Major Clock Genes Research Articles

Involvement of Bmal1 and Clock in Bromobenzene Metabolite-Induced Diurnal Renal Toxicity.

Circadian rhythms are endogenous oscillators that regulate 24 h behavioral and physiological processes. Our previous investigation demonstrated that bromobenzene metabolite (4-bromocatechol: 4-BrCA) exhibited chronotoxicity (i.e., the nephrotoxicity induced by 4-BrCA was observed during the dark phase, while not observed at light phase in mice). However, the molecular mechanism is still unknown. The aim of the present study is to investigate the cellular molecule(s) involved in the 4-BrCA-induced nephrotoxicity using mouse renal cortex tubular cell lines (MuRTE61 cells). We found that 4-BrCA showed dose dependent (0.01-1 mM) cell proliferation defect in MuRTE61 cells. By treating with 0.03 mM 4-BrCA, we demonstrated that major clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) were significantly downregulated. Interestingly, the expression levels of two genes, Bmal1 and Clock, continued to decrease after 3 h of treatment with 4-BrCA, while Cry1, Per1, and Per2 were unchanged until 24 h of treatment. Moreover, BMAL1 and CLOCK levels are higher at light phase. We speculated that BMAL1 and CLOCK might function defensively against 4-BrCA-induced nephrotoxicity since the expression levels of Bmal1 and Clock were rapidly decreased. Finally, overexpression of Bmal1 and Clock restored 4-BrCA-induced cell proliferation defect in MuRTE61 cells. Taken together, our results suggest that Bmal1 and Clock have protective roles against 4-BrCA-induced nephrotoxicity.

The Role of REV-ERB Receptors in Cancer Pathogenesis.

REV-ERB receptors are members of the nuclear receptor superfamily of proteins, which act as both intracellular receptors and transcription factors, therefore modulating the expression of target genes. REV-ERBs act as transcription repressors because of their unique structure. Their predominant role involves the control of peripheral circadian rhythmicity by participating in a transcription-translation feedback loop with other major clock genes. Regarding their role in cancer pathogenesis, recent studies in various cancerous tissues have revealed that their expression was downregulated in the majority of the cases. Dysregulation of their expression was also implicated in cancer-associated cachexia. The pharmacological restoration of their effects is feasible with synthetic agonists, which have been explored in preclinical studies but with scarce data. There is a need for further investigation, primarily with mechanistic studies, on the effect of the REV-ERB-induced circadian rhythm deregulation in carcinogenesis and cancer-related systemic effects, such as cachexia, in order to address the potential of relevant therapeutic implications.

Endogenous circadian reporters reveal functional differences of PERIOD paralogs and the significance of PERIOD:CK1 stable interaction

Adverse consequences from having a faulty circadian clock include compromised sleep quality and poor performance in the short-term, and metabolic diseases and cancer in the long-term. However, our understanding of circadian disorders is limited by the incompleteness of our molecular models and our dearth of defined mutant models. Because it would be prohibitively expensive to develop live animal models to study the full range of complicated clock mechanisms, we developed PER1-luc and PER2-luc endogenous circadian reporters in a validated clock cell model, U-2 OS, where the genome can be easily manipulated, and functional consequences of mutations can be accurately studied. When major clock genes were knocked out in these cells, circadian rhythms were modulated similarly compared with corresponding mutant mice, validating the platform for genetics studies. Using these reporter cells, we uncovered critical differences between two paralogs of PER. Although PER1 and PER2 are considered redundant and either one can serve as a pacemaker alone, they were dramatically different in biochemical parameters such as stability and phosphorylation kinetics. Consistently, circadian phase was dramatically different between PER1 and PER2 knockout reporter cells. Wefurther showed that the stable binding of casein kinase1δ/εto PER is not required for PER phosphorylation itself, but is critical for delayed timing of phosphorylation. Our system can be used as an efficient platform to study circadian disorders associated with pathogenic mutations and their underlying molecular mechanisms.

