Biological clocks and incremental growth line formation in dentine

Dentine- and enamel-forming cells secrete matrix in consistent rhythmic phases, resulting in the formation of successive microscopic growth lines inside tooth crowns and roots. Experimental studies of various mammals have proven that these lines are laid down in subdaily, daily (circadian), and multidaily rhythms, but it is less clear how these rhythms are initiated and maintained. In 2001, researchers reported that lesioning the so-called master biological clock, the suprachiasmatic nucleus (SCN), halted daily line formation in rat dentine, whereas subdaily lines persisted. More recently, a key clock gene (Bmal1) expressed in the SCN in a circadian manner was also found to be active in dentine- and enamel- secretory cells. To probe these potential neurological and local mechanisms for the production of rhythmic lines in teeth, we reexamined the role of the SCN in growth line formation in Wistar rats and investigated the presence of daily lines in Bmal1 knockout mice (Bmal1^−/−). In contrast to the results of the 2001 study, we found that both daily and subdaily growth lines persisted in rat dentine after complete or partial SCN lesion in the majority of individuals. In mice, after transfer into constant darkness, daily rhythms continued to manifest as incremental lines in the dentine of each Bmal1 genotype (wild-type, Bmal^+/–, and Bmal1^−/−). These results affirm that the manifestation of biological rhythms in teeth is a robust phenomenon, imply a more autonomous role of local biological clocks in tooth growth than previously suggested, and underscore the need further to elucidate tissue-specific circadian biology and its role in incremental line formation. Investigations of this nature will strengthen an invaluable system for determining growth rates and calendar ages from mammalian hard tissues, as well as documenting the early lives of fossil hominins and other primates.     

Involvement of the luteinizing hormone surge in the regulation of ovary and oviduct clock gene expression in mice

Circadian dysfunction perturbs the female reproductive cycle. In particular, mice lacking the clock gene Bmal1 show severe infertility, implying that BMAL1 plays roles in ovulation and luteinization. Here, we examined temporal changes in clock gene expression in the ovary and oviduct before and during gonadotropin‐induced follicular growth, ovulation, and luteinization in sexually immature mice. While the oviduct did not show a drastic change in clock gene expression, Bmal1 expression in the ovary was higher than that in control mice during the period from 4 to 16 hr after human chorionic gonadotropin (hCG) administration. Bmal1 expression reached a maximum at 16 hr after hCG administration, when follicle luteinization occurred. In an interesting manner, administration of hCG to ex vivo‐cultured oviduct triggered a shorter circadian period and inevitably resulted in phase advance. Together, our present data suggest that LH surge induces continuous expression of BMAL1 in the mouse ovary and modulates circadian phase in the mouse oviduct.     

Rhythmic adenosine triphosphate release from the cyanobacterial circadian clock protein KaiC revealed by real-time monitoring of bioluminescence using firefly luciferase

The cyanobacterial circadian clock is composed of three clock proteins, KaiA, KaiB and KaiC. This KaiABC clock system can be reconstituted in vitro in the presence of adenosine triphosphate (ATP) and Mg²⁺, and shows circadian rhythms in the phosphorylation level and ATPase activity of KaiC. Previously, we found that ATP regulates a complex formation between KaiB and KaiC, and KaiC releases ATP from KaiC itself (PLoS One, 8, 2013, e80200). In this study, we examined whether the ATP release from KaiC shows any rhythms in vitro. We monitored the release of ATP from wild-type and ATPase motif mutants of KaiC as a bioluminescence in real time using a firefly luciferase assay in vitro and obtained the following results: (a) ATP release from KaiC oscillated even without KaiA and KaiB although period of the oscillation was not 24 hr; (b) ATP was mainly released from the N-terminal domain of KaiC; and (c) the ATP release was enhanced and suppressed by KaiB and KaiA, respectively. These results suggest that KaiC can generate basal oscillation as a core clock without KaiA and KaiB, whereas these two proteins contribute to adjusting and stabilizing the oscillation.     

Establishment of human cell lines showing circadian rhythms of bioluminescence

We have established human retinal pigment epithelial cell lines stably expressing the luciferase gene, driven by the human Bmal1 promoter, to obtain human-derived cells that show circadian rhythms of bioluminescence after dexamethasone treatment. The average circadian period of bioluminescence for the obtained clones was 24.07 ± 0.48 h. Lithium (10 mM) in the medium significantly lengthened the circadian period of bioluminescence, which is consistent with previous reports, while 2 mM or 5 mM lithium had no effect. This is the first report on the establishment of human-derived cell lines that proliferate infinitely and show circadian rhythms of bioluminescence, and also the first to investigate the effects of low-dose lithium on the circadian rhythms of human-derived cells in vitro. The established cells will be useful for various in vitro studies of human circadian rhythms and for the development of new therapies for human disorders related to circadian rhythm disturbances.     

Importance of the monomer–dimer–tetramer interconversion of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria

The molecular machinery of the cyanobacterial circadian clock oscillator consists of three proteins, KaiA, KaiB and KaiC, which interact with each other to generate circadian oscillations in the presence of ATP (the in vitro KaiABC clock oscillator). KaiB comprises four subunits organized as a dimer of dimers. Our previous study suggested that, on interaction with KaiC, the tetrameric KaiB molecule dissociates into two molecules of dimeric KaiB. It is uncertain whether KaiB also exists as a monomer and whether the KaiB monomer can drive normal circadian oscillation. To address these questions, we constructed a new KaiB oligomer mutant with an N‐terminal deletion, KaiB_10–108. KaiB_10–108 was a monomer at 4 °C but a dimer at 35 °C. KaiB_10–108 was able to drive normal clock oscillation in an in vitro reconstituted KaiABC clock oscillator at 25 °C, but it was not able to drive normal circadian gene expression rhythms in cyanobacterial cells at 41 °C. Wild‐type KaiB existed in equilibrium between a dimer and tetramer at lower KaiB concentrations or in the presence of 1 m NaCl. Our findings suggest that KaiB is in equilibrium between a monomer, dimer and tetramer in cyanobacterial cells.     

Allergen‐specific basophil reactivity exhibits daily variations in seasonal allergic rhinitis

It remains poorly understood how symptoms in allergic rhinitis are most severe during overnight or early in the morning. The circadian clock consisting of a network of several ‘clock genes’ including Clock drives daily rhythms in physiology. This study showed that allergen‐induced surface CD203c expression on basophils in seasonal allergic rhinitis caused by Japanese cedar pollen exhibited a time‐of‐day‐dependent variation associated with temporal variations in canonical circadian clock gene expression. We also found that bone‐marrow‐derived basophils (BM basophils) generated from wild‐type mice exhibited a time‐of‐day‐dependent variation in IgE‐mediated IL‐4 and histamine production, which was not observed in BM basophils generated from Clock‐mutated mice. Therefore, allergen‐specific basophil reactivity shows daily variations depending on the circadian clock activity in basophils, which could partly explain temporal symptomatic variations in allergic rhinitis. Additionally, circadian variations in CD203c expression should be considered for interpretation of this biomarker in clinical research.