Daily changes in the phosphoproteome of the dinoflagellate Lingulodinium

The dinoflagellate Lingulodinium has a large number of daily rhythms, many of which have no biochemical correlates. We examined the possibility that changes in protein phosphorylation may mediate some of the rhythmic changes by comparing proteins prepared from midday (LD6) and midnight (LD18) cultures. We used two different methods, one a 2D gel protocol in which phosphoproteins were identified after staining with ProQ Diamond, and the other an LC-MS/MS identification of tryptic phosphopeptides that had been purified by TiO(2) chromatography. Two differentially phosphorylated proteins, a light harvesting complex protein and Rad24, were identified using the 2D gel protocol. Six differentially phosphorylated proteins, a polyketide synthase, an uncharacterized transporter, a LIM (actin binding) domain and three RNA binding domain proteins, were identified using the phosphopeptide enrichment protocol. We conclude that changes in protein phosphorylation may underlie some of the rhythmic behavior of Lingulodinium.     

Brefeldin a inhibits circadian remodeling of chloroplast structure in the dinoflagellate gonyaulax

Circadian increases in the rate of carbon fixation in the dinoflagellate Gonyaulax are correlated with extensive plastid remodeling. One marker for this remodeling is mobilization of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from the plastid periphery to plastid regions nearer the cell center called pyrenoids. Nuclear-encoded proteins such as Rubisco transit through the Golgi in dinoflagellates; hence, we blocked protein import into the plastids using Brefeldin A (BFA) to explore the mechanism for plastid remodeling. We find that pyrenoid formation normally occurs concurrently with increased Rubisco synthesis rates in vivo, and when BFA is given prior to the onset of Rubisco synthesis, pyrenoid formation is partially or completely inhibited by 0.1 or 0.3 microg/mL BFA, respectively. Rubisco synthesis itself is not affected, and BFA-treated cells accumulate Rubisco in novel structures we term BFA bodies. Interestingly, when given just after the onset of Rubisco synthesis, BFA delays but does not block Rubisco mobilization, suggesting that a timing signal for plastid remodeling is delivered to the organelles at the same time as newly synthesized Rubisco. BFA also inhibits the circadian increases in carbon fixation rates, supporting the hypothesis that the biochemical basis for this circadian rhythm may be Rubisco distribution within the plastid.     

Circadian rhythm of autophagy proteins in hippocampus is blunted by sleep fragmentation

Autophagy is essential for normal cellular survival and activity. Circadian rhythms of autophagy have been studied in several peripheral organs but not yet reported in the brain. Here, we measured the circadian rhythm of autophagy-related proteins in mouse hippocampus and tested the effect of sleep fragmentation (SF). Expressions of the autophagy-related proteins microtubule‐associated protein 1 light chain 3 (LC3) and beclin were determined by western blotting and immunohistochemistry. Both the hippocampal LC3 signal and the ratio of its lipid-conjugated form LC3-II to its cytosolic form LC3-I showed a 24 h rhythm. The peak was seen at ZT6 (1 pm) and the nadir at ZT16 (1 am). The LC3 immunoreactivity in hippocampal CA1 pyramidal neurons also distributed differently, with more diffuse cytoplasmic appearance at ZT16. Chronic SF had a mild effect to disrupt the 24 h rhythm of LC3 and beclin expression. Interestingly, a greater effect of SF was seen after 24 h of recovery sleep when LC3-II expression was attenuated at both the peak and trough of circadian activities. Overall, the results show for the first time that the hippocampus has a distinct rhythm of autophagy that can be altered by SF.     

