Circadian rhythms in plants: Strategies for analysis

Molecular mechanism of the circadian clock which regulates the circadian rhythms has been believed to be common in different organisms. However, recent topic about multiple oscillators in a cell is thought to suggest other possibility. We may need to reconsider effectiveness of strategies for understanding molecular mechanism of the circadian clock.     

人外周血淋巴细胞核心钟基因Clock和Bmal1的昼夜节律性表达

探讨自然光制下正常成年人外周血淋巴细胞钟基因Clock和Bmal1的昼夜节律性表达。用实时定量RT-PCR方法,测定不同昼夜时点(ZT)受试者外周血淋巴细胞总RNA中核心钟基因Clock和Bmal1的mRNA表达量,通过余弦法和Clock Lab软件获取节律参数,并经振幅检验分析是否存在昼夜节律。结果发现正常成年人外周血淋巴细胞钟基因Clock和Bmal1的mRNA表达呈昼夜节律性振荡(P0.05),Clock的峰时和谷时分别位于ZT13和ZT1,Bmal1的峰时和谷时分别位于ZT12和ZT24;两个基因在所检测的各个昼夜时点中表达水平均有明显差异(P0.05),Bmal1的表达水平较Clock降低;二者表达的峰值相位、振幅、峰时和谷时相一致(P0.05),但Bmal1转录的中值水平以及峰时mRNA水平和谷时mRNA水平降低(P0.05)。提示正常成年人外周血淋巴细胞钟基因Clock和Bmal1的表达存在同步化的昼夜节律性转录特征。     

Heat Shock Proteins and Circadian Rhythms

Significant circadian rhythms in heat shock gene expression were observed in a prokaryotic species (Synechocystis). In eukaryotes, in contrast, several heat shock genes (constitutive and inducible) were shown to be constantly expressed. A few cases of circadian expression of heat shock proteins (HSPs), however, have been reported. Significant circadian changes of thermotolerance were observed in yeast and several plant species. Higher thermo-tolerance can be attributed to a higher abundance of HSPs, but also to other adaptive mechanisms. Zeitgeber effects of temperature changes can be explained on the basis of their direct effects on the state variables of the clock gene (per, frq) expression and its negative feedback loop. Effects of increased HSP concentrations, as observed after heat shock, but also after light and serotonin (5HT), appear possible, in particular with respect to nuclear localization of the clock (PER) protein, but these effects have not been documented yet. Thus, the role of HSPs in the circadian clock system is little understood and, from our point of view, deserves more attention. (Chronobiology International, 13(4), 239-250, 1996)     

Daily Expression of Six Clock Genes in Central and Peripheral Tissues of a Night-Migratory SongBird: Evidence for Tissue-Specific Circadian Timing

In birds, independent circadian clocks reside in the retina, pineal, and hypothalamus, which interact with each other and produce circadian time at the functional level. However, less is known of the molecular clockwork, and of the integration between central and peripheral clocks in birds. The present study investigated this, by monitoring the timed expression of five core clock genes (Per2. Cry1. Cry2. Bmal1, and Clock) and one clock-controlled gene (E4bp4) in a night-migratory songbird, the redheaded bunting (rb; Emberiza bruniceps). The authors first partially cloned these six genes, and then measured their 24-h profiles in central (retina, hypothalamus) and peripheral (liver, heart, stomach, gut, testes) tissues, collected at six times (zeitgeber time 2 [ZT2], ZT6, ZT11, ZT13, ZT18, and ZT23; ZT0?=?lights on) from birds (n?=?5 per ZT) on 12?h:12?h light-dark cycle. rbPer2. rbCry1. rbBmal1, and rbClock were expressed with a significant rhythm in all the tissues, except in the retina (only rbClock) and testes. rbCry2, however, had tissue-specific expression pattern: a significant rhythm in the hypothalamus, heart, and gut, but not in the retina, liver, stomach, and testes. rbE4bp4 had a significant mRNA rhythm in all the tissues, except retina. Further, rbPer2 mRNA peak was phase aligned with lights on, whereas rbCry1. rbBmal1, and rbE4bp4 mRNA peaks were phase aligned with lights off. rbCry2 and rbClock had tissue-specific scattered peaks. For example, both rbCry2 and rbClock peaks were close to rbCry1 and rbBmal1 peaks, respectively, in the hypothalamus, but not in other tissues. The results are consistent with the autoregulatory circadian feedback loop, and indicate a conserved tissue-level circadian time generation in buntings. Variable phase relationships between gene pairs forming positive and negative limbs of the feedback loop may suggest the tissue-specific contribution of individual core circadian genes in the circadian time generation.     

