Developmental timing in C. elegans is regulated by kin-20 and tim-1, homologs of core circadian clock genes

In Caenorhabditis elegans, heterochronic genes constitute a developmental timer that specifies temporal cell fate selection. The heterochronic gene lin-42 is the C. elegans homolog of Drosophila and mammalian period, key regulators of circadian rhythms, which specify changes in behavior and physiology over a 24 hr day/night cycle. We show a role for two other circadian gene homologs, tim-1 and kin-20, in the developmental timer. Along with lin-42, tim-1 and kin-20, the C. elegans homologs of the Drosophila circadian clock genes timeless and doubletime, respectively, are required to maintain late-larval identity and prevent premature expression of adult cell fates. The molecular parallels between circadian and developmental timing pathways suggest the existence of a conserved molecular mechanism that may be used for different types of biological timing.     

Circadian rhythm of autophagy proteins in hippocampus is blunted by sleep fragmentation

Autophagy is essential for normal cellular survival and activity. Circadian rhythms of autophagy have been studied in several peripheral organs but not yet reported in the brain. Here, we measured the circadian rhythm of autophagy-related proteins in mouse hippocampus and tested the effect of sleep fragmentation (SF). Expressions of the autophagy-related proteins microtubule‐associated protein 1 light chain 3 (LC3) and beclin were determined by western blotting and immunohistochemistry. Both the hippocampal LC3 signal and the ratio of its lipid-conjugated form LC3-II to its cytosolic form LC3-I showed a 24 h rhythm. The peak was seen at ZT6 (1 pm) and the nadir at ZT16 (1 am). The LC3 immunoreactivity in hippocampal CA1 pyramidal neurons also distributed differently, with more diffuse cytoplasmic appearance at ZT16. Chronic SF had a mild effect to disrupt the 24 h rhythm of LC3 and beclin expression. Interestingly, a greater effect of SF was seen after 24 h of recovery sleep when LC3-II expression was attenuated at both the peak and trough of circadian activities. Overall, the results show for the first time that the hippocampus has a distinct rhythm of autophagy that can be altered by SF.     

OsITL1 gene encoding an inositol 1,3,4-trisphosphate 5/6-kinase is a negative regulator of osmotic stress signaling

The expression of the gene OsITL1 coding for the inositol 1,3,4-trisphosphate 5/6-kinase protein was induced by 200 mM NaCl or dehydration. The expression of OsITL1 in response to NaCl and dehydration suggests the possible functions of OsITL1 in osmotic stress responses; however, physiological tests indicate that the expression of OsITL1 in tobacco decreases tolerance to NaCl during germination and seedling development. This result was consistent with that obtained on treatment of mature tobacco seedlings with NaCl (200 mM), suggesting that OsITL1 inversely regulates plant responses to osmotic stress.     

Circadian rhythm of intracellular protein synthesis signaling in rat cardiac and skeletal muscles

Intracellular signaling exhibits circadian variation in the suprachiasmatic nucleus and liver. However, it is unclear whether circadian regulation also extends to intracellular signaling pathways in the cardiac and skeletal muscles. Here, we examined circadian variation in the intracellular mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) and extracellular signal-regulated kinase (ERK) pathways, which regulate protein synthesis in rat cardiac and skeletal muscles. Seven-week-old male Wistar rats were assigned to six groups: Zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, ZT18, and ZT22 (ZT0, lights on; ZT12, lights off). The cardiac, plantaris, and soleus muscles were removed after a 12-h fasting period, and signal transducers involved in protein synthesis (mTOR, p70S6K, and ERK) were analyzed by western blotting. Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p<0.05), but not in the soleus muscle. In the cardiac muscle, the phosphorylation rate of mTOR was significantly higher at ZT6 (peak) than at ZT18 (bottom), and the phosphorylation rate of p70S6K was significantly higher at ZT2 (peak) than at ZT18 (bottom). In contrast, in the plantaris muscle, the phosphorylation rate of ERK was significantly lower at ZT2 (bottom) than at ZT18 (peak). Our data suggested that protein synthesis via mTOR/p70S6K and ERK signaling molecules exhibits circadian variation in rat cardiac and fast-type plantaris muscles.     

The endogenous melatonin (MT) signal facilitates reentrainment of the circadian system to light-induced phase advances by acting upon MT2 receptors

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors.     

