Ase in the freshly ready two-phase Bligh-Dyer mixture (chloroform/methanol/water, two:2:1.8 (v/v/v)). The pure lipid A preparations (B. japonicum, 36 mg; B. yuanmingense, 18 mg; Bradyrhizobium sp. (Lupinus), 12 mg) were stored at 20 in CHCl3/MeOH (3:1, v/v). O-Deacylation of lipid A samples was performed by incubation (1? mg) in chloroform, methanol, 0.six M aqueous NaOH, two:3:1 (v/v/v), for 1.five h at area temperature, according to Que-Gewirth and co-workers (37). Fatty Acids, Hopanoid Lipids, and Sugars Analysis–For total fatty acid and hopanoid lipids determination, lipid A preparations were subjected to hydrolysis in four M HCl (one hundred , four h). Liberated fatty acids and hopanoids had been extracted with chloroform and converted to their methyl esters with diazomethJOURNAL OF BIOLOGICAL CHEMISTRYDECEMBER 19, 2014 ?VOLUME 289 ?NUMBERHopanoid-containing Lipid A of Bradyrhizobiumane. Just after evaporation to dryness, hydroxyl groups of fatty acids and hopanoid lipids were derivatized with BSTFA (16 h at room temperature). Neutral and amino sugar analyses have been performed in accordance with typical protocols described elsewhere (21). GC-MS analyses of fatty acids and sugars had been performed on a Hewlett Packard gas chromatograph 5890 series II and Agilent Technologies GC Program 7890A connected to a mass selective detector EI/CI MSD 5975C, equipped with a HP-5MS column (30 m 0.25 mm) with helium as a carrier gas (flow price: 0.7 ml min 1). The temperature system was as follows: 150 for three min, then raised to 250 at 3 min 1, then to 320 , 25 min 1. The final temperature was kept for 10 min for sugar analysis and 20 min for fatty acid evaluation. Mass Spectrometry–Lipid A samples obtained from B. japonicum had been analyzed on a high resolution hybrid Fourier transform ion cyclotron resonance mass spectrometry (FTICR) instrument (Apex Qe Bruker Daltonics, Billerica, MA) with electrospray ionization (ESI), equipped with a 7 tesla actively shielded magnet. Samples for analysis have been ready as described earlier (21) and measured within the negative ion mode. Mass Caspase 8 Inhibitor Biological Activity Spectra were charge deconvoluted and mass numbers provided refer towards the monoisotopic peaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed with a Bruker-Daltonics Reflex III instrument (Bruker Daltonics, Bremen, Germany) at an acceleration voltage of 20 kV and delayed ion extraction. Lipid A preparations were dispersed in methanol/water (1:1, v/v) at a concentration of 1 g/ l with addition of 20 mM EDTA. The matrix option was prepared from 2,5dihydroxybenzoic acid in 1 trifluoroacetic acid as well as the spectra had been recorded in constructive or negative ion modes. NMR Spectroscopy–For NMR analysis a sample containing 18 mg of native lipid A from B. japonicum dissolved in 0.6 ml of CDCl3/CD3OD (2:1, v/v) with five l of D2O, was applied. One- and two-dimensional NMR spectra have been recorded at 700 MHz on an AVANCE III spectrometer with Cryoprobe (Bruker) making use of Bruker computer software. Spectra have been recorded at 27 . The following two-dimensional NMR experiments were performed: COSY, DQF-COSY, TOCSY, ROESY, HSQCnd, HSQC-DEPT, and HMBC. The 1H and 13C resonances were measured relative to TMS ( H 0.0/ C 0.0).TABLE 1 Fatty acid, hopanoids, and sugar components of lipid A isolated from LPS of Bradyrhizobium strainsThe symbols represent: , present; lack of component; tr, traces. LPAR5 Antagonist MedChemExpress Component Fatty acids 12:0(3-OH) 14:0(3-OH) 26:0(25-OH) 27:0(26-OH)a 28:0(27-OH) 29:0(28-OH)a 30:0(29-OH).
