize ubiquitin promoter in isolation had very low activity. A construct with the maize alcohol dehydrogenase first exon and intron had low activity, visible in histochemical assays. Both intron sequences have promoter-like features and in the ubiquitin intron there is a sequence homologous to the opaque-2-binding box. We suggest that the combination of these elements may explain the promoter activity detected in these introns.     

ERK signaling is required for eye-specific retino-geniculate segregation

In the mammalian visual system, retinal ganglion cell (RGC) projections from each eye, initially intermixed within the dorsal-lateral geniculate nucleus (dLGN), become segregated during the early stages of development, occupying distinct eye-specific layers. Electrical activity has been suggested to play a role in this process; however, the cellular mechanisms underlying eye-specific segregation are not yet defined. It is known that electrical activity is among the strongest activators of the extracellular signal-regulated kinase (ERK) pathway. Moreover, the ERK pathway is involved in the plasticity of neural connections during development. We examine the role of ERK in the segregation of retinal afferents into eye-specific layers in the dLGN. The activation of this signaling cascade was selectively blocked along the retino-thalamic circuitry by specific inhibitors, and the distribution of RGC fibers in the dLGN was studied. Our results demonstrate that the blockade of ERK signaling prevents eye-specific segregation in the dLGN, providing evidence that ERK pathway is required for the proper development of retino-geniculate connections. Of particular interest is the finding that ERK mediates this process both at the retinal and geniculate level.