Alterations in the activity and sleep of Drosophila melanogaster under simulated microgravity

This study aimed to investigate alterations in the activity and sleep of Drosophila melanogaster under simulated microgravity, which was implemented through the random positioning machine, while different light conditions (normal photoperiod and constant dark) were set. Fruit flies of different strains and sexes were treated for 3 days, and activity and sleep were monitored using the Drosophila Activity Monitoring System. After 3 days of treatment, fruit flies were sampled to detect the relative expression levels of the major clock genes and some neurotransmitter-related genes. The results showed that for the normal photoperiod (LD) condition, the activity increased and sleep decreased under simulated microgravity, while for the constant dark (DD) condition, the activity and sleep rhythms appeared disordered and the activity increased, thus decreasing the likelihood of waking up during the day. Light conditions, strains, and sexes, individually or in combination, had impacts on the simulated microgravity effects on behaviors. The clock genes and neurotransmitter-related genes had different degrees of response among sexes and strains, although the overall changes were slight. The results indicated that the normal photoperiod could ease the effects of simulated microgravity on fruit flies’ activity and sleep and possible unidentified pathways involved in the regulatory mechanism need further exploration. This study is expected to provide ideas and references for studying the effects of microgravity on space life science.

Circadian clock regulates tear secretion in the lacrimal gland

Although diurnal variations have been observed in tear film parameters in various species, the molecular mechanisms that control circadian tear secretion remain unclear. The aim of our study was to evaluate the role of clock genes in the lacrimal gland (LG) in regulation of tear secretion. Tear volume was measured by cotton thread test in core clock genes deficient (Cry1−/−Cry2−/−-) mice which are behaviorally arrhythmic. Real-time quantitative RT-PCR was used to examine expression profiles of core clock genes in the LG including Per1, Per2, Per3, Clock, Bmal1. All experiments were performed under a 12 h of light and 12 h of darkness (LD) and constant dark (DD) conditions. Under both LD and DD conditions, diurnal and circadian rhythms were observed in tear secretion of wild-type mice with tear volume increased in the objective and subjective night while disruption in diurnal and circadian variations of tear secretion were found in Cry1−/−Cry2−/−-mice. In wild-type mice, the expression level of major clock genes in the LG showed oscillatory patterns under both LD and DD conditions. In contrast, expression clock genes in the lacrimal gland of Cry1−/−Cry2−/−- mice showed complete loss of oscillation regardless of environmental light conditions. These findings confirmed the presence of diurnal and circadian rhythms of tear secretion and provided evidences supporting a critical role for the clock in the control of tear secretion.

The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus

The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwoRNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.

Desflurane anesthesia shifts the circadian rhythm phase depending on the time of day of anesthesia

Desflurane is one of the most frequently used inhalational anesthetics in clinical practice. A circadian rhythm phase-shift after general anesthesia with sevoflurane or isoflurane has been reported in mice, but few studies have reported this effect with desflurane. In the present study, we examined the rest/activity rhythm of mice by counting the number of running wheel rotations, and we found that desflurane anesthesia caused a phase shift in the circadian rhythm that was dependent on the time of day of anesthesia. We also found that desflurane anesthesia altered the relative mRNA expression of four major clock genes (Per2, Bmal, Clock, and Cry1) in the suprachiasmatic nucleus (SCN). These results are important for elucidating the effects of desflurane on the SCN, which is the master clock for the mammalian circadian rhythm. Further studies on the relationship between anesthesia and circadian rhythm may lead to the prevention and treatment of postoperative complications related to circadian rhythms.

