CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.Almost all organisms from bacteria to mammals have developed circadian physiology and behavior to adapt to the environmental changes created by the rotation of our planet. Such daily rhythms are controlled by genetically determined and self-sustaining circadian clocks that are composed of networks of transcription-translation feedback loops involving sets of clock genes (15,30,37). In mammals, a network of feedback loops functions robustly not only in the master circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus (26), but also in most tissues (including liver, heart, lung, and muscle) and even in immortalized cell lines (1, 50, 51). These widely dispersed circadian systems are primarily synchronized by the SCN to coordinate circadian timing in vivo (29,36).One of the main questions in clock biology is how the timekeeping system is controlled at the molecular level. Intensive studies in the mouse using genetic and molecular approaches have largely clarified the structure of the central feedback loop (3,30). Two bHLH-PAS-containing transcription factors, CLOCK and BMAL1, form heterodimers that bind to E-box enhancer elements in the promoters of target genes driving the transcription of three period genes (Per1, Per2, and Per3) and two cryptochrome genes (Cry1 and Cry2) (11,14,17,18). After the PER and CRY proteins have been translated in the cytoplasm, they form heterocomplexes that translocate into the nucleus and inhibit their own transcription. CRY plays a crucial role in this negative feedback process by interacting directly with the CLOCK/BMAL1 heterodimers (12,22).Analysis of Bmal1 defective mice has revealed the indispensable role of BMAL1 as the mainspring of the molecular clockwork; thus, targeted disruption of Bmal1 results...

The development of a water oxidation catalyst has been a demanding challenge for the realization of overall water-splitting systems. Although intensive studies have explored the role of Mn element in water oxidation catalysis, it has been difficult to understand whether the catalytic capability originates mainly from either the Mn arrangement or the Mn valency. In this study, to decouple these two factors and to investigate the role of Mn valency on catalysis, we selected a new pyrophosphate-based Mn compound (Li2MnP2O7), which has not been utilized for water oxidation catalysis to date, as a model system. Due to the monophasic behavior of Li2MnP2O7 with delithiation, the Mn valency of Li(2-x)MnP2O7 (x = 0.3, 0.5, 1) can be controlled with negligible change in the crystal framework (e.g., volume change ~1%). Moreover, inductively coupled plasma mass spectrometry, X-ray photoelectron spectroscopy, ex-situ X-ray absorption near-edge structure, galvanostatic charging-discharging, and cyclic voltammetry analysis indicate that Li(2-x)MnP2O7 (x = 0.3, 0.5, 1) exhibits high catalytic stability without additional delithiation or phase transformation. Notably, we observed that, as the averaged oxidation state of Mn in Li(2-x)MnP2O7 increases from 2 to 3, the catalytic performance is enhanced in the series Li2MnP2O7 < Li(1.7)MnP2O7 < Li(1.5)MnP2O7 < LiMnP2O7. Moreover, Li2MnP2O7 itself exhibits superior catalytic performance compared with MnO or MnO2. Our study provides valuable guidelines for developing an efficient Mn-based catalyst under neutral conditions with controlled Mn valency and atomic arrangement.

No abstract

Tristetraprolin (TTP) is an AU‐rich element‐binding protein that regulates mRNA stability. Here, we report that TTP suppress the growth of human colon cancer cells both in vivo and in vitro by regulating of the expression of vascular endothelial growth factor (VEGF). TTP protein expression in human colonic tissues was markedly decreased in colonic adenocarcinoma compared with in normal mucosa and adenoma. VEGF expression was higher in colonic adenocarcinoma than in normal mucosa and adenoma. Specific inhibition of TTP expression by RNA‐interference increased the expression of VEGF in cultured human colon cancer cells, and TTP overexpression markedly decreased it. In addition, elevated expression of TTP decreased the expression level of luciferase linked to a 3′ terminal AU‐rich element (ARE) of VEGF mRNA. Colo320/TTP cells overexpressing TTP grew slowly in vitro and became tumors small in size when xenografted s.c into nude mice. These findings demonstrate that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells, suggesting that it can be used as novel therapeutic agent to treat colon cancer.

Prostaglandin E 2 (PGE 2 ) mediates the stimulatory effect of norepinephrine (NE) on the secretion of luteinizing hormonereleasing hormone (LHRH), the neuropeptide controlling reproductive function. In rodents, this facilitatory effect requires previous exposure to estradiol, suggesting that the steroid affects downstream components in the cascade that leads to PGE 2 -induced LHRH release. Because astroglia are the predominant cell type contacting LHRH-secreting nerve terminals, we investigated the involvement of hypothalamic astrocytes in the estradiol facilitation of PGE 2 -induced LHRH release. A subpopulation of LHRH neurons was found to express the mRNA encoding the PGE 2 receptor subtype EP1-R, which is coupled to calcium mobilization. The LHRH-producing cell line GT1-1 also contains EP1-R mRNA and, to a lesser extent, the three alternatively spliced forms of EP3-R mRNA (␣, ␤, and ␥) that encode receptors linked to inhibition and stimulation of cAMP formation. Hypothalamic astrocytes treated with estradiol produced a conditioned medium that when applied to GT1-1 cells resulted in a selective upregulation of EP1-R and EP3␥-R mRNAs. The conditioned medium also enhanced the LHRH response to EP1-R and EP3-R agonists and the cAMP response to EP3-R activation. Thus, one mechanism by which estradiol facilitates the effect of neurotransmitters acting via PGE 2 to stimulate LHRH release is by enhancing the glial production of substances that upregulate PGE 2 receptors on LHRH neurons. The existence of such a mechanism underscores the emerging importance of glial-neuronal communication in the control of brain neurosecretory activity.

