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Circadian rhythms help organisms adapt to changes of external environment by regulating energy metabolism and remaining the balance of homeostasis. Numerous researches have proved that the physiological function of liver was precisely controlled by circadian rhythms. Clock, one of core circadian genes, has been demonstrated to regulate the oxidative phosphorylation process of mitochondrial, which provides energy for living cells and acts as one of the hub for apoptosis. However, whether Clock gene regulates mitochondrial apoptosis pathways in liver cells remains less explored. In the present study, we used lentiviral vector to establish a stable AML12 cell lines which were capable of expressing specific shRNA to interfere the expression of Clock gene and investigated the effect of Clock on mitochondrial apoptosis pathways. Herein, we found that the interference of Clock gene could significantly suppress mitochondrial apoptosis pathways by stabilizing mitochondrial membrane potential and inhibiting mitochondria out membrane permeablization, which might be a result of lower expression of BAD and BIM proteins. Moreover, the interference of Clock gene could downregulate the expression of mitochondrial apoptosis factors, i.e. AIF, CYCS, APAF-1 and SMAC, which will suppress the formation of apoptosome and the process of DNA degradation to further inhibit apoptosis process. This work provides an insight on the important role of Clock gene participating in mitochondrial apoptosis pathways of hepatocytes and unveils a probable pathogenesis of how circadian rhythm regulates liver diseases.

Circadian rhythm has been involved in the regulation of many physiological activities. Autophagy is the metabolic process that transports substances in the cytoplasm to the lysosomes for degradatio...

Emerging evidence has demonstrated that long noncoding RNAs (lncRNAs) play critical roles in the epigenetic and transcriptional regulation of mammalian circadian systems. Circadian rhythmicity regulates many aspects of our immune system, and perturbation of the circadian clock can augment the inflammatory response. However, knowledge of the precise functions of lncRNAs in the regulation of immune functions within the circadian system is relatively limited. In this study, differentially expressed lncRNAs induced by Clock knockdown were screened via mRNA/lncRNA microarray and bioinformatic prediction analysis. We identified a Clock-regulated lncRNA, AK028245, which was correlated with the activation of the immune response. The expression levels of AK028245 were decreased in the spleen of immunosuppressed mice and elevated in immune-activated mice treated with lipopolysaccharide (LPS). Further, Clock knockdown decreased the expression of OTUD7B and A20, 2 early immune response factors acting on the NF-κB signaling pathway. Interestingly, inhibition of AK028245 increased their expression, mitigating the effects of Clock knockdown. In addition, inhibition of AK028245 downregulated the expression of tumor necrosis factor–α and interleukin-6 in the late stages of LPS stimulation and the expression of interferon-γ and Cxcl12 in the peak stages. We conclude that this newly identified lncRNA plays a role in the crosstalk between Clock and immune response regulators, likely resulting in a proinflammatory response targeting OTUD7B and A20. The lncRNA AK028245 has revealed a new mechanism of the immune response and provided new targets for the treatment of immune disorders.

Supplemental material, Supplemental_material_JBR for The Clock-Controlled lncRNA-AK028245 Participates in the Immune Response via Immune Response Factors OTUD7B and A20 by Fang Qi, Zhou Jiang, Wang Hou, Bo Peng, Shuting Cheng, Xiaolong Zhang, Zhihan Luo, Zeyong Dai, Yumeng Wang, Yanyou Liu, Yuhui Wang and Zhengrong Wang in Journal of Biological Rhythms

Colorectal carcinoma (CRC) is one of the most prevalent types of malignancy-associated mortality of the world. Recently, the studies about over-expression circadian locomotor output cycles kaput ge...

The circadian system regulates many important aspects of physiology, including the immune response to infectious agents, which is mediated by the activation of the transcription factor NF-κB. Thus, understanding the mechanisms by which circadian clocks regulate NF-κB is a necessary step toward the development of improved therapies underwritten by NF-κB manipulation. Previous reports have identified OTUD7B a deubiquitinase, as a novel regulator of noncanonical NF-κB signaling, largely through the maintenance of TRAF3, a negative regulator of NF-κB. In investigations in our laboratory, when the Clock gene was repressed by shRNA, the results of microarray analysis demonstrated that Otud7b was down-regulated. Further research by real-time PCR and western blot revealed that that Otud7b exhibits rhythmic mRNA expression. These findings confirm Otud7b as a novel clock-regulated gene. Additionally, Otud7b may be an important bridge between the circadian system and noncanonical NF-κB pathway regulation.

The circadian rhythm is one of the basic systems in an organism. It helps the organism maintain harmony with the daily changes of the external environment to ensure proper physiological activities. Previous studies from our laboratory have indicated that the miR-29a/b/c can bind to the circadian clock gene hPer1 at the 3 ′UTR region and regulate its mRNA and protein expression, affecting various organismal physiological processes. Meanwhile, it has been reported that the circadian gene Per plays a role in the regulation of the early growth response gene Egr2, which plays an important role during midbrain development. Here, we confirmed that miR-29a/b/c regulates Egr2 function through mPer1 binding, which elucidates a novel connection between mPer1 and Egr2.

In the original article, Fig. 4b was published incorrectly in which four to five lanes in Pi-ERK and Pi-CREB panels look very similar to each other (Telomerase reconstitution contributes to resetting of circadian rhythm in fibroblasts, Mol Cell Biochem, 2008, 313:11–18). Since this image was stored in The Experiment Center of the West China Second University Hospital, Sichuan University, which was dissoluted in 2012, the original data cannot be traced. Experiments were therefore redone to verify the result and the correct version of Fig. 4b is provided in this correction.