Putative Pacemakers in Crayfish Procambarus clarkii Show Circadian Oscillations in Levels of mRNA for Clock Genes under Different Light Rhythm

In the circadian rhythm mechanism, cycle (Cyc), clock (Clk), cryptochrome (Cry) are three core clock genes that constitute complex mechanism controlling organisms to follow a 24-hour oscillation cycle. In the present study, the major clock genes, i.e. PcCyc, PcClk, PcCry1 were cloned from crayfish Procambarus clarkii. The putative functions as well as physical and chemical properties of the resolved proteins were analyzed by various bioinformatic methods. Real-time quantitative PCR technique was used to explore the mRNA expression of the PcCyc, PcClk, PcCry1 in brain, eyestalk, and hepatopancreas tissues of P. clarkii. In order to examine circadian rhythm of selected clock genes in the species, the experiment was carried out under three different light conditions, namely: (a) 12 h of light and dark periods per day (12LD), (b) 24 h of constant darkness (24DD) and (c) 72 h of constant darkness (72DD). The obtained results showed essential correlation in the mRNA expression levels of PcCyc and PcClk genes under similar light conditions. It indirectly suggests that the PcCYC and PcCLK proteins might be involved in the transcriptomic regulatory mechanism of all other genes within the core-clock network. The mRNA expression levels of the PcCry1 gene were highly variable, differing significantly in relation to the various illumination conditions and sampled tissues. The obtained results seem to follow the pattern evidencing the sensitivity of PcCry1 gene to the light signals. It firmly supports the gene’s role in the conversion mechanism of light signals into optical signals, which is engaged in the generation and control of circadian rhythmicity.

Circadian rhythm of expression of core clock genes in the photosensitive pineal organ of catfish, Clarias gariepinus under different photoperiodic regimes

ABSTRACT Effects of different regimes of photoperiod (12L–12D, 16L–8D, 8L–16D, LL and DD) were studied on the circadian rhythm of expression of core clock genes (clock, bmal1, per1, per2, per3, cry1 and cry2) in the photosensitive pineal organ of air-breathing catfish, Clarias gariepinus during summer and winter seasons under both in vivo and in vitro conditions using qPCR. Our findings indicate that all major core clock genes are expressed in the photosensitive pineal organ of C. gariepinus, and exhibit significant circadian rhythm of their expression under both in vivo and in vitro conditions irrespective of photoperiods and seasons. However, photoperiods had season-dependent differential effects on mesor (24 h average), amplitude, and acrophase (peak) of the circadian rhythm of expression of the clock genes under both in vivo and in vitro conditions. Based on these findings, we suggest that the photosensitive pineal organ of the fish possesses internal autonomous clockwork composed of clock genes, and different photoperiods deferentially affect rhythm parameters of the rhythmic expression of the pineal clock genes in a season-dependent manner.

Sex-specific molecular specialization and activity rhythm-dependent gene expression in honey bee antennae.

We performed an RNA-seq-based comparison of gene expression levels in the antennae of honey bee drones and time-trained foragers (workers) collected at different times of the day and different activity states. Interestingly, olfaction-related genes [i.e. odorant receptor (Or) genes, odorant binding protein (Obp) genes, carboxyl esterase (CEst) genes, etc.] showed stable gene expression differences between drone and worker antennae. Drone antennae showed higher expression of 24 Or genes, of which 21 belong to the clade X which comprises the receptor for the major queen pheromone compound 9-ODA. This high number of drone-biased Or genes suggests that more than previously thought play a role in sex-pheromone communication. In addition, we found higher expression levels for many non-olfaction-related genes including nitric oxide synthase (NOS), and the potassium channel Shaw In contrast, workers showed higher expression of 67 Or genes, which belong to different Or clades that are involved in pheromone communication as well as the perception of cuticular hydrocarbons and floral scents. Further, drone antennae showed higher expression of genes involved in energy metabolism, whereas worker antennae showed higher expression of genes involved in neuronal communication, consistent with earlier reports on peripheral olfactory plasticity. Finally, drones that perform mating flight in the afternoon (innate) and foragers that are trained to forage in the afternoon (adapted) showed similar daily changes in the expression of two major clock genes, period and cryptochrome2 Most of the other genes showing changes with time or onset of daily flight activity were specific to drones and foragers.

