Internal noise-driven circadian oscillator in Drosophila

An internal noise-driven oscillator was studied in a two-variable Drosophila model, where both positive feedback and negative feedback are crucial to the circadian oscillations. It is shown that internal noise could sustain reliable oscillations for the parameter which produces a stable steady state in the deterministic system. The noise-sustained oscillations are interpreted by using phase plane analysis. The period of such oscillations fluctuates slightly around the period of deterministic oscillations and the coherence of oscillations becomes the best at an optimal internal noise intensity, indicating the occurrence of intrinsic coherence resonance. In addition, in the oscillatory region, the coherence of noisy circadian oscillations is suppressed by the internal noise, but the period is hardly affected, demonstrating the robustness of the Drosophila model for circadian rhythms to the intrinsic noise.     

Phase response and entrainment: Model computations on their relation using Wever's oscillator model

Abstract

Enright's theory, which explains entrainment as periodically repeated phase response, is applied to Wever's self‐sustained oscillation model of are circadian rhythm and tested by computer simulation. Ranges of phase response and entrainment are compared and the oscillatory behaviour is shown in the phase diagram for the cases of phase response and entrainment. It is shown that Enright's theory is not valid for self‐sustained oscillations in general, but it need not necessarily fail in case of the biological circadian rhythm.     

Nature of KaiB-KaiC binding in the cyanobacterial circadian oscillator

In the cyanobacteria Synechococcus elongatus and Thermosynechococcus elongatus, the KaiA, KaiB and KaiC proteins in the presence of ATP generate a post-translational oscillator (PTO) that can be reconstituted in vitro. KaiC is the result of a gene duplication and resembles a double doughnut with N-terminal CI and C-terminal CII hexameric rings. Six ATPs are bound between subunits in both the CI and CII ring. CI harbors ATPase activity, and CII catalyzes phosphorylation and dephosphorylation at T432 and S431 with a ca. 24-h period. KaiA stimulates KaiC phosphorylation, and KaiB promotes KaiC subunit exchange and sequesters KaiA on the KaiB-KaiC interface in the final stage of the clock cycle. Studies of the PTO protein-protein interactions are convergent in terms of KaiA binding to CII but have led to two opposing models of the KaiB-KaiC interaction. Electron microscopy (EM) and small angle X-ray scattering (SAXS), together with native PAGE using full-length proteins and separate CI and CII rings, are consistent with binding of KaiB to CII. Conversely, NMR together with gel filtration chromatography and denatured PAGE using monomeric CI and CII domains support KaiB binding to CI. To resolve the existing controversy, we studied complexes between KaiB and gold-labeled, full-length KaiC with negative stain EM. The EM data clearly demonstrate that KaiB contacts the CII ring. Together with the outcomes of previous analyses, our work establishes that only CII participates in interactions with KaiA and KaiB as well as with the His kinase SasA involved in the clock output pathway.     

Probing the circadian oscillator of a mammal by two-pulse perturbations

When organisms are maintained under constant conditions of light and temperature, their endogenous circadian rhythms free run, manifesting their intrinsic period. The phases of these free-running rhythms can be shifted by stimuli of light, temperature, and drugs. The change from one free-running steady state to another following a perturbation often involves several transient cycles (cycles of free-running rhythm drifting slowly to catch up with the postperturbation steady state). Although the investigation of oscillator kinetics in circadian rhythms of both insects and mammals has revealed that the circadian pacemaker phase shifts instantaneously, the phenomenon of transient cycles has remained an enigma. We probed the phases of the transient cycles in the locomotor activity rhythm of the field mouse Mus booduga, evoked by a single light pulse (LP), using LPs at critically timed phases. The results of our experiments indicate that the transient cycles generated during transition from one steady state to another steady state do not represent the state of the circadian pacemaker (basic oscillator) controlling the locomotor activity rhythm in Mus booduga. (Chronobiology International, 17(2), 129-136, 2000)     

A role for cyclic AMP in entrainment of the circadian oscillator in Xenopus retinal photoreceptors by dopamine but not by light

The circadian oscillator in Xenopus retinal photoreceptor layers can be reset in similar ways by light and agonists of D2-like dopamine receptors. Treatments that increase cyclic AMP levels act on this oscillator in an opposite fashion, mimicking darkness in the induction of phase shifts. Light and dopamine have each been reported to inhibit adenylate cyclase in photoreceptors. Together, these data suggest that the transduction pathways for entrainment by dopamine and/or light include suppression of cyclic AMP or a cyclic AMP-sensitive step. In these studies, we examined this hypothesis by measuring the effects of treatment with a cyclic AMP analogue on the phase shifts induced in photoreceptor melatonin rhythms by light or a D2 receptor agonist (quinpirole). When photoreceptor layers were treated simultaneously with 8-(4-chlorophenylthio)cyclic AMP (8-CPT-cAMP) and quinpirole at any of three different phases of the circadian cycle, the resulting phase shifts of the melatonin rhythm were always the same as those caused by 8-CPT-cAMP alone. This indicates that there is a cyclic AMP-sensitive step in the dopamine entrainment pathway. In contrast, light pulses did reset the oscillator in the presence of elevated cyclic AMP. This suggests a separate cyclic AMP-insensitive transduction pathway for entrainment by light. Quinpirole reduced basal levels of cyclic AMP in photoreceptors, but light did not. These data suggest that cyclic AMP plays a role in the entrainment pathway activated by dopamine but not in the entrainment pathway activated by light.     