Obesity, a condition characterized by increased fat content and altered secretion of adipokines, is a risk factor for postmenopausal breast cancer. Visfatin has recently been established as a novel adipokine that is highly enriched in visceral fat. Here we report that visfatin regulated proliferation of MCF-7 human breast cancer cells. Exogenous administration of recombinant visfatin increased cell proliferation and DNA synthesis rate in MCF-7 cells. Furthermore, visfatin activated G1-S phase cell cycle progression by upregulation of cyclin D1 and cdk2 expression. Visfatin also increased the expression of matrix metalloproteinases 2, matrix metalloproteinases 9, and vascular endothelial growth factor genes, suggesting that it may function in metastasis and angiogenesis of breast cancer. Taken together, these findings suggest that visfatin plays an important role in breast cancer progression.

Various pathophysiologic mechanisms leading to sickness behaviors have been proposed. For example, an inflammatory process in the hypothalamus has been implicated, but the signaling modalities that involve inflammatory mechanisms and neuronal circuit functions are ill-defined. Here, we show that toll-like receptor 2 (TLR2) activation by intracerebroventricular injection of its ligand, Pam3CSK4, triggered hypothalamic inflammation and activation of arcuate nucleus microglia, resulting in altered input organization and increased activity of proopiomelanocortin (POMC) neurons. These animals developed sickness behavior symptoms, including anorexia, hypoactivity, and hyperthermia. Antagonists of nuclear factor kappa B (NF-κB), cyclooxygenase pathway and melanocortin receptors 3/4 reversed the anorexia and body weight loss induced by TLR2 activation. These results unmask an important role of TLR2 in the development of sickness behaviors via stimulation of hypothalamic microglia to promote POMC neuronal activation in association with hypothalamic inflammation.A growing body of evidence indicates that an inflammatory process in the hypothalamus is one of the major causes of dysfunctions in energy metabolism. In particular, previous studies have focused on events of chronic inflammation, which is an important pathologic element leading to metabolic syndromes 1,2 . However, the significance of acute hypothalamic inflammation in sickness behaviors has been relatively ignored even though it is closely correlated with human diseases such as infection and cachexia 3 . Sickness responses are coordinated sets of adaptive behavioral changes including loss of appetite, hypoactivity, and hyperthermia during the course of human diseases such as cancer, acquired immune deficiency syndrome (AIDS), tuberculosis, and renal failure 4 . Although hypothalamic inflammation has been recognized as a prominent component of sickness responses 5,6 , the pathophysiologic mechanisms of sickness symptoms are poorly understood.Toll-like receptors (TLRs) are key components of the innate immune system through their role in the recognition of a variety of pathogens and inflammatory signals 7 . Recently, it has been proposed that perturbation of metabolic controls is closely associated with the function of TLRs as inflammatory modulators [8][9][10] . In the development of diet-or ageing-induced obesity, the function of TLRs in the neural cells is thought to be as important as their impact on peripheral organs 11,12 . Nevertheless, it remains unclear whether TLRs participate in the development of sickness behaviors governed by hypothalamic neural circuits. Therefore, in this study, we interrogated the role of hypothalamic TLR2 in the development of sickness behaviors caused by acute hypothalamic inflammation.We found that intracerebroventricular (icv) injection of the TLR2 ligand Pam3CSK4 led to sickness responses including anorexia, hypoactivity, and hyperthermia through stimulation of inflammatory processes in the hypothalamus. In addition, we observe...

NELL2, a protein containing EGF-like repeats, is almost exclusively expressed in the nervous system. In the mammalian brain, NELL2 expression is mostly neuronal. NELL2 was previously found to be a secreted protein that functions during embryonic development as a neuronal differentiation and survival factor. We now show that the Nell2 gene is selectively expressed in the two major subtypes of glutamatergic neurons described in the postnatal brain: those containing the vesicular glutamate transporter 1 and those expressing vesicular glutamate transporter 2. No Nell2 mRNA is detected in GABAergic neurons. Likewise, GnRH neurons are devoid of NELL2. During prepubertal development of the female rat, Nell2 mRNA abundance increases selectively in the medial basal hypothalamus, reaching maximal values at the end of the juvenile period, to decline at the time of puberty to intermediate levels. Similar, but less pronounced changes are observed in the preoptic area, but they are absent in the cerebral cortex. A well-established glutamatergic function in the neuroendocrine brain is to enhance release of GnRH, the neurohormone controlling sexual development and the time of puberty. In vivo disruption of NELL2 synthesis via intraventricular administration of antisense oligodeoxynucleotides reduced GnRH release from the medial basal hypothalamus and delayed the initiation of female puberty. These results identify NELL2 as a new component of glutamatergic neurons and provide evidence for its physiological involvement in a major, glutamate-dependent, process of neuroendocrine regulation.