Lcg is a light-inducible and clock-controlled gene expressed in the chicken pineal gland

The circadian clock is an autonomous biological clock that is entrainable to environmental 24-h cycles by receiving time cues such as light. Generally, light given at early and late subjective night, respectively, delays and advances the phase of the circadian oscillator. We previously searched for the chicken pineal genes that are induced by light in a phase-dependent manner. The present study undertook cDNA cloning and characterization of a gene whose expression was remarkably up-regulated by light at late subjective night. The mRNA level of this gene exhibited robust diurnal change in the pineal gland, with a peak in the early (subjective) day under light-dark cycles and constant dark condition, and hence it was designated Lcg (Light-inducible and Clock-controlled Gene). Chicken Lcg encodes a coiled-coil protein composed of 560 amino acid residues. Among chicken tissues, the pineal gland and the retina exhibited relatively high expression levels of LCG. LCG was colocalized with gamma-tubulin, a centrosomal protein, when expressed in COS7 cells, and LCG is the first example of a clock-related molecule being accumulated at the centrosome. Coimmunoprecipitation of LCG with gamma-tubulin in the chicken pineal lysate suggests a link between the circadian oscillator and the centrosomal function.     

Photoperiodic clock of diapause induction in Pseudopidorus fasciata (Lepidoptera: Zygaenidae)

Pseudopidorus fasciata enters diapause as fourth instar larvae at short day lengths. Using 24-h light-dark cycles, the photoperiodic response curves in this species appeared to be similar with a critical night length of 10.5h at temperatures below 30 degrees C. At an average temperature of 30.5 degrees C, the critical night length had shifted to between 15 and 17h. In experiments using non-24-h light-dark cycles, it was clearly demonstrated that the dark period (scotophase) was the decisive phase for a diapause determination. In night interruption experiments using 24-h light-dark cycles, a 1-h light pulse at LD12:12 completely reversed the long night effect and averted diapause in all treatments. At LD 9:15 light pulses of 1-h, 30- or 15-min also averted diapause effectively when both the pre-interruption (D(1)) or the post-interruption scotophases (D(2)) did not exceed the critical night length. If D(1) or D(2) exceeded the critical night length diapause was induced. The most crucial event for the photoperiodic time measurement in this species is the length of the scotophase. A 10-min light pulse placed in the most photosensitive phase reversed diapause in over 50% of the individuals. Night interruption experiments under non-24-h light-dark cycles indicated that the photoperiodic clock measured only D(1) regardless of the length of D(2), suggesting that the most inductive cycles are often those in which L+D are close to 24h. In resonance experiments, this species showed a circadian periodicity at temperatures of 24.5 or 26 degrees C, but not at 30.5 and 23.3 degrees C. On the other hand, Bünsow and skeleton photoperiod experiments failed to reveal the involvement of a circadian system in this photoperiodic clock. These results suggest the photoperiodic clock in this species is a long-night measuring hourglass and the circadian effect found in the final expression of the photoperiodic response in the resonance experiments may be caused by a disturbing effect of the circadian system in unnatural regimes.     

The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.     

Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import

We have isolated the Saccharomyces cerevisiae pex12-1 mutant from a screen to identify mutants defective in peroxisome biogenesis. The pex12delta deletion strain fails to import peroxisomal matrix proteins through both the PTS1 and PTS2 pathway. The PEX12 gene was cloned by functional complementation of the pex12-1 mutant strain and encodes a polypeptide of 399 amino acids. ScPex12p is orthologous to Pex12 proteins from other species and like its orthologues, S. cerevisiae Pex12p contains a degenerate RING finger domain of the C3HC4 type in its essential carboxy-terminus. Localization studies demonstrate that Pex12p is an integral peroxisomal membrane protein, with its NH2-terminus facing the peroxisomal lumen and with its COOH-terminus facing the cytosol. Pex12p-deficient cells retain particular structures that contain peroxisomal membrane proteins consistent with the existence of peroxisomal membrane remnants ("ghosts") in pex12A null mutant cells. This finding indicates that pex12delta cells are not impaired in peroxisomal membrane biogenesis. In immunoisolation experiments Pex12p was co-purified with the RING finger protein Pex10p, the PTS1 receptor Pex5p and the docking proteins for the PTS1 and the PTS2 receptor at the peroxisomal membrane, Pex13p and Pex14p. Furthermore, two-hybrid experiments suggest that the two RING finger domains are sufficient for the Pex10p-Pex12p interaction. Our results suggest that Pex12p is a component of the peroxisomal translocation machinery for matrix proteins.     