昼夜节律钟调控代谢的研究进展

生理和行为的昼夜节律性调控对健康生活是必需的。越来越多的流行病学和遗传学证据显示昼夜节律的破坏与代谢紊乱性疾病相关联。在分子水平上,昼夜节律受到时钟蛋白组成的转录一翻译负反馈环的调控。时钟蛋白通过以下两种途径调节代谢：首先,时钟蛋白作为转录因子直接调节一些代谢关键步骤的限速酶和代谢相关核受体的表达,其次作为代谢相关核受体的辅调节因子来激活或抑制其转录活性。虽然时钟蛋白对代谢途径的调节导致代谢物水平呈昼夜节律振荡,但是产生的代谢物反过来又可以影响昼夜节律钟基因的表达,进而影响昼夜节律钟。深入研究昼夜节律钟与代谢的交互调节可能为治疗某些代谢紊乱性疾病提供新的治疗方案。     

Circadian rhythm of a TCA cycle enzyme is apparently regulated at the translational level in the dinoflagellate Lingulodinium polyedrum

Previously, the authors have reported that intracellular amounts of several metabolic-related enzymes from the photosynthetic dinoflagellate Lingulodinium polyedrum(formerly Gonyaulax polyedra) showed a daily rhythm under a 12:12 h LD cycle. This led the authors to hypothesize that a circadian clock controls metabolism, including the tricarboxylic acid (TCA) cycle. In this study, the authors investigated daily changes in the levels of mRNA, protein, and enzyme activity of several metabolic enzymes during 12:12 h LD, 8:16 h LD, and constant light conditions. The NADP-dependent isocitrate dehydrogenase (NADPICDH) in the TCA cycle exhibited circadian changes of protein abundance and enzyme activity under all conditions, whereas its mRNA level remained constant throughout the cycle. These results indicate that the rhythm of NADPICDH is regulated by a circadian control of protein synthesis or modification rather than by message levels and suggest that the TCA cycle may be controlled by the circadian clock system.     

Influence of carbon supply on the circadian rhythmicity of photosynthesis and its stimulation by blue light in Ectocarpus siliculosus: clues to the mechanism of inorganic carbon acquisition in lower brown algae

Stimulation or light-saturated rates of photosynthesis in Ectocarpus siliculosus (Dillwyn) Lyngb. by blue light was eliminated by increasing dissolved inorganic carbon (DIC) or by lowering pH in natural seawater. The amplitude of the circadian rhythm of photosynthesis was also diminished under these conditions, and the pH compensation points in a closed system were higher in the presence of blue light and during the circadian day. These observations suggest that blue light and the circadian clock regulate the activity of a carbon acquisition system in these plants. The inhibitor of external carbonic anhydrase, acetazolamide, reduced overall rates of photosynthesis by only about 30%, but ethoxyzolamide suppressed the circadian rhythm of photosynthesis almost completely and markedly reduced the duration of responses to blue light pulses. Similar patterns were obtained when photosynthesis was measured in strongly limiting DIC concentrations (0–0.5 mol m^?3). Since blue light stimulated photosynthesis under these conditions of strong carbon limitation, we suggest that blue light activates the release of CO₂ from an internal CO₂ store. We propose a metabolic pathway with similarities to that of CAM plants. Non-photosynthetic fixation leads to the accumulation of a storage metabolite. The circadian clock and blue light control the mobilization of CO₂ at the site of decarboxylation of this metabolite. In the presence of continuous blue light the pathway is proposed to cycle and act as a pump for CO₂ into the chloroplasts. This hypothesis helps to explain a number of previously reported peculiarities of brown algal photosynthesis.     

Variations in Amazon forest productivity correlated with foliar nutrients and modelled rates of photosynthetic carbon supply

The rate of above-ground woody biomass production, W(P), in some western Amazon forests exceeds those in the east by a factor of 2 or more. Underlying causes may include climate, soil nutrient limitations and species composition. In this modelling paper, we explore the implications of allowing key nutrients such as N and P to constrain the photosynthesis of Amazon forests, and also we examine the relationship between modelled rates of photosynthesis and the observed gradients in W(P). We use a model with current understanding of the underpinning biochemical processes as affected by nutrient availability to assess: (i) the degree to which observed spatial variations in foliar [N] and [P] across Amazonia affect stand-level photosynthesis; and (ii) how these variations in forest photosynthetic carbon acquisition relate to the observed geographical patterns of stem growth across the Amazon Basin. We find nutrient availability to exert a strong effect on photosynthetic carbon gain across the Basin and to be a likely important contributor to the observed gradient in W(P). Phosphorus emerges as more important than nitrogen in accounting for the observed variations in productivity. Implications of these findings are discussed in the context of future tropical forests under a changing climate.     

