The Fas antigen is a cell surface protein that can mediate apoptosis in many cell types. Although its physiological function is still unclear, recent evidence indicates that this surface molecule is involved in apoptosis in the immune system and the liver. The epidermis is an organ that undergoes terminal differentiation with the eventual death of keratinocytes, and it has been suggested that this is a specialized form of apoptosis. In the present study, we examined whether or not the Fas antigen is involved in keratinocyte apoptosis. Immunoreactivity for the Fas antigen was found throughout the epidermis in normal human skin sections and cultured normal human keratinocytes, and mRNA for the Fas antigen was found to be constitutively expressed in normal epidermis and cultured normal keratinocytes by RT-PCR analysis. To determine whether the Fas antigen in keratinocytes is functional, we used a cytotoxic monoclonal antibody (mAb) against the Fas antigen to induce apoptosis. This antibody did not induce apoptosis of cultured keratinocytes even though they expressed the Fas antigen. We then tested the ability of several cytokines (TGF beta, TNF alpha and IFN gamma) to induce Fas-mediated keratinocyte apoptosis. Only pretreatment with IFN gamma followed by the addition of the anti-Fas mAb induced apoptosis, as assessed by cell viability, morphological changes and ultrastructural characteristics, suggesting that constitutive expression of the Fas antigen is not sufficient to induce apoptosis in keratinocytes and that keratinocyte apoptosis via the Fas antigen-mediated mechanism may require the activation of keratinocytes by IFN gamma, which is thought to be produced by activated T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Caesalpinia sappan L. (Leguminosae) is distributed inSoutheast Asia, and its heartwood, Sappan Lignum, is famous as a red dyestuff. Sappan Lignum is also used as herbal medicine for inflammation or improvement for blood circulation. 1,2) In Japan, Sappan Lignum is newly listed in the 15th Japanese Pharmacopoeia. 3)Aside from brazilin (1) and brazilein, the known constituents of Sappan Lignum, we have isolated sappanchalcone (2) and protosappanins A-E (3-7), which are dibenzoxocin derivatives, and elucidated their structures (Fig. 1). [4][5][6][7][8][9] Pharmacological studies of Sappan Lignum focusing on its vasorelaxation 10) or immunosuppressive effect 11) have concluded that 1 is the active compound. Compound 1 alone was reported to show anti-inflammatory effect, 12,13) lensaldose reductase inhibitory effect, 14) and anti-hepatotoxic effect.15) However, there are few pharmacological studies of other compounds except ours. We previously performed an in vitro assay for 1, brazilein, 2, 3, 4, and 5, and clarified such properties as inhibition of nitric oxide (NO) production and inducible NO synthase gene expression, as well as free radical scavenging and antioxidant activity.16) The results suggested that Sappan Lignum contained active compounds other than 1 that showed anti-inflammatory effect.In this study, we performed an in vitro assay for 2, 4) 3, 5)4, 6) 5, 7) 6 (isomeric mixtures), 8) and 7 (isomeric mixtures of E-1 and E-2) 9) to determine the mechanism underlying their inhibitory effects on NO and prostaglandin E 2 (PGE 2 ) production and their suppressive effects on the mRNA expression of typical chemical mediators of inflammation: tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible NO synthase (iNOS). We then performed an in vivo assay for methanolic extract of Sappan Lignum (CSE) and 1 to evaluate their anti-inflammatory effect on carrageenin-induced mouse paw edema in order to estimate whether or not 1 was the only active constituent of CSE. MATERIALS AND METHODSMaterials Brazilin (1, 5.1% from CSE), sappanchalcone (2, 0.38%), protosappanin A (3, 0.15%), protosappanin B (4, 0.80%), protosappanin C (5, 0.48%), protosappanin D (6, 0.15%), and protosappanin E (7, 0.32%) were isolated from Sappan Lignum purchased from Uchida Wakanyaku Co., Ltd. (Lot. No. 313116, December 27, 2006 We performed an in vitro assay for seven compounds from methanolic extract of Sappan Lignum (CSE) that inhibit the chemical mediators of inflammation using the J774.1 cell line: brazilin (1), sappanchalcone (2), protosappanin A (3), protosappanin B (4), protosappanin C (5), protosappanin D (6), and protosappanin E (7). Those compounds were evaluated for their inhibitory effects on nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production and their suppressive effects on tumor necrosis factor-a a (TNF-a a), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) mRNA expression. As a result, we clarified that 1 inhibited NO production, and almost no...