Melatonin accelerates reentrainment of the circadian rhythm of its own production after an 8-h advance of the light-dark cycle

Summary The rhythm in melatonin production in the rat is driven by a circadian rhythm in the pineal N-acetyltransferase (NAT) activity. Rats adapted to an artificial lighting regime of 12 h of light and 12 h of darkness per day were exposed to an 8-h advance of the light-dark regime accomplished by the shortening of one dark period; the effect of melatonin, triazolam and fluoxetine, together with 5-hydroxytryptophan, on the reentrainment of the NAT rhythm was studied.In control rats, the NAT rhythm was abolished during the first 3 cycles following the advance shift. It reappeared during the 4th cycle; however, the phase relationship between the evening rise in activity and the morning decline was still compressed.Melatonin accelerated the NAT rhythm reentrainment. In rats treated chronically with melatonin at the new dark onset, the rhythm had already reappeared during the 3rd cycle, in the middle of the advanced night, and during the 4th cycle, the phase relationship between the evening onset and the morning decline of the NAT activity was the same as before the advance shift. In rats treated chronically with melatonin at the old dark onset or in those treated with melatonin 8 h, 5 h and 2 h after the new dark onset during the 1st, 2nd and 3rd cycle, respectively, following the advance shift, the NAT rhythm reappeared during the 3rd cycle as well but in the last third of the advanced night only.Neither triazolam nor fluoxetine together with 5-hydroxytryptophan administered around the new dark onset facilitated NAT rhythm reentrainment after the 8-h advance of the light-dark cycle.Abbreviations NAT N-acetyltransferase - LD cycle light-dark cycle - CT circadian time - LD xy light dark cycle comprising x h of light and y h of darkness     

A novel mutation in kaiC affects resetting of the cyanobacterial circadian clock

Light is the most important factor controlling circadian systems in response to day-night cycles. In order to better understand the regulation of circadian rhythms by light in Synechococcus elongatus PCC 7942, we screened for mutants with defective phase shifting in response to dark pulses. Using a 5-h dark-pulse protocol, we identified a mutation in kaiC that we termed pr1, for phase response 1. In the pr1 mutant, a 5-h dark pulse failed to shift the phase of the circadian rhythm, while the same pulse caused a 10-h phase shift in wild-type cells. The rhythm in accumulation of KaiC was abolished in the pr1 mutant, and the rhythmicity of KaiC phosphorylation was reduced. Additionally, the pr1 mutant was defective in mediating the feedback inhibition of kaiBC. Finally, overexpression of mutant KaiC led to a reduced phase shift compared to that for wild-type KaiC. Thus, KaiC appears to play a role in resetting the cellular clock in addition to its documented role in the feedback regulation of circadian rhythms.     

Peroxisomal membrane protein import does not require Pex17p

Of the approximately 20 proteins required for peroxisome biogenesis, only four have been implicated in the process of peroxisomal membrane protein (PMP) import: Pex3p, Pex16p, Pex17p, and Pex19p. To improve our understanding of the role that Pex17p plays in PMP import, we examined the behavior of PMPs in a Pichia pastoris pex17 mutant. Relative to wild-type cells, pex17 cells appeared to have a mild reduction in PMP stability and slightly aberrant PMP behavior in subcellular fractionation experiments. However, we also found that the behavior of PMPs in the pex17 mutant was indistinguishable from PMP behavior in a pex5 mutant, which has no defect in PMP import, and was far different from PMP behavior in a pex3 mutant, which has a bona fide defect in PMP import. Furthermore, we found that a pex14 mutant, which has no defect in PMP import, lacks detectable levels of Pex17p. Based on these and other results, we propose that Pex17p acts primarily in the matrix protein import pathway and does not play an important role in PMP import.     

Effects of feeding and lighting stimuli on the synthesis of ornithine aminotransferase and serine dehydratase in rat liver

In a previous study we showed that rats fed ad libitum and maintained on a 12-h light/ 12-h dark cycle demonstrated out-of-phase circadian oscillations in the rates of ornithine aminotransferase and serine dehydratase synthesis. As part of an investigation of the factors regulating both the generation of these cycles and their dissimilarity, this paper ompares the circadian fluctuations in the rates of ornithine aminotransferase and serine dehydratase synthesis measured immunochemically in rats given a single 2-h daily feeding in conjunction with exposure to constant light or a 12-h light/12-h dark cycle. When the 2-hr feeding was administered to rats under constant light, reciprocal circadian oscillations in ornithine aminotransferase and serine dehydratase synthesis were observed regardless of the temporal location of the feeding interval. Ornithine aminotransferase synthesis began to increase after the feeding interval and reached a maximum 12 h later while serine dehydratase showed the opposite response. In rats maintained on both the restricted feeding regimen and a 12-h light/12-h dark cycle, however, retention of synthesis oscillations depended on the temporal location of the restricted feeding interval within the light-dark cycle. Rats fed for 2 h at the beginning of the dark phase exhibited circadian oscillations in serine dehydratase synthesis and a high nonoscillating level of ornithine aminotransferase synthesis, whereas rats fed for 2 h at the beginning of the light phase exhibited circadian oscillations in ornithine aminotransferase synthesis and a low nonoscillating level of serine dehydratase synthesis. These responses suggest the existence of meal-responsive and light-responsive regulators of ornithine aminotransferase and serine dehydratase synthesis.     