Doxorubicin (DOX) is a potent anti-neoplastic agent with cumulative cardiotoxicity. DOX-induced cardiotoxicity has been shown to depend on the different dosing times. However, the basis for determining the dosing time to minimize DOX-induced cardiotoxicity and the underlying mechanisms remain incompletely understood. Here we first showed that SIRT3, the major mitochondrial deacetylase, is negatively correlated to DOX-induced cardiotoxicity through the regulation of ATP production, mitochondrial membrane potential (MMP) level and ROS level in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Then, we used in vivo experiments to demonstrate that DOX significantly reduced the SIRT3 expression and the SIRT3 activity as reflected by the increased AcK68MnSOD/MnSOD ratio in rats after six weeks of treatment. Notably, the activity of SIRT3 had an obvious diurnal rhythm pattern in the myocardium of healthy rats. More importantly, an obvious lower AcK68MnSOD/MnSOD ratio was observed in rat hearts with DOX administrated at Zeitgeber time (ZT) 9 (ZT 0 was the time lights were turned on) than ZT1, which represent the peak and trough of SIRT3 activity. Moreover, DOX ZT9 reduced the body weight loss, extended the survival period, improved the heart function and alleviated the myocardial lesions compared to DOX ZT1. Mechanistic investigations demonstrated that DOX ZT1 significantly reduced ATP production, oxygen consumption rate (OCR) at various respiration states, MMP level and MnSOD activity and enhanced the H2O2 level compared with CON ZT1, whereas there was no significant effect for DOX ZT9 compared with CON ZT9. Taken together, dosing at the peak time of SIRT3 activity reduced DOX-induced cardiotoxicity, which may be related to the increased endogenous tolerance against the mitochondrial dysfunction and oxidative stress caused by DOX.

Gravity heavily influences living organisms on earth including their circadian rhythm, which is fundamentally important for coordinately physiology in organisms as diverse as cyanobacteria, fungus and humans. Numerous researches have revealed that microgravity in outer space can affect circadian rhythm of astronauts and rodent animals, but the mechanism remains unknown. Using rotary cell culture system to simulate microgravity environment, we investigated the role of simulated microgravity in regulating the circadian rhythm of NIH3T3 cells. Our experiments found that simulated microgravity can not only influence the mRNA level of some core circadian genes, but also modify the circadian rhythm of Per1 and Per2 synchronized after phorbol myristate acetate treatment. Remarkably, MEK/ERK pathway was transiently activated after a 2-h simulated microgravity treatment, with a significant upregulation of Kras, Raf1 and p-ERK1/ERK2. Moreover, U0126, a selective inhibitor of MEK/ERK pathway, could disrupt t...

The circadian rhythm regulates numerous physiological activities, including sleep and wakefulness, behavior, immunity and metabolism. Previous studies have demonstrated that circadian rhythm disorder is associated with the occurrence of tumors. Responsible for regulating a number of functions, the Circadian locomotor output cycles kaput (Clock) gene is one of the core regulatory genes of circadian rhythm. The Clock gene has also been implicated in the occurrence and development of tumors in previously studies. The present study evaluated the role of the Clock gene in the proliferation and migration of mouse breast cancer 4T1 cells, and investigated its possible regulatory pathways and mechanisms. It was reported that downregulation of Clock facilitated the proliferation and migration of breast cancer cells. Further investigation revealed the involvement of IQ motif containing GTPase activating protein 1 (IQGAP1) protein expression in the Clock regulatory pathway, further influencing the expression of E-cadherin, a known proprietor of tumor cell migration and invasion. To the best of our knowledge, the present study is the first to report that Clock, acting through the regulation of the scaffolding protein IQGAP1, regulates the downstream expression of E-cadherin, thereby affecting tumor cell structure and motility. These results confirmed the role of Clock in breast cancer tumor etiology and provide insight regarding the molecular avenues of its regulatory nature, which may translate beyond breast cancer into other known functions of the gene.

Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements.

We conducted a meta-analysis of eligible studies to estimate the association between gastric cancer risk and rs2275913G>A IL-17A and rs763780T>C IL-17F polymorphisms. We searched the relevant studies in both Chinese and English through PubMed, the Web of Science, the Cochrane Library, and EMBASE up to January 1, 2014, including 3939 cases and 5407 controls. Seven eligible case-control studies were selected, including seven studies on rs2275913G>A IL-17A and four studies on rs763780T>C IL-17F. The rs2275913 AG (odds ratio (OR) = 1.50, 95% confidence interval (95%CI) = 1.04-2.15) and GG (OR = 1.40, 95%CI = 1.00-1.96) genotypes were significantly associated with increased risk of gastric cancer compared with the AA genotype. The rs763780 TC (OR = 1.47, 95%CI = 1.32-1.64) and TT (OR = 1.49, 95%CI = 1.11-1.99) gen- otypes can influence gastric cancer risk. Subgroup analysis showed that rs2275913 GG (OR = 1.35, 95%CI = 1.05-1.73) and rs763780 TC (OR= 1.44, 95%CI = 1.20-1.75) genotypes were not significantly associated with increased risk of gastric cancer in Japanese populations. Our meta-analysis is the first to indicate that the rs2275913G>A and rs763780T>C polymor - phisms are risk factors for gastric cancer development.

Several essential biological progresses in mammals are regulated by circadian rhythms. Though the molecular mechanisms of oscillating these circadian rhythms have been uncovered, the specific functions of the circadian genes are not very clear. It has been reported that knocking down circadian genes by microRNA is a useful strategy to explore the function of the circadian rhythms. In this study, through a forward bioinformatics screening approach, we identified miR-29a/b/c as potent inhibitors for the human circadian gene hPER1. We further found that miR-29a/b/c could directly target hPER1 3'untranslated region (UTR) and down-regulate hPER1 at both mRNA and protein expression levels in human A549 cells. Thus, our findings suggested that the expression of hPER1 is regulated by miR-29a/b/c, which may also provide a new clue for the function of hPER1.