Circadian variation in the acetylcholinesterase activity of specific rat brain areas

Abstract

Acetylcholinesterase (AChE) activity of the adenohypophysis, cerebellum, cerebral cortex, hypothalamus, amygdala, hippocampus, midbrain, pons, medulla oblongata and caudate nucleus was determined by a spectro‐photometric method in adult, male rats adapted toan LD 12:12cycle. Results of the study show that AChE activity is highest during the light phase and lowest during the dark phase of the cycle in all the brain areas studied except the adenohypophysis, cerebellum, hippocampus and hypothalamus. These findings expand earlier observations on the circadian variation in rat brain AChE activity and suggests a relationship with reported circadian variation in the acetylcholine levels of rat brain.     

Circadian Rhythm Changes in Autotrophic Euglena Induced by Organic Carbon Sources*

SYNOPSIS. Acetate added to autotrophic Euglena cultures changed the period length of the circadian rhythm of phototaxis. Phase shifts were induced by acetate pulses. Since transition from one metabolic state to another (autotrophic/mixotrophic) caused a phase shift or a period change, such effects possibly result from switching metabolic pathways. As suggested (Brinkmann, K., 1966. Planta 70 , 344–89), differences in the temperature responses of the rhythm in mixotrophic and autotrophic cells might also be caused by participation of different metabolic pathways with different Q¹⁰ values, e.g. dark reactions vs photochemical reactions. However the Q¹⁰ of a given dark reaction, e.g. protein synthesis, can differ in the 2 states. Therefore temperature experiments alone do not suffice for deciding whether the pathways include photochemical reactions, dark reactions, or both.     

Control of photosynthetic carbon dioxide fixation by the boundary layer, stomata and ribulose 1,5-biphosphate carboxylase/oxygenase

Abstract Coefficients describing the sensitivity of the rate of photosynthetic carbon dioxide fixation to small changes in the stomatal conductance and boundary layer conductance are derived. These sensitivity or ‘control’ coefficients, together with those for the carboxylase and oxygenase activities of ribulose 1,5-bisphosphate carboxylase/oxygenase, are calculated from standard gas exchange data and apply under conditions where leaf temperature and water vapour concentration at the leaf surface remain largely constant. It is shown that the magnitude of the control coefficients depends on conditions such as photon flux density, ambient CO₂ concentration and relative humidity at the leaf surface. The extension of this analysis to encompass the sensitivity of the photosynthetic fluxes to changes in enzyme concentrations and kinetic properties is also discussed.     

Protein Synthesis in Euglena gracilis is Light-and Temperature-Dependent,Oscillating in a Circadian,Temperature-Compensated Manner

Abstract: Incorporation of radiolabelled amino acids into proteins of Euglena gracilis revealed that the amount of labelled protein depends on the conditions of illumination and temperature of cultivation. Protein synthesis was generally lower under dark conditions except at 37 °C. The largest amounts of labelled protein were measured at 21 °C and decreased at higher and lower temperatures. By separating the labelled proteins of the membraneous cell fraction from subcultures under a range of culture conditions, the synthesis of some specific proteins was found to be light- and/or temperature-dependent. On incubating cells taken at different times during a light/dark cycle and under constant conditions, a circadian rhythm of ³⁵S-methionine- as well as ³⁵S-cysteine-incorporation was detected. Thereby the cells incorporated ten-times less cysteine than methionine. Protein synthesis always peaked during the last quarter of the daily light phase, confirming the rhythmic rise in total protein. The length of the rhythm period, approximately 24 h, was nearly independent of the applied temperature in the range of 16 to 27 °C.     

