A potential EARLY FLOWERING 3 homolog in Chlamydomonas is involved in the red/violet and blue light signaling pathways for the degradation of RHYTHM OF CHLOROPLAST 15.

Light plays a major role in resetting the circadian clock, allowing the organism to synchronize with the environmental day and night cycle. In Chlamydomonas the light-induced degradation of the circadian clock protein, RHYTHM OF CHLOROPLAST 15 (ROC15), is considered one of the key events in resetting the circadian clock. Red/violet and blue light signals have been shown to reach the clock via different molecular pathways; however, many of the participating components of these pathways are yet to be elucidated. Here, we used a forward genetics approach using a reporter strain that expresses a ROC15-luciferase fusion protein. We isolated a mutant that showed impaired ROC15 degradation in response to a wide range of visible wavelengths and impaired light-induced phosphorylation of ROC15. These results suggest that the effects of different wavelengths converge before acting on ROC15 or at ROC15 phosphorylation. Furthermore, the mutant showed a weakened phase resetting in response to light, but its circadian rhythmicity remained largely unaffected under constant light and constant dark conditions. Surprisingly, the gene disrupted in this mutant was found to encode a protein that possessed a very weak similarity to the Arabidopsis thaliana EARLY FLOWERING 3 (ELF3). Our results suggest that this protein is involved in the many different light signaling pathways to the Chlamydomonas circadian clock. However, it may not influence the transcriptional oscillator of Chlamydomonas to a great extent. This study provides an opportunity to further understand the mechanisms underlying light-induced clock resetting and explore the evolution of the circadian clock architecture in Viridiplantae.

Development of pups born to rats established as a model of underweight Japanese women and the onset of impaired glucose tolerance.

An increasing number of Japanese women of childbearing age are underweight (BMI <18.5), but the association between this and the increased number of low-birth-weight babies born remains unclear. Here, a rat model was established to mimic the undernutrition (85% of the energy required for those with normal activity levels) experienced by such women and to evaluate the associated impaired glucose tolerance. The undernourished Wistar rat group showed increased serum corticosterone level reflecting stress, and greater adrenal weight and size. It also showed greater insulin resistance, higher expression of FOXO-1, a transcription factor related to muscle atrophy, and lower expression of p-Akt, an insulin-dependent signaling factor. Overall, this work shows the key role of undernutrition during pregnancy as a cause of impaired glucose tolerance and increased diabetes risk in offspring. The findings of this study may inform preemptive measures to prevent the development of metabolic syndrome in offspring of undernourished mothers.

Effects of Inadequate Folate Intake on the Onset and Progression of Hypertensive Vascular Injury.

We investigated the effects of inadequate folate intake on the onset and progression of hypertensive organ injury. In the present study, 5-wk-old male stroke-prone spontaneously hypertensive rats (SHRSP) were fed with a normal-folate (control; 160-170 µg of folate/100 g diet) or low-folate (8-10 µg of folate/100 g diet) diet until they reached 25 wk of age. After the animals reached 10 wk of age, the bodyweight of the rats in the low-folate group was lower than that of the rats in the control group. Regarding blood pressure, both groups had severe hypertension of ≥230 mmHg at 12 wk of age that was not significantly different between the groups. At 16 wk of age, the low-folate group had a low number of blood cell types. The folate levels in the serum, liver, and kidneys of these rats were significantly lower (p<0.01) and the serum homocysteine level in the low-folate group was significantly higher than in the controls. The low-folate group had a significantly lower testicular weight than the control group (p<0.05) and arterial hypertrophy, spermatogenesis arrest, and interstitial connective tissue hyperplasia were observed. However, there was no clear difference in lesions in other organs. These results indicated that under low folate status, SHRSP causes hematopoietic disorders and exacerbates hypertensive vascular injury at various degrees by organ type.

The role of ROC75 as a daytime component of the circadian oscillator in Chlamydomonas reinhardtii.

