CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms

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Oxidative stress-induced expression and modulation of Phosphatase of Regenerating Liver-1 (PRL-1) in mammalian retina

The phosphatase of regenerating liver-1, PRL-1, gene was detected in a screen for foveal cone photoreceptor-associated genes. It encodes a small protein tyrosine phosphatase that was previously immunolocalized to the photoreceptors in primate retina. Here we report that in cones and cone-derived cultured cells both PRL-1 activity and PRL-1 gene expression are modulated under oxidative stress. Oxidation reversibly inhibited the phosphatase activity of PRL-1 due to the formation of an intramolecular disulfide bridge between Cys104 within the active site and another conserved Cys, Cys49. This modulation was observed in vitro, in cell culture and in isolated retinas exposed to hydrogen peroxide. The same treatment caused a rapid increase in PRL-1 expression levels in cultured cells which could be blocked by the protein translation inhibitor, cycloheximide. Increased PRL-1 expression was also observed in living rats subjected to constant light exposure inducing photooxidative stress. We further demonstrated that both oxidation and overexpression of PRL-1 upon oxidative stress are greatly enhanced by inhibition of the glutathione system responsible for cellular redox regulation. These findings suggest that PRL-1 is a molecular component of the photoreceptor's response to oxidative stress acting upstream of the glutathione system.     

The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days

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人肝内胆管癌细胞PRL-3 的表达与侵袭转移研究

目的:探讨PRL-3在人肝内胆管癌侵袭转移中的作用.方法:利用小RNA技术干扰肝内胆管癌细胞株PRL-3表达,并采用细胞划痕实验和Transwell体外侵袭实验评价PRL-3对肝内胆管癌细胞侵袭转移能力的影响.结果:RT-PCR和Western blot结果均显示转染PRL-3特异性siRNA-2组PRL-3表达明显降低(P＜0.05).PRL-3 siRNA-2组在划痕培养24h后划痕区域宽度占初始划痕区域宽度的百分比为(62.12±6.28)％,阴性对照组为(23.88±2.55)％,空白对照HCCC-9810组为(21.20±6.07)％.PRL-3siRNA-2组细胞的划痕两端距离相比明显较宽,分别与阴性对照组细胞和空白对照HCCC-9810组细胞相比均有统计学意义(P＜0.05),后两者无显著性差异(P＞0.05).Transwell体外侵袭实验结果显示,PRL-3 SiRNA-2组细胞侵袭能力明显减弱,穿膜细胞数为(19.40±2.30)个/HP,明显少于阴性对照组(64.00±2.73)个/HP和正常HCCC-9810组(67.20±3.l1)个/HP,差异有显著性(P＜0.05);正常HCCC-9810组和阴性对照组无明显差异(P＞0.05).结论:PRL-3特异性siRNA能够抑制肝内胆管癌细胞HC-CC-9810中内源性PRL-3的表达,并可以明显抑制肝内胆管癌细胞的迁移侵袭能力.     

Photorefractoriness and Its Termination in the Subtropical House Sparrow, Passer Domesticus: Involvement of Circadian Rhythm

Groups of photorefractory female subtropical house sparrows, Passer domestkus, when treated with 6 weeks of a short photocycle (8L : 16D) showed significant ovarian growth on their return to a long photocycle (15L :9D). A 6-hr photophase coupled with scotophase of varying durations does not terminate the refractory period under photoperiod cycles of 12 (6L : 6D), 36 (6L :30D) and 60 (6L : S4D) hr but the refractory period is terminated by light-dark cycles of 24 (6L: 18D), 48 (6L :42D) and 72 (6L : 66D) hr. These results are consistent with the Biinning hypothesis of coincidence between endogenous photosensitive rhythmicity and environmental photoperiod timing that an endogenous circadian rhythm is involved in the maintenance and termination of photorefractoriness.     

Circadian and circannual changes in the testicular function of adult rhesus monkeys

The endocrine and gametogenic status of the testes were studied in 9 healthy adult rhesus monkeys of proven fertility throughout a one-year period. Testosterone levels were estimated by radioimmunoassay in blood samples collected at 4 h intervals over a 24 h period once a month. Semen samples and testicular biopsies were also examined once a month. A well-defined circandian rhythm was evident in serum levels of testosterone. The rhythmicity was less pronounced in February and September. The 24 h mean levels of serum testosterone were high between the months of August to March and low in the months of May to July. All animals did not uniformly respond to electro-ejaculation in April and May. Semen volume and total number of spermatozoa were maximal between September and March and least from April to August. Testicular biopsies indicated that all stages of spermatogenesis were evident between September and March and the spermatogenic activity was less evident between April and August. The contents of Sertoli cells showed a seasonal cyclicity; they were laden with lipid droplets during April to August when spermatogenesis was quiescent and vacuolated during September to March when spermatogenesis was active. These studies indicate that the testing of contraceptive drugs needs to be restricted to months of September to March in male rhesus monkeys otherwise, it is possible that the naturally occurring reproductive quiscence may be attributed to the effect of the drug being tested. The data accrued from the present studies also provide quantitative information on circulating levels of testosterone which could be used as a reference background while evaluating the contraceptive drug-effects in male rhesus monkeys.     

