Suspension was stirred for another 30 min. The bcr-abl Inhibitors methylated product was extracted into chloroform and washed with water. The permethylated oligosaccharides were then hydrolyzed in 2 M trifluoroacetic acid at 110 for 2 h. The partially methylated monosaccharides were reduced with 1% NaB2H4 at room temperature for 18 h. Following borate removal by drying from methanol, the partially methylated alditols were acetylated by adding pyridine/acetic anhydride and incubating at 50 for 3 h. The reagents were evaporated and the sample was dissolved in methanol. Gas chromatography/mass spectrometry The partially methylated alditol acetates were analyzed by GC/MS using a capillary column with helium as the carrier gas and a temperature program. The GC/MS analyses were carried out on a JMS K9. NMR spectroscopy Samples were exchanged twice in 2H2O with intermediate lyophilization, then dissolved at 1% in 2H2O. The NMR spectra were recorded by a JNM LA600spectrometer at 45. DQF COSY was performed using the standard pulse sequence. The proton chemical shift was referenced relative to the internal acetone at 2.225. 2,3 Mannosidase treatment Two mg of the 1,6 linkage containing oligosaccharide obtained by acetolysis, was dissolved in 100 of 50 mM sodium acetate buffer containing 5 mM CaCl2 and 10 /100 BSA. To the reaction mixture, 100 units of 2,3 mannosidase was added and digested at 37 for 48 h. The reaction products were separated using a Bio Gel P 2 column. Drug susceptibility assays Stationary phase yeast cells were diluted to 5 x 105 cells/ml, and 3 of a series of 5 fold dilutions was spotted onto YPD agar plates supplemented with various concentrations of itraconazole, micafungin, hygromycin B, and calcofluor white. Growth was scored after incubation for 2 days at 28. The concentration of drugs used was 200 ng/ml itraconazole, 6 ng/ml micafungin, 200 /ml hygromycin B, 1 M NaCl, and 400 /ml calcofluor white.
Total carbohydrate content The carbohydrate content was determined by the phenol/sulfuric acid method with D mannose as the standard. Difference in the 1H NMR spectra of the mannans from three C. glabrata strains We first checked the influence of the probe temperature on the 1H NMR chemical shift of the mannan from C. glabrata NBRC 0622. As shown in Fig. 1, the H 1 chemical shifts of the mannan were unexpectedly affected by the temperature. At 70, the 1H Vincristine NMR pattern was relatively simple and showed a sharp signal at 5.076 ppm which is consistent with previous observations. However, at 45, the H 1 signal was separated into two signals, 5.065and 5.084 ppm, and the separation of the signals was enhanced at 25. In keeping with this, the H2O signal was shifted downfield and overlapped with the signal of a 1,2 linked mannose residue, which is known to appear at 4.77 4.84 ppm. Therefore, in this experiment, we recorded the 1H NMR spectra at 45. We first compared the 1H NMR spectra and their DQF COSY of the mannans from C. glabrata NBRC 0005, 0622, and 103857 strains and the mannan of S. cerevisiae as the control. As shown in Fig. 2, there is a significant difference in the intensity of the signal at 4.788 ppm, which corresponds to the non reducing terminal 1,2 linked mannose residue, among the three C. glabrata strains.