The circadian clocks in chlorophyte algae have been studied in two model organisms, Chlamydomonas reinhardtii and Ostreococcus tauri. These studies revealed that the chlorophyte clocks include some genes that are homologous to those of the angiosperm circadian clock. However, the genetic network architectures of the chlorophyte clocks are largely unknown, especially in C. reinhardtii. In this study, using C. reinhardtii as a model, we characterized RHYTHM OF CHLOROPLAST (ROC) 75, a clock gene encoding a putative GARP DNA-binding transcription factor similar to the clock proteins LUX ARRHYTHMO (LUX, also called PHYTOCLOCK 1 [PCL1]) and BROTHER OF LUX ARRHYTHMO (BOA, also called NOX) of the angiosperm Arabidopsis thaliana. We observed that ROC75 is a day/subjective day-phase-expressed nuclear-localized protein that associates with some night-phased clock genes and represses their expression. This repression may be essential for the gating of reaccumulation of the other clock-related GARP protein, ROC15, after its light-dependent degradation. The restoration of ROC75 function in an arrhythmic roc75 mutant under constant darkness leads to the resumption of circadian oscillation from the subjective dawn, suggesting that the ROC75 restoration acts as a morning cue for the C. reinhardtii clock. Our study reveals a part of the genetic network of C. reinhardtii clock that could be considerably different from that of A. thaliana.

Publisher Correction: Methylation deficiency disrupts biological rhythms from bacteria to humans.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Methylation deficiency disrupts biological rhythms from bacteria to humans.

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies.

The CONSTANS flowering complex controls the protective response of photosynthesis in the green alga Chlamydomonas.

Light is essential for photosynthesis, but the amounts of light that exceed an organism's assimilation capacity can result in oxidative stress and even cell death. Plants and microalgae have developed a photoprotective response mechanism, qE, that dissipates excess light energy as thermal energy. In the green alga Chlamydomonas reinhardtii, qE is regulated by light-inducible photoprotective proteins, but the pathway from light perception to qE is not fully understood. Here, we show that the transcription factors CONSTANS and Nuclear transcription Factor Ys (NF-Ys) form a complex that governs light-dependent photoprotective responses in C. reinhardtii. The qE responses do not occur in CONSTANS or NF-Y mutants. The signal from light perception to the CONSTANS/NF-Ys complex is directly inhibited by the SPA1/COP1-dependent E3 ubiquitin ligase. This negative regulation mediated by the E3 ubiquitin ligase and the CONSTANS/NF-Ys complex is common to photoprotective response in algal photosynthesis and flowering in plants.

Isolation of photoprotective signal transduction mutants by systematic bioluminescence screening in Chlamydomonas reinhardtii.

In photosynthetic organisms, photoprotection to avoid overexcitation of photosystems is a prerequisite for survival. Green algae have evolved light-inducible photoprotective mechanisms mediated by genes such as light-harvesting complex stress-related (LHCSR). Studies on the light-dependent regulation of LHCSR expression in the green alga Chlamydomonas reinhardtii have revealed that photoreceptors for blue light (phototropin) and ultraviolet light perception (UVR8) play key roles in initiating photoprotective signal transduction. Although initial light perception via phototropin or UVR8 is known to result in increased LHCSR3 and LHCSR1 gene expression, respectively, the mechanisms of signal transduction from the input (light perception) to the output (gene expression) remain unclear. In this study, to further elucidate the signal transduction pathway of the photoprotective response of green algae, we established a systematic screening protocol for UV-inducible LHCSR1 gene expression mutants using a bioluminescence reporter assay. Following random mutagenesis screening, we succeeded in isolating mutants deficient in LHCSR1 gene and protein expression after UV illumination. Further characterization revealed that the obtained mutants could be separated into 3 different phenotype groups, the "UV-specific", "LHCSR1-promoter/transcript-specific" and "general photoprotective" mutant groups, which provided further insight into photoprotective signal transduction in C. reinhardtii.

ROC75 is an Attenuator for the Circadian Clock that Controls LHCSR3 Expression.

