The Food-entrainable Oscillator Is a Complex of Non-SCN Activity Bout Oscillators Uncoupled From the SCN Circadian Pacemaker.

The food-entrainable oscillator, which underlies the prefeeding activity peak developed by restricted daily feeding (RF) in rodents, does not depend on the circadian pacemaker in the suprachiasmatic nucleus (SCN) or on the known clock genes. In the present study, to clarify the roles of SCN circadian pacemaker and nutrient conditions on the development of prefeeding activity peak, RF of 3-h daily feeding was imposed on four groups of adult male mice for 10 cycles at different circadian times, zeitgeber time (ZT)2, ZT8, ZT14, and ZT20, where ZT0 is the time of lights-on in LD12:12. Seven days after the termination of RF session with ad libitum feeding in between, total food deprivation (FD) for 72 h was imposed. Wheel-running activity and core body temperature were measured throughout the experiment. Immediately after the RF or FD session, the PER2::LUC rhythms were measured in the cultured SCN slices and peripheral tissues. Not only the buildup process and magnitude of the prefeeding activity peak, but also the percentages of nocturnal activity and hypothermia developed under RF were significantly different among the four groups, indicating the involvement of light entrained circadian pacemaker. The buildup of prefeeding activity peak was accomplished by either phase-advance or phase-delay shifts (or both) of activity bouts comprising a nocturnal band. Hypothermia under FD was less prominent in RF-exposed mice than in naïve counterparts, indicating that restricted feeding increases tolerance to caloric restriction as well as to the heat loss mechanism. RF phase-shifted the peripheral clocks but FD did not affect the clocks in any tissue examined. These findings are better understood by assuming multiple bout oscillators, which are located outside the SCN and directly drive activity bouts uncoupled from the circadian pacemaker by RF or hypothermia.

Daily life habits associated with eveningness lead to a higher prevalence of dental caries in children.

BACKGROUND/PURPOSE: Circadian rhythm is an endogenous daily variation observed in most physiological functions including salivary secretion. Irregular lifestyle causes many diseases such as obesity and sleep disorders. The aim of this study is to examine the effects of the timings of sleep and meal on the prevalence of dental caries. MATERIALS AND METHODS: Study was conducted at university hospital in Japan. We asked 230 children (1-16 years old) to record the following life habits for 8 days: waking time, bedtime, mealtimes, snacking frequency, and tooth brushing frequency. We analyzed sleep habits from all data and compared dental caries and life habits using data from subjects with primary (2-7 years old) or permanent (11-16 years old) dentition period. RESULTS: The number of dental caries assessed using the decay or filled teeth (dft) index correlated with bedtime, supper time, regularity of supper time, and snacking frequency in subjects with primary dentition. Multiple regression analysis revealed that bedtime and snacking frequency were mutually independent risk factors for dental caries. No correlations were found between the prevalence of dental caries and other measurement items. The number of caries correlated with the regularity of supper time and age in subjects with permanent dentition. CONCLUSION: Children with daily life habits associated with eveningness have a higher prevalence of dental caries.

A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection.

Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.

Two coupled circadian oscillations regulate Bmal1-ELuc and Per2-SLR2 expression in the mouse suprachiasmatic nucleus.

Circadian rhythms in clock genes, Bmal1 and Per2 expression were monitored simultaneously in the cultured slice of mouse suprachiasmatic nucleus (SCN) by dual bioluminescent reporters. In the neonatal SCN, the phase-relation between the Bmal1 and Per2 rhythms were significantly changed during culture. Medium exchange produced phase-dependent phase shifts (PRCm) in the Bmal1 rhythms, but not in the Per2 rhythms. As a result, the two circadian rhythms were temporally dissociated after medium exchange. In the adult SCN, the phase-relation between the two rhythms was kept constant during culture at least up to 20 cycles. The amplitude of PRCm in the adult SCN was significantly attenuated in the Bmal1 rhythm, whereas a PRCm was developed in the Per2 rhythm. The circadian period was not systematically affected by medium exchange in either of rhythms, regardless of whether it was in the neonatal or the adult SCN. Tetrodotoxin, a sodium channel blocker, enhanced the phase-response in both rhythms but abolished the phase-dependency. In addition, tetrodotoxin lengthened the circadian period independent of the phase of administration. Thus, the Bmal1 and Per2 rhythms in the SCN are dissociable and likely regulated by distinct circadian oscillators. Bmal1 is the component of a Bmal1/REV-ERBa/ROR loop and Per2 a Per/Cry/BMAL1/CLOCK loop. Both loops could be molecular mechanisms of the two circadian oscillators that are coupled through the protein product of Bmal1. The coupling strength between the two oscillations depends on developmental stages.

