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Our previous study indicated that Clock gene could affect the thrombotic potential. In this present study, we examined the differential expression of proteins in Clock knockdown mice's plasma, including α1-antitrypsin as a potential target. The proteins were examined in AML12 cells with Clock gene being knocked down to confirm the differential expressions. RNAs of those cells were extracted every 3 h in 24 h. The transcriptional levels of α1-antitrypsin (Serpina1a) and fibronectin (Fn1) were analyzed with the least-squares fit of a 24-h cosine function by single cosinor method, but no circadian rhythm was determined in neither of these genes. The expressions of α1-antitrypsin and fibronectin in Clock knockdown cells were found upregulated in both transcriptional and translational levels. Then, we applied ELISA assay to detect the concentration of activated protein C in Clock knockdown plasma and found a risen concentration. Fibronectin was reported to play a role in inhibiting the platelet aggregation, while activated protein C was demonstrated to inhibit PAI-1. All these results indicated that downregulation of the Clock gene in the circulatory system might have effects on PAI-1 by upregulating α1-antitrypsin, and might play some roles in platelet aggregation by increased fibronectin. [ABSTRACT FROM AUTHOR]

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 degradation, and involved in the process of many diseases, including cancer. Studies have shown that autophagy activity has a circadian rhythm feature. As the core gene of the circadian rhythm system, clock has participated in the occurrence and development of cancer. The expression of clock whether regulates tumor development by autophagy has not been illustrated at present. In this study, we established a stable clock-knockdown strain of mouse breast cancer cell 4T1 to explore the changes in autophagy activity. The results showed that autophagy-related proteins ATG9A, ATG7, LC3B and Beclin1 (ATG6) were down-regulated with clock-knockdown, and the p62, the key factors of autophagy, was up-regulated. Moreover, clock-knockdown caused significant up-regulation of p65, and inhibition of IκBα and p-AKT，which were the core factors of several signaling pathways involved in autophagy. Serum rhythm-inducing experiments behaved that the expression of LC3B and Beclin1 had rhythmic characteristics，differed from LC3A. The regulative gene clock was initially established to regulate the autophagy-related genes, and the suggested direction of its regulatory pathways. [ABSTRACT FROM AUTHOR]

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 gene will promote CRC progression and inhibit tumor cell apoptosis in vitro through the AKT-pathway (an antiapoptotic pathway have been reported). However, it remains to be studied the molecular mechanism of proliferation in CRC. In our present study, we attempt to study the clock gene which inhibits proliferation of CRC CT26 cells through p53-dependent pathway when the clock gene is suppressed in the CRC cell line CT26. Lentiviral vector binds to RNA target sequence, knocking down clock genes in CT26 cells with short hairpin RNA. For detecting their proliferation rates, we used CCK8 assay, plate clone formation assay, Western blot and mouse tumorigenicity assay. The results revealed that knocking down the clock gene has a negative effect on CT26 cell proliferation, depicted that low expression of clock gene reduced cylinD1 and c-myc expression, improved P21 and P53 expression in vitro and inhibited the growth of tumor in vivo. In conclusion, while silencing the clock gene can depress CT26 cell growth through p53-dependent pathway and c-myc. [ABSTRACT FROM AUTHOR]

Social isolation usually affects the physical and mental health of the human and produces depression, whether it may relate with circadian rhythm still remains to be investigated. Two groups of mice were employed to this experiment to continuously monitoring their activities in a dark environment. Immunofluorescence was used to show the expression of the circadian clock gene in two groups of mice. The results demonstrate that the social isolation leads to decrease of mouse activities, phase advance of rhythm relatively and down-regulated of clock gene expression. We conclude that social isolation can affect the physiological function and the normal physiological rhythm of mice, which may be related to depression. [ABSTRACT FROM AUTHOR]

