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.     

Analysis of DNA Methylation of Multiple Genes in Microdissected Cells From Formalin-fixed and Paraffin-embedded Tissues

A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells. (J Histochem Cytochem 57:477–489, 2009)     

Evidence for Multiple Lateral Transfers of the Circadian Clock Cluster in Filamentous Heterocystic Cyanobacteria Nostocaceae

Cyanobacteria are the first prokaryotes reported to show circadian rhythmicity, which is regulated by a cluster of three genes: kaiA, kaiB, and kaiC. Phylogenetic analysis of the kaiBC cluster in filamentous cyanobacteria of the family Nostocaceae including Nodularia spumigena and Nostoc linckia from Arubotaim Cave, Mt. Sedom, Israel, indicated that this cluster has experienced multiple lateral transfers. The transfers have occurred in different periods of the species evolution. The data obtained suggest that lateral transfers of the circadian clock cluster in filamentous cyanobacteria have been common and might have adaptive significance.     

Epigenetic Basis of Regeneration: Analysis of Genomic DNA Methylation Profiles in the MRL/MpJ Mouse

Epigenetic regulation plays essential role in cell differentiation and dedifferentiation, which are the intrinsic processes involved in regeneration. To investigate the epigenetic basis of regeneration capacity, we choose DNA methylation as one of the most important epigenetic mechanisms and the MRL/MpJ mouse as a model of mammalian regeneration known to exhibit enhanced regeneration response in different organs. We report the comparative analysis of genomic DNA methylation profiles of the MRL/MpJ and the control C57BL/6J mouse. Methylated DNA immunoprecipitation followed by microarray analysis using the Nimblegen ‘3 × 720 K CpG Island Plus RefSeq Promoter’ platform was applied in order to carry out genome-wide DNA methylation profiling covering 20 404 promoter regions. We identified hundreds of hypo- and hypermethylated genes and CpG islands in the heart, liver, and spleen, and 37 of them in the three tissues. Decreased inter-tissue diversification and the shift of DNA methylation balance upstream the genes distinguish the genomic methylation patterns of the MRL/MpJ mouse from the C57BL/6J. Homeobox genes and a number of other genes involved in embryonic morphogenesis are significantly overrepresented among the genes hypomethylated in the MRL/MpJ mouse. These findings indicate that epigenetic patterning might be a likely molecular basis of regeneration capability in the MRL/MpJ mouse.     

Methylation profiling using degenerated oligonucleotide primer-PCR specific for genome-wide amplification of bisulfite-modified DNA

DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA, followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample, whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles, we developed degenerated oligonucleotide primer (DOP)-PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3' DOP, CTCGAGCTGHHHHHAACTAC, where H is a mixture of base consisting of 50% A, 25% T, and 25% C; and second 5' DOP, CTCGAGCTGDDDDDGTTTAG, where D is a mixture of base consisting of 50% T, 25% G, and 25% A. Our results showed that bisulfite-modified DNAs from a cell line, cord blood cells, or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template.     

Role of Patient Chronotype on Circadian Pattern of Myocardial Infarction: A Pilot Study

Population-based studies indicate the risk of acute myocardial infarction (AMI) is greatest in the morning, during the initial hours of diurnal activity. The aim of this pilot study was to determine whether chronotype, i.e., morningness and eveningness, impacts AMI onset time. The sample comprised 63 morning- and 40 evening-type patients who were classified by the Horne-Östberg Morningness-Eveningness Questionnaire (MEQ) in the hospital after experiencing the AMI. The average wake-up and bed times of morning types were ～2?h earlier than evening types. Although the lag in time between waking up from nighttime sleep and AMI onset during the day did not differ between the two chronotypes, the actual clock-hour time of the peak in the 24-h AMI pattern did. The peak in AMI of morning types occurred between 06:01 and 12:00?h and that of the evening types between 12:01 and 18:00?h. Although the results of this small sample pilot study suggest one's chronotype influences the clock time of AMI onset, larger scale studies, which also include assessment of 24-h patterning of events in neither types, must be conducted before concluding the potential influence of chronotype on the timing of AMI onset. (Author correspondence: dryavuzselvi@yahoo.com).     

Low-Carbohydrate,High-Protein Diet Affects Rhythmic Expression of Gluconeogenic Regulatory and Circadian Clock Genes in Mouse Peripheral Tissues

Recent studies have demonstrated that metabolic changes in mammals induce feedback regulation of the circadian clock. The present study evaluates the effects of a low-carbohydrate high-protein diet (HPD) on circadian behavior and peripheral circadian clocks in mice. Circadian rhythms of locomotor activity and core body temperature remained normal in mice fed with the HPD diet (HPD mice), suggesting that it did not affect the central clock in the hypothalamus. Two weeks of HPD feeding induced mild hypoglycemia without affecting body weight, although these mice consumed more calories than mice fed with a normal diet (ND mice). Plasma insulin levels were increased during the inactive phase in HPD mice, but increased twice, beginning and end of the active phase, in ND mice. Expression levels of the key gluconeogenic regulatory genes PEPCK and G6Pase were significantly induced in the liver and kidneys of HPD mice. The HPD appeared to induce peroxisome proliferator-activated receptor α (PPARα) activation, since mRNA expression levels of PPARα and its typical target genes, such as PDK4 and Cyp4A10, were significantly increased in the liver and kidneys. Circadian mRNA expression of clock genes, such as BMAL1, Cry1, NPAS2, and Rev-erbα, but not Per2, was significantly phase-advanced, and mean expression levels of BMAL1 and Cry1 mRNAs were significantly elevated, in the liver and kidneys of HPD mice. These findings suggest that a HPD not only affects glucose homeostasis, but that it also advances the molecular circadian clock in peripheral tissues. (Author correspondence: k-ooishi@aist.go.jp)     

