B-ring-homo-tonghaosu, isolated from Chrysanthemum morifolium capitulum, acts as a peroxisome proliferator-activated receptor-γ agonist.

The capitula of Chrysanthemum morifolium and C. indicum are used to prepare Chrysanthemi Flos in traditional Japanese Kampo medicine. In our previous study, we reported on the agonistic effect of methanol extract of C. indicum capitulum on peroxisome proliferator-activated receptor (PPAR)-γ. We further isolated (E)-tonghaosu from C. indicum capitulum as one of the active ingredients. In the present study, we aimed to evaluate the PPAR-γ agonistic activity of a methanol extract of C. morifolium capitulum (MCM) in which (E)-tonghaosu could not be detected. MCM exhibited PPAR-γ agonistic activity in a concentration-dependent manner, and at a dose of 100 µg/ml, it showed similar activity to pioglitazone (30 µM), a standard PPAR-γ agonist. Through activity-guided fractionation, we isolated two geometric isomers, (E)- (1) and (Z)-B-ring-homo-tonghaosu (2), as the active ingredients of MCM. Both compounds exerted concentration-dependent PPAR-γ agonistic effects, and 1 had higher activity than 2. At 1.4 µM, 1 had similar activity to pioglitazone (30 µM), which was achieved by 2 at a concentration of 140 µM. Thus, 1 has the potential to become a lead compound for the drug discovery of PPAR-γ agonists. We compared the activities and the contents of (E)-, (Z)-tonghaosu, 1, and 2 among 13 commercial samples of Chrysanthemi Flos, including those derived from both C. morifolium and C. indicum. Their PPAR-γ agonistic activities were not related to the contents of these compounds. 1 and 2 were detected in the samples derived from both species but (E)- and (Z)-tonghaosu were not detected in the samples derived from C. morifolium; hence (E)- and (Z)-tonghaosu can serve as marker compounds to identify the capitula of C. indicum in Chrysanthemi Flos samples.

Involvement of the mouse Prp19 gene in neuronal/astroglial cell fate decisions.

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.

Intrathecal high-dose morphine induces spinally-mediated behavioral responses through NMDA receptors.

Previous research has demonstrated that intrathecal i.t. morphine in a dose of 60.0 nmol into the spinal subarachnoid space of mice can evoke nociceptive behavioral responses consisting of a severe hindlimb scratching directed toward the flank followed by biting/licking of the hindpaw. The present study was undertaken to examine the involvement of spinal N-methyl-D-aspartate (NMDA) and opioid receptors on the behavioral responses evoked by high-dose i.t. morphine. Pretreatment with naloxone, an opioid receptor antagonist (1.0 and 4.0 mg/kg, s.c.), failed to reverse the morphine-evoked behavioral response, suggesting that the morphine effect is not mediated through the opioid receptors in the spinal cord. The morphine-induced behavior was dose-dependently inhibited by i.t. co-administration of the competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphonovaleric acid (D-APV) (6.25-50.0 pmol) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (3.125-25.0 pmol). The characteristic behavior was also reduced by co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801) (74.1-250 pmol), an NMDA ion-channel blocker. Ifenprodil, a competitive antagonist of the polyamine recognition site of NMDA receptor ion channel complex, produced a dose-related inhibitory effect on the behavioral response to i.t. morphine with less potency than the competitive and non-competitive antagonists examined. High doses of (+)-HA-966, a glycine/NMDA antagonist, induced a dose-dependent inhibition of morphine-induced response. The effective dose of i.t. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, needed to reduce the morphine-induced response, was approximately 10-fold greater than that of D-APV. These results suggest that spinal NMDA receptors, but not non-NMDA receptors, may be largely involved in elicitation of the behavioral episode following i.t. injection of morphine in mice.