4). Cells were post-fixed for 1 h in the dark with a solution containing 1% osmium tetroxide, 1.25% potassium ferrocyanide and 5 mM CaCl2, in 0.1 M sodium cacodylate buffer (pH 7.4). Samples were dehydrated with increasing concentrations Proteasome inhibitor of acetone, and then embedded in PolyBed (Polyscience

Inc., Warrington, PA, USA). Ultrathin sections were stained with uranyl acetate and lead citrate and then observed using a Zeiss 900 Electron Microscope (Carl Zeiss, Inc.). For detection of polysaccharide inclusions, ultrathin sections of samples prepared for transmission electron microscopy, as described above, were processed for cytochemical detection of carbohydrates (Thiéry, 1967). Tissue cysts were used as a positive control for amylopectin granules. Cysts were obtained from mice previously infected with T. gondii strain Me49 for at least 4 weeks, based on the protocol established by Freyre (1995). Ultrathin sections collected on 200-mesh gold grids were incubated in 1% periodic acid for 30 min, washed in distilled water and incubated with 1% thiosemicarbazide in 10% acetic acid for 72 h. Next, the sections were washed in 10%, 5% and 2% acetic acid and 3 times in distilled

water for 10 min. Afterwards, the sections were incubated for 30 min with 1% silver proteinate in the dark and washed abundantly in distilled water. For control assays, periodic acid was omitted. The sections were observed in a Jeol 1200 EX transmission electron microscope operating at 80 kV. For imunofluorescence assays, LLC-MK2 cells infected with tachyzoites at a ratio of 3:1 parasite/host cell were treated with compounds 1, 2 or 3 for 48 h. At the end of treatment, www.selleckchem.com/products/bmn-673.html infected cells were fixed in 3.7% freshly prepared formaldehyde, permeabilized with 0.5% Triton X-100 for 15 min and blocked with 3% bovine serum albumine in PBS pH 7.4 for 1 h at room temperature. Cells were then incubated for 1 h in the presence of Dolichos biflorus lectin conjugated with FITC (DBA-FITC) 10 μg/ml (Sigma–Aldrich Co., St. Louis, MO, USA). After lectin labeling, the coverslips were mounted

and observed in a no Zeiss Axioplan microscope using the fluorescein filters. The azasterols inhibited T. gondii proliferation with IC50 values in the micromolar range. Table 1 shows the in vitro anti-proliferative activity of the azasterols. Compound 3 was the most active, showing an IC50 at nanomolar range after 48 h. The anti-proliferative activity range of the new compounds (0.8–4.7 μM) was of the same order as that previously obtained by our group for 22,26-azasterol and 24,25-(R,S)-epiminolanosterol ( Dantas-Leite et al., 2004). These results confirm that azasterols can cause growth inhibition of T. gondii, across a variety of different structural types. Interestingly, compounds do not necessarily need to have a basic nitrogen as can be seen from compounds 2 and 3, which has implications for the mode of action. In order to investigate the selective effect of the azasterols against T.