Circadian locomotory rhythm and the influence of moulting in Australian field cricket nymphs

ABSTRACT. Evidence is presented for a circadian control of locomotory activity in the larval stadia of the cricket, Teleogryllus commodus Walker. Under light—dark cycles (LD), maximal activity occurs around the L/D transition and/or in the hours preceding it. Free-running rhythm patterns longer than 24 h are observed in constant light. Re-entrainment to phase advances in the LD cycle is also accompanied by several transient cycles. However, free-running rhythms under constant darkness or transients when exposed to LD cycle delays were not found. LD cycles during the eighth stadium set the phase of a free-running rhythm in the adult, even if the nymph does not show a rhythm. Nymphal activity is often erratic and is disrupted periodically by the moulting cycle, but moulting does not interrupt the operation of the circadian system. The daily timing of the moult itself is not under circadian control.     

Circadian Control of DRP1 Activity Regulates Mitochondrial Dynamics and Bioenergetics

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Identification of Soybean Genes Involved in Circadian Clock Mechanism and Photoperiodic Control of Flowering Time by In Silico Analyses

Glycine max is a photoperiodic short-day plant and the practical consequence of the response is latitude and sowing period limitations to commercial crops. Genetic and physiological studies using the model plants Arabidopsis thaliana and rice （Oryza sativa） have uncovered several genes and genetic pathways controlling the process, however information about the corresponding pathways in legumes is scarce. Data mining prediction methodologies, including multiple sequence alignment, phylogeneUc analysis, bioinformaUcs expression and sequence motif pattern identification, were used to identify soybean genes involved in day length perception and photoperiodic flowering induction. We have investigated approximately 330 000 sequences from open-access databases and have identified all bona fide central oscillator genes and circadian photoreceptors from A. thaliana in soybean sequence databases. We propose a working model for the photoperiodic control of flowering time in G. max, based on the identified key components. These results demonstrate the power of comparative genomics between model systems and crop species to elucidate the several aspects of plant physiology and metabolism.     

Circadian rhythm of the locomotor activity in Drosophila melanogaster and its mutants 'sine oculis' and 'small optic lobes'

ABSTRACT. The locomotor activity patterns of wildtype Drosophila melanogaster and the mutants so (sine oculis) and sol (small optic lobes) were investigated. In all strains the proportions showing circadian rhythmicity, arrhythmicity and more complex patterns which could not be thus classified were similar. The occurrence of abnormal activity patterns is thus not a property of the mutation as previously claimed (Mack & Engelmann, 1981). In flies with a distinct circadian rhythmicity, the period lengths (τ) varied between strains, τ for wildtype Italy and the mutant so was longer than for wildtype Berlin and the mutant sol. As different τ's have been reported by others, τ does not seem to be closely determined for Drosophila melanogaster. Many flies exhibited two rhythms simultaneously, one with τ shorter and one with τ longer than 24 h, apparently implying two-oscillator control of the locomotor activity. The eyeless so mutants were entrained by LD cycles, so the compound eyes are not necessary, and so must possess the relevant photoreceptor(s) elsewhere. This may therefore also be true for the wildtype. Histology of the so adults revealed no correlation between the degree of reduction in the medulla and the occurrence of abnormal activity patterns. Since the only structures common to the medulla of so and sol are known to be large tangential cells, it is concluded that either they are of importance for the rhythmic system, or the oscillator(s) controlling locomotor activity is (are) not located in the medulla.     

Influence of the Amount of Light Received during the Day and Night Times on the Circadian Rhythm of Melatonin Secretion in Women Living Diurnally

The study investigated the relationship between the circadian variation of salivary melatonin and the amount of light received during the day and night. Forty one females served as subjects. An illuminance meter worn on the wrist of the non-dominant arm measured the amount of light which subjects leading a diurnal lifestyle received during two consecutive days. Light received from the time of rising to 18:00h was defined as ‘daytime light’, and that from 18:00h to the time of retiring as ‘nighttime light’. The average amount of light over the two days was 48 × 10 4 lx during the daytime and 11 × 10 4 lx during the nighttime. Saliva was collected every 4h in order to measure melatonin secretion. Peaks of melatonin secretion were observed at 14:00h and 18:00h in the subjects who had received lesser amounts of light during the daytime and nighttime. Melatonin secretion was high around 22:00h and peaked around 02:00h in the subjects who had received greater amounts of light during the daytime and lesser amounts of light during the nighttime. Nocturnal melatonin secretion was suppressed in the subjects who received greater amounts of light during the nighttime. Thus, the amount of light received during the daytime and the nighttime during the course of a diurnal lifestyle could have a profound influence on the circadian pattern of melatonin secretion.     