Strong light intensity leads to harmful overexcitation of the photosystems in green algae. In Chlamydomonas reinhardtii, LHCSR3 is required for the rapid protective response known as energy-dependent quenching (qE). Because the majority of photoacclimation analysis has been conducted under controlled laboratory conditions, physiological responses to natural environmental changes such as light/dark cycles have not been examined in detail. Regarding fitness in higher plants and microalgae, light-dark cycles represent a major Zeitgeber for synchronizing the circadian clock to multiple physiological responses, yet there is little consensus with respect to the clock response to high-intensity light in photosynthetic organisms. In a previous study, 105 circadian rhythm insertional mutants were isolated as rhythm of chloroplast (roc) mutants. Here, we report our characterization of the roc75 mutant, which exhibited a significantly higher qE value and LHCSR3 protein accumulation when grown under red light. We performed transcript analysis of ROC75 in the pcry (plant-cryptochrome) and phot mutants and found that only the former accumulated lower levels of ROC75 mRNA, suggesting that the blue light photoreceptor pCRY positively regulates ROC75. However, the degradation of pCRY by high-light exposure contributes to prevent over-accumulation of ROC75, which in turn facilitates the PHOT mediated main activation pathway for LHCSR3. Furthermore, LHCSR3 mRNA exhibited a circadian rhythm, though its basal expression level in the roc75 mutant was higher than that in WT. We therefore conclude that ROC75 acts as an attenuator of the circadian clock to control LHCSR3 expression with blue and red light as stimuli for attenuation.

Survey on awareness of folic acid recognition and intake by female students.

To reduce the risk of neural tube defects, studies have been conducted on female students of medical services, nutritional science, and nursery education that investigated the awareness of folic acid by using questionnaires. Many investigators have suggested the need to provide detailed information about the awareness of folic acid and knowledge about folic acid intake and neural tube defect risk reduction. The dietary habits of female students showed a positive correlation with their estimated folic acid intake, suggesting that improvements in dietary habits are associated with the consumption of folic acid. The importance of folic acid intake must be more aggressively promoted among female students. Thus, many learning opportunities should be provided for such students to help increase their folic acid intake.

Neural tube defects: Risk factors and preventive measures.

For the last 25 years, it has been proven that the occurrence or recurrence of neural tube defects can be prevented with the administration of folic acid before and early pregnancy. At present, over 80 countries in the world, except Japan, have mandated the fortification of wheat flour and/or rice with folic acid, which has resulted in a significant reduction in the prevalence of neural tube defects. In 2000, the Japanese government recommended folic acid 400 µg daily for young women of childbearing age and women who are planning to conceive. In 2002, the government started to present information about the importance of folic acid in the development of fetuses in the Mother-Child Health Booklet annually. Despite these endeavors, the prevalence of neural tube defects has remained unchanged. We discuss the risk factors of neural tube defects and propose preventive measures to decrease the number of neonates with neural tube defects. We believe that the government should implement the fortification of staple food with folic acid very soon, which will eventually decrease not only the neonatal mortality and morbidity, but also the economic burden on our health care system.

CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas.

The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas.

Nitrogen starvation-induced accumulation of triacylglycerol in the green algae: evidence for a role for ROC40, a transcription factor involved in circadian rhythm.

Microalgal triacylglycerol (TAG), a promising source of biofuel, is induced upon nitrogen starvation (-N), but the proteins and genes involved in this process are poorly known. We performed isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics to identify Chlorella proteins with modulated expression under short-term -N. Out of 1736 soluble proteins and 2187 membrane-associated proteins identified, 288 and 56, respectively, were differentially expressed under -N. Gene expression analysis on select genes confirmed the same direction of mRNA modulation for most proteins. The MYB-related transcription factor ROC40 was the most induced protein, with a 9.6-fold increase upon -N. In a previously generated Chlamydomonas mutant, gravimetric measurements of crude total lipids revealed that roc40 was impaired in its ability to increase the accumulation of TAG upon -N, and this phenotype was complemented when wild-type Roc40 was expressed. Results from radiotracer experiments were consistent with the roc40 mutant being comparable to the wild type in recycling membrane lipids to TAG but being impaired in additional de novo synthesis of TAG during -N stress. In this study we provide evidence to support the hypothesis that transcription factor ROC40 has a role in -N-induced lipid accumulation, and uncover multiple previously unknown proteins modulated by short-term -N in green algae.

