The FBA motif-containing protein AFBA1 acts as a novel positive regulator of ABA response in Arabidopsis.

ABA plays a critical role in regulating seed germination and stomatal movement in response to drought stress. Screening ABA-responsive genes led to the identification of a novel Arabidopsis gene encoding a protein which contained a conserved F-box-associated (FBA) domain, subsequently named ABA-responsive FBA domain-containing protein 1 (AFBA1). Expression of ProAFBA1:GUS revealed that this gene was mainly expressed in guard cells. Expression of AFBA1 increased following the application of exogenous ABA and exposure to salt (NaCl) and drought stresses. Seed germination of the loss-of-function mutant (afba1) was insensitive to ABA, salt or mannitol, whereas AFBA1-overexpressing (Ox) seeds were more sensitive to these stresses than the wild-type seeds. The afba1 plants showed decreased drought tolerance, increased water loss rate and ABA-insensitive stomatal movement compared with the wild-type. In contrast, AFBA1-Ox plants exhibited enhanced drought tolerance and a rapid ABA-induced stomatal closure response. The expression of genes encoding serine/threonine protein phosphatases that are known negative regulators of ABA signaling increased in afba1 plants but decreased in AFBA1-Ox plants. AFBA1 was also found to be localized in the nucleus and to interact with an R2R3-type transcription factor, MYB44, leading to the suggestion that it functions in the stabilization of MYB44. Based on these results, we suggest that AFBA1 functions as a novel positive regulator of ABA responses, regulating the expression of genes involved in ABA signal transduction in Arabidopsis through its interaction with positive regulators of ABA signaling including MYB44, and increasing their stability during ABA-mediated responses.

Therapeutic effects of s-petasin on disease models of asthma and peritonitis.

To explore the anti-allergic and anti-inflammatory effects of extracts of Petasites genus, we studied the effects of s-petasin, a major sesquiterpene from Petasites formosanus (a butterbur species) on asthma and peritonitis models. In an ovalbumin-induced mouse asthma model, s-petasin significantly inhibited the accumulations of eosinophils, macrophages, and lymphocytes in bronchoalveolar fluids. S-petasin inhibited the antigen-induced degranulation of ß-hexosamidase but did not inhibit intracellular Ca(2+) increase in RBL-2H3 mast cells. S-petasin inhibited the LPS induction of iNOS at the RNA and protein levels in mouse peritoneal macrophages. Furthermore, s-petasin inhibited the production of NO (the product of iNOS) in a concentration-dependent manner in the macrophages. Furthermore, in an LPS-induced mouse model of peritonitis, s-petasin significantly inhibited the accumulation of polymorpho nuclear and mononuclear leukocytes in peritoneal cavity. This study shows that s-petasin in Petasites genus has therapeutic effects on allergic and inflammatory diseases, such as, asthma and peritonitis through degranulation inhibition in mast cells, suppression of iNOS induction and production of NO in macrophages, and suppression of inflammatory cell accumulation.

Characterizations of sphingosylphosphorylcholine-induced scratching responses in ICR mice using naltrexon, capsaicin, ketotifen and Y-27632.

Sphingosylphosphorylcholine (SPC) is upregulated in the stratum corneum of atopic dermatitis patients by sphingomyelin deacylase. We conducted an investigation, both to confirm that intradermal injection of SPC elicits scratching in mice, and to elucidate the detailed mechanism of the SPC-induced itch-scratch response. Intradermal administration of SPC increased the incidence of scratching behavior in a dose-dependent manner. SPC-induced scratching could be suppressed, significantly, by the mu-opoid receptor antagonist, naltrexon, the vaniloid receptor agonist, capsaicin, and the histamine H1 receptor antagonist ketotifen. d-erythro SPC, one of the SPC stereotypes, could elicit the scratch response, but not l-threo SPC. Y-27632 (1 mg/kg, an inhibitor of Rho-associated protein kinase (ROCK)), was found to suppress SPC-induced scratching. Both the stereospecificity of SPC and the involvement of the Rho/ROCK pathway suggested that SPC-induced scratching is related to the receptor.

Involvement of serotonin receptors 5-HT1 and 5-HT2 in 12(S)-HPETE-induced scratching in mice.

The mechanisms of 12(S)-hydroperoxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid (12(S)-HPETE)-induced scratching were studied in ICR mice. In a recent paper, we demonstrated that the 12(S)-HPETE-induced scratching was reduced not by U75302 (BLT(1) receptor antagonist), but by LY255283 (BLT(2) receptor antagonist). In the present study, we tested various compounds to elucidate the mechanism of 12(S)-HPETE-induced scratching relating to transient receptor potential vanilloid type-1 (TRPV1), histamine receptor (H(1)) and several serotonin receptors (5-HT(1), 5-HT(2), and 5-HT(3)). As a result, 12(S)-HPETE-induced scratching was suppressed by capsaicin (TRPV1 receptor agonist), but not by capsazepine (TRPV1 receptor antagonist). Additionally, chlopheniramine (H(1) receptor antagonist) did not suppress 12(S)-HPETE-induced scratching, but cyproheptadine (H(1) receptor and serotonin 5-HT(2) receptor antagonist) potently suppressed the same response. Therefore, we tested several serotonin receptor antagonists to explain the detailed mechanisms relating to serotonin receptors. The scratching was reduced by WAY100635 (5-HT(1) receptor antagonist), or ketanserin (5-HT(2) receptor antagonist), but not by ondansetron (5-HT(3) receptor antagonist), after intradermal injection of 12(S)-HPETE. These results suggest that serotonin 5-HT(1/2) receptors are implicated in 12(S)-HPETE-induced scratching in ICR mice and that the TRPV1 receptor might not be directly related to 12(S)-HPETE-induced scratching.

