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SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) is a cytoplasmic protein -tyrosine phosphatase encoded by the gene PTPN11. It plays a crucial role in regulating cell growth and differentiation. Specifically, SHP2 is an oncoprotein associated with developmental pathologies and several different cancer types, including gastric, leukemia and breast cancer and is of great therapeutic interest. Given these roles, current research efforts have focused on developing SHP2 inhibitors. Allosteric SHP2 inhibitors have been shown to be more selective and pharmacologically appealing compared to competitive catalytic inhibitors targeting SHP2. Nevertheless, there remains a need for novel allosteric inhibitor scaffolds targeting SHP2 to develop compounds with improved selectivity, cell permeability, and bioavailability. Towards this goal, this study applied various computational tools to screen over 6 million compounds against the allosteric site within SHP2. The top-ranked hits from our in-silico screening were validated using protein thermal shift and biolayer interferometry assays, revealing three potent compounds. Kinetic binding assays were employed to measure the binding affinities of the top-ranked compounds and demonstrated that they all bind to SHP2 with a nanomolar affinity. Hence the compounds and the computational workflow described herein provide an effective approach for identifying and designing a generation of improved allosteric inhibitors of SHP2.

Introduction:T cell functions are altered during chronic viral infections and tumor development. This is mainly manifested by significant changes in T cells’ epigenetic and metabolic landscapes, pu...

Off-target interactions of drugs with the human ether-à-go-go related gene 1 (hERG1) channel have been associated with severe cardiotoxic conditions leading to the withdrawal of many drugs from the market over the last decades. Consequently, predicting drug-induced hERG-liability is now a prerequisite in any drug discovery campaign. Understanding the atomic level interactions of drug with the channel is essential to guide the efficient development of safe drugs. Here we utilize the recent cryo-EM structure of the hERG channel and describe an integrated computational workflow to characterize different drug-hERG interactions. The workflow employs various structure-based approaches and provides qualitative and quantitative insights into drug binding to hERG. Our protocol accurately differentiated the strong blockers from weak and revealed three potential anchoring sites in hERG. Drugs engaging in all these sites tend to have high affinity towards hERG. Our results were cross-validated using a fluorescence polarization kit binding assay and with electrophysiology measurements on the wild-type (WT-hERG) and on the two hERG mutants (Y652A-hERG and F656A-hERG), using the patch clamp technique on HEK293 cells. Finally, our analyses show that drugs binding to hERG disrupt and hijack certain native—structural networks in the channel, thereby, gaining more affinity towards hERG.

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has emerged as a powerful strategy in cancer immunotherapy. Recently, there have been enormous efforts to develop potent PD-1/PD-L1 inhibitors. In particular, Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies have individually disclosed several promising PD-1/PD-L1 inhibitors, whose detailed experimental data are not publicly disclosed. In this work, we report the rigorous and systematic in vitro characterization of a selected set of potent PD-1/PD-L1 macrocyclic peptide (BMSpep-57) and small-molecule inhibitors (BMS-103, BMS-142) from BMS and a peptidomimetic small-molecule inhibitor from Aurigene (Aurigene-1) using a series of biochemical and cell-based assays. Our results confirm that BMS-103 and BMS-142 are strongly active in biochemical assays; however, their acute cytotoxicity greatly compromised their immunological activity. On the other hand, Aurigene-1 did not show any activity in both biochemical and immunological assays. Furthermore, we also report the discovery of a small-molecule immune modulator, whose mode-of-action is not clear; however, it exhibits favorable drug-like properties and strong immunological activity. We hope that the results presented here will be useful in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors.

Off-target interactions of drugs with the human ether-à-go-go related gene 1 (hERG1) channel have been associated with severe cardiotoxic conditions leading to the withdrawal of many drugs from the market over the last decades. Consequently, predicting drug-induced hERG-liability is now a prerequisite in any drug discovery campaign. Understanding the atomic level interactions of drug with the channel is essential to guide the efficient development of safe drugs. Here we utilize the recent cryo-EM structure of the hERG channel and describe an integrated computational workflow to characterize different drug-hERG interactions. The workflow employs various structure-based approaches and provides qualitative and quantitative insights into drug binding to hERG. Our protocol accurately differentiated the strong blockers from weak and revealed three potential anchoring sites in hERG. Drugs engaging in all these sites tend to have high affinity towards hERG. Our results were cross-validated using a fluorescence polarization kit binding assay and with electrophysiology measurements on the wild-type (WT-hERG) and on the two hERG mutants (Y652A-hERG and F656A-hERG), using the patch clamp technique on HEK293 cells. Finally, our analyses show that drugs binding to hERG disrupt and hijack certain native-structural networks in the channel, thereby, gaining more affinity towards hERG.

Domestically developed concrete storage casks include an internal canister to maintain the confinement integrity of radioactive materials. In this study, we analyzed the depth of flaws caused by loads that propagate canister weld cracks under normal, off-normal and accident conditions, and evaluated the maximum allowable weld flaw depth needed to secure the structural integrity of the canister weld and to reduce the welding time of the internal canister lid of the concrete storage cask. Structural analyses for normal, off-normal and accident conditions were performed using the general-purpose finite element analysis program ABAQUS; the allowable flaw depth was assessed according to ASME B&PV Code Section XI. Evaluation results revealed an allowable canister weld flaw depth of 18.75 mm for the concrete storage cask, which satisfies the critical flaw depth recommended in NUREG-1536.