Transactivator (Tat), a regulatory protein of HIV-1, plays a very important role in HIV-1 infection by promoting the rapid replication of HIV. Research surrounding Tat protein function has mainly focused on inhibition of the immune system, promotion of growth of vascular endothelial cells, and nervous system damage. To date, very little research has addressed the role of Tat in circadian rhythms. Previous studies in our lab have found that the concentration of Tat protein in HIV patients’ blood was positively correlated with patients’ sleep quality and melatonin concentrations. In this study, we applied a Tat expression plasmid in mice. Result demonstrated that the locomotor activities of mice and the concentration of melatonin were significantly increased. Alternatively, the expression ofClockgene was markedly decreased. On the other hand, the expression of theCry1gene was significantly increased, while the expression ofBmal1andPer1genes exhibited no significant difference. [ABSTRACT FROM PUBLISHER]

Circadian locomotor output cycles kaput (Clock) gene is a core gene in the circadian rhythm system that is involved in cancer cell proliferation. However, the molecular mechanism ofClockgene participate in the cancer cell proliferation is unclear. Previous studies demonstrated that cell proliferation could be regulated by the canonical Wnt pathway (also known as the Wnt/β-catenin pathway), and the Wnt/β-catenin pathway had a relation with the circadian system. To investigate whether theClockgene affects the proliferation of breast cancer cell by regulating the expression ofβ-catenin, we knocked down theClockexpression of mouse breast cancer cells (4T1) by RNA interference. Then detected their proliferation rates using CCK8 assay and the expression of theβ-cateningene by real-time PCR and Western blot. The results showed that the proliferation of theClockknocked down 4T1 cells is slower than the control. The expression level ofβ-cateninof these 4T1 cells is reduced. Our study showed thatClockgene knocked down inhibiting the proliferation of the 4T1 cells, probably by suppressing the expression of β-catenin. [ABSTRACT FROM PUBLISHER]

Emerging evidences have shown that circadian rhythm can also be regulated by a nontranscriptional mechanism. In this study, we investigated thein vitrorhythmic characters of peroxiredoxin2 (PRX2) and nicotinamide adenine dinucleotide (NAD) in red blood cells (RBCs) and/or in heparin anticoagulated plasma under different light conditions. Our results showed that NAD had a free-running rhythm in accordance with that of PRX2 in RBCs, and both rhythms were liable to light entrainment. A decreased expression of PRX2 in RBCs and a notable phase delay of locomotor activity were found in mice intravenously injected with NAD, suggesting that NAD in nontranscriptional oscillating systems could receive the light zeitgeber signal and influence the expression of PRX2. NAD may also act as a nonphotic zeitgeber, which could make an advanced phase shift ofper2, entraining the transcriptional oscillating system to make these two oscillating systems synchronized in a very short time. This zeitgeber might be of great significance as a mediator between these two oscillating systems. [ABSTRACT FROM AUTHOR]

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.Clockis 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 ofClockandfviigenes 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 thatClockgene could only regulatefviigene of some specific organ when the exposure to EMR in a certain range of power density. [ABSTRACT FROM PUBLISHER]

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 carriers of A/A heterozygote (1.27 ± 0.69 vs. 1.76 ± 1.22; P = 0.01). The Clif (rs2289709), the level of triglycerides and LDL cholesterol in carriers of G/A heterozygote wassignificantly different from carriers of G/G heterozygote (2.31 ± 1.81 vs. 1.11 ± 0.32; P = 2.33 × 10−8; 2.83 ± 0.98 vs. 1.63 ± 0.69; P = 5.83 × 10−15, respectively). Our findings indicate that Clock/Clif genetic variations might be associated with hyperlipidemia. [ABSTRACT FROM AUTHOR]

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 and hormones, and the change of growth rate in different LD cycles may be related to the level of corticosterone and growth hormone concentrations. The results also suggest that both the long-period LD cycle and short-period LD cycles can improve the growth of mice, but they disturbed the biorhythm stabilization and affected hormone secretion; in general, these conditions would not promote the animals' survival. [ABSTRACT FROM AUTHOR]