The exposure to electromagnetic radiation (EMR) would increase the risk of cardiovascular diseases. Central nervous system, especially the suprachiasmatic nucleus in hypothalamus, is a sensitive target of EMR. Clock is a main circadian gene which plays an important role in the circadian system, while factor VII (FVII) plays a central role in the coagulation cascade in the cardiovascular system. To investigate the effect of a continuous EMR exposure on the circadian and cardiovascular systems, we had mature Institute of Cancer Research (ICR) mice exposed to EMR of different power densities. The intake of food and water, the physiological signs, and the motor activity were monitored to have no significant difference. And their expressions of Clock and fvii genes were detected. Our study showed that a series of EMR exposure to ICR mice would have some key gene expression changed without visible changes in physiological signs and activities. We assume that Clock gene could only regulate fvii gene of some spec...

To study the effect of L-arginine on circadian system, ICR mice and AR4-2J cells were treated with L-arginine. The locomotor activity of the mice intraperitoneally injected with a high dose of L-arginine was observed. We also detected effects of arginine on Per1 and Per2 gene expressions in AR4-2J cells. Double-plotted actograms were applied to analyze the locomotor activity of the mice, and reverse transcription-polymerase chain reaction (RT-PCR) was used in analysis of the expression of the major circadian genes Per1 and Per2 in AR4-2J cells. The results presented here indicate that the intraperitoneal injection of L-arginine would strongly disrupt the circadian rhythm of locomotor activity in mice. While the co-culture of L-arginine would also disturb the circadian rhythm of Per1 and Per2 genes which were induced by PMA in AR4-2J cells, the expression levels of both genes greatly decreased.

Nutrition is essential for health, and it has been widely studied and demonstrated to be related with circadian rhythm. To verify the effect of nutrient deficiency on the in vitro cultured cells, we had NIH3T3 cells cultured in diluted medium for 5 days, and detected the expression levels of Bmal1, Clock and Cry1 on each day. Phorbol 12-myristate 13-acetate (1PMA) was also applied to induce the circadian expression of Period genes, and the temporal expression levels before and after undernutrition treatment were analyzed by cosinor method. Our study showed that the expressions of circadian genes would be affected as soon as a nutritional deficiency occurred. This influence was not only on the expression level of Bmal1, Clock, and Cry1 genes but also on the PMA induction of circadian expression of Per1 and Per2 in the NIH3T3 cells.

Mice homozygous for a dominant-negative allele of the Clock gene ( ClockΔ 19/Δ 19) have slightly but significantly decreased male fertility. The molecular mechanism for this reduction in fertility is unknown. In the present study, we used a small hairpin RNA (shRNA) strategy to specifically knock down the Clock gene expression in the testes of male mice and determined its effect on male fertility. Clock knockdown led to smaller litter size, a lower in vitro fertility rate, lower blastula formation rate, and lower acrosin activity of the knockdown sperm. Locomotor activity analysis of the Clock knockdown mice revealed that Clock knockdown in testes did not alter their circadian rhythm. Taken together, these results provide the first evidence that Clock gene expression in round spermatids is essential for maintaining male reproductivity and suggest that acrosin may be a novel regulatory target of the Clock gene that would regulate the fertilization and early embryonic development to blastula. These findings may provide new clues for development of novel male contraceptive strategies.

It has been widely studied and demonstrated that circadian rhythm, as well as circadian clock genes, plays an important role in many physiological and pathological processes. Period1 (Per1) gene is a core gene in the circadian clock system that is involved in cell differentiation and apoptosis. To verify the role of PER1 on the proliferation of normal cells, we chose NIH3T3 cells to have their PER1 up-regulated by transfecting a Per1 expression plasmid and determining their proliferation rates. Previous studies demonstrated that cell migration could be regulated by matrix metallopeptidase 2 (MMP2), and this MMP2 protein had a relation with the circadian system. To investigate the function of PER1 on tumor cell migration, Lewis lung carcinoma (LLC) cells were chosen to have their PER1 up-regulated, and the MMP2 level and the migration of LLC cells were tested. Our study showed that PER1 overexpression led to MMP2 down regulation in both NIH3T3 and CCL cells and inhibited the proliferation rate in NIH3T3. M...

Alterations in metabolism could be due to cell-autonomous effects associated with altered expression of Clock in central nervous system feeding centers and/or peripheral tissues involved in metabolism. Clock mutant mice are hyperphagic and obese, which indicates that Clock is related to obesity. In the present study, we used intracerebroventricular injection of recombinant adenoviral vector harboring Clock genes to explore the role of Clock on diet induced obesity and the mechanisms involved in leptin resistance and leptin signaling in mice. The results demonstrated that expression of Clock in the arcuate nucleus of diet induced obesity mice was down-regulated. The recombinant adenoviral vector harboring Clock genes could reduce obesity indexes of diet induced obesity mice including body weight, BMI and total fat mass, attenuate hyperleptinemia, increase leptin sensitivity and decrease accumulated suppressor of cytokine signaling-3 in the arcuate nucleus. These results indicate that Clock plays an important role on obesity, which may be involved in leptin resistance and regulation of suppressor of cytokine signaling-3 in arcuate nucleus.

Results from previous studies on the association between methylenetetrahydrofolate reductase (MTHFR) polymorphisms C677T and A1298C and lung cancer have been conflicting. The aim of this meta-analysis was to clarify the effect of MTHFR polymorphisms on the risk of lung cancer. An electronic search of PubMed, EMBASE, the Cochrane library, and the China Knowledge Resource Integrated Database for papers on C677T and A1298C and susceptibility to lung cancer was performed. The STATA software (Version 13.0) was used for statistical analysis. Statistical heterogeneity, tests of publication bias, and a sensitivity analysis were performed. Twenty-six studies on C677T (12,324 cases and 12,532 controls) and thirteen studies on A1298C (6773 cases and 8207 controls) were included in the meta-analysis. The MTHFR C677T polymorphism showed significant pooled ORs for the homozygote comparison (TT versus CC: OR = 1.518, 95%CI = 1.220-1.890), heterozygote comparison (CT versus CC: OR = 1.053, 95%CI = 0.940-1.179), dominant model (CT + TT versus CC: OR = 1.143, 95%CI = 1.013-1.291), recessive model (TT versus CT + CC: OR = 1.435, 95%CI = 1.190-1.730), and additive model (T versus C: OR = 1.176, 95%CI = 1.066-1.298). In summary, our meta-analysis showed that the MTHFR C677T polymorphism is associated with a significant increase in lung cancer risk in Asian and overall populations, but not in Caucasian populations. However, no significant association between the MTHFR A1298C polymorphism and lung cancer risk was found in either the Caucasian or Asian group with any genetic models.

