05. Data BTK high throughput screening was manually checked for validation. The N-terminal sequences of French bean thaumatin-like protein, French bean antifungal peroxidase, pinto bean chitinase (phasein A), and pea defensins (PSDs) were taken from [28]. The alignment of these sequences with the major urease of C. ensiformis (NCBI gi 167228) was performed with the ClustalW program [21], using the BLOSUM matrix [19]. The regions of urease which are similar to these antifungal proteins were colored

manually with the UCSF Chimera molecular viewer [30]. The growth assays were performed according to [34]. Yeast cells of C. tropicalis, C. albicans, C. parapsilosis, S. cerevisiae, K. marxianus and Pichia membranisfaciens were set to multiply in Petri dishes containing Sabouraud agar for 24 or 48 h at 30 °C. For the assay, cells were removed with the aid of a sowing handle, and added to 10 mL of Sabouraud culture medium. The test samples were added to cells (1 × 104 per mL) http://www.selleckchem.com/products/gsk269962.html and growth was evaluated by turbidity readings at a wavelength of 620 nm for a period of 24–48 h. The tests were performed in 96 well plates, U-bottom and read in a plate reader (Reader 400 EZ – Biochrom). To evaluate the reversibility of the antifungal effect and discriminate fungistatic

versus fungicidal activity, yeasts (104) were incubated with 0.36 μM JBU or buffer for 24 h at 28 °C. Then 10-fold serial dilutions of the incubated yeasts were made in fresh Sabouraud medium and plated in Sabouraud agar. The number of CFU PLEK2 in the 106-fold dilution after 24 h at 28 °C was determined under a microscope. The fungi

were grown for 14 d on PDA at 28 °C. To obtain the spores, 5 mL of sterile saline were added to each Petri dish and the colonies gently washed with the tip of a pipette. To evaluate the hyphal growth, the experiment was made according to [7]. The spore suspension (1 × 106 spores per mL) was inoculated into 96 well plates containing potato dextrose broth (PDB), incubated at 28 °C for 16 h, and then the test samples (up to 80 μL) were added. The final volume in each well was 200 μL. The dialysis buffer (Tris 10 mM pH 6.5) was used as negative control and 0.1% hydrogen peroxide (H2O2), as a positive control. The plates were incubated at 28 °C and monitored turbidimetrically at 620 nm at 12 h intervals for 96 h. Alternatively, spores were incubated with the samples for 96 h at 28 °C and then germination was monitored by turbidity. The tests were performed in triplicate and data presented as means and standard deviations. Glucose-stimulated acidification of the medium results from extrusion of H+ by the cells, through a H+-ATPase pump in the plasma membrane [18]. We evaluated the effects of JBU and peptide(s) on this metabolic activity of S. cerevisiae and C. albicans, as described in [34].