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note
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3): δ 138.5, 134.9, 124.4, 124.3, 59.3, 59.1, 45.3, 39.5, 37.2, 26.2, 25.5, 16.3, 16.2.
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42
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33845800025
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note
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4) at a flow rate of 0.7 ml/min and a sample injection volume of 20 μl, λ = 220 nm. For all compounds, eluants A and B were delivered isocratically at a 20:80 ratio. All four compounds displayed a purity of at least 95%.
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43
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33845742786
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note
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The S. cerevisiae strains: SMY8[pBJ1.21], expressing the OSC of T. cruzi; SMY8[pSM61.21], expressing the wild-type yeast OSC; SMY8[pBJ4.21], expressing the P. carinii OSC were kindly provided by Professor S. P. T. Matsuda [Department of Chemistry and Biochemistry and Cell Biology, Rice University, Houston, TX, USA. For references and cultural conditions see Refs. 11, 13, and 24. The enzymatic activity was determined as described in Refs. 13 and 24.
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44
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45
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33845756541
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note
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Pichia pastoris cells expressing human OSC were kindly provided by Ralf Thoma from Hoffmann-La Roche Ltd, Pharma Research Discovery (CH-4070 Basel, Switzerland). Cultural conditions were as in Ref. 22 and cell-free homogenates were prepared as described for S. cerevisiae cells in Ref. 13. Enzymatic assays were determined in phosphate buffer, pH 8, at 45 °C as described in Ref. 13.
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46
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11844279023
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48
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33845763147
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note
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4 cells were suspended and grown for 24 h in 950 μl DMEM containing 10% (v/v) lipid-depleted serum. After a 3 h preincubation in the presence of inhibitor, labeled acetate (1 μCi/well) was added. After further 3 h incubation, the medium was removed and the cells were lysed with 500 μl of 0.1 N NaOH for 30 min. After saponification with 1.5 ml of 10% (w/v) methanolic KOH for 1 h at 80 °C, nonsaponifiable lipids were extracted with petroleum ether and separated on TLC plates (Alufolien Kieselgel 60F254, Merck, Darmstadt, Germany) using n-hexane/ethyl acetate (85:15 v/v) as a developing solvent and authentic standards of cholesterol, lanosterol, dioxidosqualene, oxidosqualene, and squalene as reference compounds. Radioactivity in separated bands was measured using a System 200 Imaging Scanner (Hewlett-Packard, Palo Alto, CA, USA).
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