Clock is a core, well-established, circadian gene, and clif is a newly discovered circadian gene, both of which not only maintain the circadian rhythm of cells but also regulate some organic functions. The purpose of this study was to determine whether genetic variations (single nucleotide polymorphisms – SNPs) in clock and/or clif genes associate with coronary artery disease (CAD). We silenced the clock and clif genes in cells and mice. The mRNA levels of clock, clif, pai-1, and tm of mice were examined using Reverse transcription polymerase chain reaction (RT-PCR). Plasminogen activator inhibitor 1 (PAI-1) and thrombomodulin (TM) levels will be examined using enzyme-linked immunosorbent assay. The clock SNPs (rs3840267, rs3749474, and rs1402963), and clif SNPs (rs62758860 and rs2289709) were analyzed in 116 patients with CAD and 95 healthy controls by sequence-specific primer-polymerase chain reaction. Data were analyzed by χ2 analysis. The mRNA levels of pai-1 and tm was decreased in the clock or clif ...

Circadian gene, Clock and Clif, not only maintain the circadian rhythm of cells but also regulate lipid and glucose metabolism in peripheral organs. The purpose of this study was to determine whether genetic variations (single nucleotide polymorphisms – SNPs) in the Clock and/or Clif genes were associated with hyperlipidemia in Chinese Han descent. The Clock variants (rs3840267, rs3749474, rs1052925) and the Clif variant (rs2289709) were analyzed in 205 patients with hyperlipidemia and 281 healthy controls by sequence-specific primer polymerase chain reaction (SSP-PCR). The Clock variant (rs1052925) showed significant association to hyperlipidemia (P alleo = 0.003, ORalleo = 0.529). We also found correlations between the Clock (rs1052925), the Clif (rs2289709), and types of hyperlipidemia (P alleo-IV = 0.043, ORalleo-IV = 0.525; P alleo-IIb = 4.738 × 10−4, OR alleo-IIb = 2.959). In addition, the Clock (rs1052925), the level of triglycerides in carriers of A/- heterozygote was significantly different from...

This study examined the association between hypertension and AD by using a quantitative meta-analysis of longitudinal studies. EMBASE and MEDLINE were searched for articles published up to February 2011. All studies that examined the association of hypertension or antihypertensive medication use with the onset of AD were included. Pooled relative risks (RR) were calculated using fixed and random effects models. Twelve studies met our inclusion criteria for this meta-analysis. All subjects were without dementia at baseline. Among them, 9 studies compared the incidence of AD between subjects with (7,270) and without (8,022) hypertension. The quantitative meta-analysis showed that there was no significant difference in incidence of AD (RR: 1.02, 95% confidence interval (CI): 0.91-1.14) between subjects with and without hypertension. Seven studies compared the incidence of AD between subjects with (8,703) and without (13,041) antihypertensive medication use. The quantitative meta-analysis showed that there was no significant difference in incidence of AD (RR: 0.90, 95% CI: 0.79-1.03) between subjects with and without antihypertensive medication use. The quantitative meta-analysis showed that neither hypertension nor antihypertensive medication use was associated with risk for incident AD.

Chronic morphine addiction may trigger functional changes in the mesolimbic dopamine system, which is believed to be the neurobiological substrate of opiate addiction. Brain derived neurotrophic factor (BDNF) has been implicated in addiction-related pathology in animal studies. Our previous studies have shown that RACK1 is involved in morphine reward in mice. The recent research indicates nuclear RACK1 by localizing at the promoter IV region of the BDNF gene and the subsequent chromatin modifications leads to the activation of the promoter and transcription of BDNF. The present study was designed to investigate if shRACK1 (a short hairpin RNA of RACK1) could reverse the mice's behavioral responses to morphine and BDNF expression in hippocampus and prefrontal cortex. No significant changes were observed in vehicle-infused mice which received no morphine treatment (CONC) and shRACK1-infused mice which received no morphine treatment (CONR), whereas vehicle-infused mice preceded the morphine injection (MIC) showed increased BDNF expression in hippocampus and prefrontal cortex, as compared to vehicle-infused mice which received no morphine treatment (CONC). Intracerebroventricular shRACK1 treatment reversed these, and in fact, ShRACK1-infused mice preceded the morphine injection (MIR) showed reduced BDNF expression in hippocampus and prefrontal cortex, as compared to MIC. In the conditioned place preference (CPP) test, inactivating RACK1 markedly reduces morphine-induced conditioned place preference. Non-specific changes in CPP could not account for these effects since general CPP of shRACK1- and vehicle-infused animals was not different. Combined behavioral and molecular approaches have support the possibility that the RACK1-BDNF system plays an important role in the response to morphine-induced reward.

