Search

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.

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.

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.

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.

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.

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 (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.

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.

In our ongoing search for anti-inflammatory agents originating from Korean medicinal plants, we found that the hexane and BuOH fractions of the MeOH extract from the whole plants of Melandrium firmum Rohrbach inhibited 5-lipoxygenase (5-LOX) activity. By activity-guided fractionation, eleven compounds, alpha-spinaterol (1), ursolic acid (2), ergosterol peroxide (3), alpha-spinaterol glucoside (4), 2-methoxy-9-beta-D-ribofuranosyl purine (5), aristeromycin (6), ecdysteron (7), polypodoaurein (8), (-)-bornesitol (9), mannitol (10) and cytisoside (11) were isolated from the hexane and BuOH fractions using column chromatography. Compounds 2, 5, 6, 8, 9, 10 and 11 were isolated for the first time from this plant. Compounds 1, 3, 4 and 7 inhibited 5-LOX activity with IC50 values of 21.04 microM, 42.30 microM, 32.82 microM, and 17.18 microM, respectively.

Ochnaflavone (OC), a naturally occurring biflavonoid with anti-inflammatory activity [S.J. Lee, J.H. Choi, H.W. Chang, S.S. Kang, H.P. Kim. Life Sci. 57(6), 1995, 551-558], was isolated from Lonicera japonica and its effects on inducible nitric oxide synthase (iNOS) gene expression was examined in RAW264.7 cells. U0126, an inhibitor of the extracellular signal-regulated kinase (ERK), significantly down-regulated lipopolysaccharide (LPS)-induced iNOS expression and promoter activity. Transactivation of LPS-stimulated NF-kappaB was inhibited by U0126. These results suggest that the transcription factor NF-kappaB is involved in ERK-mediated iNOS regulation and that activation of the Ras/ERK pathway contributes to the induction of iNOS expression in RAW264.7 cells in response to LPS. OC treatment inhibited the production of nitric oxide in a concentration-dependent manner and also blocked the LPS-induced expression of iNOS. These inhibitory effects were associated with reduced ERK1/2 activity. OC inhibited the phosphorylation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase. The findings herein show that the inhibition of LPS-induced ERK1/2 activation may be a contributing factor to the main mechanisms by which OC inhibits RAW264.7. To clarify the mechanistic basis for its ability to inhibit iNOS induction, we examined the effect of OC on the transactivation of the iNOS gene by luciferase reporter activity using the -1588 flanking region. OC potently suppressed reporter gene activity. We also report here, for the first time, that LPS-induced iNOS expression was abolished by OC in RAW264.7 cells through by blocking the inhibition of transcription factor NF-kappaB binding activities. These activities are associated with the down-regulation of inhibitor kappaB (IkappaB) kinase (IKK) activity by OC (6 microM), thus inhibiting LPS-induced phosphorylation as well as the degradation of IkappaBalpha. These findings suggest that the inhibition of LPS-induced NO formation by OC is due to its inhibition of NF-kappaB, which may be the mechanistic basis for the anti-inflammatory effects of OC.

Deoxypodophyllotoxin (Anthricin) is a medicinal herbal product isolated from Anthriscus sylvestris HOFFM. that inhibits cyclooxygenase-2 (COX-2) and COX-1-dependent phases of prostaglandin D(2) (PGD(2)) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC(50) values of 1.89 microM and 65.3 microM, respectively. This study also found that this compound inhibited COX-1 and 2-dependent conversion of the exogenous arachidonic acid to PGD(2) in a dose-dependent manner with an IC(50) values of 0.01 microM and 12.1 microM, respectively using a COX enzyme assay kit. However, this compound did not inhibit COX-2 protein expression up to a concentration of 30 microM in the BMMC, indicating that deoxypodophyllotoxin directly inhibits COX-2 activity. Furthermore, this compound consistently inhibited the production of leukotriene C(4) (LTC(4)) in a dose dependent manner, with an IC(50) value of 0.37 microM. These results demonstrate that deoxypodophyllotoxin has a dual cyclooxygenase-2 selective/5-lipoxygenase inhibitory activity, and therefore this compound might provide a basis for novel anti-inflammatory drugs.

Platelet activating factor acetylhydrolase (PAF-AH) activity has been identified in cerebrospinal fluid (CSF) samples taken from children with meningitis. We reported that PAF-AH activity is significantly increased, by about 3 fold, in patients with meningitis compared to control subjects. Because of limited knowledge about this enzyme in CSF, we examined the biochemical properties of CSF PAF-AH. PAF-AH of CSF was calcium independent, showed a broad pH spectrum and was relatively heat stable. In addition, this enzyme activity was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), partially inhibited by p-bromophenacylbromide (p-BPB), uninhibited by iodoacetamide, and moderately stimulated by dithiothreitol (DTT). PAF-AH of CSF did not degrade phospholipid with a long chain fatty acyl group at sn-2 position. This enzyme hydrolyzed PAF and oxidatively modified phosphatidylcholine. Furthermore, we identified a monomeric polypeptide with a molecular weight of approximately 45 kDa by Western blot using human plasma PAF-AH antibody. These results suggested that plasma type PAF-AH activity exist in CSF taken from children with meningitis.