A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells.

Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.

Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.

Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.

Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells.

Although the co-development of companion diagnostics with molecular targeted drugs is desirable, truly efficient diagnostics are limited to diseases in which chromosomal translocations or overt mutations are clearly correlated with drug efficacy. Moreover, even for such diseases, few methods are available to predict whether drug administration is effective for each individual patient whose disease is expected to respond to the drug(s). We have previously developed a biosensor based on the principle of Förster resonance energy transfer to measure the activity of the tyrosine kinase BCR-ABL and its response to drug treatment in patient-derived chronic myeloid leukemia cells. The biosensor harbors CrkL, one of the major substrates of BCR-ABL, and is therefore named Pickles after phosphorylation indicator of CrkL en substrate. The efficacy of this technique as a clinical test has been demonstrated, but the number of cells available for analysis is limited in a case-dependent manner, owing to the cleavage of the biosensor in patient-derived leukemia cells. Here, we describe an improved biosensor with an amino acid substitution and a nuclear export signal being introduced. Of the two predicted cleavage positions in CrkL, the mutations inhibited one cleavage completely and the other cleavage partially, thus collectively increasing the number of cells available for drug evaluation. This improved version of the biosensor holds promise in the future development of companion diagnostics to predict responses to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.

Receptor activator of NF-κB ligand induces cell adhesion and integrin α2 expression via NF-κB in head and neck cancers.

Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-κB ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin α2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin α2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-κB signaling mediated integrin α2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin ß1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin α2 and endocytosis. Moreover, the RANK-integrin α2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.

Attenuation of ligand-induced activation of angiotensin II type 1 receptor signaling by the type 2 receptor via protein kinase C.

Angiotensin II (AII) type 2 receptor (AT2R) negatively regulates type 1 receptor (AT1R) signaling. However, the precise molecular mechanism of AT2R-mediated AT1R inhibition remains poorly understood. Here, we characterized the local and functional interaction of AT2R with AT1R. AT2R colocalized and formed a complex with AT1R at the plasma membrane, even in the absence of AII. Upon AII stimulation, the spatial arrangement of the complex was modulated, as confirmed by Förster resonance energy transfer (FRET) analysis, followed by AT2R internalization along with AT1R. AT2R internalization was specifically observed only in the presence of AT1R; AT2R alone could not be internalized. The AT1R-specific inhibitor losartan completely inhibited both the conformational change and the internalization of AT2R with AT1R, whereas the AT2R-specific inhibitor PD123319 partially hindered these phenomena, demonstrating that the activation of both receptors was indispensable for these effects. In addition, treatment with the protein kinase C (PKC) inhibitors inhibited the ligand-dependent accumulation of AT2R but not that of AT1R in the endosomes. A mutation in the putative phosphorylation sites of AT2R also abrogated the co-internalization of ATR2 with AT1R and the inhibitory effect of ATR2 on AT1R. These data suggest that AT2R inhibits ligand-induced AT1R signaling through the PKC-dependent pathway.

Morning and evening physical exercise differentially regulate the autonomic nervous system during nocturnal sleep in humans.

Effects of daily physical exercise in the morning or in the evening were examined on circadian rhythms in plasma melatonin and core body temperature of healthy young males who stayed in an experimental facility for 7 days under dim light conditions (<10 lux). Sleep polysomnogram (PSG) and heart rate variability (HRV) were also measured. Subjects performed 2-h intermittent physical exercise with a bicycle ergometer at ZT3 or at ZT10 for four consecutive days, where zeitgeber time 0 (ZT0) was the time of wake-up. The rising phase of plasma melatonin rhythm was delayed by 1.1 h without exercise. Phase-delay shifts of a similar extent were detected by morning and evening exercise. But the falling phase shifted only after evening exercise by 1.0 h. The sleep PSG did not change after morning exercise, while Stage 1+2 sleep significantly decreased by 13.0% without exercise, and RE sleep decreased by 10.5% after evening exercise. The nocturnal decline of rectal temperature was attenuated by evening exercise, but not by morning exercise. HRV during sleep changed differentially. Very low frequency (VLF) waves increased without exercise. VLF, low frequency (LF), and high frequency (HF) waves increased after morning exercise, whereas HR increased after evening exercise. Morning exercise eventually enhanced the parasympathetic activity, as indicated by HRV, while evening exercise activated the sympathetic activity, as indicated by increase in heart rate in the following nocturnal sleep. These findings indicated differential effects of morning and evening exercise on the circadian melatonin rhythm, PSG, and HRV.