As a main component of circadian genes, clock plays not only an important role in circadian rhythm but also in the regulation of many physiological systems. The dysfunction of clock genes is associated with the development of various disorders. Many studies have investigated the association between clock genes and blood coagulation and the fibrinolytic system. The present study was designed to investigate the effect of downregulation of circulatory Clock on blood coagulation and fibrinolysis at the initial stage of active phase in male mice. Downregulation of the expression of the Clock gene by siRNA and, subsequently, its effect on the thrombotic potential and the expression of relative coagulative and/or fibrinolytic factors were investigated. It was found that the Clock interfered mice were less liable to thrombosis and showed prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) at Zeitgeber time (ZT) 15. Meanwhile, these mice also showed an increase in factor VII (FVII) and a decrease in thrombomodulin (TM) and plasminogen activator inhibitor 1 (PAI-1) at ZT 15 at both transcriptional and translational levels. PT, APTT and mRNA expressions of fvii, tm and pai-1 were analyzed with the least-squares fit of a 24-h cosine function by single cosinor method; no circadian rhythm was determined in PT and APTT, and a higher amplitude of fvii in the Clock RNAi group was found with a circadian phase shift, while lower amplitudes of tm and pai-1 were found in the Clock RNAi group with nearly no phase shift. All these results suggest that downregulation of the Clock gene in circulatory system has an effect on factors involved in both blood coagulation and fibrinolysis resulting in an enhancement in mice. This may be considered as an indication that Clock regulates thrombotic homeostasis through the fibrinolytic system.

It has been shown that circadian genes not only play an important role on circadian rhythms, but also participate in other physiological and pathological activities, such as drug dependence, cancer development and radiation injury. The Per2, an indispensable component of the circadian clock, not only modulates circadian oscillations, but also regulates organic function. In the present study, we applied mPER2-upregulated NIH3T3 cells to reveal the relationship of mPer2 and the cells damaged by ultraviolet C (UVC). NIH3T3 cells at the peak of the expression of mPer2 induced by phorbol 12-myristate 13-acetate (PMA) demonstrated little damage by UVC evaluated by MTT assay, cell growth curves and cell colony-forming assay, compared with that at the nadir of the expression of mPer2. Overexpression of mPER2, accompanied p53 upregulated, also demonstrated protective effect on NIH3T3 cells damaged by UVC. These results suggest that mPer2 plays a protective effect on cells damaged by UVC, whose mechanism may be involved in upregulated p53.

Many reports have shown that the biologic rhythm could be altered due to mutations of circadian gene hClock or hPeriod, and the mutations of circadian genes have some relationship with psychosis according to recent studies. A preliminary study has been conducted to examine wether the T3111C single nucleotide polymorphism of the hClock gene or the length polymorphism of the hPer3 gene is associated with the development of schizophrenia. The samples from schizophrenics (n=148, male: 57.4%, female: 42.6%) and normal controls (n=199, male: 59.3%, female: 40.7%) were examined. Allele frequencies of T3111C SNP of hClock were significantly different between schizophrenics and controls (χ2=19.738, P0.05). Schizophrenics had a significantly higher frequency of the C allele compared with controls (OR=2.613, 95% CI=1.693-4.034). On the other hand, there is no significant difference of allele frequencies of 18 exon of hper3 between schizophrenics and controls (χ2=0.192, P0.05). Our results suggest that the T3111C (RS1801260) polymorphism of hClock gene is associated with schizophrenia, but it seems that the length polymorphism of 18 exon of hPer3 may not be associated with schizophrenia. It is important to address of the relationship between circadian gene polymorphisms and dopamine functions in further study.

Opiate addiction is associated with upregulation of cAMP signaling in the brain. cAMP-responsive element binding protein (CREB), a nuclear transcription factor, is a downstream component of the extracellular signal-regulated protein kinase (ERK) pathway, which has been shown to regulate different physiological and psychological responses of drug addiction. RACK1, the protein receptor for activated C kinase 1, is a multifunctional scaffolding protein known to be a key regulator of various signaling cascades in the CNS. RACK1 functions specifically in integrin mediated activation of ERK cascade and targets active ERK. We examined if RACK1 is involved in the mechanism of drug addiction by regulating CREB in mouse hippocampus and prefrontal cortex. Several expressions were observed. Chronic administration of morphine made the expression of RACK1 and CREB mRNA increase in hippocampus and prefrontal cortex. The expression of RACK1 and CREB protein was strongly positive in CA1, CA3 and dentate gyrus (DG) of the hippocampus of morphine-treated mice brain, especially the pyramidal neurons in the DG of the hippocampus. Using the small interfering RNA technology, we determined that the expression of CREB mRNA was decreased in hippocampus and prefrontal cortex of morphine-treated mice. The expression of RACK1 and CREB protein was negative in CA1, CA3 and DG of hippocampus. These findings suggest that morphine reward can influence the expression of RACK1 in mouse hippocampus and prefrontal cortex through regulating CREB transcription.

Previous studies indicated that recovery of brain injury involves in circadian system and circadian system also modulates cardiovascular function. The present study was focused on the circadian characteristic of blood pressure (BP) and heart rate (HR) of patients with hypertensive intracerebral hemorrhage (HICH) after neurosurgical operation at the first 24-hour and its relationship to prognosis of HICH. This retrospective study was based on the analysis of circadian rhythm of postoperative patients with HICH who received no anti-hypertensive treatment before operation and during the first 24-hour after operation. Series data of BP and HR after operation were analyzed with cosinor method to determine whether circadian rhythms were present or absent. The patients were divided into two groups, including presence of circadian rhythm group and absence of circadian rhythm group. The basic clinical characteristics of two groups were analyzed with Student's t-test. The percentage of good prognosis in two groups was analyzed with Pearson's Chi-squared test. Statistical results indicated that the percentage of good prognosis was significantly different between the presence and absence group. More cases of good prognosis in presence group of Systolic Blood Pressure (SBP) was discovered than in absence group (p=0.032). The results of Diastolic Blood Pressure (DBP) and HR were similar as SBP was observed in presence group for DBP (p=0.002) and for HR (p=0.001), respectively. We conclude that the presence or absence of circadian rhythm after operation would be an early predictor of the postoperative prognosis from hypertensive intracerebral hemorrhage.

