A novel binding site between the voltage-dependent calcium channel Ca1.2 subunit and Caβ2 subunit discovered using a new analysis method for protein-protein interactions.

We developed a new method to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein-protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein-protein interactions would affect toxin activity, resulting in colony formation.

Analysis of circadian rhythm components in EEG/EMG data of aged mice.

Aging disrupts circadian clocks, as evidenced by a reduction in the amplitude of circadian rhythms. Because the circadian clock strongly influences sleep-wake behavior in mammals, age-related alterations in sleep-wake patterns may be attributable, at least partly, to functional changes in the circadian clock. However, the effect of aging on the circadian characteristics of sleep architecture has not been well assessed, as circadian behaviors are usually evaluated through long-term behavioral recording with wheel-running or infrared sensors.

A simple, dual direct expression plasmid system in prokaryotic and mammalian cells.

We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic () and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.

Structure and Function of Neuronal Circuits Linking Ventrolateral Preoptic Nucleus and Lateral Hypothalamic Area.

To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPO and VLPO neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with hybridization (ISH) in male and female mice, we revealed that the vesicular GABA transporter ()- and galanin ()-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA.

Rapid method for plasmid DNA recombination (Murakami-system).

DNA recombination is a useful technology for cloning and subsequent functional analysis, while standard techniques for plasmid DNA recombination have remained unchanged. In the present study, we introduced rapid method for plasmid DNA recombination, which we named "Murakami-system", to complete the experiments in under 33 h. For this purpose, we selected the following: PCR amplification with 25 cycles and strain with rapid growth (incubation time of 6-8 h).

Muscarinic Acetylcholine Receptors Chrm1 and Chrm3 Are Essential for REM Sleep.

Sleep regulation involves interdependent signaling among specialized neurons in distributed brain regions. Although acetylcholine promotes wakefulness and rapid eye movement (REM) sleep, it is unclear whether the cholinergic pathway is essential (i.e.

Oscillatory gene expression and somitogenesis.

A bilateral pair of somites forms periodically by segmentation of the anterior ends of the presomitic mesoderm (PSM). This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic gene expression. Expression of her1 and her7 in zebrafish and Hes7 in mice oscillates by negative feedback, and mathematical models have been used to generate and test hypotheses to aide elucidation of the role of negative feedback in regulating oscillatory expression.

Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis.

Somitogenesis is controlled by cyclic genes such as Notch effectors and by the wave front established by morphogens such as Fgf8, but the precise mechanism of how these factors are coordinated remains to be determined. Here, we show that effectors of Notch and Fgf pathways oscillate in different dynamics and that oscillations in Notch signaling generate alternating phase shift, thereby periodically segregating a group of synchronized cells, whereas oscillations in Fgf signaling released these synchronized cells for somitogenesis at the same time. These results suggest that Notch oscillators define the prospective somite region, while Fgf oscillators regulate the pace of segmentation.

Ultradian oscillations in Notch signaling regulate dynamic biological events.

Notch signaling regulates many dynamic processes; accordingly, expression of genes in this pathway is also dynamic. In mouse embryos, one dynamic process regulated by Notch is somite segmentation, which occurs with a 2-h periodicity. This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the Notch effector gene Hes7.

Rhythmic gene expression in somite formation and neural development.

In mouse embryos, somite formation occurs every two hours, and this periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the basic helix-loop-helix gene Hes7. Hes7 expression oscillates by negative feedback and is cooperatively regulated by Fgf and Notch signaling. Both loss of expression and sustained expression of Hes7 result in severe somite fusion, suggesting that Hes7 oscillation is required for proper somite segmentation.

Mesp2 and Tbx6 cooperatively create periodic patterns coupled with the clock machinery during mouse somitogenesis.

The metameric structures in vertebrates are based on the periodicity of the somites that are formed one by one from the anterior end of the presomitic mesoderm (PSM). The timing and spacing of somitogenesis are regulated by the segmentation clock, which is characterized by the oscillation of several signaling pathways in mice. The temporal information needs to be translated into a spatial pattern in the so-called determination front, at which cells become responsive to the clock signal.

The initiation and propagation of Hes7 oscillation are cooperatively regulated by Fgf and notch signaling in the somite segmentation clock.

Periodic formation of somites is controlled by the segmentation clock, where the oscillator Hes7 regulates cyclic expression of the Notch modulator Lunatic fringe. Here, we show that Hes7 also regulates cyclic expression of the Fgf signaling inhibitor Dusp4 and links Notch and Fgf oscillations in phase. Strikingly, inactivation of Notch signaling abolishes the propagation but allows the initiation of Hes7 oscillation.

Oscillator mechanism of Notch pathway in the segmentation clock.

Somites are formed by periodic segmentation of the presomitic mesoderm (PSM). This periodic event is controlled by the segmentation clock, where Notch signaling plays an essential role. The basic helix-loop-helix factor Hes7, a Notch effector, is cyclically expressed by negative feedback and regulates cyclic expression of Lunatic fringe (Lfng), a Notch modulator.