This article is available online at http://www.jlr.org role in the formation of membrane microdomains ( 1 ). Until now, only very limited and expensive sources of labeled cholesterol were available. Cholesterol enriched at carbons 3 and 4 or 23-27 are commercially available, but other positions require de novo biosynthesis. Small amounts of low-enrichment cholesterol have been obtained in vivo by skin injection of enriched mevalonate in rat ( 2 ) or feeding mammals and humans with 13 C enriched precursors ( 3, 4 ). An in vitro alternative based on human hepatoma Hep G2 cells cultures ( 5 ) produces small amounts of cholesterol with higher enrichment levels, but if these methods can produce samples for MS analysis and 14 C labeling, they are not effi cient enough for NMR applications. We have previously engineered a Saccharomyces cerevisiae strain by deleting the ERG5 and ERG6 genes and introducing plasmids that express the DHCR7 and DHCR24 genes from Danio rerio , leading to the synthesis of cholesterol ( 6 ). Subsequently, we integrated cassettes expressing DHCR7 and DHCR24 into the ERG5 and ERG6 loci, creating deletions of the latter genes, to create a more stable strain that effi ciently produces cholesterol as its major (<95%) sterol ( 7 ). The metabolically engineered yeast use the same biosynthesis pathway as animals ( Fig. 1 ) and effi ciently produce 13 C-enriched cholesterol with different labeling pat terns with a yield of ‫ف‬ 1 mg of cholesterol per gram of glucose in 100 ml of culture medium. EXPERIMENTAL PROCEDURES MaterialsThe Saccharomyces cervisiae strain used was RH6829 (MATa ura3 leu2 his3 trp1 bar1 erg5 ⌬ ::HIS5-GPD-DHCR24 erg6 ⌬ ::TRP1-GPD-DHCR7 ( 7 ). Yeast nitrogen base and yeast extracts were obtained from US Biological and Difco, respectively. D-glucose (99%), leucine (95%), and uracil (99%) were obtained from Aristar, Fluka, and Sigma, respectively. The pyrogallol and petroleum ether Sterols are important lipids in most eukaryotes. In particular, cholesterol has attracted a lot of attention because of its involvement in cardiovascular diseases in humans and because it has been suggested to play an important

Abstract:TheNMRmethodologybasedonspectralaliasingdevelopedattheUniversityofGenevaisreviewed. Differentapproachesaimedatincreasingtheresolutionintheindirectcarbondimensionof2Dheteronuclear experimentsarepresentedwiththeirrespectiveadvantages.ApplicationstoHSQC,HMBCandother2Dhet-eronuclearexperimentstothestudyofnaturalproductsandsynthesisintermediatesareshown.HSQC-based experimentsfordiffusionmeasurements,kineticsstudiesandtitrationsexperimentsalltakeadvantageofspectralaliasingtoreducetheexperimentaltimefromunrealisticallylongacquisitiontimestoovernightexperiments. TherolesofcomputationalmethodssuchasDFT/GIAOandLogicforStructureDetermination(LSD)instructure determinationarediscussed.

Glucose is an essential nutrient for Plasmodium falciparum and robust glycolytic activity is indicative of viable parasites. Using NMR spectroscopy, we show that P. falciparum infected erythrocytes consume ~20 times more glucose, and trophozoites metabolize ~6 times more glucose than ring stage parasites. The glycolytic activity, and hence parasite viability, can be measured within a period of 2 h to 5 h, using this method. This facilitates antimalarial bioactivity screening on ring and trophozoite stage parasites, exclusively. We demonstrate this using potent and mechanistically distinct antimalarial compounds such as chloroquine, atovaquone, cladosporin, DDD107498 and artemisinin. Our findings indicate that ring stage parasites are inherently more tolerant to antimalarial inhibitors, a feature which may facilitate emergence of drug resistance. Thus, there is a need to discover novel antimalarial compounds, which are potent and fast acting against ring stage parasites. The NMR method reported here can facilitate the identification of such molecules.

NMR titrations can be used to simultaneously determine the pKa's of mixtures of organic acids and bases provided chemical shifts are sensitive to their change of protonation states. Carbons are in principle better probes than protons because their chemical shifts are typically more sensitive to the changes in the electronic densities of the functional groups they are bound to. They are also less prone to overlap, do not suffer from second-order effects and are less sensitive to changes in solvent conditions. Recording HSQC spectra satisfactorily reduces the lack of sensitivity of carbon-detected experiments and the signal dispersion in two dimensions allows to automatically and finely track the drifts of a large number of signals simultaneously. The application of spectral aliasing to the carbon dimensions allows to quickly reach the high resolution required to precisely measure variations in carbon chemical shifts. The paper extends the methodology to the treatment of the data stemming from the simultaneous analysis of mixtures of compounds. It also introduces methods to improve the normalization process and deal with missing points. The application is not limited to the determination of pKa and can be readily extended to the determination of other chemical constants in experiments where the components of the mixture dynamically compete for substrates other than protons

A new indole alkaloid, calanthumindole (4), and three known biflavonoids, amentoflavone, sequoiaflavone, and podoscarpusflavone B, were isolated from Campylospermum calanthum (Ochnaceae). Calanthumindole is a new indole alkaloid of the serotobenine family characterized by the presence of a CC bond between atoms C(7′) and C(8′) of the furan ring. This is the first compound to have a fully unsaturated furan ring among the members of this family. The combination of NMR and DFT allowed the determination and comparison of the 3D structures and relevant conformational characteristics of serotobenine (1), flavumindole (2), and calanthumindole (4).