Mast cells are critical for initiating innate immune and inflammatory responses by releasing a number of pro-inflammatory mediators. The potential immunomodulatory properties of hydrogenated aromatic hydrocarbons have been the subject of extensive investigation, as the immune system is a sensitive target for hydrogenated aromatic hydrocarbon toxicity. In this report, the effects of polychlorinated biphenyl (PCB) on the expression of cyclooxygenase-2 and pro-inflammatory cytokines such as interleukin-1beta (IL-1beta), IL-6 and tumor necrosis factor (TNF)-alpha in the human leukemic mast cell line were investigated. TNF-alpha and IL-1beta expressed their respective mRNA in the presence or absence of PCB, while cyclooxygenase-2 (COX-2) and IL-6 mRNA expression were highly induced by PCB after 2 h. Moreover, pre-treatment with the nuclear factor (NF)-kappaB pathway inhibitor, pyrrolidine dithiocarbamate, suppressed COX-2, TNF-alpha and IL-1beta induction and reduced the IL-6 mRNA levels induced by PCB. The NF-kappaB activity was determined by electrophoretic mobility shift analysis (EMSA) using an oligonucleotide containing a consensus NF-kappaB binding sequence. Stimulating the cells with PCB activated NF-kappaB. However, pre-treating them with a NF-kappaB pathway inhibitor, pyrrolidine dithiocarbamate, suppressed PCB-induced NF-kappaB activation. This suggests that PCB induces cycloxoygenase-2 and pro-inflammatory cytokine expression, and that this induction occurs through NF-kappaB.

Platelet-activating factor acetylhydrolase was analyzed in cerebrospinal fluid samples taken from children with a variety of neurological conditions (85 patients; mean age, 3.8 years) to determine it is involved in the defense mechanism against the toxic effect of inflammatory mediators in the central nervous system. A significant increase in cerebrospinal fluid activity was seen in the patients with meningitis and acute febrile illness in comparison with the control subjects. The activity was also significantly higher in the patients with meningitis than in the patients with inflammatory neurological diseases. In addition, the biochemical profile of cerebrospinal fluid platelet-activating factor acetylhydrolase was different from other known acetylhydrolases. These findings suggest that cerebrospinal fluid platelet-activating factor acetylhydrolase activity may be a sensitive marker of the host response to central nervous system infections.

PGD2 has long been implicated in allergic diseases. Recent cloning of a second PGD2 receptor, DP2 (also known as CRTh2), led to a greater understanding of the physiological and pathophysiological implications of PGD2. PGD2 signaling through DP1 and 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 little information about their potential expression of DP2 and its functional significance. In this study, we show that tissue MC in human nasal polyps express DP2 protein, and that human MC lines and primary cultured human MC express mRNA as well as protein of DP2. By immunohistochemistry, we detected that 34% of MC in human nasal polyps expressed DP2. In addition, flow cytometry showed that 87% of the LAD2 human MC line and 98% of primary cultured human MC contained intracellular DP2. However, we could not detect surface expression of DP2 on human MC by single cell analysis using imaging flow cytometry. Blocking of endogenous PGD2 production with aspirin did not induce surface expression of DP2 in human MC. Two DP2 selective agonists, DK-PGD2 and 15R-15-methyl PGD2 induced dose-dependent intracellular calcium mobilization that was abrogated by pertussis toxin, but not by three DP2 selective antagonists. MC mediator release including degranulation was not affected by DP2 selective agonists. Thus, human MC express DP2 intracellularly rather than on their surface, and the function of DP2 in human MC is different than in other immune cells such as Th2 cells, eosinophils and basophils where it is expressed on the cell surface and induces Th2 cytokine and/or granule associated mediator release. Further studies to elucidate the role of intracellular DP2 in human MC may expand our understanding of this molecule and provide novel therapeutic opportunities.

Two types of phospholipase A2 (PLA2) inhibitory protein (PLIP-I, PLIP-II) were detected and isolated from human amniotic fluid by Sephacryl S300 gel filtration chromatography. The lower molecular weight-fraction (PLIP-II) was further purified by Sephadex G75 gel filtration and analyzed by SDS-PAGE. Its molecular weight was estimated to be approximately 18 kDa, and it was sensitive to heat treatment. Inhibition of phospholipase A2 (sPLA2 type IIA) by PLIP-II occurred in a dose-dependent manner (IC50 about 0.82/microm), and the effect was stronger on sPLA2 IIA than on pancreatic sPLA2 (IC50 about 3.11 microL). The ratio of the inhibitions of the sPLA2 IIA by PLIP-II remained consistent over an entire range of substrate concentrations. Furthermore, addition of excess Ca2+ at concentrations of up to 10 mm did not antagonize the inhibitory activity of PLIP-II.

We investigated the effect of a new PAF antagonist pinusolide, isolated from the leaves of Biota orientalis, on PAF-induced [3H]serotinin release from rabbit platelets, hypotension and vascular permeability. Pinusolide (IC50, about 5 x 10(-6) M) inhibited specifically [3H]serotinin release from rabbit platelets when stimulated with PAF (5 x 10(-8) M), but showed no effect when induced by ADP, collagen, and thrombin. It also inhibited PAF-induced hypotension in a dose-dependent manner in rats with no effect on the hypotension induced by acetylcholine, histamine and serotonin. The inhibitory effect of pinusolide on the PAF-induced vascular permeability is less specific than the induced hypotension. These results suggest that pinusolide may prove of therapeutic value in the treatment of hypotension and a molecular design of pinusolide analogues may provide the possibility of a new PAF specific antagonists.