Fluorescent protein-based biosensors to visualize signal transduction beneath the plasma membrane.

In response to extracellular stimuli, cells display a variety of behaviors, including proliferation, differentiation, morphological changes and migration. The analysis of the spatiotemporal regulation of signal transduction in living cells is needed for a better understanding of such behaviors, and such investigations have been greatly accelerated by the development of fluorescent protein-based biosensors. Currently, by using these biosensors a range of molecular actions, including lipid metabolism, protein activation, and ion dynamics, can be visualized in living cells. We recently reported that intracellular calcium, with its relevant downstream signaling pathways consisting of the small GTPase Ras and the lipid kinase phoshoinositide-3-kinase (PI3K), can be exploited in an efficient incorporation of influenza A viruses into host cells via endocytosis using a set of biosensors based on fluorescent proteins and the principle of Förster resonance energy transfer. Here, we focus this review on fluorescent protein-based biosensors that have been utilized in our recent research reports.

Differential regulation of circadian melatonin rhythm and sleep-wake cycle by bright lights and nonphotic time cues in humans.

Our previous study demonstrated that physical exercise under dim lights (<10 lux) accelerated reentrainment of the sleep-wake cycle but not the circadian melatonin rhythm to an 8-h phase-advanced sleep schedule, indicating differential effects of physical exercise on the human circadian system. The present study examined the effects of bright light (>5,000 lux) on exercise-induced acceleration of reentrainment because timed bright lights are known to reset the circadian pacemaker. Fifteen male subjects spent 12 days in temporal isolation. The sleep schedule was advanced from habitual sleep times by 8 h for 4 days, which was followed by a free-run session. In the shift session, bright lights were given during the waking time. Subjects in the exercise group performed 2-h bicycle running twice a day. Subjects in the control kept quiet. As a result, the sleep-wake cycle was fully entrained by the shift schedule in both groups. Bright light may strengthen the resetting potency of the shift schedule. By contrast, the circadian melatonin rhythm was phase-advanced by 6.9 h on average in the exercise group but only by 2.0 h in the control. Thus physical exercise prevented otherwise unavoidable internal desynchronization. Polysomnographical analyses revealed that deterioration of sleep quality by shift schedule was protected by physical exercise under bright lights. These findings indicate differential regulation of sleep-wake cycle and circadian melatonin rhythm by physical exercise in humans. The melatonin rhythm is regulated primarily by bright lights, whereas the sleep-wake cycle is by nonphotic time cues, such as physical exercise and shift schedule.

Organ-specific development characterizes circadian clock gene Per2 expression in rats.

To explore developmental changes in circadian organization of central and peripheral oscillators, circadian rhythms in clock gene expression were examined in 12 organs in transgenic rats carrying a bioluminescence reporter for Per2. Organ slices were obtained from different developmental stages starting at postnatal day 5 and tissue was cultured for more than 6 days. In addition, four organs were examined from embryonic day 20. Robust circadian rhythms in bioluminescence were detected in all organs examined. The circadian period in vitro was specific to each organ and remained essentially the same during development. The circadian peak phase on the first day of culture was significantly different not only among organs but also in the same organ. Three patterns in circadian phase were detected during development. Thus, during development, circadian phase did not change in the suprachiasmatic nucleus, adrenal gland, and liver, whereas delay shifts were seen in the pineal, lung, heart, kidney, spleen, thymus, and testis. Finally, circadian phase advanced at postnatal day 10-15 and subsequently delayed in skeletal muscle and stomach.Circadian amplitude also showed developmental changes in several organs. These findings indicate that the temporal orders of physiological functions of various organs change during development. Such age-dependent and organ-specific changes in the phase relationship among circadian clocks most likely reflect entrainment to organ-specific time cues at different developmental stages.