As a part of innate immune defense, the role of mast cells during viral replication has been incompletely understood. In this study, we characterized and compared the responses of the human mast cell line, LAD2, and human lung epithelial cell line, Calu-3, against three influenza A virus strains; A/PR/8/34 (H1N1), A/WS/33 (H1N1) and A/HK/8/68 (H3N2). We found that there were strain-dependent mast cell responses, and different profiles of cytokine, chemokine and antiviral gene expression between the two cell types. All three strains did not induce histamine or β-hexosaminidase release in LAD2. A/HK/8/68 induced release of prostaglandin D2 in LAD2, whereas A/PR/8/34 and A/WS/33 did not. We found that, among those examined, only CCL4 (by A/PR/8/34) was statistically significantly released from LAD2 cells. Furthermore, there was increased mRNA expression of viral recognition receptors (RIG-I and MDA5) and antiviral protein, viperin, but levels and kinetics of the expression were different among the cell types, as well as by the strains examined. Our findings highlight the variability in innate response to different strains of influenza A virus in two human cell types, indicating that further investigation is needed to understand better the role of mast cells and epithelial cells in innate immunity against influenza A viruses.

인양장비는 원자력발전소에서 발생하는 사용후핵연료를 운반하는 운반용기를 인양하기 위해 사용된다. 본 연구는 원자력 안전위원회고시 제2013-27호와 미국 10CFR Part 71 §71.45에서 규정하는 기술수준에 따라 이론적인 방법과 유한요소방법 으로 인양장비의 구조적안전성을 평가하였다. 이론적으로 평가한 결과 모든 구성 요소에서의 응력이 응력제한치 내에 있어 운영상 발생하는 구조적 안전성을 확보하고 있는 것으로 판단하였다. 또한 유한요소해석을 통한 평가결과, 항복과 극한조건 모두에서 설계기준을 만족하는 것으로 평가되었다. 모든 구성요소에서 충분한 안전여유도(항복조건에서 3 이상의 안전율, 극한조건에서 5 이상의 안전율)를 갖는 것으로 나타나 구조적으로 안전하다고 판단하였다.

Mast cells are important in innate immunity and protective against certain bacterial infections. However, there is limited evidence that mast cells respond to viruses. As mast cells are abundant in mucosal tissues of the lung, they are in a prime location to detect and respond to influenza virus. In this study, we characterized for the first time the replication cycle of influenza A virus in human mast cells by measuring influenza A virus transcription, RNA replication, protein synthesis, and formation of infectious virus as compared to the replication cycle in epithelial cells. We detected the presence of influenza A viral genomic RNA transcription, replication, and protein synthesis in human mast cells and epithelial cells. However, there was no significant release of infectious influenza A virus from mast cells, whereas epithelial cells produce ~100-fold virus compared with the inoculating dose. We confirmed that influenza A virus infects human mast cells, begins to replicate, but the production of new virus is aborted. Thus, mast cells may lack critical factors essential for productive infection or there are intrinsic or inducible anti-influenza A mechanisms in mast cells.

In addition to its well-known role in relaxation of vascular smooth muscle, NO modulates immune responses in a concentration- and location-specific manner. For MC, it is well accepted that exogenous NO regulates their function. However, there are inconsistencies in the literature of whether MC express NOS and make NO. MC progenitors mature in peripheral tissues, but the factors that influence MC maturation and their specific phenotype, such as whether they express NOS, are not well understood. To study microenvironmental conditions that could be “permissive” for NOS expression, we cultured BMMC in various conditions—BMMCIL-3, BMMCSCF/IL-3, or BMMCSCF/IL-4—for >3 weeks and examined NOS expression. We detected Nos2 mRNA in BMMCSCF/IL-4 but not BMMCIL-3 or BMMCSCF/IL-3. After stimulation with IFN-γ and/or LPS, NOS2 expression and NO production were detected in BMMCSCF/IL-4 but rarely detected in BMMC cultured with other conditions. Confocal microscopic analysis showed that NOS2 expression induced by IFN-γ colocalized in CD117+ BMMC. NO production, after activation with IFN-γ and LPS in BMMCSCF/IL-4, was abrogated by pretreatment with the NOS2-specific inhibitor. In addition to NOS2 expression, BMMCSCF/IL-4 were distinguished from BMMCIL-3 in heparin and MMCP expression. Thus, MC progenitors that develop in SCF + IL-4 can be induced to express NOS2 after receiving appropriate signals, such as IFN-γ, and subsequently produce NO. Microenvironmental conditions during their development can influence whether MC are capable of NOS expression and of NO production.

We evaluated the ability of saucerneol D (SD), a tetrahydrofuran-type sesquilignan isolated from Saururus chinensis, to regulate the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells. SD consistently inhibited nitric oxide (NO) production in a dose-dependent manner, with an IC(50) of 2.62 microM, and also blocked LPS-induced iNOS expression. SD potently suppressed both the reporter gene expression and DNA-binding activity of nuclear factor-kappaB (NF-kappaB). In addition, SD inhibited IkappaB-alpha degradation in a concentration- and time-dependent manner. SD also inhibited LPS-induced activation of various mitogen-activated protein kinases, including extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These findings suggest that SD may inhibit LPS-induced iNOS expression by blocking NF-kappaB and MAPK activation.

Meso-dihydroguaiaretic acid (MDGA) is a medicinal herbal product isolated from the aerial parts of Saururus chinensis that inhibits the cyclooxygenase-2 (COX-2)-dependent phase of prostaglandin D(2) (PGD(2)) generation in bone marrow-derived mast cells (BMMC) (IC(50) 9.8 microM). However, this compound did not inhibit COX-2 protein expression in BMMC at concentrations up to 30 microM, indicating that MDGA directly inhibits COX-2 activity. In addition, this compound consistently inhibited the production of leukotriene C(4) (IC(50) 1.3 microM). These results demonstrate that MDGA inhibits both COX-2 and 5-lipoxygenase. Furthermore, this compound strongly inhibited the degranulation reaction in BMMC (IC(50) 11.4 microM). Therefore, this compound might provide a basis for novel anti-inflammatory drug development.