Transcriptome analysis of Oncidium petals provides new insights into the initiation of petal senescence

ABSTRACTThe Oncidium Gower Ramsey orchid is both an important cut flower plant and a potential good model plant for study the ethylene-sensitive floral senescence. Though significant progress has been made in understanding the physiological and molecular events occurred in petal senescence, little is known regarding the regulatory mechanism underlying onset of petal senescence. In this study, RNA sequencing and transcriptome analysis of petals during the transition from Oncidium flower opening to senescence were employed to identify novel regulatory genes and pathways in the initiation of ethylene-sensitive petal senescence. The petal senescence is a very active process accompanying differential expressions of a large number of genes. The key genes involved in the biosynthesis of gibberellin (GA), brassinosteroid (BR) and salicylic acid (SA), cytokinin (CK) homeostasis, CK and jasmonate (JA) signalling were found to be down-regulated in opening stage but up-regulated in senescent stage, and a gene in ethylene signalling was the reverse, suggesting that these hormones might play a role in the start of petal senescence. The transition-pattern expressions of major circadian clock genes showed that the timing of petal senescence might be under the circadian clock regulation. Moreover, epigenetic and transcriptional regulations were involved in the onset of petal senescence.

PD70-11 CIRCADIAN RHYTHM COORDINATES ATP RELEASE IN THE UROTHELIUM VIA CONNEXIN43 HEMICHANNELS.

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology III1 Apr 2017PD70-11 CIRCADIAN RHYTHM COORDINATES ATP RELEASE IN THE UROTHELIUM VIA CONNEXIN43 HEMICHANNELS. Atsushi Sengiku, Hiromitsu Negoro, Takeshi Sano, Masakatsu Ueda, Jin Kono, Louis Liou, Hitoshi Okamura, and Osamu Ogawa Atsushi SengikuAtsushi Sengiku More articles by this author , Hiromitsu NegoroHiromitsu Negoro More articles by this author , Takeshi SanoTakeshi Sano More articles by this author , Masakatsu UedaMasakatsu Ueda More articles by this author , Jin KonoJin Kono More articles by this author , Louis LiouLouis Liou More articles by this author , Hitoshi OkamuraHitoshi Okamura More articles by this author , and Osamu OgawaOsamu Ogawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3165AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We have previously reported that a peripheral circadian clock exists in the bladder and that connexin 43 (Cx43), a major gap junction protein, oscillates and is a regulator of circadian functional bladder capacity. On the other hand, it has been reported that Cx43 forms hemichannels involved in the release of adenosine triphosphate (ATP) in other cell types. Moreover, it has been also known that extracelluar ATP activates the afferent nerves, which leads to the sensation of bladder fullness. We thus hypothesized that the urothelium had circadian rhythm, which regulated the Cx43 oscillation and function as the hemichannels for ATP release to coordinate the diurnal urination rhythm. METHODS We measured bioluminescence of cultured urothelium from Per2::luc clock gene reporter mice to identify whether the urothelium had circadian rhythms. We also quantified the expression levels of the major clock genes and Cx43 in the urothelium of 11-week-old C57BL/6 female mice at seven consecutive points every 4 hours by real-time polymerase chain reaction and immunoblotting methods. The concentrations of ATP released into the bladder were measured after bladder distention with 30 cm H2O for 10 minutes at zeitgeber time (ZT) 7, a sleeping phase, and ZT19, an active phase for mice. Additionally, we analyzed the circadian rhythm and Cx43 functions with immortalized human urothelial cells. RESULTS The cultured urothelium from Per2::luc mice showed robust oscillations of bioluminescence, while such rhythms were completely lost in that from Per2::luc mice with Bmal1 knockout having a dysfunctional clock. Cx43 in the urothelium demonstrated a circadian rhythm in conjunction with major clock genes with a peak time at ZT19 and with a nadir time at ZT7. The concentration of ATP released into the bladder showed diurnal changes along with the Cx43 expression. In addition, the circadian rhythms of major clock genes and Cx43 were also observed in immortalized human urothelial cells. The concentration of mechanically-induced ATP release had oscillations in correlation with the Cx43 expression. Furthermore, it was significantly higher in the Cx43-overexpressed cells, whereas it was significantly lower in Cx43- knockdown ones or in the presence of GAP19 peptide, a selective Cx43 hemichannel blocker. CONCLUSIONS A functional circadian rhythm existed in the urothelium, and coordinated the Cx43 expression and function as hemichannels which provided a direct pathway of ATP release for mechanotransduction and signaling in the urothelium. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1354 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Atsushi Sengiku More articles by this author Hiromitsu Negoro More articles by this author Takeshi Sano More articles by this author Masakatsu Ueda More articles by this author Jin Kono More articles by this author Louis Liou More articles by this author Hitoshi Okamura More articles by this author Osamu Ogawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Circadian regulation of cardiovascular and kidney function.