β-carotene ameliorates CUS-induced circadian alternations of locomotor activity and melatonin patterns in rats

Circadian and stress systems have a crucial role in adaptation of organisms to environmental challenges. This study investigates the ability of Oscillatoria brevis (O. brevis) β-carotene extract (βC) in modulating the circadian alternations of locomotor activity (LA) and serum melatonin (M) rhythms under chronic unpredictable stress (CUS) in rats. Twenty rats (5 rats/group) were used in monitoring LA using running wheels. Eighty rats (20 rats/group) were used in observing circadian serum M profile. Rats were randomly divided into four groups, viz. control, βC-treated, CUS-exposed, and “βC-treated&CUS-exposed” groups. CUS-exposure was applied for 21 days. One hour before exposure, βC was daily administered (10 mg/kg), intraperitoneally (IP). Blood was sampled at 6-h intervals for 5 rats/time point at Zeitgeber (ZT) 3, 9, 15, and 21. Results demonstrated that unstressed rats exhibited circadian M pattern and nocturnal LA rhythm with acrophase around ZT 21 and ZT 15, respectively. CUS-exposure revealed a disturbance in these patterns. Phase shifting of M and LA profiles was recorded. A decrease M acrophase and a significant decrease in LA (p < 0.05) were recorded at ZT 9. Daily βC administration in stressed rats modulates the CRs alternation induced by CUS. It may be concluded that βC ameliorated the induced alternations in circadian rhythms.     

Circadian physiology is a toxicity target of the anticancer drug gemcitabine in mice

The circadian timing system determines the optimal timing and waveform of drug tolerability, yet treatment itself can alter this system. Gemcitabine is an antimetabolite agent that is active against lung and pancreatic cancers. Tolerability for this drug is best following dosing at ZT 11 in mice. The authors investigated the effects of gemcitabine on the circadian rhythms in body temperature and rest activity as physiological markers of the circadian timing system. Healthy unrestrained B6D2F(1) mice implanted with radiotelemetry transmitters were kept in LD 12:12 prior to receiving a single intravenous dose of gemcitabine (200, 400, or 600 mg/kg) at ZT 11 or 23. Gemcitabine (400 mg/kg) transiently suppressed the body temperature rhythm in 50% of the mice dosed at ZT 23, as compared to none of the mice treated at ZT 11 within the 2 days following drug dosing (Fisher 's exact test p = 0.04). The rest-activity circadian rhythm was suppressed in 40% (ZT 11) and 50% (ZT 23) of the mice, respectively. In the mice with persistent circadian rhythms, gemcitabine delivery at ZT 23 resulted in more prominent decreases and slower recovery of circadian mesor and amplitude of both rhythms as compared to mice treated at ZT 11. Gemcitabine also induced a transient internal desynchronization between temperature and activity rhythms following dosing at ZT 23 but not at ZT 11. The delivery of a single therapeutic dose of gemcitabine near its time of least toxicity produced least alterations in circadian physiological outputs, a finding that suggests that the extent of circadian disruption contributes to toxicokinetic processes.     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

The Endogenous Melatonin (MT) Signal Facilitates Reentrainment of the Circadian System to Light-Induced Phase Advances by Acting Upon MT2 Receptors

The indolamine melatonin is an important rhythmic endocrine signal in the circadian system. Exogenous melatonin can entrain circadian rhythms in physiology and behavior, but the role of endogenous melatonin and the two membrane-bound melatonin receptor types, MT1 and MT2, in reentrainment of daily rhythms to light-induced phase shifts is not understood. The present study analyzed locomotor activity rhythms and clock protein levels in the suprachiasmatic nuclei (SCN) of melatonin-deficient (C57BL/6J) and melatonin-proficient (C3H/HeN) mice, as well as in melatonin-proficient (C3H/HeN) mice with targeted deletion of the MT1, MT2, or both receptors, to determine effects associated with phase delays or phase advances of the light/dark (LD) cycle. In all mouse strains and genotypes, reentrainment of locomotor activity rhythms was significantly faster after a 6-h phase delay than a 6-h phase advance. Reentrainment after the phase advance was, however, significantly slower than in melatonin-deficient animals and in mice lacking functional MT2 receptors than melatonin-proficient animals with intact MT2 receptors. To investigate whether these behavioral differences coincide with differences in reentrainment of clock protein levels in the SCN, mPER1, mCRY1 immunoreactions were compared between control mice kept under the original LD cycle and killed at zeitgeber time 04 (ZT04) or at ZT10, respectively, and experimental mice subjected to a 6-h phase advance of the LD cycle and sacrificed at ZT10 on the third day after phase advance. This ZT corresponds to ZT04 of the original LD cycle. Under the original LD cycle, the numbers of mPER1- and mCRY1-immunoreactive cell nuclei were low at ZT04 and high at ZT10 in the SCN of all mouse strains and genotypes investigated. Notably, mouse strains with intact melatonin signaling and functional MT2 receptors showed a significant increase in the number of mPER1- and mCRY1-immunoreactive cell nuclei at the new ZT10 as compared to the former ZT04. These data suggest the endogenous melatonin signal facilitates reentrainment of the circadian system to phase advances on the level of the SCN molecular clockwork by acting upon MT2 receptors. (Author correspondence: M.Pfeffer@em.uni-frankfurt.de)     