5-Methoxy-8-(2-hydroxy-3-buthoxy-3-methylbutyloxy)-psoralen (MP) is a medicinal herbal product isolated from Angelica dahurica that inhibits the cyclooxygenase-2 (COX-2)-dependent phase of prostaglandin D(2) (PGD(2)) generation in bone marrow-derived mast cells (IC(50), 23.5 microM). Western blotting with specific anti-COX-2 antibodies showed that the decrease in PGD(2) production was accompanied by a decrease in COX-2 protein levels. In addition, this compound consistently inhibited the production of leukotriene C(4) in a dose dependent manner, (IC(50), 2.5 microM). These results demonstrate that MP inhibits both cyclooxygenase-2 and 5-lipoxygenase activity. Furthermore, this compound also inhibited the degranulation reaction (IC(50), 4.1 microM). Therefore, this compound might provide a basis for novel anti-inflammatory drug development.

Isoimperatorin (4-[(3-Methyl-2-butenyl)oxy]-7H-furo[3,2-g][1]benzopyran-7-one) is a medicinal herbal product that is isolated from the dried roots of Angelicae dahuricae. Isoimperatorin inhibits the cyclooxygenase-2 (COX-2) and COX-1-dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner, with IC50 values of 10.7 microM and 24 microM, respectively. However, this compound was not able to inhibit COX-1 and 2 protein expression in BMMC that were treated with concentrations of up to 50 microM, which indicates that isoimperatorin directly inhibits COX-2 activity. Furthermore, this compound consistently inhibited the production of leukotriene C4 (LTC4), as well as the degranulation reaction in BMMC, with an IC50 value of 5.7 microM and 9 microM, respectively, and these effects occurred in a dose dependent fashion. These results demonstrate that isoimperatorin has a dual cyclooxygenase-2 selective/5-lipoxygenase inhibitory activity, and therefore may provide the basis for novel anti-inflammatory drugs.

The present study aims to design a multilayer microstrip antenna with composite sandwich construction and investigate fatigue behavior of this multilayer SAS (surface antenna structure) that was asymmetric sandwich structure for the next generation of structural surface technology. This term, SAS, indicates that the structural surface becomes an antenna. Constituent materials were selected considering electrical properties, dielectric constant and tangent loss as well as mechanical properties. For the antenna performance, antenna elements inserted into structural layers were designed for satellite communication at a resonant frequency of 12.2 GHz. From electrical measurements, it was shown that antenna performances were in good agreement with design requirements. In cyclic 4-point bending, flexure behavior was investigated by static and fatigue test. Fatigue life curve of the SAS was obtained. The experimental results of bending fatigue were compared with single load level fatigue life prediction equation...

Ym1 and Ym2 (Ym1/2) are chitinase-like proteins and we reported previously that IL-4 induced Ym1/2 in mouse bone marrow-derived mast cells. In the present study, ovalbumin-induced asthmatic mice were used to investigate the effect of glucocorticoids on Ym1/2 expression. Ym1/2 were highly induced in bronchoalveolar lavage fluid (BALF) and the lung. Ym1/2 expression was completely inhibited by dexamethasone (Dex) in BALF and weakly inhibited in the lung. Primary cultured macrophages were used to investigate the inhibition of Ym1/2 expression at the cellular level. Although Dex pretreatment inhibited the Ym1/2 expression level in an animal model, it did not reduce IL-4 induction of Ym1/2 expression in vitro. Next, we tested whether Dex blocks IL-4 induced STAT6 signaling and found that it had no inhibitory effect on the phosphorylation level of STAT6 in macrophages. The luciferase reporter assay also revealed that Dex did not inhibit IL-4 induction of Ym1/2 promoter activity. These results indicate that the inhibitory effect of Dex on Ym1/2 protein expression in the murine model of asthma does not involve the STAT6 signaling pathway.

Anti-bone resorption properties of the Korean herbal formulation, Gami-Honghwain (HJ), which comprises Carthamus tinctorius L. seed and hominis placenta, were investigated. We demonstrate that the production of PGE2 is inhibited by 20-100 microg/ml HJ in nontransformed osteoblastic cells (MC3T3-E1 cells), indicating that HJ inhibits PGE2 production. The effect of HJ on the proliferation and osteoblastic differentiation in MC3T3-E1 was also studied. HJ dose-dependently increased DNA synthesis (significant at 20-100 microg/ml), and increased alkaline phosphatase (ALP) and prolyl hydroxylase activities of MC3T3-E1 cells (20-100 microg/ml), while anti-estrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1 which was induced by HJ. These results indicate that HJ directly stimulates cell proliferation and differentiation of osteoblasts. Also, when we assessed the effects of HJ on osteoblastic differentiation in MC3T3-E1, HJ enhanced ALP activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the HJ was observed at relatively low doses (significant at 20-100 microg/ml and maximal at 100 microg/ml). Northern blot analysis showed that the HJ (60 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. HJ (100 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that HJ has anabolic effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases.

