Embryonic stem (ES) cells are pluripotent-undifferentiated cells that have a great interest for the investigation of developmental biology. Murine ES cells maintain their pluripotency by the supplementation of the leukemia inhibitory factor (LIF). LIF is reported to act as a matrix-anchored form, and immobilized cytokines are useful to sustain their signaling on target cells. In this study, we used the immobilizable fusion protein composed of LIF and IgG-Fc region, which was used as a model of the matrix-anchored form of LIF to establish a novel system for ES cell culture and to investigate the effect of immobilized LIF on maintenance of ES cell pluripotency. Mouse ES cells maintained their undifferentiated state on the surface coated with LIF-Fc. Furthermore, when cultured on the co-immobilized surface with LIF-Fc and E-cadherin-Fc, mouse ES cells showed characteristic scattering morphologies without colony formation, and they could maintain their undifferentiated state and pluripotency without additional LIF supplementation. The activation of LIF signaling was sustained on the co-immobilized surface. These results indicate that immobilized LIF and E-cadherin can maintain mouse ES cells efficiently and that the immobilizable LIF-Fc fusion protein is useful for the investigation of signaling pathways of an immobilized form of LIF in the maintenance of ES cell pluripotency. ES3 cells are pluripotent-undifferentiated cells derived from the inner cell mass of a blastocyst, and they retain the potentiality of multilineage differentiation in vitro and self-renewal activity (1, 2). For the maintenance of pluripotency, mouse ES cells are cultured on a feeder layer of mouse embryonic fibroblasts (MEF) or incubated with LIF that is produced by MEF.LIF is a pleiotropic cytokine, which induces the differentiation of leukemia cell lines into macrophages (3) and the expression of acute phase proteins in hepatocytes (4), as well as inhibits proliferation of endothelial cells (5).Several studies suggest that the biological signals of growth factors and cytokines are mediated by two different forms, the secreted form and the cell membrane-or matrix-anchored forms, which stimulate different signal transduction cascades (6 -8). It is reported that the LIF signal is mediated by either a soluble form or by a form bound to extracellular matrices, both of which induce different effects on cells (9, 10).Recently, we reported the application of immobilizable fusion proteins for the analysis of cell function, and we also reported the establishment of a culture system for stem cells (11-13). When cells were cultured on the fusion protein of E-cadherin and IgG Fc domain (E-cad-Fc), they showed scattering behavior and high proliferative ability (11). We also reported the novel method of immobilizing epidermal growth factor as a juxtacrine model, and we applied this system to the analysis of the function of immobilized epidermal growth factor on hepatocytes (13).In this study, we focused on the effect of the immobilized form of LIF on the m...

Functions and the presence of 5-hydroxytryptamine (5-HT) receptors in the fundus, corpus and antrum of the guinea pig stomach were examined by measuring contractile force and acetylcholine (ACh) release. Stimulation of the 5-HT1 receptor caused tetrodotoxin (TTX)-insensitive relaxations in the preparations from 3 regions. Stimulation of the 5-HT2 receptor caused TTX-insensitive contractions in the preparations of fundus and antrum. Stimulation of 5-HT3 receptors caused contractions that were sensitive to TTX and atropine and enhanced the outflow of [3H]ACh from preparations of only antrum. Stimulation of 5-HT4 receptors caused contractions of antral strips and decreased relaxations of corporal strips and enhanced the outflow of [3H]ACh from the preparations of both corpus and antrum. In the guinea pig stomach, the fundus possesses relaxant 5-HT1 receptor < contractile 5-HT2 receptors and caused the contractile response to 5-HT. The corpus possesses relaxant 5-HT1 receptors and relaxant receptors other than 5-HT1, 5-HT2, 5-HT3 and 5-HT4 receptors > contractile 5-HT4 receptor, and therefore 5-HT caused relaxations. The antrum possesses relaxant 5-HT1 receptor < contractile 5-HT2, 5-HT3 and 5-HT4 receptors, and thus 5-HT caused contractions.

Ability of mosapride, a gastrokinetic agent, to bind to 5-HT4 receptor was examined in the stomach of human and guinea pig by in vitro receptor autoradiography. [125I]SB207710 binding sites were detected in the muscle layer including the myenteric plexus of the stomach from both humans and guinea pigs, although the binding was observed more clearly and densely in the stomach of guinea pigs than humans. Mosapride as well as SB204070 inhibited the binding of [125I]SB207710. Thus, mosapride possesses the ability to bind to 5-HT4 receptors of human stomach and may modulate the motility, as in the case of guinea pig stomach.

1. The role of synaptophysin in the exocytotic release of dopamine (DA) was examined in Xenopus laevis oocytes injected with rat brain mRNA. 2. The mRNA-injected oocytes showed DA uptake which depended on the incubation time and external DA concentrations. 3. Stimulation with KCl (10-50 mM) of mRNA-injected oocytes preloaded with DA evoked external Ca2+ -dependent release of DA. The noninjected and water-injected oocytes did not produce uptake of DA and stimulation-evoked release of DA. 4. The high-KCl (50 mM)-stimulated release of DA decreased in the oocytes injected with rat brain mRNA together with antibody to synaptophysin. 5. Immunoblot analysis demonstrated that synaptophysin was expressed in the brain mRNA-injected oocytes but not in the noninjected and water-injected oocytes. 6. Thus, uptake and release machinery similar to native dopaminergic nerve terminals was expressed in Xenopus oocytes by injecting mRNA-extracted from the rat brain, and synaptophysin may play a role in the exocytotic release of DA.

Olefins with a phenathroline ring and a pyrrole ring were prepared and hydrogen atom transfer reaction in the excited singlet state was observed. The tautomer produced in the excited singlet state has lifetime of 30–50 ps for cis‐1 and c,t‐2 and with fluorescence maximum at longer wavelength region at 640 nm. The compound 1 exhibited photochromic behavior with its absorption maximum changing between 380 nm and 440 nm, while 2 exhibited only the small change of the absorption spectra on photoirradiation. Copyright © 2007 John Wiley & Sons, Ltd.