Developmental Regulation of Protein Phosphatase Types 1 and 2A in Post-Hatch Chicken Brain

The activity and subcellular distribution of protein phosphatases 1 and 2A were measured in chicken forebrain and cerebellum during post-hatch development. At all post-hatch ages, a large proportion of PP1 and PP2A was membrane bound and these enzymes were less active than their cytosolic counterparts. The protein concentration of PP1 in the membranes increased 40% between 2 and 14 days and a further 60% between 14 days and adult, whereas the PP1 enzyme activity in the membranes progressively decreased. In contrast to PP1, the protein concentration of PP2A remained constant in all fractions during post-hatch development, and the enzyme activity of PP2A did not change except for a decrease in the membrane-bound activity between 2 and 14 days. These results show that the subcellular distribution and activity of PP1 is selectively regulated during post-hatch development and that membrane association and inactivation of PP1 are independent events.     

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms^1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night ³. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons³. Drosophila locomotor activity is a well known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms⁴. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR³. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies^5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.     

PRR5 regulates phosphorylation,nuclear import and subnuclear localization of TOC1 in the Arabidopsis circadian clock

Many core oscillator components of the circadian clock are nuclear localized but how the phase and rate of their entry contribute to clock function is unknown. TOC1/PRR1, a pseudoresponse regulator (PRR) protein, is a central element in one of the feedback loops of the Arabidopsis clock, but how it functions is unknown. Both TOC1 and a closely related protein, PRR5, are nuclear localized, expressed in the same phase, and shorten period when deficient, but their molecular relationship is unclear. Here, we find that both proteins interact in vitro and in vivo through their conserved N‐termini. TOC1–PRR5 oligomerization enhances TOC1 nuclear accumulation two‐fold, most likely through enhanced nuclear import. In addition, PRR5 recruits TOC1 to large subnuclear foci and promotes phosphorylation of the TOC1 N‐terminus. Our results show that nuclear TOC1 is essential for normal clock function and reveal a mechanism to enhance phase‐specific TOC1 nuclear accumulation. Interestingly, this process of regulated nuclear import is reminiscent of similar oligomeric pairings in animal clock systems (e.g. timeless/period and clock/cycle), suggesting evolutionary convergence of a conserved mechanism across kingdoms.     

Circadian Rhythm of Prohormone Atrial Natriuretic Peptides 1-30, 31-67 and 99-126 in Man

Two peptides consisting of amino acids 1-30 and 31-67 of the N-terminal end of the prohormone of the atrial natriuretic factor (pro ANF), vasodilate aortas in vitro, lower blood pressure in vivo, and have natriuretic properties similar to the atrial natriuretic factor (ANF, amino acids 99-126 of the prohormone). It has been recently discovered that pro ANF 1-30 and pro ANF 31-67 as well as ANF circulate in man. To determine if these three peptide hormones have a circadian variation in their circulating plasma concentrations, eight housestaff volunteers were studied on a day when they were in the hospital for 24 hr. These 5 men and 3 women, ages 25 to 39 had blood samples taken at 0800, 1200, 1600, 2000, 0000, 0400 and 0800 on the following day. One-half of these house officers were up all night while the other half went to sleep from midnight to 0800 and had their 0400 plasma samples drawn while in a supine position. The peak level for all three peptide hormones was at 0400 for both supine and upright subjects. It was concluded that there are circadian rhythms in normal, active people of these three peptide hormones, whose peak levels are at 0400 irrespective of posture.     

Circadian Rhythm of Prohormone Atrial Natriuretic Peptides 1-30, 31-67 and 99-126 in Man

Two peptides consisting of amino acids 1-30 and 31-67 of the N-terminal end of the prohormone of the atrial natriuretic factor (pro ANF), vasodilate aortas in vitro, lower blood pressure in vivo, and have natriuretic properties similar to the atrial natriuretic factor (ANF, amino acids 99-126 of the prohormone). It has been recently discovered that pro ANF 1-30 and pro ANF 31-67 as well as ANF circulate in man. To determine if these three peptide hormones have a circadian variation in their circulating plasma concentrations, eight housestaff volunteers were studied on a day when they were in the hospital for 24 hr. These 5 men and 3 women, ages 25 to 39 had blood samples taken at 0800, 1200, 1600, 2000, 0000, 0400 and 0800 on the following day. One-half of these house officers were up all night while the other half went to sleep from midnight to 0800 and had their 0400 plasma samples drawn while in a supine position. The peak level for all three peptide hormones was at 0400 for both supine and upright subjects. It was concluded that there are circadian rhythms in normal, active people of these three peptide hormones, whose peak levels are at 0400 irrespective of posture.