Caffeic acid phenyl ester (CAPE), a biologically active ingredient of propolis, has several interesting biological properties including antioxidant, anti-inflammatory, antiviral, immunostimulatory, anti-angiogenic, anti-invasive, anti-metastatic and carcinostatic activities. Recently, several groups have reported that CAPE is cytotoxic to tumor cells but not to normal cells. In this study, we investigated the mechanism of CAPE-induced apoptosis in human myeloid leukemia U937 cells. Treatment of U937 cells with CAPE decreased cell viability in a dose-dependent and time-dependent manner. DNA fragmentation assay revealed the typical ladder profile of oligonucleosomal fragments in CAPE-treated U937 cells. In addition, as evidenced by the nuclear DAPI staining experiment, we observed that the nuclear condensation, a typical phenotype of apoptosis, was found in U937 cells treated with 5 microg/ml of CAPE. Therefore, it was suggested that CAPE is a potent agent inducing apoptosis in U937 cells. Apoptotic action of the CAPE was accompanied by release of cytochrome C, reduction of Bcl-2 expression, increase of Bax expression, activation/cleavage of caspase-3 and activation/cleavage of PARP in U937 cells, but not by Fas protein, an initial mediator in the death signaling, or by phospho-eIF2 alpha and CHOP, crucial mediators in ER-mediated apoptosis. From the results, it was concluded that CAPE induces the mitochondria-mediated apoptosis but not death receptors- or ER-mediated apoptosis in U937 cells.

Several prototypes of C–C biflavones ( a – f ) were synthesized and evaluated their inhibitory activity against phospholipase A 2 s (PLA 2 s) activity. The synthetic C–C biflavones ( a – f ) showed rather different inhibitory activity against various PLA 2 s. Most synthetic C–C biflavonoids exhibited potent and broad inhibitory activity against various sPLA 2 s and cPLA 2 tested regardless of their structural array. In particular, of natural and synthetic biflavonoids tested, the synthetic C–C biflavonoid ( d ) only showed inhibitory activity against sPLA 2 X. None of the natural and synthetic biflavonoids tested showed inhibitory activity against sPLA 2 IB. Further chemical modification of these basic structures will be carried out in order to investigate the synthetic C–C biflavones which possess more selective inhibitory activity against isozymes of PLA 2 .

The anti-allergic action of various Oriental medicinal herbs was investigated using in vitro and in vivo experimental models. Of these extracts, the ethanol extract of Meliae cortex (MC) exhibited the most potent activity in mast cells; its IC(50) values were 29+/-1.5 microg/ml for antigen stimulation and 57+/-3.4 microg/ml for thapsigargin stimulation. It inhibited compound-48/80-induced systemic anaphylaxis by 52.9% at a dose of 300 mg/kg in mice; it also inhibited the expression of the proinflammatory mediator TNF-alpha. With regard to its mechanism of action, MC suppressed the activating phosphorylation of Syk, a key enzyme in mast-cell signaling processes and that of Akt in a dose-dependent manner. It also inhibited the MAP kinase ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of the activating phosphorylation of ERK1/2. Taken together, these results suggest that the anti-allergic activity of MC may be due to the inhibition of histamine secretion and cytokine expression through the Syk inhibition in mast cells.

Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetylhydrolase (PAF-AH) activity has been identified in bovine colostrum and high levels of this activity are found in early colostrum (within 24 h after parturition). In this study, PAF-AH in early colostrum was purified by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl 650M, DEAE-Sepharose, heparin-Sepharose, hydroxyapatite, chelating-Sepharose and Mono Q HPLC column chromatography. This enzyme is a monomeric polypeptide with a molecular weight of approximately 45 kDa on 12.5% SDS-PAGE. The V(max) and K(m) for PAF-AH were 87.6 microM and 7.96 nmol/min/mg respectively. This enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetamide and p-bromophenacylbromide, suggesting that both serine and histidine residues are required for enzyme activity. It was not inactivated by NaF or dithiothreitol. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. Accordingly, this enzyme is distinct from phospholipase A(2). In addition, PAF-AH selectively hydrolyzed oxidatively modified phosphatidylcholine. Furthermore, this enzyme was shown by Western blot analysis using antibody to human plasma PAF-AH to be plasma type PAF-AH. These results clearly demonstrate that 45 kDa plasma type PAF-AH activity exists in bovine colostrum.

Methyl gallate (MG) is a medicinal herbal product that is isolated from Paeonia lactiflora that inhibits cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with an IC50 values of 17.0 microM. This compound also found inhibited the COX-2-dependent conversion of the exogenous arachidonic acid to PGD2 in a dose-dependent manner with an IC50 values of 19.0 microM, using a COX enzyme assay kit. However, at concentrations up to 80 microM, MG did not inhibit COX-2 protein expression in BMMC, indicating that MG inhibits COX-2 activity directly. Furthermore, MG consistently inhibited the production of leukotriene C4 (LTC4) in a dose dependent manner, with an IC50 value of 5.3 microM. These results demonstrate that MG has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity, which might provide the basis for novel anti-inflammatory drugs.

Ochnaflavone is a medicinal herbal product isolated from Lonicera japonica that inhibits cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC50 values of 0.6 microM. Western blotting probed with specific anti-COX-2 antibodies showed that the decrease in quantity of the PGD2 product was accompanied by a decrease in the COX-2 protein level. In addition, this compound consistently inhibited the production of leukotriene C4 (LTC4) in a dose dependent manner, with an IC50 value of 6.56 microM. These results demonstrate that ochnaflavone has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity. Furthermore, this compound strongly inhibited degranulation reaction in a dose dependent manner, with an IC50 value of 3.01 microM. Therefore, this compound might provide a basis for novel anti-inflammatory drugs.

A series of substituted rutaecarpines were prepared by employing Fischer indole synthesis as key step and their inhibitory activities on COX-1 and 2 as well as selectivity on COX-2 were evaluated. The compounds with a methanesulfonyl and a bromo group at C10 showed promising inhibitory activity (IC 5 0 = 0.27, 0.35 μM, respectively) with selectivity.