The Circadian Clock Machinery During Early Development of Senegalese sole (Solea senegalensis): Effects of Constant Light and Dark Conditions

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0–4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae. (Author correspondence: munoz.cueto@uca.es; carlos.pendon@uca.es)     

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5''-CpG-3'' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.¹ and Clark et al.², methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.     

多糖的甲基化方法及图谱解析

本文对微量多糖的甲基化方法进行了研究。0.1 mg多糖样品用0.3 mL二甲亚砜溶解后,加入10 mg氢氧化钠粉末,室温下超声20 min。冰浴冷却后加入0.1 mL碘甲烷1,8～20℃下超声20 min,再加入0.1 mL碘甲烷,超声20 min。加入1 mL含4 mmol/L Na2S2O3的水,终止甲基化反应。反应液用0.5 mL氯仿提取4次,氯仿提取液用0.5 mL水处理5次。氯仿相用无水硫酸钠脱水后,氮气吹干。该法简便易行,甲基化程度高,适用于易溶(或难溶)于水和二甲亚砜的多糖。此外,本文对部分甲基化的糖醇乙酰酯衍生物的质谱图解析进行了阐述,并对特殊糖类样品的甲基化方法进行了说明。     

Circadian Rhythm in Dihydropyrimidine Dehydrogenase Activity and Reduced Glutathione Content in Peripheral Blood of Nasopharyngeal Carcinoma Patients

Dihydropyrimidine dehydrogenase (DPD) is a rate‐limiting enzyme of 5‐fluorouracil (5‐FU) catabolism. Glutathione (GSH) is a tripeptide involved in platinum complex detoxification. This study explored the circadian rhythms of DPD activity and GSH concentration in the peripheral blood of 16 patients with histologically proven nasopharyngeal carcinoma (NPC) in order to guide the establishment of chronotherapeutic schedules for this cancer. DPD activity and GSH concentration were determined by high performance liquid chromatography (HPLC). Both variables displayed significant circadian rhythms (Cosinor analysis: p=0.009 and 0.012, respectively). Peak DPD activity occurred at about 02:30 h; whereas, peak GSH concentration occurred around 12:40 h. The differences between the peak and nadir mean values were 25.5% and 38.7%, respectively. The study showed that the circadian rhythms in DPD activity and GSH concentration in Chinese NPC are similar to those reported for western patients with colorectal cancer, despite the differences in race and kinds of cancer. These findings imply that the chronotherapeutic schedule of 5‐FU and platinum used to treat European colorectal cancer patients probably is applicable to Chinese NPC patients.     

Circadian rhythm in dihydropyrimidine dehydrogenase activity and reduced glutathione content in peripheral blood of nasopharyngeal carcinoma patients

Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme of 5-fluorouracil (5-FU) catabolism. Glutathione (GSH) is a tripeptide involved in platinum complex detoxification. This study explored the circadian rhythms of DPD activity and GSH concentration in the peripheral blood of 16 patients with histologically proven nasopharyngeal carcinoma (NPC) in order to guide the establishment of chronotherapeutic schedules for this cancer. DPD activity and GSH concentration were determined by high performance liquid chromatography (HPLC). Both variables displayed significant circadian rhythms (Cosinor analysis: p=0.009 and 0.012, respectively). Peak DPD activity occurred at about 02:30 h; whereas, peak GSH concentration occurred around 12:40 h. The differences between the peak and nadir mean values were 25.5% and 38.7%, respectively. The study showed that the circadian rhythms in DPD activity and GSH concentration in Chinese NPC are similar to those reported for western patients with colorectal cancer, despite the differences in race and kinds of cancer. These findings imply that the chronotherapeutic schedule of 5-FU and platinum used to treat European colorectal cancer patients probably is applicable to Chinese NPC patients.     

Epigenetics of Osteoporosis: Critical Analysis of Epigenetic Epidemiology Studies

Osteoarthritis (OA) is an age-related disease with poorly understood pathogenesis. Recent studies have demonstrated that miRNA might play a key role in OA initiation and development. We reviewed recent publications and elucidated the connection between miRNA and OA cartilage anabolic and catabolic signals, including four signaling pathways: TGF-β/Smads and BMPs signaling, associated with cartilage anabolism; and MAPK and NF-KB signaling, associated with cartilage catabolism. We also explored the relationships with MMP, ADAMTS and NOS (NitricOxide Synthases) families, as well as with the catabolic cytokines IL-1 and TNF-α. The potential role of miRNAs in biological processes such as cartilage degeneration, chondrocyte proliferation, and differentiation is discussed. Collective evidence indicates that miRNAs play a critical role in cartilage degeneration. These findings will aid in understanding the molecular network that governs articular cartilage homeostasis and in to elucidate the role of miRNA in the pathogenesis of OA.     