Diversity of plant circadian clocks: Insights from studies of Chlamydomonas reinhardtii and Physcomitrella patens.

Arabidopsis thaliana has long been the model plant of choice for elucidating the mechanisms of the circadian clock. Recently, relevant results have accumulated in other species of green plant lineages, including green algae. This mini-review describes a comparison of the mechanism of the A. thaliana clock to those of the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens, focusing on commonalities and divergences of subsystems of the clock. The potential of such an approach from an evolutionary viewpoint is discussed.

Comparative metabolome analysis of cultured fetal and adult hepatocytes in humans.

The liver is the central organ of metabolism, but its function varies during development from fetus to adult. In this study, we comprehensively analyzed and compared metabolites in fetal and adult hepatocytes, the major parenchymal cell in the liver, from human donors. We identified 211 metabolites (116 anions and 95 cations) by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) in the hepatocytes cultured in vitro. Principal component analysis and hierarchical clustering analysis of the relative amounts of metabolites clearly classified hepatocytes into 2 groups that were consistent with their origin, i.e., the fetus and adult. The amounts of most metabolites in the glycolysis/glyconeogenesis pathway, tricarboxylic acid cycle and urea cycle were lower in fetal hepatocytes than in adult hepatocytes. These results suggest different susceptibility of the fetal and adult liver to toxic insults affecting energy metabolism.

High-throughput phenotyping of chlamydomonas swimming mutants based on nanoscale video analysis.

Studies on biflagellated algae Chlamydomonas reinhardtii mutants have resulted in significant contributions to our understanding of the functions of cilia/flagella components. However, visual inspection conducted under a microscope to screen and classify Chlamydomonas swimming requires considerable time, effort, and experience. In addition, it is likely that identification of mutants by this screening is biased toward individual cells with severe swimming defects, and mutants that swim slightly more slowly than wild-type cells may be missed by these screening methods. To systematically screen Chlamydomonas swimming mutants, we have here developed the cell-locating-with-nanoscale-accuracy (CLONA) method to identify the cell position to within 10-nm precision through the analysis of high-speed video images. Instead of analyzing the shape of the flagella, which is not always visible in images, we determine the position of Chlamydomonas cell bodies by determining the cross-correlation between a reference image and the image of the cell. From these positions, various parameters related to swimming, such as velocity and beat frequency, can be accurately estimated for each beat cycle. In the examination of wild-type and seven dynein arm mutants of Chlamydomonas, we found characteristic clustering on scatter plots of beat frequency versus swimming velocity. Using the CLONA method, we have screened 38 Chlamydomonas strains and detected believed-novel motility-deficient mutants that would be missed by visual screening. This CLONA method can automate the screening for mutants of Chlamydomonas and contribute to the elucidation of the functions of motility-associated proteins.

Melanoma transition is frequently accompanied by a loss of cytoglobin expression in melanocytes: a novel expression site of cytoglobin.

The tissue distribution and function of hemoglobin or myoglobin are well known; however, a newly found cytoglobin (CYGB), which also belongs to the globin family, remains to be characterized. To assess its expression in human malignancies, we sought to screen a number of cell lines originated from many tissues using northern blotting and real time PCR techniques. Unexpectedly, we found that several, but not all, melanoma cell lines expressed CYGB mRNA and protein at much higher levels than cells of other origins. Melanocytes, the primary origin of melanoma, also expressed CYGB at a high level. To verify these observations, immunostaining and immunoblotting using anti-CYGB antibody were also performed. Bisulfite-modified genomic sequencing revealed that several melanoma cell lines that abrogated CYGB expression were found to be epigenetically regulated by hypermethylation in the promoter region of CYGB gene. The RNA interference-mediated knockdown of the CYGB transcript in CYGB expression-positive melanoma cell lines resulted in increased proliferation in vitro and in vivo. Flow cytometric analysis using 2'-, 7'-dichlorofluorescein diacetate (DCFH-DA), an indicator of reactive oxygen species (ROS), revealed that the cellular ROS level may be involved in the proliferative effect of CYGB. Thus, CYGB appears to play a tumor suppressive role as a ROS regulator, and its epigenetic silencing, as observed in CYGB expression-negative melanoma cell lines, might function as an alternative pathway in the melanocyte-to-melanoma transition.