KaiA调控蓝藻生物钟的分子机制研究进展

蓝藻生物钟系统主要包括输入途径、核心振荡器和输出途径3部分,核心振荡器主要由时钟蛋白KaiA、KaiB、KaiC构成。3种蛋白之间的相互作用产生节律信号及调控输入、输出信号进而维持生物振荡的精确与稳定。文中围绕蓝藻生物钟核心振荡器及核心振荡器组成蛋白的结构、功能与相互作用特点,结合本实验室近期取得的研究成果,针对时钟蛋白KaiA调节KaiC的酶活性、介导核心振荡器的时相重置、与CikA竞争KaiB的结合位点等方面近年来的研究进展进行了综述。     

Coupling-induced synchronization in multicellular circadian oscillators of mammals

In mammals, circadian rhythms are controlled by the neurons located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Each neuron in the SCN contains an autonomous molecular clock. The fundamental question is how the individual cellular oscillators, expressing a wide range of periods, interact and assemble to achieve phase synchronization. Most of the studies carried out so far emphasize the crucial role of the periodicity imposed by the light-dark cycle in neuronal synchronization. However, in natural conditions, the interaction between the SCN neurons is non-negligible and coupling between cells in the SCN is achieved partly by neurotransmitters. In this paper, we use a model of nonidentical, globally coupled cellular clocks considered as Goodwin oscillators. We mainly study the synchronization induced by coupling from an analytical way. Our results show that the role of the coupling is to enhance the synchronization to the external forcing. The conclusion of this paper can help us better understand the mechanism of circadian rhythm.     

Influence of light and food on the circadian clock in liver of rainbow trout,Oncorhynchus mykiss

Several reports support the existence of multiple peripheral oscillators in fish, which may be able to modulate the rhythmic functions developed by those tissues hosting them. Thus, a circadian oscillator has been proposed to be located within fish liver. In this vertebrate group, the role played by the circadian system in regulating metabolic processes in liver is mostly unknown. We, therefore investigated the liver of rainbow trout (Oncorhynchus mykiss) as a potential element participating in the regulation of circadian rhythms in fish by hosting a functional circadian oscillator. The presence and expression pattern of main components of the circadian molecular machinery (clock1a, bmal1, per1 and rev-erbβ-like) were assessed. Furthermore, the role of environmental cues such as light and food, and their interaction in order to modulate the circadian oscillator was also assessed by exposing animals to constant conditions (absence of light for 48 h, and/or a 4 days fasting period). Our results demonstrate the existence of a functional circadian oscillator within trout liver, as demonstrated by significant rhythms of all clock genes assessed, independently of the environmental conditions studied. In addition, the daily profile of mRNA abundance of clock genes is influenced by both light (mainly clock1a and per1) and food (rev-erbβ-like), which is indicative of an interaction between both synchronizers. Our results point to rev-erbβ-like as possible mediator between the influence of light and food on the circadian oscillator within trout liver, since its daily profile is influenced by both light and food, thus affecting that of bmal1.     

Unique self-sustaining circadian oscillators within the brain of Drosophila melanogaster

In Drosophila circadian rhythms persist in constant darkness (DD). The small ventral Lateral Neurons (s-LN_v) mainly control the behavioral circadian rhythm in consortium with the large ventral Lateral Neurons (l-LN_v) and dorsal Lateral Neurons (LN_d). It is believed that the molecular oscillations of clock genes are the source of this persistent behavior. Indeed the s-LN_v, LN_d, Dorsal Neurons (DN)-DN₂ and DN₃ displayed self-sustained molecular oscillations in DD both at RNA and protein levels, except the DN₂ oscillates in anti-phase. In contrast, the l-LN_v and DN₁ displayed self-sustained oscillations at the RNA level, but protein oscillations quickly dampened. Having self-sustained and dampened molecular oscillators together in the DN groups suggested that they play different roles. However, all DN groups seemed to contribute together to the light-dark (LD) behavioral rhythm. The LD entrainment of LN oscillators is achieved through Rhodopsin (RH) and Cryptochrome (CRY). CRY's expression in all DN groups implicates also its role in LD entrainment of DN, like in DN₁. However, mutations in cry and glass that did not inflict LD synchronization of the DN₂, DN₃ oscillator implicate the existence of a novel photoreceptor at least in DN₃.     

