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Background Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. Methods Upstream signaling cascades mediated by several known receptors in bone marrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. Results All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1. Conclusions Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose-dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag-, SCF-, or HSP70-stimulated mast cells occur at the level of Lck-LAT phosphorylation.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-β-d-glucopyranosyl-(1→4)-β-d-xylopyranosyl)-28-β-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

Background In mast cells, induction of HSP70 expression during antigen stimulation has not been reported. Methods Mouse bone marrow derived mast cells (BMMC) were stimulated with IgE/Ag or HSP70. Induction of HSP70 expression, and signaling protein phosphorylation, were evaluated by immunoblotting. Results HSP70 expression is induced in BMMC at an early stage of IgE/Ag-dependent stimulation, some of which is released from the cells in a granule-associated form. Induction of HSP70 expression was also observed with an IgE/Ag-stimulated human basophilic cell line, indicating that the phenomenon is not restricted to mouse BMMC. The induction of HSP70 expression, and its release, followed a similar time course to that of degranulation. Released HSP70 seems to be responsible for degranulation and production of eicosanoids, at least in part, since a neutralizing anti-HSP70 antibody mitigated these activities, and since exogenous HSP70 not only induced immediate degranulation followed by autocrine HSP70 expression but also enhanced degranulation in IgE/Ag stimulated BMMC. Extracellular HSP70 was found to induce phosphorylation of Linker for activation of T cells (LAT) and a series of downstream signaling molecules in BMMC. We further found that Fyn, Lyn and spleen tyrosine kinase (Syk), which are known to concern LAT phosphorylation in IgE/Ag stimulated BMMC, were not phosphorylated in HSP70-stimulated BMMC, whereas lymphocyte-specific-protein tyrosine kinase (Lck) was phosphorylated. Conclusion FceRI stimulation in BMMC and basophils induces HSP70 expression and its release. Extracellular HSP70 induces degranulation and mediator release via phosphorylation of LAT. This article is protected by copyright. All rights reserved.

A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 μM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1β and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

Background IgE/Ag-stimulated mast cells release various pro-allergic inflammatory mediators, including histamine, eicosanoids, and pro-inflammatory cytokines. NecroX-5, a cell permeable necrosis inhibitor, showed cytoprotective effects in both in vitro and in vivo models. However, the anti-allergic effect of NecroX-5 has not yet been investigated. The aims of this study were to evaluate the anti-allergic activity of NecroX-5 in vivo and to investigate the underlying mechanism in vitro. Methods The anti-allergic activity of NecroX-5 was evaluated in vitro using bone marrow-derived mast cells (BMMCs) and IgE receptor-bearing RBL-2H3 or KU812 cells and in vivo using a mouse model of passive anaphylaxis. The levels of histamine, eicosanoids (PGD2 and LTC4), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured using enzyme immunoassay kits. The mechanism underlying the action of NecroX-5 was investigated using immunoblotting, immunoprecipitation, and gene knockdown techniques. Results NecroX-5 markedly inhibited mast cell degranulation and the synthesis of eicosanoids, TNF-α, and IL-6 by suppressing the activation of Syk, LAT, phospholipase Cγ1, MAP kinases, the Akt/NF-κB pathway, and intracellular Ca2+ mobilization via the activation of phosphatase SHP-1. Oral administration of NecroX-5 effectively suppressed mast cell-dependent passive cutaneous and systemic anaphylactic reactions in a dose-dependent manner. Conclusions NecroX-5 might be a potential candidate for the development of a novel anti-allergic agent that suppresses IgE-dependent mast cells signaling.

Background and Purpose Endoplasmic reticulum (ER) stress has been implicated in the pathogeneses of insulin resistance and type 2 diabetes, and extracellular signal-regulated kinase (ERK) antagonist is an insulin sensitizer that can restore muscle insulin responsiveness in both tunicamycin-treated muscle cells and type 2 diabetic mice. The present study was undertaken to determine whether the chemical or genetic inhibition ER stress pathway targeting by ERK results in metabolic benefits in muscle cells.

Rutaecarpine is a pentacyclic indolopyridoquinazolinone alkaloid found in Evodia rutaecarpa and other related herbs. It has a variety of intriguing biological properties, which continue to attract the academic and industrial interest. Studies on rutaecarpine have included isolation from new natural sources, development of new synthetic methods for its total synthesis, the discovery of new biological activities, metabolism, toxicology, and establishment of analytical methods for determining rutaecarpine content. The present review focuses on the synthesis, biological activities, and structure-activity relationships of rutaecarpine derivatives, with respect to their antiplatelet, vasodilatory, cytotoxic, and anticholinesterase activities.

