Growth and morphogenesis ofBotrytis cinerea. Effects of exogenous calcium ions,calcium channel blockers and cyclosporin A

Calcium channel blockers, verapamil, nitrendipin and nifedipin, and cyclosporin A inhibited growth of colonies ofBotrytis cinerea in a concentration-dependent manner and simultaneously induced morphological changes of its hyphal tips. Exogenous calcium at the concentration of 100 mmol/L decreased the growth-inhibitory effects of channel blockers and cyclosporin A; however, at the concentration of 500 mmol/L Ca²⁺ their inhibitory effects were increased. At the latter concentration, calcium partly reversed the morphogenic effects of the blockers but not of cyclosporin A.     

Regulation of periodicity and intensity of photo-induced conidiation ofTrichoderma viride

After a short irradiation at 366 nm with 200 lx, the intensity of conidiation ofTrichoderma viride colonies grown in the dark increased for the first 10 s proportionally with time. The increase slowed down after 10 s—6 min of exposure and after 10–60 min of irradiation the conidiation intensity began to decrease. When photo-induced by daylight, the conidiation started at a high rate after 25 h and persisted even after 48 h. The conidiation had no circadian character and its periodicity depended on the periodicity of photo-induction. Its intensity was also influenced by the carbon sources used, a maximum being reached with glucose (1–2 %). Higher glucose concentrations inhibited conidiation but had no influence on growth of colonies.     

Effects of agents affecting Ca2+ homeostasis onTrichoderma viride growth and conidiation

Agents known to influence Ca²⁺ homeostasis affected significantly the vegetative growth and starvation-induced conidiation ofTrichoderma viride. Ca²⁺ in millimolar concentrations stimulated both growth and conidiation; a Ca²⁺ deprivation of the fungus by the chelation of extracellular Ca²⁺ (not Mg²⁺ or divalent trace metals) with EGTA (ethyleneglycolbis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid) restricted both the vegetative growth rate and starvation-induced conidiation. Both processes were affected by either Ca²⁺ or EGTA with different efficiencies. Divalent cations (Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺, Mg²⁺, Cd²⁺, Cu²⁺, Mn²⁺) and La³⁺ (inorganic Ca²⁺ blockers) in millimolar concentrations exerted complex (stimulatory, inhibitory, or biphasic) effects on growth and conidiation. In general, their effects on the two processes were mutually different either qualitatively, or quantitatively, or both. Organic Ca²⁺ antagonists (verapamil and dihydropyridines) inhibited the vegetative growth. The results show that Ca²⁺ is required for vegetative growth and conidiation, and that different Ca²⁺-dependent mechanisms may be involved in the two processes. Divalent cations could serve as a tool for investigating the relationship between growth and conidiation.     

Vanadate inhibits the production and/or release of secondary metabolites without impairing growth in several fungal species

Vanadate (NaVO₃) in concentrations between 0.1–3.0 mmol/L inhibited the production of secondary metabolites (SMs) of strains of the following species:Trichoderma viride, Penicillium purpurogenum, Penicillium citrinum, Talaromyces avellaneus, andVerticillium psalliotœ. Growth was either not affected by NaVO₃, or the inhibition of the SM production occurred at lower NaVO₃, concentrations than that of the growth. Thus, at some NaVO₃ concentration the SM production was inhibited but the growth remained unaffected. The results suggest that NaVO₃ exerts a specific action either on the SM biosynthetic pathway(s) or on the export of SMs from cells.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Structural and biochemical characterization of toad liver fatty acid-binding protein

Two paralogous groups of fatty acid-binding proteins (FABPs) have been described in vertebrate liver: liver FABP (L-FABP) type, extensively characterized in mammals, and liver basic FABP (Lb-FABP) found in fish, amphibians, reptiles, and birds. We describe here the toad Lb-FABP complete amino acid sequence, its X-ray structure to 2.5 A resolution, ligand-binding properties, and mechanism of fatty acid transfer to phospholipid membranes. Alignment of the amino acid sequence of toad Lb-FABP with known L-FABPs and Lb-FABPs shows that it is more closely related to the other Lb-FABPs. Toad Lb-FABP conserves the 12 characteristic residues present in all Lb-FABPs and absent in L-FABPs and presents the canonical fold characteristic of all the members of this protein family. Eight out of the 12 conserved residues point to the lipid-binding cavity of the molecule. In contrast, most of the 25 L-FABP conserved residues are in clusters on the surface of the molecule. The helix-turn-helix motif shows both a negative and positive electrostatic potential surface as in rat L-FABP, and in contrast with the other FABP types. The mechanism of anthroyloxy-labeled fatty acids transfer from Lb-FABP to phospholipid membranes occurs by a diffusion-mediated process, as previously shown for L-FABP, but the rate of transfer is 1 order of magnitude faster. Toad Lb-FABP can bind two cis-parinaric acid molecules but only one trans-parinaric acid molecule while L-FABP binds two molecules of both parinaric acid isomers. Although toad Lb-FABP shares with L-FABP a broad ligand-binding specificity, the relative affinity is different.     

Inhibition of photo-inducedTrichoderma viride conidiation by inhibitors of RNA synthesis

The photo-induced conidiation ofTrichoderma viride is suppressed by ethidium bromide, acriflavin, lomofungin and 8-quinolinol at concentrations which do not inhibit the colony growth of this deuteromycete.     

Screening for alkaline keratinolytic activity in fungi isolated from soils of the biosphere reserve “Parque Costero del Sur” (Argentina)

A screening was carried out on 69 fungal strains isolated from alkaline-calcareous, neutral and alkaline-sodic soils, as well as from their associated plant material, to determine their ability to grow at alkaline pH. A total of 32 fungi were selected for their ability to produce alkaline keratinase activity in submerged shaken cultures supplemented with soybean meal (SM) and tryptone and on cow hair (CH) under solid state fermentation conditions. Although several fungal strains produced keratinolytic activity on both SM and CH, they differed in the levels detected. Among them, Aspergillus niger, Cladosporium cladosporioides, Metarrhizium anisopliae, Neurospora tetrasperma and Westerdikella dispersa were the best producers, with levels higher than 1.2 U ml⁻¹. Different fungal species are here reported for the first time for their ability to produce keratinolytic activity at alkaline pH.     

The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.