Ginkgetin, a biflavone from Ginkgo biloba leaves, was previously reported to be a phospholipase A2 inhibitor and this compound showed the potent antiarthritic activity in rat adjuvant-induced arthritis as well as analgesic activity. This investigation was carried out to find effects on cyclooxygenase-2 (COX-2) in vitro effect. Ginkgetin inhibits COX-2 dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC50 values of 0.75 μM. Western blotting probed with specific anti-COX-2 antibodies showed that the decrease in quantity of the PGD2 product was accompanied by a decrease in the COX-2 protein level. In addition, this compound consistently inhibited the production of leukotriene C4 (LTC4) in a dose dependent manner, with an IC50 value of 0.33 μM. These results demonstrate that ginkgetin has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity. Furthermore, this compound also inhibited degranulation reaction in a dose dependent manner, with an IC50 value of 6.52 μM. Therefore, this compound might provide a basis for novel anti-inflammatory agents.

Patridoids I, II and IIA, are new iridoids isolated from the whole plants of Patrinia saniculaefolia. These compounds were examined by assessing their effects on the production of tumor necrosis factor-alpha (TNF-alpha) as well as by investigating the expression of two enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in the lipopolysaccharide (LPS)-stimulated murine macrophage-like cell line, Raw 264.7. Among them, patridoid II consistently inhibited nitric oxide (NO) and TNF-alpha production in a dose-dependent manner, with IC (50) values of 14.1 and 17.6 microM, respectively. Western Blotting probed with specific anti-iNOS antibodies showed that the decrease in the quantity of the NO product was accompanied by a decrease in the iNOS protein level. However, all three patridoids did not affect the COX-2 protein expression level in the LPS-stimulated macrophages. In addition, the C-5 isomer of patridoid II, patridoid I, only slightly affected the production of NO. Moreover, the C-3 isomer of patridoid II, patridoid IIA, did not inhibit proinflammatory cytokines and NO production. These results suggest that the orientations of the C-3 and C-5 methoxy groups in the patridoids have a significant role in the expression of their biological activity.

A new non-destructive fatigue prediction model for composite laminates is developed. The natural frequencies of fatigue-damaged laminates under extensional loading are related to the fatigue life of the laminates by establishing the equivalent flexural stiffness reduction as a function of the elastic properties of sublaminates. In particular, the stiffness degradation in 90° laminae is recast by exploring the 90°-ply elastic modulus reduction, and by using the concept of a linear fracture mechanism for the cracks in 90° plies, where the crack-growth displacement is defined to give the correct average crack density in the cross-ply laminate. This crack density is then used in the stiffness reduction model. The flexural stiffness is derived by relating the 90°-ply elastic modulus reduction, and using the laminate plate theory to the degraded elastic modulus and the intact elastic modulus of other laminae. The natural-frequency reduction model, in which the dominant fatigue mode can be identified from the sensitivity scale factors of sublaminate elastic properties, provides natural frequency vs. fatigue curves for the composite laminates. Vibration tests were also conducted on [90 2 /0 2 ] s carbon/epoxy laminates to verify the natural-frequency reduction model. Correlations between the predictions of the model and experimental results are good.

Ethnopharmacological relevance 15,16-Dihydrotanshinone I (DHT-I), isolated from the dried root of Salvia miltiorrhiza Bung, which is traditionally used to treat cardiovascular and inflammatory diseases agent in Chinese medicine. DHT-I has been reported to have a broad range of biological activities, including antibacterial activity, and has been used to treat circulatory disorders, hepatitis, inflammation, cancer, and neurodegenerative diseases. Aim of the study The aim of this study was to evaluate the anti-allergic inflammatory effects of DHT-I on degranulation and on the generation of eicosanoids, such as, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4), in IgE/Ag-stimulated bone marrow-derived mast cells (BMMCs). Materials and methods The anti-allergic inflammatory activity of DHT-I was evaluated using BMMCs. The effects of DHT-I on mast cell activation were investigated by following degranulation and eicosanoid generation using ELISA and immunoblotting and immunoprecipitation techniques. Results DHT-I at a concentration of 20 μM markedly inhibited degranulation and the generation of PGD2 and LTC4 in IgE/Ag-stimulated BMMCs (about 90% inhibitions, respectively). Analyses of FceRI-mediated signaling pathways demonstrated that DHT-I inhibited the phosphorylations of spleen tyrosine kinase (Syk) and linker for activation of T cells (LAT), and inhibited downstream signaling process, including [Ca2+]i mobilization induced by the phosphorylation of phospholipase Cγ1 (PLCγ1), and the activations of mitogen-activated protein kinases (MAPKs) and the Akt-nuclear factor-κB (NF-κB) pathway. Conclusions DHT-1 inhibits the release of allergic inflammatory mediators from IgE/Ag-stimulated mast cells by suppressing a FceRI-mediated Syk-dependent signal pathway. This result suggests DHT-I offers a novel developmental basis for drugs targeting allergic inflammatory diseases.

The production of nitric oxide in mast cells has been difficult to measure due to the low amounts made by mast cells, as well as limitations in the specificity and sensitivity of the assays available. We present here a sensitive and specific 96-well plate-based method to directly measure NO using the cell-permeable fluorescent compound DAF-FM diacetate.