The stem and root barks of Ulmus davidiana var. japonica (Ulmaceae) have been used to treat inflammatory diseases including mastitis, rhinitis, sinusitis, and enteritis. In an ongoing study focused on the discovery of natural anti-inflammatory compounds from natural products, a methanol extract of the stem and root barks of U. davidiana var. japonica showed anti-inflammatory activities. Activity-guided fractionation of the methanol extract yielded a new trihydroxy fatty acid, 9,12,13-trihydroxyoctadeca-10(Z),15(Z)-dienoic acid (1), and a known compound, pinellic acid (2). These two trihydroxy fatty acids 1 and 2 inhibited prostaglandin D2 production with IC50 values of 25.8 and 40.8 μM, respectively. These results suggest that 9,12,13-trihydroxyoctadeca-10(Z),15(Z)-dienoic acid (1) and pinellic acid (2) are among the anti-inflammatory principles in this medicinal plant. Copyright © 2012 John Wiley & Sons, Ltd.

Ascochlorin, a prenylphenol antitumor antibiotic, profoundly increases the expression of endogenous p53 by increasing protein stability in the human osteosarcoma cells and human colon cancer cells. Ascochlorin also increases DNA binding activity to the p53 consensus sequence in nuclear extract and enhances transcription of p53 downstream targets. Ascochlorin specifically induces p53 phosphorylation at ser 392 without affecting ser 15 or 20, whereas DNA damaging agents typically phosphorylate these serines. Moreover, ascochlorin does not induce phosphorylation of ATM and CHK1, an established substrate of ATR that is activated by genotoxins, nor does it increase DNA strand break, as confirmed by comet assay. The structure-activity relationship suggests that p53 activation by ascochlorin is related to inhibition of mitochondrial respiration, which is further supported by the observation that respiratory inhibitors activate p53 in a manner similar to ascochlorin. These results suggest that ascochlorin, through the inhibition of mitochondrial respiration, activates p53 through a mechanism distinct from genotoxins.

Mast cells play an important role in allergic inflammation by releasing various bioactive mediators. The function of mast cells is enhanced by various stimuli, partly due to the induction of specific genes and their products. Although many inducible genes have been identified, a significant number of genes remain to be identified. Therefore, this study used PCR-selected cDNA subtraction to establish the profile of induced genes in the connective tissue (CT) type-like mast cells derived from bone marrow cells cultured in the presence of IL-4 and stem cell factor. Two hundred and fifty cDNA clones were obtained from the CT type-like mast cells by PCR-selected cDNA subtraction. Among them, Ym1/2, a chitinase-like protein, is one of the most abundantly induced genes. Ym1 is produced by activated macrophages in a parasitic infection, whereas its isotype, Ym2, is highly upregulated in allergic lung disease. In order to differentiate which isotype is expressed in bone marrow cells, specific primers for bone marrow-derived mast cells (BMMC), and CT type-like mast cells were used for RT-PCR. The results showed that Ym1 was constitutively expressed in bone marrow cells and gradually decreased in the presence of IL-3, whereas Ym2 was induced only in the presence of IL-4. CT type-like mast cells from bone marrow cells expressed Ym1 throughout the culture period and Ym2 was induced only by the addition of IL-4 into BMMC, indicating that IL-4 is essential for the expression of Ym1/2 genes.

Synurus deltoides Aiton, Nakai, is an edible plant that has been used as a folk medicine for treating inflammatory disorders. This investigation was carried out to establish the antiinflammatory activity of this plant material using a 75% ethanol extract from the aerial part of S. deltoides. Against the acute inflammatory animal model of mouse croton oil-induced ear oedema assay, the extract did not show significant inhibition at 100–800 mg/kg by oral administration. On the other hand, the extract showed considerable inhibition against the chronic inflammatory animal model of rat adjuvant-induced arthritis (25% inhibition at 100 mg/kg/day) while prednisolone exerted 40% inhibition at 10 mg/kg/day. In addition, S. deltoides possessed strong analgesic activity (IC50 = 50 mg/kg) in the acetic acid-induced writhing test. From the extract, ursolic acid and scopoletin were successfully isolated and their contents were found to be 0.31% and 0.37% (w/w), respectively, based on the dried extract by HPLC analysis. All the results obtained indicate that this plant material may be used beneficially as an antiinflammatory agent having analgesic action. Copyright © 2004 John Wiley & Sons, Ltd.