Monitoring circadian time in rat plasma using a secreted Cypridina luciferase reporter.

A firefly luciferase reporter enabled us to monitor promoter activity in vivo as well as ex vivo; however, this requires a sufficient supply of the substrate luciferin and specific monitoring devices. To overcome these disadvantages, we developed transgenic rats carrying a secreted enzyme Cypridina luciferase (CLuc) reporter under the promoter of clock gene Per2 (Per2-CLuc). Per2-CLuc activity in serially sampled blood from freely moving rats exhibited robust circadian rhythms with a peak at early morning. The Per2-CLuc bioluminescence could be quantified even with approximately 100pl of plasma. Plasma Per2-CLuc rhythms were phase reversed, and the level was reduced by restricting food access for 2h during the light phase, suggesting that the plasma Per2-CLuc rhythms reflect the phase of peripheral clocks entrained to feeding cues as well as fuel metabolism. Fasting for 2days did not alter the circadian Per2-CLuc rhythms in rats, suggesting that feeding per se did not affect the circadian Per2-CLuc rhythms. Tissue-specific Per2-CLuc rhythms were observed in culture medium of peripheral tissues. The Per2-CLuc reporter is a powerful tool to access gene expression in vivo as well as ex vivo with ordinary laboratory equipment.

Daily exposure to cold phase-shifts the circadian clock of neonatal rats in vivo.

Maternal rhythms entrain the prenatal and neonatal circadian clock in the suprachiasmatic nucleus (SCN) before light entrainment is established. However, the responsible time cues for maternal entrainment are not identified. To examine the role of cyclic changes of ambient temperature in maternal entrainment, blind neonatal rats carrying a clock gene (Per2) bioluminescence reporter were exposed to either of three ambient temperatures (10, 20 or 30 °C) during 6-h maternal separation in the early light phase. Cold exposure was performed from postnatal day 1 (P1) to P5. On P6, the SCN was harvested and cultured for photometric monitoring of the circadian rhythm in Per2 expression. Here we demonstrate that the daily cold exposure phase-delayed the circadian Per2 expression rhythms at P6 in a temperature-dependent manner. Exposure to 10 °C produced the largest phase-shift of 12.7 h, and exposure to 20 and 30 °C yielded moderate shifts of 4.1 and 4.5 h, respectively. There was no significant difference in the phase-shifts between the latter two temperatures, indicating that ambient temperature is not the sole factor for the phase-shift. Behavioral rhythms that developed after weaning reflected the phase-shift of clock gene expression rhythm in the SCN. These findings indicate that a daily exposure to an ambient temperature of 10 °C during the neonatal period is capable of resetting the circadian clock in the SCN, but other factors yet unidentified are also involved in maternal entrainment.

JNK regulates the photic response of the mammalian circadian clock.

The posttranslational regulation of mammalian clock proteins has been assigned a time-keeping function, but seems to have more essential roles. Here we show that c-Jun N-terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron-specific isoform JNK3 have altered behavioural rhythms, with longer free-running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light.

De novo synthesis of PERIOD initiates circadian oscillation in cultured mouse suprachiasmatic nucleus after prolonged inhibition of protein synthesis by cycloheximide.

The circadian oscillation is known to stop with prolonged inhibition of protein synthesis and to restart from a particular phase after the removal of inhibition. In order to know the underlying molecular mechanisms, the mouse suprachiasmatic nucleus was cultured and treated with a protein synthesis inhibitor, cycloheximide (CHX), for various durations. Circadian rhythms in Bmal1 expression and PER2 protein were monitored by means of bioluminescence reporters. Bmal1-LUC and PER2::LUC bioluminescence decreased to basal levels after CHX application. CHX washout restarted the circadian rhythms from a fixed phase when CHX treatment exceeded 18 h. mRNA of Per1, Per2 and Rev-erbα increased and reached high plateau levels in 18 h after CHX application, which continued for 48 h; whereas Bmal1 mRNA increased for the first 18 h but then decreased to the basal level. Immunoblot analysis showed a decreased PER2::LUC level at 24 h after CHX application, indicating that the transcription of Pers and Rev-erbα was disinhibited by CHX. CHX washout increased PER2::LUC bioluminescence and protein level in a few hours. High Per mRNA levels induced the rapid increases of their proteins, which might trigger the restarting of circadian oscillation. These findings indicate that the circadian oscillation is stopped by disinhibition of Per1 and Per2 transcriptions, and restarted upon the recovery of the PER mediated auto-feedback loop. De novo synthesis of PER protein is a key factor to initiate the circadian oscillation after prolonged inhibition of protein synthesis.

