Summary Cellular proliferation requires formation of additional cellular membrane material, and the current thinking in the field is that lipids required for this new membrane formation are mostly synthesized de novo. Here we measured the contribution of de novo lipid synthesis in proliferating and contact-inhibited fibroblasts and find that proliferating fibroblasts prefer exogenous palmitate over de novo synthesis. We determined that when exogenous palmitate is provided in culture media at physiological concentrations, de novo synthesis accounts for only ~10% of intracellular palmitate in proliferating fibroblasts, as well as HeLa and H460 lung cancer cells. Blocking fatty acid uptake decreased the rate of fibroblast, HeLa, and H460 cell proliferation, while supplementing media with exogenous palmitate resulted in decreased glucose uptake and rendered cells less sensitive to glycolytic inhibition. Thus, our results suggest that cells scavenging exogenous lipids may be less susceptible to both glycolytic and lipogenic inhibitors.

SummaryWe labeled soybean (Glycine max) leaves with 200 and 600 ppm 13 CO 2 spiked with 11 CO 2 and examined the effects of light intensity and water stress on metabolism by using a combination of direct positron imaging and solid-state 13 C nuclear magnetic resonance (NMR) of the same leaf.We first made 60-min movies of the transport of photosynthetically assimilated 11 C labels. The positron imaging identified zones or patches within which variations in metabolism could be probed later by NMR. At the end of each movie, the labeled leaf was frozen in liquid nitrogen to stop metabolism, the leaf was lyophilized, and solid-state NMR was used either on the whole leaf or on various leaf fragments.The NMR analysis determined total 13 C incorporation into sugars, starch, proteins, and protein precursors.The combination of 11 C and 13 C analytical techniques has led to three major conclusions regarding photosynthetically heterogeneous soybean leaves: transient starch deposition is not the temporary storage of sucrose excluded from a saturated sugar-transport system; peptide synthesis is reduced under high-light, high CO 2 conditions; and all glycine from the photorespiratory pathway is routed to proteins within photosynthetically active zones when the leaf is water stressed and under high-light and low CO 2 conditions.

The DNA repair enzyme uracil DNA glycosylase (UDG) utilizes base flipping to recognize and remove unwanted uracil bases from the genome but does not react with its structural congener, thymine, which differs by a single methyl group. Two factors that determine whether an enzyme flips a base from the duplex are its shape and hydrogen bonding properties. To probe the role of these factors in uracil recognition by UDG, we have synthesized a DNA duplex that contains a single difluorophenyl (F) nucleotide analogue that is an excellent isostere of uracil but possesses no hydrogen bond donor or acceptor groups. By using binding affinity measurements, solution (19)F NMR, and solid state (31)P[(19)F] rotational-echo double-resonance (REDOR) NMR measurements, we establish that UDG partially unstacks F from the duplex. However, due to the lack of hydrogen bonding groups that are required to support an open-to-closed conformational transition in UDG, F cannot stably dock in the UDG active site. We propose that F attains a metastable unstacked state that mimics a previously detected intermediate on the uracil-flipping pathway and suggest structural models of the metastable state that are consistent with the REDOR NMR measurements.