The purpose of this study was to investigate the effect of green tea catechin on the cyclooxygenase and lipoxygenase pathways in chronic cadmium-poisoned rats. Sprague-Dawley male rats weighing 100 +/- 10 g were randomly assigned to one normal and three cadmium-poisoned groups. The cadmium groups were classified as catechin-free diet group (Cd-0C), 0.25% catechin diet group (Cd-0.25C) and 0.5% catechin diet group (Cd-0.5C), in accordance with the level of catechin supplement. The phospholipase A2 activity was remarkably increased 117% in the Cd-0C group and 60% in the Cd-0.25C group compared with the normal group, and the level in the Cd-0.5C group was the same as the normal group. Activity of platelet cyclooxygenase increased 284% in the Cd-0C group, 147% in the Cd-0.25C group and 193% in the Cd-0.5C group. The synthesis of platelet thromboxane A2 (TXA2) increased 157% in the Cd-0C group and 105% in the Cd-0.25C group, compared with the normal group. The Cd-0.5C group showed the same level as the normal group. Prostacyclin (PGI2) formation in the aorta decreased 24% in the Cd-0C group and 18% in the Cd-0.25C group. The ratio of PGI2/TXA2, the thrombocyte synthesis index, decreased 70% in the Cd-0C group and 59% in the Cd-0.25C group. The activity of 5'-lipoxygenase in the polymorphonuclear leukocyte was increased 40% in the Cd-0C group as compared with the normal group. Catechin-supplemented Cd-0.25C and Cd-0.5C groups showed the level of the normal group. In this study, the observed content of leukotriene B4, which induces the inflammatory process, increased 54% in the Cd-0C group, and in catechin-supplemented groups, showed the same level as in the normal group. The serum peroxide value increased 60% in the Cd-0C group compared with the normal group; but in the Cd-0.5C group, it showed the level of the normal group. These results indicate that chronic cadmium poisoning in rats accelerates arachidonic acid metabolism. Inhibition of arachidonic acid metabolism due to catechin supplementation, however, decreases platelet aggregation and inflammatory action. In conclusion, it would appear that green tea catechin supplementation in chronic cadmium-poisoned rats inhibits the arachidonic acid cascade by regulating the activity of phospholipase A2.

Group IID secretory phospholipase A2 (sPLA2-IID), a heparin-binding sPLA2 that is closely related to sPLA2-IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA2-IIA. Here we identified the residues of sPLA2-IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA2-IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus-induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA2s, which were equally active on A23187- and IL-1-primed cellular membranes, sPLA2-IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA2-IID and sPLA2-X constitutively, the former of which was negatively regulated by IL-1. sPLA2-IID, but not other sPLA2 isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA2-IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA2-IID may be involved in inflammation in cell- and tissue-specific manners under particular conditions.

Inhibitors of the A1 adenosine receptor were isolated from the skin extract of Korean frog, Rana rugosa. The frog-skin extract was prepared by an electrical shock and fractionated with C4 followed by C18 reverse-phase HPLC. Two A1 receptor inhibitors were isolated using a filter binding assay and the molecular masses of the proteins were estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be 15 347 and 15 404 Da, respectively. The inhibitory activity was also measured against other membrane receptors, such as the A2 adenosine receptor, muscarinic acetylcholine receptor and capsaicin receptor. Ligand binding to the A2 and muscarinic receptors was also severely inhibited by these proteins. However, they did not inhibit the functional activation of the capsaicin receptor by its ligand, capsaicin, suggesting that inhibition of ligand–receptor binding occurs specifically. Their N-terminal sequences were determined by Edman degradation. Surprisingly, they showed sequence similarity to the secretory protein, phospholipase A2 from various organisms. The phospholipase A2 activity of both proteins was tested using Dole's assay technique. Both proteins showed phospholipase A2 activity, and therefore, they were designated as PLA2-R1 and PLA2-R2, respectively. In addition, their ligand-binding inhibitory activity depended on their phospholipase A2 activity. This is the first finding that the frog secretes a phospholipase A2 similar to that of snake venoms, which posess inhibitory activity against the adenosine A1, adenosine A2 and muscarinic receptors.

H COSY spectrum indicated coupling between the oxygenated methine at δ 4.25 (H-7) and the methine signal at δ 1.75 (H-8), which was also coupled to the methyl signal at δ 0.54 (H-9). On the other hand, the methine signal at δ 2.39 (H-8′) was coupled to the methyl signal at δ 0.81 (H-9 ′). These observations are consistent with a lignan-7-olic skeleton. The positions of each functional group were determined by a HMBC experiment. One set of correlations was observed between the oxygenated methine H-7 (δ 4.25) and C-1, C-2, C-6, C-8, C-8′ and C-9 and the other set was between H-7 ′ (δ 2.46) and C-1 ′, C-2′, C-6′, C-8, C-8′ and C-9′. Additionally, the correlations between two methylenedioxy signal at δ 5.88 and 5.89 and C-3′/C-3 and C-4′/C-4 supported the proposed link the 3,4,3′,4′-dimethy-lenedioxy phenyl moiety. The absolute stereochemistry at C-7

Polyunsaturated fatty acid-derived monoglycerides were characterized from the marine brown alga Sargassum sagamianum, collected from Jeju Island, Korea. A new compound of this structural class was isolated and determined to be 1-octadecatetraenoyl glycerol, by combined spectroscopic methods. Based on the structures and bioactivity of these compounds, a series of monoglycerides were synthesized using glycerol and various fatty acids. Several compounds exhibited moderate to significant inhibition of phospholipase A(2) and cyclooxygenase-2.