Mast cells (MC) are widely distributed throughout the body and are common at mucosal surfaces, a major host-environment interface. MC are functionally and phenotypically heterogeneous depending on the microenvironment in which they mature. Although MC have been classically viewed as effector cells of IgE-mediated allergic diseases, they are also recognized as important in host defense, innate and acquired immunity, homeostatic responses, and immunoregulation. MC activation can induce release of pre-formed mediators such as histamine from their granules, as well as release of de novo synthesized lipid mediators, cytokines, and chemokines that play diverse roles, not only in allergic reactions but also in numerous physiological and pathophysiological responses. Indeed, MC release their mediators in a discriminating and chronological manner, depending upon the stimuli involved and their signaling cascades (e.g., IgE-mediated or Toll-like receptor-mediated). However, the precise mechanisms underlying differential mediator release in response to these stimuli are poorly known. This review summarizes our knowledge of MC mediators and will focus on what is known about the discriminatory release of these mediators dependent upon diverse stimuli, MC phenotypes, and species of origin, as well as on the intracellular synthesis, storage, and secretory processes involved.

The production of nitric oxide in mast cells has been difficult to measure due to the low amounts made by mast cells, as well as limitations in the specificity and sensitivity of the assays available. We present here a sensitive and specific 96-well plate-based method to directly measure NO using the cell-permeable fluorescent compound DAF-FM diacetate.

Objective and Design: We investigated the effect of a new class of COX-2 inhibitor, rutaecarpine, on the production of PGD2 in bone marrow derived mast cells (BMMC) and PGE2 in COX-2 transfected HEK293 cells. Inflammation was induced by γ-carrageenan in male Splague-Dawley (SD) rats.¶Material: Rutaecarpine (8,13-Dihydroindolo[2',3':3,4]pyridol[2,1-b]quinazolin-5(7H)-one) was isolated from the fruits of Evodia rutaecarpa. BMMC were cultured with WEHI-3 conditioned medium. c-Kit ligand and IL-10 were obtained by their expression in baculovirus.¶Methods: The generation of PGD2 and PGE2 were determined by their assay kit. COX-1 and COX-2 protein and mRNA expression was determined by BMMC in the presence of KL, LPS and IL-10.¶Treatment: Rutaecarpine and indomethacin dissolved in 0.1% carboxymethyl cellulose was administered intraperitoneally and, 1h later, γ-carrageenan solution was injected to right hind paw of rats. Paw volumes were measured using plethysmometer 5h after γ-carrageenan injection.¶Results: Rutaecarpine inhibited COX-2 and COX-1 dependent phases of PGD2 generation in BMMC in a concentration-dependent manner with an IC50 of 0.28 μM and 8.7 μM, respectively. It inhibited COX-2-dependent conversion of exogenous arachidonic acid to PGE2 in a dose-dependent manner by the COX-2-transfected HEK293 cells. However, rutaecarpine inhibited neither PLA2 and COX-1 activity nor COX-2 protein and mRNA expression up to the concentration of 30 μM in BMMC, indicating that rutaecarpine directly inhibited COX-2 activity. Furthermore, rutaecarpine showed in vivo anti-inflammatory activity on rat γ-carrageenan induced paw edema by intraperitoneal administration.¶Conclusion: Anti-inflammatory activity of Evodia rutaecarpa could be attributed at least in part by inhibition of COS-2.

A series of disubstituted 4,5-polymethylenepyrazoles were synthesized and evaluated their inhibitory activities against COX-2. Some compounds showed strong (0.3 nM) inhibitory activity on COX-2 and were found somewhat selective (up to 16) on COX-2 over COX-1.

Emerging evidence has suggested the pivotal role of mast cells in a host defense against bacterial infection. In this paper, we report that bacterial lipopolysaccharide (LPS) is a potent enhancer of the cytokine- and IgE-dependent delayed responses of IL-3-dependent mouse bone marrow-derived cultured mast cells (BMMC). LPS, although showing minimal effects, significantly augmented the c-kit ligand (KL)- or IgE-dependent expression of cyclooxygenase (COX)-2 and the attendant delayed PGD2 generation, with IL-10 and IL-4 acting as potentiating and inhibitory cytokines, respectively. The COX-2-inducing activity of LPS was mimicked by exogenous IL-1 beta. Assessment of endogenous cytokine induction revealed that IL-1 beta expression was stimulated by either LPS or exogenous IL-1 beta. IL-6 expression occurred in parallel with COX-2 expression. IL-10 expression, which lagged behind COX-2 expression, depended on exogenous IL-10, but not on LPS and IL-1 beta. Thus, LPS and IL-1 beta exhibited similar biological activities in terms of COX-2 and endogenous cytokine expression. However, adding an antibody against the type I IL-1 receptor to BMMC, which abrogated the effects of IL-1 beta, failed to neutralize the effects of LPS. These results suggest that LPS activates BMMC through the signal transduction pathway shared with exogenous IL-1 beta, rather than exerting its action indirectly via the production of endogenous IL-1 beta.

This study investigated the protective effects of a group IIA secretory phospholipase A2 (sPLA2-IIA) inhibitor, ochnaflavone, on the progression of carbon tetrachloride (CCl4)-induced acute liver injury in rat liver microsomes in vitro. When rat liver was incubated at 37 degrees C in the presence of CCl4, the level of phosphatidylethanolamine (PE) degradation increased markedly compared with the control. The rat 14 kDa platelet PLA2 antibody, R377, suppressed the degradation of PE. Pretreating the microsome with ochnaflavone (2-16 microM) reduced the level of PE degradation in a dose dependent manner. In addition, p-bromophenacy bromide (p-BPB), which is a PLA2 inhibitor, also inhibited PE degradation. However, the inhibitory activity was weaker than that of ochnaflavone. Further investigation showed that ochnaflavone not only inhibited the purified rat platelet sPLA2 activity in a dose dependent manner with an IC50 value of 3.45 microM, when arachidonyl PE was used as a substrate, but also inhibited lipid peroxidation in a dose dependent manner with an IC50 value of 7.16 microM. This result suggests that ochnaflavone prevents the progression of CCl4-induced PE hydrolysis by inhibiting the endogenous sPLA2 activity.