CLOCK is required for maintaining the circadian rhythms of Opsin mRNA expression in photoreceptor cells

In zebrafish, the expression of long-wavelength cone (LC) opsin mRNA fluctuated rhythmically between the day and night. In a 24-h period, expression was high in the afternoon and low in the early morning. This pattern of fluctuation persisted in zebrafish that were kept in constant darkness, suggesting an involvement of circadian clocks. Functional expression of Clock, a circadian clock gene that contributes to the central circadian pacemaker, was found to play an important role in maintaining the circadian rhythms of LC opsin mRNA expression. In zebrafish embryos, in which the translation of Clock was inhibited by anti-Clock morpholinos, the circadian rhythms of LC opsin mRNA expression diminished. CLOCK may regulate the circadian rhythms of LC opsin mRNA expression via cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. In control retinas, the concentration of cAMP was high in the early morning and low in the remainder of the day and night. Inhibition of Clock translation abolished the fluctuation in the concentration of cAMP, thereby diminishing the circadian rhythms of opsin mRNA expression. Transient increase of cAMP concentrations in the early morning (i.e. by treating the embryos with 8-bromo-cAMP) restored the circadian rhythms of LC opsin mRNA expression in morpholino-treated embryos. Together, the data suggest that Clock plays important roles in regulating the circadian rhythms in photoreceptor cells.     

The effect of insulin on circadian rhythm of voluntary locomotor activity in rats

Effects of single intranasal administration of 0.2 ng insulin at different moments of the projected daily cycle (ZT = 1, ZT = 7, ZT = 13 and ZT = 19) on the circadian rhythms of voluntary locomotor activity (wheel-running) were studied in Wistar male rats. Insulin administered at ZT-7 or ZT-13 induced a statistically significant phase advance by 4.4 and 5.5 hours, respectively. The administration of insulin at ZT-13 additionally induced a reduction of the period of the circadian rhythm of voluntary locomotor activity. Intranasal administration of insulin at other moments of the projected daily cycle (ZT = 1 or ZT = 19) did not induce any statistically significant change in phase or period duration of the circadian rhythms. Insulin did not cause changes in total daily activity irrespective of administration time. The results of the study suggest the role of endogenous insulin as entrainment factor for circadian oscillator in absence of the main physiological zeitgeber--cyclic afferent input from retina photoreceptors.     

Circadian rhythm of the cyanobacterium Synechocystis sp. strain PCC 6803 in the dark.

The cyanobacterium Synechocystis sp. strain PCC 6803 exhibited circadian rhythms in complete darkness. To monitor a circadian rhythm of the Synechocystis cells in darkness, we introduced a PdnaK1::luxAB gene fusion (S. Aoki, T. Kondo, and M. Ishiura, J. Bacteriol. 177:5606-5611, 1995), which was composed of a promoter region of the Synechocystis dnaK1 gene and a promoterless bacterial luciferase luxAB gene set, as a reporter into the chromosome of a dark-adapted Synechocystis strain. The resulting dnaK1-reporting strain showed bioluminescence rhythms with a period of 25 h (on agar medium supplemented with 5 mM glucose) for at least 7 days in darkness. The rhythms were reset by 12-h-light-12-h-dark cycles, and the period of the rhythms was temperature compensated for between 24 and 31 degrees C. These results indicate that light is not necessary for the oscillation of the circadian clock in Synechocystis.