In mammals, circadian oscillators reside not only in the suprachiasmatic nucleus of the brain, which harbors the central pacemaker, but also in most peripheral tissues. Here, we show that the glucocorticoid hormone analog dexamethasone induces circadian gene expression in cultured rat-1 fibroblasts and transiently changes the phase of circadian gene expression in liver, kidney, and heart. However, dexamethasone does not affect cyclic gene expression in neurons of the suprachiasmatic nucleus. This enabled us to establish an apparent phase-shift response curve specifically for peripheral clocks in intact animals. In contrast to the central clock, circadian oscillators in peripheral tissues appear to remain responsive to phase resetting throughout the day.

The circadian clock orchestrates many aspects of human physiology, and disruption of this clock has been implicated in various pathologies, ranging from cancer to metabolic syndrome and diabetes. Although there is evidence that metabolism and the circadian clockwork are intimately linked on a transcriptional level, whether these effects are directly under clock control or are mediated by the rest-activity cycle and the timing of food intake is unclear. To answer this question, we conducted an unbiased screen in human subjects of the metabolome of blood plasma and saliva at different times of day. To minimize indirect effects, subjects were kept in a 40-h constant routine of enforced posture, constant dim light, hourly isocaloric meals, and sleep deprivation. Under these conditions, we found that ∼15% of all identified metabolites in plasma and saliva were under circadian control, most notably fatty acids in plasma and amino acids in saliva. Our data suggest that there is a strong direct effect of the endogenous circadian clock on multiple human metabolic pathways that is independent of sleep or feeding. In addition, they identify multiple potential small-molecule biomarkers of human circadian phase and sleep pressure.metabolomics | LC/GC-MS | metabolite profiling | sleep-wake regulation T he circadian clock has been shown to modulate many aspects of behavior and physiology (1). It is thought to be an important regulator of metabolism, and disruption of the clock and sleep is associated with obesity, metabolic syndrome, and type 2 diabetes, as well as other disorders (2-4). In the last decade, ample data on the circadian transcriptome (5, 6) and the even larger circadian proteome (7) have been compiled. These datasets are directly dependent on the genome of a particular species and cannot be compared easily between model systems. However, changes in physiology and metabolism governed by these genes and proteins ultimately affect the abundance of small metabolites that are quite conserved among species and fewer in number (50-fold fewer than transcripts and 500-fold fewer than proteins).The relationship between metabolism and the clock is not unidirectional, and the two processes are intertwined (8). For example, metabolic status feeds back to the clock, so that feeding behavior directly entrains molecular clock function (9). Likewise, obesity is correlated with poor sleep (2), and in mice 80% of circadian transcription in the brain is dependent on the rest-activity cycle (10). Given these feedback mechanisms, it is unclear what proportion of circadian metabolic control is directly clockregulated and what proportion is controlled by circadian restactivity and food intake.In plants, the metabolome approach has been used to characterize the effects of clock disruption on general metabolism (11). The circadian metabolome also has been characterized in CBA/N mice, and ∼20% of the recorded molecules were found to vary in abundance with time of day (12). Similarly, the urine and saliva metabolomes of human subjects diff...

Changes in chromatin structure have frequently been correlated with changes in transcription. However, the cause-and-effect relationship between chromatin structure and transcription has been hard to determine. In addition, identifying the proteins that regulate chromatin structure has been difficult. Recent evidence suggests that a functionally related set of yeast transcriptional activators {SNF2/SWI2, SNF5, SNF6, SWII, and SWI3), required for transcription of a diverse set of genes, may affect chromatin structure. We now present genetic and molecular evidence that at least two of these transcriptional activators, SNF2/SWI2 and SNF5, function by antagonizing repression mediated by nucleosomes. First, the transcriptional defects in strains lacking these SNF genes are suppressed by a deletion of one of the two sets of genes encoding histones H2A and H2B, lhtal-htbl)A. Second, at one affected promoter (SUC2}, chromatin structure is altered in sn[2/swi2 and sn[5 mutants, and this chromatin defect is suppressed by {htal-htbl)A. Finally, analysis of chromatin structure at a mutant SUC2 promoter, in which the TATA box has been destroyed, demonstrates that the differences in SUC2 chromatin structure between SNF5 + and sn[5 mutant strains are not simply an effect of different levels of SUC2 transcription. Thus, these results strongly suggest that SNF2/SWI2 and SNF5 cause changes in chromatin structure and that these changes allow transcriptional activation.[Key Words: Yeast; chromatin; transcriptional activation; SNF and SWI proteins] Received August 10, 1992; revised version accepted September 17, 1992. Understanding the mechanisms by which genes are transcriptionally activated and repressed is central to understanding gene regulation. Numerous studies have shown a correlation between changes in transcription and alterations in chromatin structure (for review, see van Holde 1988;Grunstein 1990a). These studies have suggested that the positions of nucleosomes, the primary components of chromatin, may affect transcription. However, the cause-and-effect relationship between changes in transcription and alterations in chromatin structure is unclear.Recent genetic and biochemical data strongly support the view that nucleosomes play an important role in transcriptional regulation. Studies in the yeast Saccharomyces cerevisiae have provided genetic evidence that histones, the protein components of nucleosomes, affect transcription. For example, alterations in histone stoichiometry restore transcription to promoters disrupted by certain transposon insertion mutations (Clark-Adams et el. 1988). In addition, depletion of histone H4 or mutations in genes encoding histone H4 can result in in- creased or decreased levels of transcription (for review, see Grunstein 1990b). Genetic and biochemical data also suggest that some transcriptional activators may function to alleviate the repressive effects of chromatin structure (Fascher et el. 1990; Croston et el. 1991; Workman et el. 1991 }. However, aside from histones, the pro...