This study examined the effect of astilbic acid (3beta,6beta-dihydroxyolean-12-en-27-oic acid), which is a herbal medicine isolated from Astilbe chinensis. Astilbic acid inhibited 5-lipoxygenase (5-LOX)-dependent leukotriene C4 (LTC4) generation in bone marrow-derived mast cells in a concentration-dependent manner with an IC50 value of 2.1 microM. In addition, astilbic acid was tested in a rat passive cutaneous anaphylaxis (PCA) reaction assay by administering 10 to 50 mg/kg i.p. Astilbic acid dose dependently inhibited the PCA reaction, which was activated by anti-dinitrophenyl (DNP) IgE. Furthermore, this compound inhibited mouse acetic acid-induced writhing (47-62% inhibition at 0.4-50 mg/kg) after being administered orally, while aspirin (200 mg/kg) showed 62% inhibition. These results suggest that astilbic acid may be beneficial in regulating various inflammatory processes.

Background Extracellular signal-regulated kinases 1/2 (ERK1/2) make important contributions to allergic responses via their regulation of degranulation, eicosanoid production, and cytokine expression by mast cells, yet the mechanisms underlying their positive effects on FceRI-dependent signaling are not fully understood. Recently, we reported that mast cell activation and anaphylaxis are negatively regulated by AMP-activated protein kinase (AMPK). However, little is known about the relationship between ERK1/2-mediated positive and the AMPK-mediated negative regulation of FceRI signaling in mast cells. Objective We investigated possible interactions between ERK1/2 and AMPK in the modulation of mast cell signaling and anaphylaxis. Methods Wild-type or AMPK α 2 −/− mice, or bone marrow–derived mast cells obtained from these mice, were treated with either chemical agents or small interfering RNAs that modulated the activity or expression of ERK1/2 or AMPK to evaluate the functional interplay between ERK1/2 and AMPK in FceRI-dependent signaling. Results The ERK1/2 pathway inhibitor U0126 and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside similarly inhibited FceRI-mediated mast cell signals in vitro and anaphylaxis in vivo . ERK1/2-specific small interfering RNA also mimicked this effect on FceRI signals. Moreover, AMPK α 2 knockdown or deficiency led to increased FceRI-mediated mast cell activation and anaphylaxis that were insensitive to U0126 or activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside, suggesting that the suppression of FceRI signals by the inhibition of the ERK1/2 pathway relies largely on AMPK activation. ERK1/2 controlled AMPK activity by regulating its subcellular translocation. Conclusions ERK1/2 ablated the AMPK-dependent negative regulatory axis, thereby activating FceRI signals in mast cells.

This study examined the effect of a podophyllotoxin derivative, deoxypodophyllotoxin (anthricin), which is a medicinal herb product isolated from Anthriscus sylvestris Hoffm. Deoxypodophyllotoxin was tested in a rat PCA (passive cutaneous anaphylaxis) assay by administering deoxypodophyllotoxin intraperitoneally (1.0 to 10 mg/kg, i.p.) and intravenously (0.25 to 1.0 mg/kg, i.v.). Deoxypodophyllotoxin dose-dependently inhibited the PCA reaction activated by anti-dinitrophenyl (DNP) IgE. The PCA inhibitory activity of deoxypodophyllotoxin was stronger than those of prednisolone and indomethacin, which were used as positive controls. These results suggest that deoxypodophyllotoxin may be beneficial in regulating the immediate-type allergic reaction.

Background Nitric oxide (NO) is a short-lived, reactive molecule generated by nitric oxide synthase (NOS). Three isoforms of NOS have been identified including: neuronal NOS (NOS1), endothelial NOS (NOS3) and inducible NOS (NOS2). Objective To identify expression of isoforms of NOS in nasal polyps (NP) and normal nasal tissue, and to determine if differences exist in NOS expression in NP rich in eosinophils (Eo-hi) or with few eosinophils (Eo-low). Methods NP were obtained after endoscopic surgery for chronic rhinosinusitis, and separated into two categories based on eosinophil density. Nasal middle turbinates (MT) were also collected as normal controls from patients who had undergone surgery for pituitary adenomas. To identify cell types expressing isoforms of NOS, double immunostaining was performed using anti-NOS and anti-leukocyte antibodies (e.g. mast cells [MC], eosinophils, T cells and macrophages). Results Expression of NOS isoforms in all cell types was greater in NP than in MT. Number of NOS2 positive cells in Eo-hi NP was higher than in Eo-low NP. However, there were no differences in the numbers of NOS1 and NOS3 positive cells between Eo-hi and Eo-low NP. Both NOS2 positive MC and NOS2 positive Eo were significantly greater in NP than in MT. In addition, the percentage of NOS2 positive MC in Eo-hi NP was significantly higher than in Eo-low NP. Conclusion Elevation in NOS2 expression in several cell types might be an important factor in the life history of NP, especially those with an abundance of eosinophils.

Background Prostaglandin D2 (PGD2) has long been implicated in allergic diseases such as asthma by contributing to bronchoconstriction, vasodilation, and vascular permeability. Recently, cloning of a second novel PGD2 receptor CRTh2, led to a greater understanding of the physiological and pathophysiological implications of PGD2. PGD2 signaling through DP1 and CRTh2 (DP2) mediates different and often opposite effects in many cell types of the immune system. Although mast cells (MC) are the largest source of PGD2 in the body, there is lack of information about their expression and the role of PGD2 receptors.

