Glyceraldehyde-3-phosphate dehydrogenase is regulated on a daily basis by the circadian clock

Circadian clocks function to govern a wide range of rhythmic activities in organisms. An integral part of rhythmicity is the daily control of target genes by the clock. Here we describe the sequence and analysis of a novel clock-controlled gene, ccg-7, showing similarity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme widely used as a constitutive control in a variety of systems. That ccg-7 encodes GAPDH was confirmed by demonstrating that in vitro synthesized CCG-7 possesses GAPDH activity. Rhythms in both ccg-7 mRNA accumulation and CCG-7 (GAPDH) activity are observed in a clock wild-type strain where the peak in GAPDH activity lags several hours behind the peak in ccg-7 mRNA accumulation in the late night. Together with our previous observation that ccg-7 mRNA is not developmentally regulated, we show that ccg-7 is not induced by environmental stresses such as glucose or nitrogen deprivation (which also trigger development), heat shock, or osmotic stress. Thus, the finding that GAPDH is clock-regulated points to a specific role for the circadian clock in controlling aspects of general metabolism and provides evidence for circadian regulation of a gene found in most living organisms.     

Resynchronisation of a diurnal rodent circadian clock accelerated by a melatonin agonist

Using 'jet lag' paradigms involving phase shifts in the light-dark (LD) cycle, we studied the effects of S-20098 on the circadian clock of a diurnal rodent. Arvicanthis mordax, entrained to a regular LD cycle, were subjected to advance shifts (i.e. 4, 6 or 8 h) in the LD cycle and injected with vehicle or the melatonin agonist S-20098 (20 mg/kg) the day of the shift (and also on subsequent days in the 6 h or 8 h shift paradigms). In each condition, S-20098 accelerated by about 30% resynchronization to the new LD cycle. These data, which are the first to demonstrate the chronobiotic effects of a melatonin agonist in a diurnal rodent, provide new insights for the design of human chronopharmacological protocols.     

Fos protein expression in the circadian clock is not associated with phase shifts induced by a nonphotic stimulus, triazolam

A mutant human lysozyme, designated as C77A-a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)-lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h-lysozyme C77A-a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.     

Rapid resetting of the mammalian circadian clock

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. We used a two-pulse paradigm, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrated that resetting of the oscillator occurred within 2 hr. This was the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse showed resetting within 2 hr and in addition showed that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal.     

Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain

The period (per) gene, controlling circadian rhythms in Drosophila, is expressed throughout the body in a circadian manner. A homolog of Drosophila per was isolated from rat and designated as rPer2. The rPER2 protein showed 39 and 95% amino acid identity with mPER1 and mPER2 (mouse homologs of per) proteins, respectively. A robust circadian fluctuation of rPer2 mRNA expression was discovered not only in the suprachiasmatic nucleus (SCN) of the hypothalamus but also in other tissues including eye, brain, heart, lung, spleen, liver, and kidney. Furthermore, the peripheral circadian expression of rPer2 mRNA was abolished in SCN-lesioned rats that showed behavioral arrhythmicity. These findings suggest that the multitissue circadian expression of rPer2 mRNA was governed by the mammalian brain clock SCN and also suggest that the rPer2 gene was involved in the circadian rhythm of locomotor behavior in mammals.     

GABAB receptor stimulation phase-shifts the mammalian circadian clock in vitro

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) generates 24-h rhythms in vitro. Here we show that the GABAB agonist baclofen resets the SCN pacemaker in vitro in a phase-dependent manner: advances were induced at zeitgeber time (ZT) 6, and delays were induced at ZT 22. Both effects were blocked the GABAB antagonist, 2-hydroxysaclofen, while the GABAA antagonist, bicuculline was ineffective. Thus, the SCN pacemaker is sensitive to resetting by GABAB stimulation.     

Neuropeptide Y phase shifts the circadian clock in vitro via a Y2 receptor

The suprachiasmatic nuclei (SCN) contain a circadian clock whose activity can be recorded in vitro for several days. This clock can be reset by the application of neuropeptide Y. In this study, we focused on determination of the receptor responsible for neuropeptide Y phase shifts of the hamster circadian clock in vitro. Coronal hypothalamic slices containing the SCN were prepared from Syrian hamsters housed under a 14 h:10 h light:dark cycle. Tissue was bathed in artificial cerebrospinal fluid (ACSF), and the firing rates of individual cells were sampled throughout a 12 h period. Control slices received either no application or application of 200 nl ACSF to the SCN at zeitgeber time 6 (ZT6; ZT12 was defined as the time of lights off). Application of 200 ng/200 nl of neuropeptide Y at ZT6 resulted in a phase advance of 3.4 h. Application of the Y2 receptor agonist, neuropeptide Y (3-36), induced a similar phase advance in the rhythm, while the Y1 receptor agonist, [Leu31, Pro34]-neuropeptide Y had no effect. Pancreatic polypeptide (rat or avian) also had no measurable phase-shifting effect. Neuropeptide Y applied at ZT20 or 22 had no detectable phase-shifting effect. These results suggest that the phase-shifting effects of neuropeptide Y are mediated through a Y2 receptor, similar to results found in vivo.     

Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock

Circadian clocks are synchronized by environmental cues such as light. Photoreceptor-deficient Arabidopsis thaliana mutants were used to measure the effect of light fluence rate on circadian period in plants. Phytochrome B is the primary high-intensity red light photoreceptor for circadian control, and phytochrome A acts under low-intensity red light. Cryptochrome 1 and phytochrome A both act to transmit low-fluence blue light to the clock. Cryptochrome 1 mediates high-intensity blue light signals for period length control. The presence of cryptochromes in both plants and animals suggests that circadian input pathways have been conserved throughout evolution.     