Temporal organization of chilling resistance in cotton seedlings: effects of low temperature and relative humidity

The effect of temperature and relative humidity (RH) on the time course of the rhythmic endogenous changes of chilling resistance was studied in cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 12:12 h. The resistant phase to 5° C, 85% RH lasted during most of the dark period while to 5° C, 100% RH it was longer and extended into the last half of the light period because a transient phase advance occurred when chilling started at the middle of the light period. Seedlings acclimated by low temperature were resistant throughout the light-dark cycle. A treatment with 100% RH before chilling to acclimated seedlings introduced a sensitive phase that corresponded to that of non-acclimated seedlings. In non-acclimated seedlings, this treatment decreased the resistance but the basic pattern of the rhythm was sustained. Changes in chilling resistance were analyzed under fluctuating temperatures and RHs, and explained taking into consideration the functioning of the circadian clock and environmental induction of resistance.Abbreviations CR chilling resistance - LDC light-dark cycle of 24 h - RH relative humidity     

ldpA encodes an iron-sulfur protein involved in light-dependent modulation of the circadian period in the cyanobacterium Synechococcus elongatus PCC 7942

We generated random transposon insertion mutants to identify genes involved in light input pathways to the circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942. Two mutants, AMC408-M1 and AMC408-M2, were isolated that responded to a 5-h dark pulse differently from the wild-type strain. The two mutants carried independent transposon insertions in an open reading frame here named ldpA (for light-dependent period). Although the mutants were isolated by a phase shift screening protocol, the actual defect is a conditional alteration in the circadian period. The mutants retain the wild-type ability to phase shift the circadian gene expression (bioluminescent reporter) rhythm if the timing of administration of the dark pulse is corrected for a 1-h shortening of the circadian period in the mutant. Further analysis indicated that the conditional short-period mutant phenotype results from insensitivity to light gradients that normally modulate the circadian period in S. elongatus, lengthening the period at low light intensities. The ldpA gene encodes a polypeptide that predicts a 7Fe-8S cluster-binding motif expected to be involved in redox reactions. We suggest that the LdpA protein modulates the circadian clock as an indirect function of light intensity by sensing changes in cellular physiology.     

Amplitude of circadian oscillations entrained by 24-h light-dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light-dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light-dark cycles is maximized when the free-running period is smaller than 24h. Moreover, if the amplitude of the light-dark cycle is very strong, complex oscillations occur when the free-running period is close to 24h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light-dark cycles is also maximized if the free-running period is smaller than 24h, the range of entrainment is centered around 24h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.     

Preperoxisomal vesicles can form in the absence of Pex3

We demonstrate that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells. Notably, pex3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex—Pex13 and Pex14—as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in pex3 cells. Instead, we find that reintroduced Pex3 sorts to the preperoxisomal structures in pex3 cells, after which these structures mature into normal peroxisomes.     

Effects of photoperiod reduction on rat circadian rhythms of BP, heart rate, and locomotor activity

The effects of a photoperiod reduction in the entrainment of circadian rhythms of systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and spontaneous locomotor activity (SLA) were determined in conscious Wistar rats by using radiotelemetry. Two groups of seven rats were maintained in a 12:12-h light-dark (12L/12D) photoperiod for 11 wk and then placed in a reduced photoperiod of 8:16-h light-dark (8L/16D) by advancing a 4-h darkness or by advancing and delaying a 2-h darkness for 6 wk. Finally, they were resynchronized to 12L/12D. Advancing a 4-h dark phase induced a 1-h advance of acrophase for SBP, DBP, and HR, but not for SLA. The percent rhythm, amplitude, and the 12-h mean values of all parameters were significantly decreased by the photoperiod reduction. When symmetrically advancing and delaying a 2-h dark phase, a 1 h 20 min delay of acrophases and a decrease in percent rhythms and amplitudes of SBP, DBP, HR, and SLA were observed. Only the 12-h mean values of HR and SLA were decreased. Our findings show that the cardiovascular parameters differ from SLA in phase-shift response to photoperiod reduction and that the adjustment of circadian rhythms to change from 12L/12D to 8L/16D photoperiod depends on the direction of the extension of the dark period.     

Diapause induction and clock mechanism in the cabbage beetle, Colaphellus bowringi (Coleoptera: Chrysomelidae)

Photoperiodic control of diapause induction was investigated in the short-day species, Colaphellus bowringi, which enters summer and winter diapause as adult in the soil. Photoperiodic responses at 25 and 28 degrees C revealed a critical night length between 10 and 12 h; night lengths > or =12 h prevented diapause, whereas night lengths <12 h induced summer diapause in different degree. Experiments using non-24-h light-dark cycles showed that the duration of scotophase played an essential role in the determination of diapause. Night-interruption experiments with T=24 h showed that diapause was effectively induced by a 2-h light pulse in most scotophases; whereas day-interruption experiments by a 2-h dark break had a little effect on the incidence of diapause. The experiments of alternating short-night cycles (LD 16:8) and long-night cycles (LD 12:12) during the sensitive larval period showed that the information of short nights as well as long nights could be accumulated. Nanda-Hamner experiments showed three declining peaks of diapause at 24 h circadian intervals. Bünsow experiments showed two very weak peaks for diapause induction, one being 8 h after lights-off, and another 8 h before lights-on, but it did not show peaks of diapause at a 24 h interval. These results suggest that the circadian oscillatory system constitutes a part of the photoperiodic clock of this beetle but plays a limited role in its photoperiodic time measurement.