We postulate that both endogenous and environmental temperature cycles can participate in the synchronization of peripheral clocks in mammals.

Peripheral cells from mammalian tissues, while perfectly capable of circadian rhythm generation, are not light sensitive and thus have to be entrained by nonphotic cues. Feeding time is the dominant zeitgeber for peripheral mammalian clocks: Daytime feeding of nocturnal laboratory rodents completely inverts the phase of circadian gene expression in many tissues, including liver, heart, kidney, and pancreas, but it has no effect on the SCN pacemaker. It is thus plausible that in intact animals, the SCN synchronizes peripheral docks primarily through temporal feeding patterns that are imposed through behavioral rest-activity cycles. In addition, body temperature rhythms, which are themselves dependent on both feeding patterns and rest-activity cycles, can sustain circadian, clock gene activity in vivo and in vitro. The SCN may also influence the phase of rhythmic gene expression in peripheral tissues through direct chemical pathways. In fact, many chemical signals induce circadian gene expression in tissue culture cells. Some of these have been shown to elicit phase shifts when injected into intact animals and are thus candidates for physiologically relevant timing cues. While the response of the SCN to light is strictly gated to respond only during the night, peripheral oscillators can be chemically phase shifted throughout the day. For example, injection of dexamethasone, a glucocorticoid receptor agonist, resets the phase of circadian liver gene expression during the entire 24-h day. Given the bewildering array of agents capable of influencing peripheral clocks, the identification of physiologically relevant agents used by the SCN to synchronize peripheral clocks will clearly be an arduous undertaking. Nevertheless, we feel that experimental systems by which this enticing problem can be tackled are now at hand.

The fluoroquinolones are a series of synthetic antibacterial agents that are undergoing extensive investigation for both human and veterinary use in the treatment of a variety of bacterial infections. These agents work through the inhibition of DNA gyrase, interfering with the supercoiling of bacterial chromosomal material. As a result, these agents are rapidly bactericidal primarily against gram‐negative bacteria, mycoplasma, and some grampositive bacteria, with most having little to no activity against group D streptococci and obligate anaerobic bacteria. Resistance develops slowly and is almost always chromosomal and not plasmid‐mediated. However, development of resistance to the fluoroquinolones and transfer of that resistance among animal and human pathogens have become a hotly debated issue among microbiologists. The fluoroquinolones are a current antimicrobial class whose use in veterinary medicine is being scrutinized. From a pharmacokinetic perspective, these agents are variably but well absorbed from the gastrointestinal tract and almost completely absorbed from parenteral injection sites, and they are well distributed to various tissues in the body. The fluoroquinolones are metabolized and renally excreted, with many of them having approximately equal excretion by the hepatic and the renal excretory systems. The primary toxicity observed at therapeutic doses involves the gastrointestinal system and phototoxicity, although at higher doses central nervous system toxicity and ocular cataracts are observed. Administration to immature animals may result in erosive arthropathies at weight‐bearing joints, and administration of high doses to pregnant animals results in maternotoxicity and occasionally embryonic death. The fluoroquinolones are approved for indications such as urinary tract infections and soft tissue infections in dogs and cats and colibacillosis in poultry. Approval for bovine respiratory disease in the United States is being sought. Other indications for which the fluoroquinolones have been used in animal health include deep‐seated infections, prostatitis, and other bacterial infections resistant to standard antimicrobial therapy.

Mammalian circadian behavior is governed by a central clock in the suprachiasmatic nucleus of the brain hypothalamus, and its intrinsic period length is believed to affect the phase of daily activities. Measurement of this period length, normally accomplished by prolonged subject observation, is difficult and costly in humans. Because a circadian clock similar to that of the suprachiasmatic nucleus is present in most cell types, we were able to engineer a lentiviral circadian reporter that permits characterization of circadian rhythms in single skin biopsies. Using it, we have determined the period lengths of 19 human individuals. The average value from all subjects, 24.5 h, closely matches average values for human circadian physiology obtained in studies in which circadian period was assessed in the absence of the confounding effects of light input and sleep–wake cycle feedback. Nevertheless, the distribution of period lengths measured from biopsies from different individuals was wider than those reported for circadian physiology. A similar trend was observed when comparing wheel-running behavior with fibroblast period length in mouse strains containing circadian gene disruptions. In mice, inter-individual differences in fibroblast period length correlated with the period of running-wheel activity; in humans, fibroblasts from different individuals showed widely variant circadian periods. Given its robustness, the presented procedure should permit quantitative trait mapping of human period length.

Many physiological processes in organisms from bacteria to man are rhythmic, and some of these are controlled by self-sustained oscillators that persist in the absence of external time cues. Circadian clocks are perhaps the best characterized biological oscillators and they exist in virtually all light-sensitive organisms. In mammals, they influence nearly all aspects of physiology and behavior, including sleep-wake cycles, cardiovascular activity, endocrinology, body temperature, renal activity, physiology of the gastro-intestinal tract, and hepatic metabolism. The master pacemaker is located in the suprachiasmatic nuclei, two small groups of neurons in the ventral part of the hypothalamus. However, most peripheral body cells contain self-sustained circadian oscillators with a molecular makeup similar to that of SCN (suprachiasmatic nucleus) neurons. This organization implies that the SCN must synchronize countless subsidiary oscillators in peripheral tissues, in order to coordinate cyclic physiology. In this review, we will discuss some recent studies on the structure and putative functions of the mammalian circadian timing system, but we will also point out some apparent inconsistencies in the currently publicized model for rhythm generation.