The synchronization of the circadian signals to external or suprachiasmatic nucleus stimulation in the peripheral clocks is essential for maintaining the usual function of human body. However, aging will disrupt the synchronization of peripheral circadian rhythms, thus leading to some age-associated diseases. Up to now, little is known about the modification of the oscillatory rhythms in aged cells. A recent report showed that cell senescence in vascular human smooth muscle cells (HSMCs) altered circadian rhythms by a dysregulation of rhythmic gene expression. Furthermore, this alteration could be reversed by telomerase reconstitution. To test whether telomerase reconstitution can restore disrupted circadian rhythm in other types of senescent cells, we used fibroblasts as cell models to profoundly investigate the relationship between cell senescence and circadian rhythm modulation. We found that the response of rhythmic gene expression to serum stimulation was markedly attenuated in senescent fibroblasts, telomerase-reconstituted fibroblasts reset the circadian oscillation of rhythmic gene expression, and the activation of pERK-CREB and p38-CREB pathways might be involved in the circadian rhythm resetting. These findings suggested that telomerase reconstitution might be a good way to reset synchronization of peripheral circadian rhythms disrupted in senescent tissues.

Objective. This study investigates how the shortening or lengthening of the circadian period of the environmental lighting schedule affects the circadian and extra-circadian structure of locomotor activity in mice, with particular focus on the circasemiseptan, circaseptan, and circadecadian components with anticipated periods of about 3.5, 7, and 10 days, respectively. Methods. The locomotor activity of mice kept on different light – dark (LD) cycles was automatically monitored around the clock for about five weeks. The data were analyzed by linear – nonlinear rhythmometry. Results. As anticipated, in LD12:12, a prominent circadian rhythm was observed, with a marked 12-hour component qualifying the circadian waveform. By comparison, in LD10:10 and in LD14:14, the circadian rhythm had a reduced amplitude and a period close to that of the environmental synchronizer. Extra-circadian variation was then also demonstrated. In conclusion, circadian lighting schedules affect a broader-than-circadian time...

Biological clocks are intrinsic time-keeping systems that regulate behavior and physiological functions in most living organisms. Previous works suggested a possible link between the endogenous circadian clock and cell cycle regulation. The mammalian Period-2 gene (mPer2), an important component of the circadian clock mechanism, is recently demonstrated to play an important role in repressing tumor growth. In this study, we found that polyethylenimine-mediated intratumoral Per2 gene delivery had significant antitumor effects in C57BL/6 mice transplanted with Lewis lung carcinoma. Our data illustrated that the Per2 gene delivery inhibited PCNA expression and induced apoptosis. Our results support the emerging role of the circadian clock in critical aspects of tumorigenesis. These findings underscore the potential use of Per2 gene delivery as a novel therapeutic intervention for the treatment of malignant tumors.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neurophysiological technique. Pre-clinical and clinical studies supported that rTMS might have antidepressant effects. However, whether antidepressant effect of chronic rTMS is gender-dependent is still unknown. In this study, male and female Wistar rats received 10-day rTMS (4 trains of 15 Hz; 200 stimuli/day; 1.0 T) or control condition, and then were subjected to the forced-swim test (FST). We found that female rats consistently showed higher activity levels than males in FST and revealed the significant effects of gender and rTMS as well as the interaction of gender and rTMS. The result suggested the antidepressant-like effects of chronic rTMS on behavioral components in FST are gender-dependent. The gender discrepancy related to rTMS should not be neglected in antidepressant treatment of rTMS.

Although there are clear interactions between circadian rhythms and drug addiction, mechanisms for such interactions remain unknown. Studies have shown that the circadian clock gene Period in Drosophila melanogaster could influence behavioral responses to cocaine, and the mouse homologues, mPer1 and mPer2, modulate cocaine sensitization and reward. In the present study, we applied DNAzyme targeting mPer1 to interfere the expression of mPer1 in CNS in mice, and studied its effects on morphine-induced reward and its molecular mechanism. The results demonstrated that the DNAzyme could attenuate the expression of mPer1 in CNS in mice and downregulate the increased extracellular signal-regulated kinase (ERK) activity induced by morphine in whole brain and the nucleus accumbens, the key region of drug addiction. Mice treated with morphine and injected intracerebroventricularly with DNAzyme did not show preference to the morphine-trained side. These results indicate that drug dependence seems to be influenced at least partially by mPer1 and its mechanism may involve the ERK signal pathway.

Increased expression of pituitary tumor-transforming gene 1 (PTTG1) is expressed in many tumors and regulates tumor growth and progression. However, the precise function of PTTG1 in the tumorigenesis of lung adenocarcinoma (LAC) is not defined yet. Here, we examined the expression of PTTG1 in human LAC tissues by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was carried out to investigate the effects of lentiviral vector-mediated PTTG1 shRNA (shPTTG1) on cell growth and invasive potential in LAC cell lines (A549 and LETPα-2), assessed by MTT and Transwell assays. As a consequence, we found that the expression of PTTG1 protein was markedly upregulated in LAC tissues compared with the adjacent non-cancerous tissues (ANCT) (54.0% vs. 28.0%, P = 0.008), and was positively associated with the lymphatic invasion of the tumor ( P = 0.01). Moreover, knockdown of PTTG1 expression inhibited tumor proliferation and invasion of LAC cells, companied by the decreased expression of CyclinD1 and MMP-2 and increased expression of p-TGFβ1 and p-SMAD3. Collectively, our findings indicate that high expression of PTTG1 is correlated with the tumor metastasis of LAC patients, and knockdown of PTTG1 suppresses the growth and invasion of LAC cells through upregulation of the TGFβ1/SMAD3 signaling, suggesting that PTTG1 may be a potential target for developing an effective immunotherapeutic strategy for LAC.

Studies on Drosophila melanogaster have shown that the period gene, a circadian clock gene and essential component of the underlying molecular feedback loop, can influence behavioural responses to cocaine. The mouse homologues, mPer1 and mPer2, were also shown to modulate cocaine sensitization and reward. Reportedly, normal expression of the per gene is important for the development of cocaine sensitization in Drosophila and in the mouse. This study examines the role of mPer1 on morphine addiction by applying a ribozyme to interfere with the expression of mPer1 in the central nervous system of mice. A plasmid based on pcDNA3.1 was constructed, using the expression of a hammerhead ribozyme that can selectively cleave the mPer1 mRNA. The ribozyme's presence was associated with a statistically significant reduction in mPer1 mRNA. Mice treated with the ribozyme and morphine synchronously did not show any preference for the morphine-paired side, whereas the control mice did. These results suggest that...