Circadian expression of connexins in the mouse heart

Significant circadian variations exist in the frequency of cardiac arrhythmia; however, the underlying mechanism is largely unknown. Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is related to the genesis of cardiac arrhythmia. In this study, we investigated if cx40 and cx43 expressed circadianly in the mouse heart using suprachiasmatic nuclei (SCN) lesion and pharmacological autonomic nervous system block mouse. Significant 24-h variations were observed in the expression of cx40 and cx43 in the sham-operated mice. In the SCNX mouse, all genes examined lost circadian rhythm. In the ANSB mouse, the expressions of Bmal1 was dampened significantly but still had circadian rhythm, whereas the two connexin gene expressions lost rhythm. These results suggest that cx40 and cx43 gene expressions have clear circadian rhythm and might be regulated by the central clock in the SCN through the ANS.     

Relationship between photosynthetic and respiratory carbon metabolism in freshwater phytoplankton

Measurements of algal carbon metabolism in the light and the dark were conducted in (1) short-term (3-h) light and dark incubations, (2) a diel (24-h) experiment, and (3) a longer-term (4-d) carbon accumulation experiment to examine the relationship between photosynthetic rates, photosynthetic carbon metabolism in the light, and respiration and carbon metabolism in the ensuing dark period in natural assemblages of freshwater phytoplankton. High rates of photosynthesis and polysaccharide synthesis in the light were followed by high rates of respiration and polysaccharide utilization in the dark. Polysaccharide was the major respiratory substrate in the dark, and small molecular weight metabolites, lipids, and protein were less important sources of metabolic energy. The protein pool accumulated carbon during dark incubations, but more slowly than during active photosynthesis in the light. Because the intracellular macromolecular pools turn over at very different rates (polysaccharide > protein and lipid), patterns of short-term photosynthetic carbon metabolism are not necessarily indicative of the biochemical composition of the phytoplankton.     

Circadian rhythms of indoleamines and serotonin N-acetyltransferase activity in the pineal gland

Conclusion The circadian rhythm of melatonin synthesis in the pineal glands of various species has been summarized. The night-time elevation of melatonin content is in most if not all cases regulated by the change of N-acetyltransferase activity. In mammals, the N-acetyltransferase rhythm is controlled by the central nervous system, presumably by suprachiasmatic nuclei in hypothalamus through the superior cervical ganglion. In birds, the circadian oscillator that regulates the N-acetyltransferase rhythm is located in the pineal glands. The avian pineal gland may play a biological clock function to control the circadian rhythms in physiological, endocrinological and biochemical processes via pineal hormone melatonin.     

Adaptations by macroalgae to low carbon availability. II. Ultrastructural specializations, related to the function of a photosynthetic buffer system in the Fucaceae

Abstract Among the brown algae, species of the Fucaceae (Pelvetia, Fucus and Ascophyllum) were found to have a ‘photosynthetic buffering’ system, allowing the algae to carry out oxygen production without a concomitant uptake of inorganic carbon. This system was not found in other brown algae examined (e.g. Halidrys, Laminaria and Desmarestia) nor in 16 examined species of red and green algae. Pelvetia, Fucus and Ascophyllum belong to the littoral algae which are periodically emersed. In the Fucaceae, the meristodermal cells were found to have a special organization of organelles. Towards the outer cell wall there was a prominent layer of mitochondria while the chloroplasts were concentrated towards the inner and side walls. Between the mitochondria and the chloroplasts there was a large number of physodes. This arrangement of organelles was not found in the other brown algae examined nor in red or green algae. The significance of this organization of the mitochondria is discussed in connection with the function of the ‘photosynthetic buffering’ system.     

Circadian rhythms in the retina of rats with photoreceptor degeneration

Previous studies have demonstrated that the mammalian retina contains a circadian clock system that controls several retinal functions. In mammals the location of the retinal circadian clock is unknown whereas, in non-mammalian vertebrates, earlier work has demonstrated that photoreceptor cells contain the circadian clock. New experimental evidence has suggested that in mammals the retinal circadian clock may be located outside the photoreceptor cells. In this study we report that circadian rhythms in Aa-nat mRNA (in vivo) and melatonin synthesis (in vitro) are still present in the retina of rats lacking photoreceptors. The circadian pacemaker(s) controlling such rhythms is probably located in kainic acid sensitive neurons in the inner retina since kainic acid injections abolished the rhythmicity. These data are the first direct demonstration that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors and the suprachiasmatic nuclei of the hypothalamus.