Exogenous Fatty Acids Are the Preferred Source of Membrane Lipids in Proliferating Fibroblasts.

Cellular proliferation requires the formation of new membranes. It is often assumed that the lipids needed for these membranes are synthesized mostly de novo. Here, we show that proliferating fibroblasts prefer to take up palmitate from the extracellular environment over synthesizing it de novo. Relative to quiescent fibroblasts, proliferating fibroblasts increase their uptake of palmitate, decrease fatty acid degradation, and instead direct more palmitate to membrane lipids. When exogenous palmitate is provided in the culture media at physiological concentrations, de novo synthesis accounts for only a minor fraction of intracellular palmitate in proliferating fibroblasts as well as proliferating HeLa and H460 cells. Blocking fatty acid uptake decreased the proliferation rate of fibroblasts, HeLa, and H460 cells, while supplementing media with exogenous palmitate resulted in decreased glucose uptake and rendered cells less sensitive to glycolytic inhibition. Our results suggest that cells scavenging exogenous lipids may be less susceptible to drugs targeting glycolysis and de novo lipid synthesis.

Carbon partitioning in soybean (Glycine max) leaves by combined (11) C and (13) C labeling.

We 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.

Oxygen-17 appears only in protein in water-stressed soybean leaves labeled by (17)O2.

We have used a rotational-echo adiabatic-passage double-resonance (13)C{(17)O} solid-state NMR experiment to prove that the glycine produced in the oxygenase reaction of ribulose bisphosphate carboxylase-oxygenase is incorporated exclusively into protein (or protein precursors) of intact, water-stressed soybean leaves exposed to (13)CO(2) and (17)O(2). The water stress increased stomatal resistance and decreased gas exchange so that the Calvin cycle in the leaf chloroplasts was no more than 35% (13)C isotopically enriched. Labeled O(2) levels were sufficient, however, to increase the (17)O isotopic concentration of oxygenase products 20-fold over the natural-abundance level of 0.04%. The observed direct incorporation of glycine into protein shows that water stress suppresses photorespiration in soybean leaves.

Variability in C(3)-plant cell-wall biosynthesis in a high-CO(2) atmosphere by solid-state NMR spectroscopy.

We have used a frequency-selective rotational-echo double-resonance (REDOR) solid-state NMR experiment to measure the concentrations of glycine-glycine pairs in proteins (and protein precursors) of intact leaves of plants exposed to both high- and low-CO(2) atomospheres. The results are interpreted in terms of differences in cell-wall biosynthesis between plant species. We illustrate this variability by comparing the assimilation of label in cheatgrass and soybean leaves labeled using (15)N-fertilizer and (13)CO(2) atmospheres. Cheatgrass and soybean are both C(3) plants but differ in their response to a high-CO(2) environment. Based on REDOR results, we determined that cheatgrass (a plant that seems likely to flourish in future low-water, high-CO(2) environments) routes 2% of the assimilated carbon label that remains in the leaf after 1 h in a 600-ppm (13)CO(2) atmosphere to glycine-rich protein (or its precursors), a structural component of cell walls cross-linked to lignins. In contrast, soybean under the same conditions routes none of its assimilated carbon to glycine-rich protein.

Recognition of an unnatural difluorophenyl nucleotide by uracil DNA glycosylase.

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.