Anti-inflammatory effect of kamebakaurin in in vivo animal models.

We have identified kamebakaurin as an inhibitor of NF-KB and elucidated its molecular mechanism as a specific inhibitor in the DNA-binding activity of the p50 subunit of NF-KB. Here, we describe its anti-inflammatory activity in in vitro and in vivo models. Kamebakaurin dose-dependently inhibited not only the expression of inflammatory NF-KB target genes such as iNOS,COX-2, and TNF-x, but also the production of PGE2 and TNF-a in LPS-stimulated RAW264.7 cells. Moreover, in an air pouch model of inflammation, it suppressed the recruitment of neutrophils,production of TNF-a as well as PGE2 in the pouch exudates induced by carrageenan. In addition, kamebakaurin dose-dependently suppressed the inflammation in an adjuvant arthritis model. Oral administration of 20 mg/kg kamebakaurin resulted in the 75% decrease of paw volume. Taken together, kamebakaurin, a specific inhibitor of DNA-binding activity of the p50 subunit, is a valuable candidate for the intervention in NF-KB-dependent pathological conditions such as inflammation.

2,3-diarylpyran-4-ones: a new series of selective cyclooxygenase-2 inhibitors.

A new series of cyclooxygenase-2 (COX-2) inhibitors with gamma-pyrone as central scaffold unit has been synthesized and their biological activities were evaluated against cyclooxygenase inhibitory activity. The changes of physical properties of the molecules were performed according to the medicinal chemistry principles and moderate oral anti-inflammatory activity was obtained with this series of inhibitors.

In vitro structure-activity relationship and in vivo studies for a novel class of cyclooxygenase-2 inhibitors: 5-aryl-2,2-dialkyl-4-phenyl-3(2H)furanone derivatives.

5-Aryl-2,2-dialkyl-4-phenyl-3(2H)furanone derivatives were studied as a novel class of selective cyclooxygenase-2 inhibitors with regard to synthesis, in vitro SAR, antiinflammatory activities, pharmacokinetic considerations, and gastric safety. 1f, a representative compound for methyl sulfone derivatives, showed a COX-2 IC(50) comparable to that of rofecoxib. In case of 20b, a representative compound for sulfonamide derivatives, a potent antiinflammatory ED(50) of 0.1 mg kg(-1) day(-1) was observed against adjuvant-induced arthritis by a preventive model, positioning 20b as one of the most potent COX-2 inhibitors ever reported. Furthermore, 20b showed strong analgesic activity as indicated by its ED(50) of 0.25 mg/kg against carrageenan-induced thermal hyperalgesia in the Sprague-Dawley rat. 3(2H)Furanone derivatives showed due gastric safety profiles as selective COX-2 inhibitors upon 7-day repeat dosing. A highly potent COX-2 inhibitor of the 3(2H)furanone scaffold could be considered suitable for a future generation COX-2 selective arthritis medication with improved safety profiles.

Torilin from Torilis japonica, as a new inhibitor of testosterone 5 alpha-reductase.

The methanolic extract of the fruits of Torilis japonica showed a potent inhibition against 5 alpha-reductase activity in vitro. Bioassay-guided fractionation of the methanol extract of the fruits followed by repeated silica gel chromatography led to the isolation of an active principle and its structure was identified as torilin on the basis of spectroscopic data. Torilin (IC50 = 31.7 +/- 4.23 microM) showed a stronger inhibition of 5 alpha-reductase than alpha-linolenic acid (IC50 = 160.3 +/- 24.62 microM) but was weaker than finasteride. (IC50 = 0.38 +/- 0.06 microM). Simple guaiane-type compounds, such as (-)-guaiol and guaiazulene showed weak inhibitory effects on the 5 alpha-reductase activity with IC50 values of f 81.6 microM and 100.8 microM, respectively, while azulene was not active. These results suggest that both degrees of unsaturation and the side-chain in the guaiane skeleton are important for the manifestation of 5 alpha-reductase inhibition.

2,3-Diarylbenzopyran derivatives as a novel class of selective cyclooxygenase-2 inhibitors.

A new series of cyclooxygenase-2(COX-2) inhibitors with naturally occurring flavone as the main skeleton has been synthesized and their biological activities were evaluated for cyclooxygenase inhibitory activity. Rational structural modifications were applied to potent COX-2 inhibitors to obtain the desired pharmacokinetic profiles for improved oral anti-inflammatory activity.

Inhibitory effects of natural sesquiterpenoids isolated from the rhizomes of Curcuma zedoaria on prostaglandin E2 and nitric oxide production.

Beta-turmerone and ar-turmerone, sesquiterpenoids isolated from the rhizome of Curcuma zedoaria, inhibited lipopolysaccharide (LPS)-induced prostaglandin E 2 production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC 50 = 7.3 microM for beta-turmerone; IC 50 = 24.0 microM for ar-turmerone). In addition, these compounds exhibited inhibitory effects on LPS-induced nitric oxide production in the cell system.