An Example of Circular Statistics in Chronobiological Studies: Analysis of Polymorphism in the Emergence Rhythms of Schistosoma Mansoni Cercariae

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals. (Chronobiology International, 17(4), 433–451, 2000)     

Human Intestinal Circadian Clock: Expression of Clock Genes in Colonocytes Lining the Crypt

Biological clock components have been detected in many epithelial tissues of the digestive tract of mammals (oral mucosa, pancreas, and liver), suggesting the existence of peripheral circadian clocks that may be entrainable by food. Our aim was to investigate the expression of main peripheral clock genes in colonocytes of healthy humans and in human colon carcinoma cell lines. The presence of clock components was investigated in single intact colonic crypts isolated by chelation from the biopsies of 25 patients (free of any sign of colonic lesions) undergoing routine colonoscopy and in cell lines of human colon carcinoma (Caco2 and HT29 clone 19A). Per‐1, per‐2, and clock mRNA were detected by real‐time RT‐PCR. The three‐dimensional distributions of PER‐1, PER‐2, CLOCK, and BMAL1 proteins were recorded along colonic crypts by immunofluorescent confocal imaging. We demonstrate the presence of per‐1, per‐2, and clock mRNA in samples prepared from colonic crypts of 5 patients and in all cell lines. We also demonstrate the presence of two circadian clock proteins, PER‐1 and CLOCK, in human colonocytes on crypts isolated from 20 patients (15 patients for PER‐1 and 6 for CLOCK) and in colon carcinoma cells. Establishing the presence of clock proteins in human colonic crypts is the first step toward the study of the regulation of the intestinal circadian clock by nutrients and feeding rhythms.     

Methylation analyses on promoters of mPer1, mPer2, and mCry1 during perinatal development

Recent studies revealed dramatic changes in circadian clock genes’ expression during the perinatal period. In this study, we characterized DNA methylation for three clock genes mPer1, mPer2, and mCry1 at their selected promoter regions during development. Results for the suprachiasmatic nucleus (SCN) and liver (at embryonic day 19, postnatal day 1 and postnatal day 7) were compared to those of sperm. Few methylations were detected for the mPer2 and mCry1 promoters. The 3rd E-box region of the mPer1 promoter exhibited methylation only in sperm. Significant demethylation was observed in the 4th E-box region of the mPer1 promoter between E19 and P1 in the SCN but not in liver tissue. This demethylation state was maintained at P7 for the SCN. Luciferase reporter assays using in vitro methylated promoters revealed an inhibitory effect of promoter methylation on mPer1 expression. The results suggested that epigenetic mechanisms such as DNA methylation might contribute to the developmental expression of clock genes.     

Circadian Discrimination of Reward: Evidence for Simultaneous Yet Separable Food- and Drug-Entrained Rhythms in the Rat

A unique extra-suprachiasmatic nucleus (SCN) oscillator, operating independently of the light-entrainable oscillator, has been hypothesized to generate feeding and drug-related rhythms. To test the validity of this hypothesis, sham-lesioned (Sham) and SCN-lesioned (SCNx) rats were housed in constant dim-red illumination (LL_red) and received a daily cocaine injection every 24?h for 7 d (Experiment 1). In a second experiment, rats underwent 3-h daily restricted feeding (RF) followed 12 d later by the addition of daily cocaine injections given every 25?h in combination with RF until the two schedules were in antiphase. In both experiments, body temperature and total activity were monitored continuously. Results from Experiment 1 revealed that cocaine, but not saline, injections produced anticipatory increases in temperature and activity in SCNx and Sham rats. Following withdrawal from cocaine, free-running temperature rhythms persisted for 2–10 d in SCNx rats. In Experiment 2, robust anticipatory increases in temperature and activity were associated with RF and cocaine injections; however, the feeding periodicity (23.9?h) predominated over the cocaine periodicity. During drug withdrawal, the authors observed two free-running rhythms of temperature and activity that persisted for >14 d in both Sham and SCNx rats. The periods of the free-running rhythms were similar to the feeding entrainment (period?=?23.7 and 24.0?h, respectively) and drug entrainment (period?=?25.7 and 26.1?h, respectively). Also during withdrawal, the normally close correlation between activity and temperature was greatly disrupted in Sham and SCNx rats. Taken together, these results do not support the existence of a single oscillator mediating the rewarding properties of both food and cocaine. Rather, they suggest that these two highly rewarding behaviors can be temporally isolated, especially during drug withdrawal. Under stable dual-entrainment conditions, food reward appears to exhibit a slightly greater circadian influence than drug reward. The ability to generate free-running temperature rhythms of different frequencies following combined food and drug exposures could reflect a state of internal desynchrony that may contribute to the addiction process and drug relapse. (Author correspondence: heiko@vetmed.wsu.edu)