A Proton Pump Inhibitor in the Reformulation Setting: Bioequivalence and Potential Implications for Long-Term Safety.

Proton pump inhibitors (PPIs) have become known for both their therapeutic effect and good safety profile. An application was submitted to the US Food and Drug Administration for approval of a reformulated PPI product that failed bioequivalence testing, but was submitted on the basis of the long history of PPI use as a "surrogate" for equivalence. This review evaluates the safety data for PPIs, discuss variability of pharmacokinetic parameters of PPIs in the reformulation setting, and potential implications of those changes for long-term safety.

Altered entrainment and feedback loop function effected by a mutant period protein.

The period (per) and timeless (tim) genes encode interacting components of the circadian clock. Levels and phosphorylation states of both proteins cycle with a circadian rhythm, and the proteins drive cyclic expression of their RNAs through a feedback mechanism that is, at least in part, negative. We report here that a hypophosphorylated mutant PER protein, produced by creating a small internal deletion, displays increased stability and low-amplitude oscillations, consistent with previous reports that phosphorylation is required for protein turnover. In addition, this protein appears to be defective in feedback repression because it is associated with relatively high levels of RNA and high levels of TIM. Transgenic flies carrying the mutant PER protein display a temperature-dependent shortening of circadian period and are impaired in their response to light, particularly to pulses of light in the late night that normally advance the phase of the rhythm. Interestingly, per RNA is induced by light in these flies, most likely because of the removal of the light-sensitive TIM protein, thus implicating a more direct role for TIM in transcriptional inhibition. These data have relevance for mechanisms of feedback repression, and they also address existing models for the differential behavioral response to light at different times of the night.

Alterations of per RNA in noncoding regions affect periodicity of circadian behavioral rhythms.

Circadian rhythms in Drosophila depend on a molecular feedback loop that includes products of the period (per) and timeless (tim) genes. RNA and protein products of both genes cycle with a circadian period and the proteins feedback to inhibit expression of their own mRNAs. While cyclic expression of PER protein appears to be necessary for rhythmic behavior, the function of per RNA cycling is somewhat controversial. Rhythmic transcription accounts, in part, for cycling of per RNA, but it is clear now that posttranscriptional mechanisms also contribute to the cyclic expression of both per RNA and protein. As posttranscriptional mechanisms, such as mRNA stability and translation, are frequently mediated by 3' untranslated regions (UTR) of genes, the authors examined the role of this region of per in the regulation of circadian rhythms. Removal of most of per's 3' UTR had a small effect on the function of a per transgene. However, replacement of per's 3'UTR with corresponding sequences of the tubulin gene led to the rescue of behavioral rhythms in per01 flies with periods that were 3 h shorter than those generated by a wild-type per transgene. The hybrid RNA cycles, but the protein produced by it accumulates earlier in a day-night cycle than the PER protein produced by a control per transgene carrying its own 3'UTR, perhaps because the tubulin sequences counteract the effect of destabilizing elements in the per RNA at earlier points in the circadian cycle. These data indicate that the appropriate regulation of per RNA expression, effected by transcriptional as well as posttranscriptional mechanisms, is critical for the determination of circadian period.

What makes the circadian clock tick: genes that keep time?

Intense activity in the field of circadian rhythms has led in recent years to a basic understanding of how an endogenous clock is generated. Oscillating products of the period (per) and timeless (tim) genes, which feed back to regulate their own synthesis, and transcription factors, which activate these genes, combine to generate a molecular loop that apparently drives behavioral and physiological rhythms. The best-characterized component of this system is the per gene, with considerable effort directed towards identifying the mechanisms that regulate cyclic expression of RNA and protein. Since the cycling of PER protein is controlled largely by post-transcriptional mechanisms, the relative importance of RNA versus protein cycling has been addressed in several studies that are discussed in this chapter. However, it now is clear that regulation of per cannot be dissociated from that of tim, since they are co-dependent components. The overt behavioral phenotype likely depends upon the effect that any perturbation has on both components, rather than on either alone. Major features of the feedback loop appear to be conserved, from fruit flies to mammals. One difference between the two systems is the manner in which the "molecular clock" responds to light. In flies, levels of TIM protein are reduced in response to light, while in mammals, per RNA is induced. The pathway that conducts light to the clock is poorly understood but there is increasing evidence in support of a dedicated pathway for circadian photoreception, as opposed to the sole use of the visual transduction system.