, 1997). Phylogenetic trees were constructed using the neighbor-joining method (Saitou & Nei, 1987). The robustness of the tree topology was calculated from bootstrap

analysis using 1000 resamplings of the sequences (Felsenstein, 1985). The 16S rRNA gene sequences of the six hmgr gene-positive strains were Selleckchem PARP inhibitor submitted to the DNA Data Bank of Japan and were assigned the following accession numbers: SpC080624SC-11 (AB514576), Sp080513SC-18 (AB498736), Se080624GE-07 (AB514578), SpA080624GE-02 (AB514579), SpC080624GE-05 (AB514580), and Sp080513GE-23 (AB498636); the accession numbers for their corresponding hmgr genes are AB514576, AB514577, AB514578, AB514579, AB514580, and AB514581, respectively. The production medium for SpC080624SC-11 and SpA080624GE-02 consisted of 2% glycerol (Nacalai Tesque, Kyoto, Japan), 1% molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan), 0.5% casein (Kanto Chemical, Tokyo, Japan), 0.1% polypeptone (Nihon Pharmaceutical, Tokyo, Japan), 0.4% CaCO3 (Kozaki Pharmaceutical, Tokyo, Japan), 1% HP-20 (Mitsubishi Chemical, Tokyo, Japan), and 1.75% Sealife (pH 7.2 before sterilization). The

production medium for Sp080513GE-23 and SpC080624GE-05 contained 2.5% starch (Kosokagaku, Tokyo, Japan), 1.5% soybean meal (Nisshin Oillio, Tokyo, Japan), 0.2% dry yeast (Mitsubishi Tanabe Pharma, Osaka, check details Japan), and 0.4% CaCO3 (Kozaki Pharmaceutical) (pH 6.2 before sterilization). These strains were cultured on a rotary shaker (180 r.p.m.) at 27 °C for 5 days in 500-mL Erlenmeyer flasks containing 100 mL of the production Orotidine 5′-phosphate decarboxylase medium. The mycelial extract or the supernatant of the fermentation broth of Actinobacteria was extracted with ethyl acetate, and the organic layer was evaporated to dryness. The dried residue was separated using normal-phase medium-pressure liquid chromatography (LC). The fractions containing isoprenoids were further purified by preparative reversed-phase HPLC. The structures of active compounds were determined on the basis of HPLC-MS and nuclear magnetic resonance spectroscopic data. Human acute myelogenous leukemia HL-60 cells were cultured in Roswell Park Memorial Institute medium

(Nacalai Tesque) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 U mL−1), and streptomycin (100 μg mL−1) at 37 °C in a humidified incubator with 5% CO2. The cytotoxic activity was estimated by a WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] colorimetric assay. HL-60 cells were incubated on 384-well plates at a density of 1 × 103 cells per well in 20 μL of medium overnight, and then treated with compounds at various concentrations for 48 h. Next, 2 μL of WST-8 reagent solution (Cell Counting Kit, Dojindo, Kumamoto, Japan) was added and incubated for an hour at 37 °C in a humidified incubator with 5% CO2. The A450 nm of the formazan dye formed was measured.