Physical exercise accelerates reentrainment of human sleep-wake cycle but not of plasma melatonin rhythm to 8-h phase-advanced sleep schedule.

Effects of timed physical exercise were examined on the reentrainment of sleep-wake cycle and circadian rhythms to an 8-h phase-advanced sleep schedule. Seventeen male adults spent 12 days in a temporal isolation facility with dim light conditions (<10 lux). The sleep schedule was phase-advanced by 8 h from their habitual sleep times for 4 days, which was followed by a free-run session for 6 days, during which the subjects were deprived of time cues. During the shift schedule, the exercise group (n = 9) performed physical exercise with a bicycle ergometer in the early and middle waking period for 2 h each. The control group (n = 8) sat on a chair at those times. Their sleep-wake cycles were monitored every day by polysomnography and/or weight sensor equipped with a bed. The circadian rhythm in plasma melatonin was measured on the baseline day before phase shift: on the 4th day of shift schedule and the 5th day of free-run. As a result, the sleep-onset on the first day of free-run in the exercise group was significantly phase-advanced from that in the control and from the baseline. On the other hand, the circadian melatonin rhythm was significantly phase-delayed in the both groups, showing internal desynchronization of the circadian rhythms. The sleep-wake cycle resynchronized to the melatonin rhythm by either phase-advance or phase-delay shifts in the free-run session. These findings indicate that the reentrainment of the sleep-wake cycle to a phase-advanced schedule occurs independent of the circadian pacemaker and is accelerated by timed physical exercise.

The circadian pacemaker in the cultured suprachiasmatic nucleus from pup mice is highly sensitive to external perturbation.

The circadian clock in the suprachiasmatic nucleus of the hypothalamus (SCN) entrains to non-photic maternal rhythms in the fetal and neonatal periods of rodents but this capacity disappears in later life. In order to understand the mechanism behind the non-photic entrainment in the early postnatal period, the phase response of the clock gene (Bmal1) expression rhythm to external stimuli was examined in cultured SCN harvested at postnatal day 6. The SCN was obtained from transgenic mice carrying a bioluminescence reporter for Bmal1 expression. Phase-dependent phase shifts of circadian rhythm were detected in the pup as well as in the adult for culture medium exchange but the amount of phase shift was significantly larger in the pup than in the adult SCN. Half of the pup SCNs did not show integrated circadian rhythmicities in the first few days in culture. In pups, the circadian period of Bmal1 expression rhythm was shorter and the amplitude of circadian rhythm was much lower than in adults. It is concluded that the responsiveness of cultured SCN to medium exchange is much larger in pups than in adult mice. Immaturity of the structural organization in the circadian system seems to underlie the high responsiveness of the pup SCN.

New reporter system for Per1 and Bmal1 expressions revealed self-sustained circadian rhythms in peripheral tissues.

A new reporter system for monitoring expressions of two clock genes, Per1 and Bmal1, from a single tissue in culture was developed in mice. Reporters are Vargula hilgendorfii luciferase (VL) and firefly luciferase (FL), whose activities are increased in parallel with Per1 and Bmal1 expressions, respectively. Formal properties of the circadian system in transgenic mice are indistinguishable from those in wild-type animals. Circadian rhythms in Per1-VL and Bmal1-FL in the suprachiasmatic nucleus (SCN) were robust and anti-phasic, although they were phase delayed by 4-8 h as compared with circadian rhythms in respective transcript levels in vivo. In peripheral tissues such as liver, circadian rhythms in Bmal1-FL persisted for more than 3 weeks. In the course of prolonged culture, circadian rhythms apparently damped out, but were restored immediately by refreshment of the culture medium. Restoration of the circadian rhythm is unlikely to be due to resetting of desynchronized population oscillation, because peripheral circadian rhythms did not show a type 0 phase response curve (PRC) for medium refreshment, a requirement for instantaneous resetting of circadian oscillation. Long-term persistence of circadian oscillation in spite of external perturbations supports an idea that circadian oscillations in peripheral tissues are self-sustained.