NO is a short-lived free radical that plays a critical role in the regulation of cellular signaling. Mast cell (MC)-derived NO and exogenous NO regulate MC activities, including the inhibition of MC degranulation. At a molecular level, NO acts to modify protein structure and function through several mechanisms, including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MCs, we investigated protein tyrosine nitration in human MC lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione. Using two-dimensional gel Western blot analysis with an anti-nitrotyrosine Ab, together with mass spectrometry, we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MCs. The nitration of aldolase A was associated with a reduction in the maximum velocity of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance analysis showed that despite these changes in the activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that S-nitrosoglutathione treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose 1,6-bisphosphate. Interestingly, fructose 1,6-bisphosphate inhibited IgE-mediated MC degranulation in LAD2 cells. Thus, for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target. This posttranslational nitration of aldolase A may be an important pathway that regulates MC phenotype and function.

Mast cells are classically viewed as effector cells of IgE-mediated allergic diseases. However, over the last decade our understanding has been enriched about their roles in host defense, innate and adaptive immune responses, and in homeostatic responses, angiogenesis, wound healing, tissue remodeling, and immunoregulation. Despite impressive progress, there are large gaps in our understanding of their phenotypic heterogeneity, regulatory mechanisms involved, and functional significance. This review summarizes our knowledge of mast cells in innate and acquired immunity, allergic inflammation and tissue homeostasis, as well as some of the regulatory mechanisms that control mast cell development, phenotypic determination, and function, particularly in the context of mucosal surfaces.

Mast cells (MC) originate from CD34+ progenitor cells in bone marrow, but they are highly heterogeneous depending on local microenvironmental factors where they finally mature. The local microenvironment can markedly alter gene expression, responsiveness to secretagogues, MC phenotype and function. Although there is unequivocal evidence that exogenous NO has effects on MC function such as degranulation, and cytokine, chemokine and eicosanoid production, there has been conflicting reports about nitric oxide synthase (NOS) expression and endogenous production of NO in MC. The mechanism of action of NO on MC activities is mediated by posttranslational modification of target proteins through nitration and nitrosylation. We propose a new conceptual model of NOS expression in MC and provide an overview of the literature on the NOS expression and NO production by MC that may help resolve differences in the literature.

The matrix metalloproteinases (MMP-9 and MMP-2) in aortic smooth muscle cells (SMC) play key roles in the pathogenesis atherosclerosis. The SMC migration into the vascular wall via the bloodstream is directly linked with MMP-9 expression. Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan with anti-inflammatory activity, was isolated from Anthriscus sylvestris Hoffm. and has been known inhibit the expression of MMP-9 in tumor necrosis factor-alpha (TNF-alpha) stimulated human aortic smooth muscle cells (HASMC). In this study, DPT was purified and demonstrated to inhibit the MMP-9/2 activities in TNF-alpha-induced HASMC. In addition, MMP-9 expression and migration was strongly inhibited by DPT in TNF-alpha-induced HASMC. To examine whether TNF-alpha-induced MMP-9 expressions are involved with migrations of HASMC, reverse transcription-polymerase chain reaction (RT-PCR) and luciferase-tagged promoter analysis were applied. These experiments revealed that DPT inhibited the mRNA transcription of MMP-9 gene expression. Furthermore, Western blot analysis indicated that the TNF-alpha-induced phosphorylation of extracellular signal regulated kinase 1 and 2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) were strongly inhibited by DPT. From these results, it is concluded that DPT has an inhibitory activities on migration and MMP-2/9 activities, and MMP-9 transcription in HASMC.

Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan with anti-inflammatory activity, was isolated from Anthriscus sylvestris HOFFM., and we examined its effects on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated, murine macrophage-like RAW264.7 cells. Western blot analysis performed with specific anti-iNOS antibodies showed that a decrease in nitric oxide (NO) was accompanied by a decrease in the iNOS protein level. To clarify the mechanistic basis for DPT's ability to inhibit iNOS induction, we examined the effect of DPT on nuclear factor (NF)-kappaB transcriptional activity and DNA binding activity. DPT potently suppressed both reporter gene activity and DNA binding activity. These findings suggest that DPT in RAW264.7 cells abolished LPS-induced iNOS expression by inhibiting the transcription factor, NF-kappaB.

Nitric oxide (NO) is an important signaling molecule that regulates MC function. However, the involvement of NO in an important lipid mediator, prostaglandin (PG) D(2) production by MC, is unclear. The role of NO in cyclooxygenase (COX)-2 expression and PGD(2) generation as well as IL-6 production in mouse bone marrow-derived MC (BMMC) was investigated using NO donors. Exogenous NO augmented COX-2 protein expression and increased COX-2-dependent PGD(2) generation in response to SCF, IL-10, and IL-1beta, or antigen activation in combination with IL-10 and IL-1beta after sensitization with IgE. The increased expression of COX-2 by NO donors was inhibited by hemoglobin. Moreover it was not affected by soluble guanylyl cyclase inhibitor, but reduced by the p38 MAPK inhibitor, SB202190. Downstream of p38 MAPK, NO donors augmented not only COX-2 mRNA transcription but also its stability. Exogenous NO also augmented IL-6 production by SCF, IL-10, and IL-1beta. These results show that exogenous NO can increase COX-2-dependent PGD(2) and IL-6 production by MC in inflammatory environments through the p38 MAPK pathway. Therefore, our novel observations suggest that the effect of NO on MC is not limited to the suppression of their activation as has been the emphasis previously, but can also augment certain MC responses.