C677T mutation in methylenetetrahydrofolate reductase gene and neural tube defects: should Japanese women undergo gene screening before pregnancy?

We analyzed the role of maternal C677T mutation in methylenetetrahydrofolate reductase (MTHFR) gene on spina bifida development in newborns. A total of 115 mothers who had given birth to a spina bifida child (SB mothers) gave 10 mL of blood together with written informed consent. The genotype distribution of C677T mutation was assessed and compared with that of the 4517 control individuals. The prevalence of the homozygous genotype (TT) among SB mothers was not significantly different from that among the controls (odds ratio [OR] = 0.65; 95% confidence interval [CI] = 0.31-1.25; P = 0.182), suggesting that MTHFR 677TT genotype in Japan is not associated with spina bifida development in newborns. The T allele frequency was not increased in SB mothers (34.8%) as compared to that of the control individuals (38.2%). Further, the internationally reported association between the two groups was found to be similar in all 15 countries studied except the Netherlands, where the TT genotype was found to be a genetic risk factor for spina bifida. For the prevention of affected pregnancy every woman planning to conceive has to take folic acid supplements 400 µg a day and the government is asked to take action in implementing food fortification with folic acid in the near future. In conclusion, it is not necessary for Japanese women to undergo genetic screening C677T mutation of the MTHFR gene as a predictive marker for spina bifida prior to pregnancy, because the TT genotype is not a risk factor for having an affected infant.

Phase-resetting mechanism of the circadian clock in Chlamydomonas reinhardtii.

Although the circadian clock is a self-sustaining oscillator having a periodicity of nearly 1 d, its period length is not necessarily 24 h. Therefore, daily adjustment of the clock (i.e., resetting) is an essential mechanism for the circadian clock to adapt to daily environmental changes. One of the major cues for this resetting mechanism is light. In the unicellular green alga Chlamydomonas reinhardtii, the circadian clock is reset by blue/green and red light. However, the underlying molecular mechanisms remain largely unknown. In this study, using clock protein-luciferase fusion reporters, we found that the level of RHYTHM OF CHLOROPLAST 15 (ROC15), a clock component in C. reinhardtii, decreased rapidly after light exposure in a circadian-phase-independent manner. Blue, green, and red light were able to induce this process, with red light being the most effective among them. Expression analyses and inhibitor experiments suggested that this process was regulated mainly by a proteasome-dependent protein degradation pathway. In addition, we found that the other clock gene, ROC114, encoding an F-box protein, was involved in this process. Furthermore, we demonstrated that a roc15 mutant showed defects in the phase-resetting of the circadian clock by light. Taken together, these data strongly suggest that the light-induced degradation of ROC15 protein is one of the triggers for resetting the circadian clock in C. reinhardtii. Our data provide not only a basis for understanding the molecular mechanisms of light-induced phase-resetting in C. reinhardtii, but also insights into the phase-resetting mechanisms of circadian clocks in plants.

N-terminal acetyltransferase 3 gene is essential for robust circadian rhythm of bioluminescence reporter in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a model species of algae for studies on the circadian clock. Previously, we isolated a series of mutants showing defects in the circadian rhythm of a luciferase reporter introduced into the chloroplast genome, and identified the genes responsible for the defective circadian rhythm. However, we were unable to identify the gene responsible for the defective circadian rhythm of the rhythm of chloroplast 97 (roc97) mutant because of a large genomic deletion. Here, we identified the gene responsible for the roc97 mutation through a genetic complementation study. This gene encodes a protein that is homologous to the subunit of N-terminal acetyltransferase (NAT) which catalyzes N-terminal acetylation of proteins. Our results provide the first example of involvement of the protein N-terminal acetyltransferase in the circadian rhythm.