The adaptation of the endogenous rhythm of an organism to external cycles may influence the development of physiological processes in animals. Light not only synchronizes the circadian system, but also exerts profound direct effects: the immediate reduction of melatonin release at night-time and the inhibition of locomotor activity in nocturnal rodents after a light pulse are well-known examples, yet little is known about effects of different light/dark (LD) cycles on the level of corticosterone, growth hormone and growth rate. Mice were raised under different period length of LD cycle including LD5:5 (light: 5 h; dark: 5 h), LD12:12 (light: 12 h; dark: 12 h) and LD16:16 (light: 16 h; dark: 16 h) for four weeks. Mice in LD5:5 and LD16:16 groups manifested higher locomotor activity, plasma corticosterone and growth hormone concentrations and growth rate than the LD12:12 group. The results suggest that different LD cycles may affect many physiological processes including growth rate, food intake an...

PER1, an important component of circadian clock systems, plays a critical role in regulating the period length and maintaining the precision and stability of the period of circadian rhythms. RACK1 (receptor for activated protein kinase C-1), a member of the WD-40 family of proteins, can interact with numerous signaling proteins and is regarded as a scaffolding, anchor, or adaptor protein in multiple intracellular signal transduction pathways. In the present study, we identified and confirmed RACK1 as a novel protein interacting with human clock protein, hPER1, using the yeast two-hybrid system and co-immunoprecipitation experiment. Further study by RT-PCR showed that RACK1 was expressed widely in tissues and there was no obvious expressional rhythmicity. However, RNA interfering plasmid inhibiting, hPER1 (pTER/hPER1-II) could not interfere expression of RACK1. These results together suggested that RACK1 might act as a novel signal molecule to mediate or regulate the functions of PER1 through, protein interaction.

To investigate the effect of two deoxyribozymes targeting period1 (per1) mRNA in vitro for exploring a novel gene therapy approach about circadian rhythm diseases, the specific deoxyribozymes targeting per1 were designed and synthesized chemically following MFold analysis according to its mRNA secondary structure. per1 RNA fragments were prepared by in vitro transcription of pcDNA3.1(+)-per1(164:256). The cleavage reactions containing deoxyribozymes and per1 RNA fragments were performed under certain conditions. With the transfection technique mediated by LipofectAMINE, pcDNA3-per1 and DRz164 or DRz256 were introduced into NIH3T3 cells. The effects of deoxyribozymes on per1 were studied by reverse transcript-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). When deoxyribozymes and RNA transcripts were incubated under the adopted conditions at 37 degrees C for 2 h, about 63% of per1(164:256) RNA transcripts were cleaved by DRz164 and about 50.5% by DRz256. After cotransfecting pcDNA3-per1 with DRz164 or DRz256, the expression of per1 mRNA was decreased, as indicated by RT-PCR semi-quantity analysis. FCM analysis showed that Per1 protein was inhibited. Both DRz164 and DRz256 targeting per1 have the specific cleavage activity toward per1 mRNA in vitro and can highly block the expression of per1 gene in cellular milieu.

Ten alleles with length and nucleotide sequence variations were identified in the regulatory region of human tissue kallikrein gene. This present study aimed to study the polymorphisms of the regulatory region of human tissue kallikrein gene of the Chinese and investigate the relationship of the polymorphisms with essential hypertension. A case-control study was conducted in 200 hypertensive and 200 normotensive subjects of unrelated Chinese Han origin. All subjects were aged from 30 to 70 years and had no history of diabetes mellitus, kidney failure, or thyroid gland disease. The alleles were detected by polymerase chain reaction (PCR) and genotyping was performed with allele-specific oligonucleotide analysis (ASO). Data from the essential hypertensive and control subjects were statistically analysed by the Student's t-test and chi2-test. The age- and gender-matching of the groups were accurate. The case group and the control group were in Hardy-Weinberg equilibrium at this locus (cases, P=0.313; control subjects, P=0.457). There were nine alleles among the case and control groups, and the allele frequencies were found to be significantly different between cases and controls (chi2=25.701, P

Several essential biological progresses in mammals are regulated by circadian rhythms. Though the molecular mechanisms of oscillating these circadian rhythms have been uncovered, the specific functions of the circadian genes are not very clear. It has been reported that knocking down circadian genes by microRNA is a useful strategy to explore the function of the circadian rhythms. In this study, through a forward bioinformatics screening approach, we identified miR-29a/b/c as potent inhibitors for the human circadian gene hPER1. We further found that miR-29a/b/c could directly target hPER1 3'untranslated region (UTR) and down-regulate hPER1 at both mRNA and protein expression levels in human A549 cells. Thus, our findings suggested that the expression of hPER1 is regulated by miR-29a/b/c, which may also provide a new clue for the function of hPER1.

We previously demonstrated that receptor for activated C kinase 1 (RACK1) inhibited phosphorylated extracellular signal-regulated kinase (p-ERK) during morphine reward in mice. In the present study, we examined the role of Src in regulating the inhibition of p-ERK in the brain following RACK1 over-expression during morphine reward.Mice were subcutaneously injected with morphine on days 2, 4, 6, and 8 after pre-test (day 1), and saline was delivered the following day. After mice showed place preference, RACK1 over-expression plasmid was administered by intraventricular injection 20 minutes after morphine injection on days 11 and 13. Conditioned place preference (CPP) was measured on days 14, 15, 19, and 20.Chronic morphine injection increased Src and p-ERK expression in cortex and hippocampus, and mice exhibited increased place preference. Intraventricular administration of RACK1 reduced Src and p-ERK levels in cortex and hippocampus, as well as morphine reward. At 7 days of final RACK1 administration, the effects of RACK1 on Src and p-ERK disappeared, and morphine place preference was restored.We demonstrated that RACK1 acts on ERK activation via Src in morphine reward in mice.

