Metastable States in the Hinge-Bending Landscape of an Enzyme in an Atomistic Cytoplasm Simulation.

Many enzymes undergo major conformational changes to function in cells, particularly when they bind to more than one substrate. We quantify the large-amplitude hinge-bending landscape of human phosphoglycerate kinase (PGK) in a human cytoplasm. Approximately 70 µs of all-atom simulations, upon coarse graining, reveal three metastable states of PGK with different hinge angle distributions and additional substates. The "open" state was more populated than the "semi-open" or "closed" states. In addition to free energies and barriers within the landscape, we characterized the average transition state passage time of ≈0.3 µs and reversible substrate and product binding. Human PGK in a dilute solution simulation shows a transition directly from the open to closed states, in agreement with previous SAXS experiments, suggesting that the cell-like model environment promotes stability of the human PGK semi-open state. Yeast PGK also sampled three metastable states within the cytoplasm model, with the closed state favored in our simulation.

The hedgehog signaling pathway in the mouse ovary.

The hedgehog (HH) signaling pathway plays an essential role in the Drosophila ovary, regulating cell proliferation and differentiation, but a role in the mammalian ovary has not been defined. Expression of components of the HH pathway in the mouse ovary and effects of altering HH signaling in vitro were determined. RT-PCR analyses show developmentally regulated expression of sonic (Shh), indian (Ihh) and desert (Dhh) HH in the ovary. Expression is detected in whole ovary, granulosa cells, and corpora lutea. The mRNAs for the two receptors, patched homolog 1 and 2 (Ptch1, Ptch2), and the signal transducer, smoothened (Smo), are also expressed. Immunohistochemistry using an antibody that detects all three HH ligands demonstrated HH protein primarily in granulosa cells of follicles from primary to antral stages of development. Follicles also stained for PTCH1 and SMO in both granulosa and theca cells. Treatment of cultured preantral follicles and granulosa cells with recombinant SHH increased growth and proliferation while treatment with the HH pathway inhibitor, cyclopamine, had no effect. Therefore, activation of HH signaling can increase cell proliferation and follicle growth but is not essential for these processes in vitro. Treatment of granulosa cells with SHH increased levels of mRNA for Gli1, a transcriptional target of HH signaling, while cyclopamine decreased expression. SHH had no effect on production of progesterone by cultured granulosa cells, while cyclopamine increased progesterone production. The results demonstrate a functional HH pathway in the follicle and identify granulosa cells as at least one of the potential targets of HH signaling.

Diets deficient in indispensable amino acids rapidly decrease the concentration of the limiting amino acid in the anterior piriform cortex of rats.

Diets deficient in an indispensable amino acid have long been known to suppress food intake in rats. Detection of dietary deficiency takes place in the anterior piriform cortex (APC). Recent studies showed that the response to amino acid deficiency takes as little as 15 min to develop, but few data exist to correlate the concentration of amino acids in the APC with this rapid response. The purpose of this study was to measure the concentration of amino acids in the APC in a behaviorally relevant time frame. Rats were preconditioned by consumption of a basal diet for 7-10 d, and then given a test diet with either a control or deficient amino acid profile. Both the threonine- and leucine-deficient diets reliably depleted threonine and leucine concentration in the APC within 30 min, respectively. The control diets and a diet lacking the dispensable amino acid glycine did not lead to amino acid depletion. In combination with previous studies, the present results show that the decrease in the concentration of indispensable amino acids in the APC may be the initial sensory signal for recognition of dietary amino acid deficiency.

Rats rapidly reject diets deficient in essential amino acids.

Omnivores must obtain diets balanced with respect to amino acids to support growth and protein synthesis. The standard paradigm used to study behavioral responses to amino acid deficiency combines deficient diets with dietary novelty. The objective of this study was to examine the effects of amino acid deficiency on the first meal of rats without the confounding effects of novelty. We report on a series of five studies of feeding behavior in rats. Rats were fed low protein diets for 5-7 d and then exposed to diets with and without essential amino acids. Rats consistently demonstrated recognition of essential amino acid deficiency within the first meal by a significant reduction in first meal duration, rejecting the deficient diets after just 12-16 min exposure. This is the first report of a rapid effect of amino acid-deficient diets without the confounding effects of dietary novelty.

The rapid anorectic response to a threonine imbalanced diet is decreased by injection of threonine into the anterior piriform cortex of rats.

Rats quickly recognize and reject diets deficient in an essential amino acid. The purpose of this study was to determine whether the anterior piriform cortex (APC), the site traditionally recognized as the amino acid chemosensor, plays a role in this early behavior. Rats had cannulae implanted bilaterally into the APC, and were injected with either saline vehicle or 2 nmoles of threonine (n = 6 per group). All rats were then fed a diet imbalanced with respect to threonine. The threonine-injected group had first meals of longer duration and consumed more food. These data conformed to expectations derived from earlier studies of responses to the first meal of an amino acid imbalanced diet. We conclude that the concentration of the dietary limiting amino acid in the APC regulates acceptance and rejection of amino acid deficient diets.