Immortal time: circadian clock properties of rat suprachiasmatic cell lines

Cell lines derived from the rat suprachiasmatic nucleus (SCN) were screened for circadian clock properties distinctive of the SCN in situ. Immortalized SCN cells generated robust rhythms in uptake of the metabolic marker 2-deoxyglucose and in their content of neurotrophins. The phase relationship between these rhythms in vitro was identical to that exhibited by the SCN in vivo. Transplantation of SCN cell lines, but not mesencephalic or fibroblast lines, restored the circadian activity rhythm in arrhythmic, SCN-lesioned rats. Thus, distinctive oscillator, pacemaker, and clock properties of the SCN are not only retained but also maintained in an appropriate circadian phase relationship by immortalized SCN progenitors.     

A neuronal ryanodine receptor mediates light-induced phase delays of the circadian clock

Circadian clocks are complex biochemical systems that cycle with a period of approximately 24 hours. They integrate temporal information regarding phasing of the solar cycle, and adjust their phase so as to synchronize an organism's internal state to the local environmental day and night. Nocturnal light is the dominant regulator of this entrainment. In mammals, information about nocturnal light is transmitted by glutamate released from retinal projections to the circadian clock in the suprachiasmatic nucleus of the hypothalamus. Clock resetting requires the activation of ionotropic glutamate receptors, which mediate Ca2+ influx. The response induced by such activation depends on the clock's temporal state: during early night it delays the clock phase, whereas in late night the clock phase is advanced. To investigate this differential response, we sought signalling elements that contribute solely to phase delay. We analysed intracellular calcium-channel ryanodine receptors, which mediate coupled Ca2+ signalling. Depletion of intracellular Ca2+ stores during early night blocked the effects of glutamate. Activators of ryanodine receptors induced phase resetting only in early night; inhibitors selectively blocked delays induced by light and glutamate. These findings implicate the release of intracellular Ca2+ through ryanodine receptors in the light-induced phase delay of the circadian clock restricted to the early night.     

IL-6 expression by oral fibroblasts is regulated by androgen

The leucine-to-alanine mutation at residue 201 of D-amino acid aminotransferase provides a unique enzyme which gradually loses its activity while catalyzing the normal transamination; the co-enzyme form is converted from pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate upon the inactivation [Kishimoto,K., Yoshimura,T., Esaki,N., Sugio,S., Manning,J.M. and Soda,K. (1995) J. Biochem., 117, 691-696]. Crystal structures of both co-enzyme forms of the mutant enzyme have been determined at 2.0 A resolution: they are virtually identical, and are quite similar to that of the wild-type enzyme. Significant differences in both forms of the mutant are localized only on the bound co-enzyme, the side chains of Lys145 and Tyr31, and a water molecule sitting on the putative substrate binding site. Detailed comparisons of the structures of the mutant, together with that of the pyridoxamine-5'-phosphate form of the wild-type enzyme, imply that Leu201 would play a crucial role in the transamination reaction by keeping the pyridoxyl ring in the proper location without disturbing its oscillating motion, although the residue seems to not be especially important for the structural integrity of the enzyme.     

Cyclic AMP resets the circadian clock in cultured Xenopus retinal photoreceptor layers

The Xenopus retinal photoreceptor layer contains a circadian oscillator that regulates melatonin synthesis in vitro. The phase of this oscillator can be reset by light or dopamine. The phase-response curves for light and dopamine are similar, with transitions from phase delays to phase advances in the mid-subjective night. Light and dopamine each can inhibit adenylate cyclase in retinal photoreceptors, suggesting cyclic AMP as a candidate second messenger for entrainment of the circadian oscillator. We report here that treatments that increase intracellular cyclic AMP reset the phase of the photoreceptor circadian oscillator, and that the phase-response curves for these treatments are 180 degrees out of phase with the phase-response curves for light and dopamine. Activation of adenylate cyclase by forskolin during the late subjective day or early subjective night caused phase advances. The same treatment during the late subjective night or early subjective day caused phase delays. Similar phase shifts were induced by 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) or 8-(4-chlorophenylthio)cyclic AMP. All of these treatments also acutely increased melatonin release. Forskolin and 3-isobutyl-1-methylxanthine increased the accumulation of intracellular cyclic AMP, but not cyclic GMP, in photoreceptor layers. The results indicate that cyclic AMP-dependent pathways regulate the photoreceptor circadian oscillator and suggest that a decrease in cyclic AMP may be involved in circadian entrainment by light and/or dopamine.     

Flicker is a primitive visual attribute in visual search.

At the earliest processing stages, visual stimuli are decomposed by a set of filters tuned to specific values of such attributes as colour, orientation, and motion. These filters have been characterised both neurophysiologically and behaviourally. The single exception is the attribute of flicker that has been characterised neurophysiologically but not behaviourally. Using a visual search paradigm, the authors provide the first behavioural demonstration that flicker is indeed a primitive attribute used by the visual system in stimulus encoding. Consistent with the temporal contrast-sensitivity function, sensitivity to flicker was highest at about 10 Hz and decreased as the flicker rate was either increased or decreased. (PsycINFO Database Record (c) 2010 APA, all rights reserved)