DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1 ؊/؊ mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.The mammalian molecular clock system consists of various clock genes and their protein products involved in interlocked feedback loops of transcriptional and translational regulation through clock elements such as CACGTG E-box, D-box, and ROR/REV-ERB binding elements (RORE) (18,24). Among these regulatory sequences, the E box is thought to be the most important element in the molecular oscillatory system, since it is the binding site for the CLOCK/BMAL1 heterodimer, which up-regulates various clock genes, including Dec1, Dec2, Per1, Dbp, and Rev-erb␣. In this regulatory system, PER, CRY, and DEC serve as negative factors for transcription from E-boxdriven promoters, and the E-box-like element EЈ box (CAC GTT) was recently shown to be involved in the direct regulation of Per2 and Cry1 genes by CLOCK/BMAL1 (1, 31, 34). RORE, on the other hand, is a clock element to which the transcriptional activators-ROR␣/ROR␤/ROR␥-and repressors-REV-ERB␣/REV-ERB␤-bind, and ROR and REV-ERB regulate the circadian expression of Bmal1, Clock, Npas2, and Cry1 via RORE (31).In the mammalian clock system, DEC1 (also known as BHLHB2, STRA13, or SHARP2) and DEC2 (BHLHB3 or SHARP1) serve as transcriptional repressors for CLOCK/ BMAL1-enhanced promoter activity, through binding to E boxes or interaction with BMAL1 (12,14,18,26). Among suppressive factors for E boxes, DEC1 and DEC2 can bind directly to E boxes through their basic helix-loop-helix DNA binding domains (18,26), although it remains unclear whether DEC1 and DEC2 also bind to the EЈ boxes. In contrast, PER and CRY interact with the CLOCK/BMAL1 heterodimer but cannot bind directly to E/EЈ boxes, since they have no DNA binding domain.Dec1 expression shows...

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Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have regenerative capability and exert paracrine actions on damaged tissues. Since peritoneal fibrosis is a serious complication of peritoneal dialysis, we tested whether MSCs suppress this using a chlorhexidine gluconate model in rats. Although MSCs isolated from green fluorescent protein–positive rats were detected for only 3 days following their injection, immunohistochemical staining showed that MSCs suppressed the expression of mesenchymal cells, their effects on the deposition of extracellular matrix proteins, and the infiltration of macrophages for 14 days. Moreover, MSCs reduced the functional impairment of the peritoneal membrane. Cocultures of MSCs and human peritoneal mesothelial cells using a Transwell system indicated that the beneficial effects of MSCs on the glucose-induced upregulation of transforming growth factor-β1(TGF-β1) and fibronectin mRNA expression in the human cells were likely due to paracrine actions. Preincubation in MSC-conditioned medium suppressed TGF-β1-induced epithelial-to-mesenchymal transition, α-smooth muscle actin, and the decrease in zonula occludens-1 in cultured human peritoneal mesothelial cells. Although bone morphogenic protein 7 was not detected, MSCs secreted hepatocyte growth factor and a neutralizing antibody to this inhibited TGF-β1 signaling. Thus, our findings imply that MSCs ameliorate experimental peritoneal fibrosis by suppressing inflammation and TGF-β1 signaling in a paracrine manner.

High-efficiency light-driven hydrogen evolution from water was demonstrated by using poly(phenyleneethynylene) bearing negatively charged, [G3] poly(benzyl ether) dendrimeric side groups 3(L4) as photosensitizer. Three-dimensional wrapping of the conjugated backbone suppressed self-quenching of the photoexcited state, while methyl viologen (MV(2+)), a positively charged electron acceptor, was trapped on its negatively charged surface, to form a spatially separated donor-acceptor supramolecular complex. Studies with time-resolved fluorescence spectroscopy showed that the quenching rate constant (k(q) = 1.2 x 10(15) M(-1) s(-1)) is much greater than diffusion control rate constants. Upon excitation of 3(L4) in the presence of a mixture of MV(2+), triethanolamine (TEOA; sacrificial electron donor), and a colloidal PVA-Pt, hydrogen evolution took place with an overall efficiency of 13%, 1 order of magnitude better than precedent examples. Comparative studies with several reference sensitizers showed that spatial isolation of the conjugated backbone and its long-range pi-electronic conjugation, along with electrostatic interactions on the exterior surface, play important roles in achieving the efficient photosensitized water reduction.

The basic helix-loop-helix transcription factor DEC1 is expressed in a circadian manner in the suprachiasmatic nucleus where it seems to play a role in regulating the mammalian circadian rhythm by suppressing the CLOCK/BMAL1-activated promoter. The interaction of DEC1 with BMAL1 has been suggested as one of the molecular mechanisms of the suppression [Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y. & Honma, K. (2002) Nature 419, 841-844]. Deletion analysis of DEC1 demonstrated that its N-terminal region, which includes the basic helix-loophelix domain, was essential for both the suppressive activity and the interaction with BMAL1, as DEC1 lacking the basic region did not show any suppression or interaction. Furthermore, we found that Arg65 in the basic region, which is conserved among group B basic helix-loop-helix proteins, was responsible for the suppression, for the interaction with BMAL1 and for its binding to CACGTG E-boxes. However, substitution of His57 for Ala significantly reduced the E-box binding activity of DEC1, although it did not affect the interaction with BMAL1 or suppression of CLOCK/BMAL1-induced transcription. On the other hand, the basic region-deleted DEC1 acted in a dominant-negative manner for DEC1 activity, indicating that the basic region was not required for homodimer formation of DEC1. Moreover, mutant DEC1 also counteracted DEC2-mediated suppressive activity in a dominant-negative manner. The heterodimer formation of DEC1 and DEC2 was confirmed by pull-down assay. These findings suggest that the basic region of DEC1 participates in the transcriptional regulation through a protein-protein interaction with BMAL1 and DNA binding to the E-box.Keywords: DEC1; DEC2; BMAL1; circadian rhythm; clock.Circadian rhythms are regulated by a molecular clock(s), which has an endogenous period of 24 h and synchronizes to the 24 h period after light entrainment. In mammals, the clock genes Clock, Bmal1, Per and Cry, and their protein products, comprise a molecular feedback loop in which a CLOCK/BMAL1 heterodimer binds to a CACGTG E-box and activates transcription of Per and Cry [1,2]; protein products of Per and Cry in turn suppress the transactivation by CLOCK/BMAL1 [3,4]. This core feedback loop apparently generates a 24 h period in the molecular oscillator. Furthermore, another feedback loop has been reported to control the rhythmic expression of Bmal1: expression of Rev-Erba is inducible by the CLOCK/BMAL1 heterodimer, and its protein product suppresses the expression of Bmal1 [5,6]. These two feedback loops may be interlocked to stabilize the circadian core loop system. DEC1 (bhlhb2) and DEC2 (bhlhb3) are basic helix-loophelix (bHLH) transcription factors which bind to CAC-GTG E-boxes and suppress transcription from target genes [7][8][9][10][11][12]. Expression of DEC1 and DEC2 showed circadian rhythms in most organs, including the suprachiasmatic nucleus (SCN) [7,13], and Dec1 expression in the SCN was enhanced by a light pulse in a phase-dependent manner sim...