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We investigated the vasorelaxant activity of the methanolic extracts of Sappan Lignum (CSE) and its constituents, brazilin, sappanchalcone, and protosappanins A-E, on rat aorta and mesenteric artery. By comparing the vasorelaxant activity of CSE and brazilin on both blood vessels, we found that CSE contained active constituents other than brazilin. When added to brazilin, sappanchalcone and protosappanin D showed vasorelaxant activity on both blood vessels precontracted with phenylephrine. We clarified that the vasorelaxant activity of brazilin was endothelium-independent, while that of sappanchalcone was endothelium-dependent, on both blood vessels. On the other hand, the vasorelaxant activity of protosappanin D was independent of the endothelium of the aorta and dependent on the endothelium of the mesenteric artery. Experiments on sappanchalcone and protosappanin D using N G -nitro-L-arginine and indomethacin revealed the involvement of nitric oxide and prostaglandin as endothelium-derived relaxation factors (EDRFs). The anti-oketsu effect of Sappan Lignum might be attributable to the interaction of those compounds. We could partly evaluate the anti-oketsu activity of Sappan Lignum using both the aorta and the mesenteric artery. Through this study, we showed the importance of comparing the effects on the aorta and the mesenteric artery as we found that natural compounds showed different mechanisms of action on the two blood vessels.

Background and purpose The circadian clock governs endogenous day-night variations. In bone, the metabolism and growth show diurnal rhythms. The circadian clock is based on a transcription-translation feedback loop composed of clock genes including Period2 (Per2), which encodes the protein period circadian protein homolog 2. Because plasma parathyroid hormone (PTH) levels show diurnal variation, we hypothesized that PTH could carry the time information to bone and cartilage. In this study, we analyzed the effect of PTH on the circadian clock of the femur.Patients and methods Per2::Luciferase (Per2::Luc) knock-in mice were used and their femurs were organ-cultured. The bioluminescence was measured using photomultiplier tube-based real-time bioluminescence monitoring equipment or real-time bioluminescence microscopic imaging devices. PTH or its vehicle was administered and the phase shifts were calculated. Immunohistochemistry was performed to detect PTH type 1 receptor (PTH1R) expression.Results Real-time bioluminescence monitoring revealed that PTH reset the circadian rhythm of the Per2::Luc activity in the femurs in an administration time-dependent and dose-dependent manner. Microscopic bioluminescence imaging revealed that Per2::Luc activity in the growth plate and the articular cartilage showed that the circadian rhythms and their phase shifts were induced by PTH. PTH1R was expressed in the growth plate cartilage.Interpretation In clinical practice, teriparatide (PTH (1-34)) treatment is widely used for osteoporosis. We found that PTH administration regulated the femoral circadian clock oscillation, particularly in the cartilage. Regulation of the local circadian clock by PTH may lead to a more effective treatment for not only osteoporosis but also endochondral ossification in bone growth and fracture repair.

The circadian clock contains clock genes including Bmal1 and Period2, and it maintains an interval rhythm of approximately 24 hours (the circadian rhythm) in various organs including growth plate and articular cartilage. As endochondral ossification is involved not only in growth plate but also in fracture healing, we investigated the circadian clock functions in fracture sites undergoing healing. Our fracture models using external fixation involved femurs of Period2::Luciferase knock-in mice which enables the monitoring of endogenous circadian clock state via bioluminescence. Organ culture was performed by collecting femurs, and fracture sites were observed using bioluminescence imaging systems. Clear bioluminescence rhythms of 24-hour intervals were revealed in fracture healing sites. When parathyroid hormone (PTH) was administered to fractured femurs in organ culture, peak time of Period2::Luciferase activity in fracture sites and growth plates changed, indicating that PTH-responsive circadian clock functions in the mouse femur fracture healing site. While PTH is widely used in treating osteoporosis, many studies have reported that it contributes to improvement of fracture healing. Future studies of the role of this local clock in wound healing may reveal a novel function of the circadian timing mechanism in skeletal cells.