To compare the circadian variation of blood pressure (BP) between patients with intra-cerebral haemorrhage (ICH) and with cerebral infarction (CI), around-the-clock BP measurements were obtained from 89 hypertensive patients with ICH, from 63 patients with CI and from 16 normotensive volunteers. The single and population-mean cosinor yielded individual and group estimates of the MESOR (Midline Estimating Statistic Of Rhythm, a rhythm-adjusted mean value), circadian double amplitude and acrophase (measures of extent and timing of predictable daily change). Comparison shows that without any difference in BP MESOR, the circadian amplitude of systolic (S) BP was larger in ICH than CI patients (P

It has been suggested that some animals are much more capable of perceiving certain kinds of geophysical stimuli which may precede earthquakes than humans, but the anecdotal phenomena or stories about unusual animal behaviors prior to an earthquake should be interpreted with objective data. During the Wenchuan magnitude 8.0 earthquake that happened in Wenchuan county (31.0 degrees north latitude, 103.4 degrees east longitude) of Sichuan province, China, on May 12, 2008, eight mice were monitored for locomotor activity and circadian rhythm in constant darkness with temperature 22-24 degrees C and humidity 55-65% for 38 days. The ongoing monitoring of locomotor activity of mice in our laboratory made it possible to design a posteriori study investigating whether the earthquake was associated with any change in animal behavior. Based on analyzing the recorded data with single cosinor, we found that the locomotor activity dramatically decreased in six of these eight mice on day 3 before the earthquake, and the circadian rhythm of their locomotor activity was no longer detected. The behavioral change lasted for 6 days before the locomotor activity returned to its original state. Analyses of concurrent geomagnetic data showed a higher total intensity during the span when the circadian rhythm in locomotor activity weakened. These results indicated that the behaviors, including circadian rhythm and activity, in these mice indeed changed prior to the earthquake, and the behavioral change might be associated with a change of geomagnetic intensity.

Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to 60Co-gamma-rays. NIH 3T3 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with 60Co-gamma-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 +/- 6.51 vs 66.0 +/- 3.51 and 67.7 +/- 7.37; transfection: 121.7 +/- 6.50 vs 65.3 +/- 3.51 and 69.0 +/- 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, c-myc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferation- and apoptosis-related genes and DNA repair-related genes.

The circadian clock is the central timing system that controls numerous physiologic processes. The current model of these oscillators is based on autoregulatory transcription and translation feedback loops of these circadian genes in which Period1 (Per1) gene occupies a central position. The laminin receptor 1 (Lamr1) and its precursor are expressed in most tissues and play important roles in several physiologic and pathologic processes, including cell differentiation, growth, migration and cancer invasion. The present study showed that Lamr1 was a novel protein that interacted with human circadian clock protein hPer1 by the yeast two-hybrid system and co-immunoprecipitation, which was expressed in many tissues and did not display circadian rhythm. The expression of hPer1 was knocked down to 84.9% by the hPer1 RNA interfering test, but the expression levels of Lamr1 was not depressed by the hPer1 RNA interfering test. The results suggest that Lamr1 is a novel protein that interacts with human circadian clock protein hPer1 and Lamr1 is not a direct efferent element of circadian clock.

Circadian clock genes have the function of producing circadian rhythm. They are also implicated in the origin or development of many diseases such as cancers and neuropsychiatric diseases. The purpose of this study is to determine whether the 54-nucleotide repeat polymorphism of hPer3, one of the circadian clock genes, associates with heroin dependence. DNA samples were obtained from 209 Chinese heroin-dependent subjects and 249 Chinese healthy controls. The 54-nucleotide repeat polymorphism was detected by polymerase chain reaction and DNA agarose gel electrophoresis. The frequency of four-repeat allele was significantly higher (chi(2)= 10.64, P = 0.001; corrected for multiple tests, P = 0.003) in the mixed gender heroin-dependent subject group (four repeat: 0.89, five repeat: 0.11) than in the mixed gender control group (four repeat: 0.81, five repeat: 0.19); the frequency of four-repeat allele was also significantly higher (chi(2)= 10.00, P = 0.002; corrected for multiple tests, P = 0.006) in the male heroin-dependent subject group (four repeat: 0.89, five repeat: 0.11) than in the male control group (four repeat: 0.81, five repeat: 0.19); for females, no significant trend was observed with the 54-nucleotide repeat polymorphism between the heroin-dependent subject group and the control group. Our results suggest that the 54-nucleotide repeat polymorphism of hPer3 significantly associates with heroin dependence at the allele frequency level and may be a potential risk factor for the development of heroin dependence.

Aims: To characterize and optimize a novel Bacillus pumilus strain isolated from biological waste which produces protease with excellent dehairing effect. This newly isolated strain could be utilized in the industrial leather dehairing process. Methods and Results: Bacterial strains secreting proteases were screened from biological wastes. Positive clones were further characterized by analysing their efficacy in dehairing and effects on collagen integrity. Among 171 colonies tested, a strain BA06, identified as B. pumilus, was picked owing to its efficient dehairing capabilities with minimal impact on collagen. By combined mutagenesis using UV, N-methyl-N′-nitro-N-nitrosdguanidine and Co60−γ-rays, this strain was further improved with regard to its alkaline protease production. The alkaline protease activity of the mutant strain SCU11was greatly improved up to 6000 U ml−1, in comparison with its parent strain BA06 of 1200 U ml−1. Conclusions: By using screening and mutagenesis methods, we have successfully created a B. pumilus strain that can produce high levels of alkaline proteases that are able to efficiently remove hair from skin with minimal damage on the collagen. Significance and Impact of the Study: This strain could be used in commercial alkaline protease production for leather dehairing.