A series of C-C biflavones was designed to investigate the relationship between structural array of different flavone-flavone subunit linkage and the inhibitory activity against phospholipase A2 (PLA2). Among six classes of C-C biflavones designed, four classes of C-C biflavones, which have flavone-flavone subunit linkages at A ring-A ring, A ring-B ring, B ring-B ring, and B ring-C ring, were synthesized. The synthetic biflavones exhibited somewhat different inhibitory activities against sPLA2-IIA. Among them, the biflavone a having a C-C 4'-4' linkage showed comparable inhibitory activity with that of the natural biflavonoid, ochnaflavone, and 7-fold stronger activity than that of amentoflavone. Further chemical modification is being carried out in order to obtain the chemically optimized biflavonoids.

College of Pharmacy, Catholic University of Daegu, Gyongsan 712-702, KoreaReceived October 7, 2003Key Words : Juglans mandshurica, Juglandaceae, DiarylheptanoidThe roots of Juglans mandshurica Maximowicz(Juglandaceae) have been used as a folk medicine for thetreatment of cancer in Korea. Several naphthoquinones,naphthalenyl glucosides, tetralones, flavonoids, diarylheptanoid,and galloyl glycosides have been isolated from Juglansspecies.

1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OMe) has been reported to possess definite antitumor activity in vivo. Twenty-two alkyl lysophospholipid analogs were chemically synthesized, and their antitumor activity against mouse experimental tumors (Sarcoma 180, MM46, P388) was examined. Among them, 1-O-octadecyl-2-O-acetoacetyl-rac-glycerol-3-phosphocholine was found to show antitumor activity similar to ET-18-OMe with less acute toxicity. Intravenous injection of the ET-18-OMe withsn-3 configuration retarded the subcutaneous growth of Sarcoma 180 cells effectively, while the growth inhibition by thesn-1 isomer was much less effective. This stereospecificity was similar to that observed in their activities as platelet-activating factor (PAF) agonists. The acetoacetyl compound, another PAF agonist, showed similar stereospecific antitumor action in vivo. These findings suggest that some alkyl lysophospholipids may activate host cells to a cytostatic stage against tumor cells in vivo through binding to a PAF receptor. Our preliminary results indicated that the responsible cells under these conditions might be primarily immature macrophages present in the bone marrow. No appreciable or even adverse stereospecificity was observed in the different sets of experiments where the activity of ET-18-OMe against MM46 tumor cells in vivo or the direct cytotoxicity against human promyelocytic leukemia HL-60 cells in vitro was examined. Under, some conditions, the antitumor activity of ET-18-OMe in vivo may be revealed through direct cytotoxicity and/or modulation of the host defense system by “nonspecific” mechanisms. Some alkylphospholipids without PAF activity may also show antitumor activity through similar, “nonspecific” mechanisms.

1. The pharmacological action of the L- and D-enantiomers of lysophosphatidylserine has been studied in vivo by following the increase in blood and brain glucose content caused by this phospholipid in mice. Preliminary experiments have confirmed that these effects are the consequence of lysophosphatidylserine-induced mast cell activation since they are not observed in mast cell-deficient mice bearing the W/Wv genotype. 2. Maximal hyperglycaemic response and brain glucose accumulation occur at 10 mg kg-1 lysophosphatidyl-L-serine (i.v.). Half-maximal effect is at 3.5 mg kg-1. Lysophosphatidyl-D-serine at doses of up to 25 mg kg-1 i.v. elicits 40% (blood glucose) and 60% (brain glucose) of the maximal effect. The difference in activity between the two enantiomers is also observed in the desensitization to lysophosphatidylserine occurring when this phospholipid is administered by the oral route. 3. Lysophosphatidyl-L-serine is more active than the D-enantiomer in mouse isolated peritoneal mast cells. Activity ratios of 10 are observed between 20 and 50% histamine release. Similar results are obtained with rat isolated peritoneal mast cells. 4. It is concluded that the configuration of the alpha carbon atom of serine influences the activity of lysophosphatidylserine in vivo and in vitro. Thus, the appropriate position of the serine amino group is required for optimal interaction of the phospholipid head group and a receptor in the mast cell membrane.