Genetics of Circadian Clocks

The isolation of circadian clock mutants in Neurospora crassa and Drosophila melanogaster have identified numerous genes whose function is necessary for the normal operation of the circadian clock. In Neurospora many of these mutants map to a single locus called frq, whose properties suggest that its gene product is intimately involved in clock function. In Drosophila mutations at the per locus also suggest a significant role for the product of this gene in the insect clock mechanism. The per gene has been cloned and its gene product identified as a proteoglycan, most likely a membrane protein involved in affecting the ionic or electrical properties of cells in which it is located. Future progress in elucidating the mechanisms of circadian clocks are likely to come from continued analysis of clock mutants, both at the genetic and molecular levels.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Rubidium Chloride Fuses Split Circadian Activity Rhythms in Hamsters Housed in Bright Constant Light

Chronotypic effects of rubidium (Rb) were examined in hamsters whose circadian activity rhythms had split into two components while they were housed in bright constant light. Seven of 12 hamsters receiving RbCl in drinking water for 10 weeks showed fusing of the components into an intact rhythm compared with none of 7 control hamsters (p = 0.016). Rb may modify coupling between circadian oscillators via reduced photic input to the suprachiasmatic nuclei. Alternative mechanisms include changes in potassium metabolism or endocrine function or behavioral changes that in turn alter circadian function. This normalization of a circadian anomaly by a putative antidepressant suggests that Rb may be valuable in strengthening coupling between oscillators in cases of human chronopathology, including those implicated in the etiology of some affective disorders.     

Rubidium Chloride Fuses Split Circadian Activity Rhythms in Hamsters Housed in Bright Constant Light

Chronotypic effects of rubidium (Rb) were examined in hamsters whose circadian activity rhythms had split into two components while they were housed in bright constant light. Seven of 12 hamsters receiving RbCl in drinking water for 10 weeks showed fusing of the components into an intact rhythm compared with none of 7 control hamsters (p = 0.016). Rb may modify coupling between circadian oscillators via reduced photic input to the suprachiasmatic nuclei. Alternative mechanisms include changes in potassium metabolism or endocrine function or behavioral changes that in turn alter circadian function. This normalization of a circadian anomaly by a putative antidepressant suggests that Rb may be valuable in strengthening coupling between oscillators in cases of human chronopathology, including those implicated in the etiology of some affective disorders.     

Circadian Rhythms in Amphibians and Reptiles: Ecological Implications

Circadian rhythms of amphibians and reptiles in the field and under semi-natural conditions and the underlying mechanisms, including the ways of entrainment to environmental cues and the oscillators driving the rhythms, have been reviewed. Studies on the behavioral rhythms in the field are meager in both amphibians and reptiles. In anuran amphibians, Xenopus adults showed more robust nocturnal locomotor activity than did tadpoles. This indicates the ecological significance of the differences in activity pattern shown by amphibians at different life stages, because differences between adults and young in the same environment may serve to isolate partially the young from the adults' cannibalism. In reptiles, free-running rhythms are more robust and continue for a longer time compared to amphibians. In both amphibians and reptiles, multi-photoreceptors are involved in photo-entrainment of circadian rhythms. The eyes, pineal complex and deep brain comprise a multi-oscillator system as well as a multi-photoreceptor system.     

Circadian Rhythms of Circulating NK Cells in Healthy and Human Immunodeficiency Virus-Infected Men

Antiviral immunity involves NK cells, which circulate rhythmically every 24 hours. We have investigated circadian and 12-hour rhythms in the peripheral count of circulating NK cells in 15 men infected with human immunodeficiency virus (HIV) and 13 healthy controls. We analyzed three phenotypes using double-labeling with monoclonal antibodies and flow cytometry assessment: CD3^? CD16⁺, CD3^?CD57⁺, and CD2⁺CD3^?. A statistical validation of time-dependent differences was achieved if significance (p < 0.05) was validated both with analysis of variance and cosinor. The circadian rhythm had a similar asymmetric waveform for the three phenotypes and is homogeneous on an individual basis. The circulating NK cell count peaked in the early morning and was low at night. A circadian rhythm and a circahemidian harmonic characterized all phenotypes in healthy subjects. We considered two groups of HIV-infected men: those who were asymptomatic (eight) and those with acquired immune deficiency syndrome (AIDS) (seven). Circadian changes in NK cell count were similar in both subgroups and in healthy controls. The circadian pattern was also consistent among individual patients. Asymptomatic HIV-infected men (early-stage disease) exhibited more pronounced 12-hour rhythmicity than did patients with AIDS or controls. The circulation of NK cells does not appear to share the same synchro-nizer(s) as other circulating T- or B-lymphocyte subsets. Thus, HIV infection gradually abolished circadian rhythmicity in circulating T and B cells, whereas it did not disturb that in NK cells.