Video slideshow introduction to the symposium by symposium organiser David Pollock can be found here.

MP68-04 CLOCK GENES REGULATE CIRCADIAN RHYTHM OF VNUT EXPRESSION, CONNEXIN26 EXPRESSION AND STRETCH-EVOKED ATP RELEASE IN THE CULTURED UROTHELIAL CELLS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology II1 Apr 2016MP68-04 CLOCK GENES REGULATE CIRCADIAN RHYTHM OF VNUT EXPRESSION, CONNEXIN26 EXPRESSION AND STRETCH-EVOKED ATP RELEASE IN THE CULTURED UROTHELIAL CELLS Tatsuya Ihara, Satoru Kira, Tatsuya Miyamoto, Norifumi Sawada, Hiroshi Nakagomi, Takahiko Mitsui, Hideki Kobayashi, Mitsuharu Yoshiyama, Masayuki Takeda, Yuki Nakamura, Atsuhito Nakao, Eiji Shigetomi, Yohichi Shinozaki, and Shuichi Koizumi Tatsuya IharaTatsuya Ihara More articles by this author , Satoru KiraSatoru Kira More articles by this author , Tatsuya MiyamotoTatsuya Miyamoto More articles by this author , Norifumi SawadaNorifumi Sawada More articles by this author , Hiroshi NakagomiHiroshi Nakagomi More articles by this author , Takahiko MitsuiTakahiko Mitsui More articles by this author , Hideki KobayashiHideki Kobayashi More articles by this author , Mitsuharu YoshiyamaMitsuharu Yoshiyama More articles by this author , Masayuki TakedaMasayuki Takeda More articles by this author , Yuki NakamuraYuki Nakamura More articles by this author , Atsuhito NakaoAtsuhito Nakao More articles by this author , Eiji ShigetomiEiji Shigetomi More articles by this author , Yohichi ShinozakiYohichi Shinozaki More articles by this author , and Shuichi KoizumiShuichi Koizumi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1341AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Clock genes exist in almost all the cells and organs, and the products of Per, Cry, Bmal and Clock have the most important role to regulate circadian rhythm as representative clock genes. It has been known that lower urinary tract function is also regulated by clock genes. We previously reported that the Clock mutant mouse (ClockΔ19/Δ19) showed phenotype of nocturia (NOC) and nocturnal polyuria (NP). We hypothesis that clock genes regulate circadian rhythm of perception of the bladder fullness through the circadian expression of the ATP transmitters such as VNUT and Connexin26 in urothelium, which release ATP after mechanical stretch stimulation and send signals of urine perception to the CNS. In this study, we investigated the circadian rhythm of urine perception in mouse primary urothelial cell cultures (MPUCC), measuring the gene and protein expression of VNUT, Connexin26, and the ATP release after stretch stimulation. METHODS MPUCC isolated from C57BL/6 mice (WT) and C57BL/6 ClockΔ19/Δ19 were used. To reset and synchronize the gene expression rhythms in each cells, 50% horse serum shock (HSS) were added for 2hrs. Samples were collected every 4hrs from 12hrs later after HSS. That time was defined as 0. The expression of major clock genes, VNUT, and Connexin26 in MPUCC was investigated by RT-PCR and Western blot. Released ATP after stretch stimulation was quantified using luciferin-luciferase bioluminescence assay. Statistical analyses were done by one-way ANOVA. RESULTS In addition to major clock genes (e.g. Per2 and Bmal1), the expressions of mRNA and proteins of VNUT and Connexin26, showed circadian rhythm in WT. Meanwhile, these circadian rhythm were disrupted in ClockΔ19/Δ19 mice. ATP release after stretch stimulation showed circadian rhythm in WT, but ClockΔ19/Δ19lost this circadian rhythm. These oscillation changes were almost same as circadian rhythm of VNUT- and Connexin26-mRNA (Fig.1), which indicated that CLOCK could regulate the perception of bladderfullness. CONCLUSIONS Clock genes regulate circadian rhythm of perception of the bladder fullness, and disruption of clock genes could induce NOC and NP. Redressing of abnormalities of clock genes can lead to novel treatment of NOC/NP. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e890 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Tatsuya Ihara More articles by this author Satoru Kira More articles by this author Tatsuya Miyamoto More articles by this author Norifumi Sawada More articles by this author Hiroshi Nakagomi More articles by this author Takahiko Mitsui More articles by this author Hideki Kobayashi More articles by this author Mitsuharu Yoshiyama More articles by this author Masayuki Takeda More articles by this author Yuki Nakamura More articles by this author Atsuhito Nakao More articles by this author Eiji Shigetomi More articles by this author Yohichi Shinozaki More articles by this author Shuichi Koizumi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effect of spaceflight on the circadian rhythm, lifespan and gene expression of Drosophila melanogaster.