Differential effect of lithium on the circadian oscillator in young and old hamsters

Lithium is one of the most commonly used drugs in the prophylaxis and treatment of bipolar disorder. It is also known to lengthen circadian period in several organisms. Previously, we reported that there was the association between lengthening circadian period by lithium and GSK-3 protein and its enzyme activity in the mouse suprachiasmatic nucleus (SCN). In this study, we show that lithium affects the circadian oscillator in young and old hamster SCN, in an age-dependent manner. We found that basal levels of phosphorylated GSK-3 (pGSK-3) protein expression in old hamsters are much lower than that in young hamsters. Furthermore, in the old hamsters, lithium did not affect the period of the locomotor activity rhythm or pGSK-3 expression, while changing period and pGSK-3 in the younger animals. These results indicate that the content of pGSK-3 in the SCN has an important role in age-dependent effects of lithium on the circadian oscillator.     

In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB

Biochemical circadian oscillation of KaiC phosphorylation, by mixing three Kai proteins and ATP, has been proven to be the central oscillator of the cyanobacterial circadian clock. In vivo, the intracellular levels of KaiB and KaiC oscillate in a circadian fashion. By scrutinizing KaiC phosphorylation rhythm in a wide range of Kai protein concentrations, KaiA and KaiB were found to be “parameter-tuning” and “state-switching” regulators of KaiC phosphorylation rhythm, respectively. Our results also suggest a possible entrainment mechanism of the cellular circadian clock with the circadian variation of intracellular levels of Kai proteins.     

Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate

The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ~24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we performed autodephosphorylation assays using (32)P-labeled KaiC. The residual radioactive ATP bound to subunit interfaces was removed using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from (32)P-labeled KaiC were examined. We detected the transient formation of [(32)P]ATP preceding the accumulation of (32)P(i). Together with kinetic analyses, our data demonstrate that KaiC undergoes dephosphorylation via a mechanism that differs from those of conventional protein phosphatases. A phosphate group at a phosphorylation site is first transferred to KaiC-bound ADP to form ATP as an intermediate, which can be regarded as a reversal of the autophosphorylation reaction. Subsequently, the ATP molecule is hydrolyzed to form P(i). We propose that the ATPase active site mediates not only ATP hydrolysis but also the bidirectional transfer of the phosphate between phosphorylation sites and the KaiC-bound nucleotide. On the basis of these findings, we can now dissect the dynamics of the KaiC phosphorylation cycle relative to ATPase activity.     

Feeding entrainment of locomotor activity rhythms in the goldfish is mediated by a feeding-entrainable circadian oscillator

Periodic food availability can act as a potent zeitgeber capable of synchronizing many biological rhythms in fishes, including locomotor activity rhythms. In the present paper we investigated entrainment of locomotor rhythms to scheduled feeding under different light and feeding regimes. In experiment 1, fish were exposed to a 12:12?h light/dark cycle and fed one single daily meal in the middle of the light phase. In experiment 2, we tested the effect of random versus scheduled feeding on the daily distribution of activity. During random feeding, meals were randomly scheduled with intervals ranging from 12 to 36?h, while scheduled feeding consisted of one single daily meal set in the middle of the light or dark phase. Finally, in experiment 3, we studied the synchronization of activity rhythms to feeding under constant darkness (DD) and after shifting the feeding cycle by either advancing or delaying the feeding cycle by 9?h. The results revealed that goldfish synchronized to feeding, overcame light entrainment and significantly changed their daily distribution of activity according to their feeding schedule. In addition, the daily activity pattern modulated by feeding differed between layers: a peak of activity being noticeable directly after feeding at the bottom, while an anticipatory behaviour was obvious at the surface of the tank. Under DD and no food, free-running rhythms averaging 25.5?± 1.9?h (mean?±?SD) were detected. In conclusion, some properties of feeding entrainment (e.g. anticipation of the feeding time, free-running rhythms following termination of periodic feeding, and the stability of ø after shifting the feeding cycle) suggested that goldfish have (a) separate but tightly coupled light- and food-entrainable oscillators, or (b) a single oscillator that is entrainable by both light and food (one synchronizer being eventually stronger than the other).     

The cyanobacterial alkaloid nostocarboline: an inhibitor of acetylcholinesterase and trypsin

Preselected cyanobacterial strains (available from culture collections and our own isolates), belonging primarily to the heterocystous cluster, were screened for inhibitors against butyrylcholinesterase. About one-half of the extracts exhibited inhibitory activity. Nostocarboline, the responsible metabolite in Nostoc 78–12A, was studied in more detail as an acetylcholinesterase (AChE) inhibitor. The compound showed potent activity against this enzyme (IC₅₀ = 5.3 μM), and the Michaelis-Menten kinetics indicated a non-competitive component in the inhibitory mechanism. In addition, nostocarboline turned out to be a potent inhibitor of trypsin (IC₅₀ = 2.8 μM), and thus is the first described cyanobacterial serine protease inhibitor of an alkaloid structure. The function of nostocarboline in aquatic ecosystems and its potential as a lead compound for the development of useful therapeutic AChE inhibitors is discussed.