Space travelers are reported to experience circadian rhythm disruption during spaceflight. However, how the space environment affects circadian rhythm is yet to be determined. The major focus of this study was to investigate the effect of spaceflight on the Drosophila circadian clock at both the behavioral and molecular level. We used China’s Shenzhou-9 spaceship to carry Drosophila. After 13 days of spaceflight, behavior tests showed that the flies maintained normal locomotor activity rhythm and sleep pattern. The expression level and rhythm of major clock genes were also unaffected. However, expression profiling showed differentially regulated output genes of the circadian clock system between space flown and control flies, suggesting that spaceflight affected the circadian output pathway. We also investigated other physiological effects of spaceflight such as lipid metabolism and lifespan, and searched genes significantly affected by spaceflight using microarray analysis. These results provide new information on the effects of spaceflight on circadian rhythm, lipid metabolism and lifespan. Furthermore, we showed that studying the effect of spaceflight on gene expression using samples collected at different Zeitgeber time could obtain different results, suggesting the importance of appropriate sampling procedures in studies on the effects of spaceflight.

Clock gene expression in different synovial cells of patients with rheumatoid arthritis and osteoarthritis

Patients with rheumatoid arthritis (RA) show modulated circadian rhythms of inflammatory cytokines and cortisol, which may be associated with a modified expression of clock genes. The expression of major clock genes was previously studied in synovial tissues and fibroblasts of patients with RA and osteoarthritis (OA). We therefore especially aimed to examine the localization of clock genes at the cellular level in synovial tissue. Furthermore we were interested in studying the expression of the D site of albumin promoter (albumin D-box) binding protein (DBP) at the immunohistochemical level in human samples. Methods used include the in situ expression of the clock genes Brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal 1), Circadian Locomotor Output Cycles Kaput (Clock), Period 1 and 2 (Per 1 and Per 2), and DBP was examined by immunohistochemistry in synovial tissues of patients with RA or OA. Additionally, expression profiles of different clock genes were determined over 24h by real time PCR in synovial fibroblasts (SFs) after a 2h serum shock or TNF-α. Results show that all clock genes investigated were found to be expressed both in RA and OA synovial tissues. Double staining against cell specific markers revealed that clock proteins were especially seen in macrophages, SFs and B-lymphocytes. Cell counting showed that clock proteins were found in approximately 5–20% of cells. Additionally, preliminary cell culture experiments showed that TNF-α treatment resulted in differential 24h expression profiles between RA and OA samples and also compared to the results obtained from the serum shock experiments. From our study we conclude that the major clock genes, including DBP, are expressed in samples from RA and OA patients, especially in macrophages and synovial fibroblasts, but also in B-lymphocytes. Preliminary experiments suggest that TNF-α seems to be able to modify clock gene expression in synovial fibroblasts.

Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis.

BackgroundThe circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level.ResultsWe systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD.ConclusionTaken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected.

Cadmium-induced changes in Per 1 and Per 2 gene expression in rat hypothalamus and anterior pituitary: Effect of melatonin

Chronic cadmium (Cd) administration affects the circadian release of pituitary hormones in rats. To assess whether Cd modifies expression of two major clock genes, period (Per) 1 and Per 2, in the hypothalamic–pituitary unit and to what extent the changes could be prevented by melatonin, rats were exposed to CdCl 2 (5 ppm in drinking water) with or without melatonin (3 μg/mL drinking water) for 1 month and were killed at two time intervals, i.e. a the beginning of the rest span (09:00 h) and at the middle of the activity span (01:00 h). Hypothalamic and pituitary mRNA levels encoding Per 1 and Per 2 were measured by real-time PCR analysis. Cd treatment decreased expression of hypothalamic Per 1 gene at both time intervals, of hypothalamic Per 2 gene at 01:00 h, and of adenohypophysial Per 1 and Per 2 genes at 09:00 h. Melatonin administration counteracted most of the effects of Cd and augmented hypothalamic Per 2, and adenohypophysial Per 1 and Per 2 gene expression. The results indicate that Cd administered chronically in the drinking water to rats affected expression of clock genes in the hypothalamic–pituitary unit, an effect prevented by melatonin.

Clock gene expression in purified mouse hematopoietic stem cells

Circadian genes have recently been characterized in many tissues, but not in hematopoietic stem cells. These cells are rare in the bone marrow (BM), which makes it difficult to collect enough cells for detailed molecular analysis in a short period of time without reduced RNA quality. The aim was to improve methodology and reliability of clock gene expression analysis in purified mouse hematopoietic stem cells. Stem cells were highly enriched by high-speed flow cytometric cell sorting of the side population (SP) cells from Hoechst 33342 (Hoechst)-stained mouse BM. Total RNA was isolated from sorted SP and whole BM cells and exposed to DNase treatment. The relative mRNA levels of major clock genes mPer1, mPer2, mBmal1, mCry1, mClock, and mRev-erb alpha were measured with real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) and normalized to m36B4, used as a reference gene. The clonogenity of sorted SP cells and whole BM; cells taken before and after sorting, were tested in colony-formation assay. Clock gene activity in sorted SP cells showed pronounced relative differences compared with whole BM for mPer1 and mCry1. The high-speed sorting procedure did not influence clock gene expression or cell clonogenity, even when this was performed with a delay period up to 24 hours. We demonstrated expression of six clock genes in mouse hematopoietic stem cells. A combination of high-speed flow cytometric sorting and Q-RT-PCR was shown to be useful and reliable for analysis of clock gene activity in small stem cell fractions.

Indication of circadian oscillations in the rat pancreas

The central circadian oscillator of the suprachiasmatic nucleus controls diurnal rhythmicity of the body with light as its dominant zeitgeber. Recently, peripheral oscillators have been detected in liver and heart, which follow as yet unidentified cues. In this study real-time reverse transcription-polymerase chain reaction (RT-PCR) was used in analysis of the expression of the major clock genes Per1, Per2, Bmal1, Cry1, Tim ( timeless) and Clock, as well as of the output genes Dbp and Rev-erbα in the pancreatic tissue of rats. The results presented here indicate a robust circadian expression of clock genes (e.g. Per1 and Bmal1) and the probable existence of a peripheral oscillator in the pancreas. Whether this oscillator regulates the diverse functions of the islets of Langerhans remains to be elucidated.