Immunomodulation of Zerumbone via Decreasing the Production of Reactive Oxygen Species from Immune Cells.

BACKGROUND AND OBJECTIVE: Zerumbone has been reported to exert anti-inflammatory, anti-ulcer and anti-hyperglycemic effects but the specific mechanism through which zerumbone exerts its anti-inflammatory action through inhibiting reactive oxygen species was not well studied. Hence, this paper studied the zerumbone capacity to inhibit intracellular and extracellular Reactive Oxygen Species (ROS) produced by whole blood cell, polymorphoneutrophil (PMNs) and macrophage cells due to the zymogen and phorbolmyristerate acetate (PMA) oxidant effect. MATERIALS AND METHODS: Zymogen and PMA based chemiluminescence assay were used to determine the immunomodulatory effect of zerumbone at concentrations (100, 10 and 1 µg mL-1) toward production of Reactive Oxygen Species (ROS) from whole blood, PMNs and macrophage. RESULTS: Zerumbone significantly inhibited intracellular and extracellular ROS production by the zymosan/PMA-activated phagocyte cells with IC50 values of (16.3±0.1, 23.7±0.1 and 4.97±0.1 µg mL-1) against whole blood, PMNs and macrophage respectively. CONCLUSION: The anti-inflammatory activity of zerumbone was so much significant that even strong oxidant (zymogen and PMA) were not able to produce reactive oxygen species when incubated together in phagocytic cells, thus suppress production of ROS. Therefore, it is highly used in herbal medicine as a potent immunomodulatory therapy in various inflammation associated diseases.

PMA induces vaccine adjuvant activity by the modulation of TLR signaling pathway.

Toll-like receptor (TLR) ligands are being developed for use as vaccine adjuvants and as immunomodulators because of their ability to stimulate innate and adaptive immune responses. Flagellin, a TLR5 ligand, was reported to show potent mucosal vaccine adjuvant activity. To identify ligands that potentiate the adjuvant activity of flagellin, we screened a plant library using HEK293T cells transiently cotransfected with phTLR5 and pNF- κ B-SEAP plasmids. The 90% EtOH extract from Croton tiglium showed significant NF- κ B transactivation in a TLR5-independent manner along with the increase of a flagellin activity. We have studied to characterize an active component from Croton tiglium and to elucidate the action mechanisms. Phorbol 12-myristate 13-acetate (PMA) was isolated as an active component of Croton tiglium by activity-guided fractionation, column chromatography, HPLC, NMR, and MS. PMA at a range of nM induced PKC-dependent NF- κ B activation and IL-8 production in both TLR5- and TLR5+ assay systems. In in vivo mouse vaccination model, PMA induced antigen-specific IgG and IgA antibody responses and increased IL-12 production corresponding to T cell responses in spleen lymphocytes. These results suggest that PMA would serve as an efficacious mucosal vaccine adjuvant.

Structure determination of inonotsuoxides A and B and in vivo anti-tumor promoting activity of inotodiol from the sclerotia of Inonotus obliquus.

Two new lanostane-type triterpenoids, inonotsuoxides A (1) and B (2) along with three known lanostane-type triterpenoids, inotodiol (3), trametenolic acid (4), and lanosterol (5), were isolated from the sclerotia of Inonotus obliquus (Pers.: Fr.) (Japanese name: Kabanoanakake) (Russian name: Chaga). Their structures were determined to be 22R,25-epoxylanost-8-ene-3beta,24S-diol (1) and 22S,25-epoxylanost-8-ene-3beta,24S-diol (2) on the basis of spectral data including single crystal X-ray analysis. These compounds except for 2 were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), as a test for potential cancer chemopreventive agents. The most abundant triterpene, inotodiol (3), was investigated for the inhibitory effect in a two-stage carcinogenesis test on mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. Compound 3 was found to exhibit the potent anti-tumor promoting activity in the in vivo carcinogenesis test.

Development of a generic dual-reporter gene assay for screening G-protein-coupled receptors.

Multiple assay formats have been developed for the pharmacological characterization of G-protein-coupled receptors (GPCRs) and for screening orphan receptors. However, the increased pace of target identification and the rapid expansion of compound libraries present the need to develop novel assay formats capable of screening multiple GPCRs simultaneously. To address this need, the authors have developed a generic dual-reporter gene assay that can detect ligand activity at 2 GPCRs within the same assay. Two stable HEK293 cell lines were generated expressing either a firefly (Photinus) luciferase gene under the control of multiple cAMP-response elements (CREs) or a Renilla luciferase gene under the control of multiple 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive elements (TREs). Coseeded reporter cells were used to assess ligand binding activity at both Galphas-and Galphaq-coupled receptors. By selectively coexpressing receptors with a chimeric G-protein, agonist activity was assessed at Galphai/o-coupled receptors in combination with either Galphas-or Galphaq-coupled receptors. The dual-reporter gene assay was shown to be capable of simultaneously performing duplexed screens for a variety of agonist and/or antagonist combinations. The data generated from the duplexed reporter assays were pharmacologically relevant, and Z' factor analysis indicated the suitability of both agonist and antagonist screens for use in high-throughput screening.

Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C.

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGK as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK translocation from the cytoplasm to the plasma membrane within 2-6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.

NKIAMRE, a novel conserved CDC2-related kinase with features of both mitogen-activated protein kinases and cyclin-dependent kinases.

We report the cloning of the NKIAMRE gene located on human chromosome 5q31.1. It encodes a novel 52kDa Cdc2-related kinase with a 1.5kb open reading frame. Like MAP kinases, NKIAMRE contains a Thr-X-Tyr (TXY) motif in the activation loop domain. Similar to cdks, NKIAMRE contains the putative negative regulatory Ser14 and Tyr15 residues and the cyclin-binding motif, NKIAMRE, from which it derives its name. Human NKIAMRE has significant amino acid identity to related kinases in rat, mouse, Caenorhabditis elegans, and Drosophila, and is widely expressed in human tissues and cell lines. Confocal microscopy demonstrates that NKIAMRE localizes to the cytoplasm. NKIAMRE is activated by treatment of cells with phorbol 12-myristate 13-acetate. Mutation of the ATP-binding Lys-33 to arginine and the Thr-Glu-Tyr motif to Ala-Glu-Phe abolished its ability to phosphorylate myelin basic protein. NKIAMRE is a member of a conserved family of kinases with homology to both MAP kinases and cyclin-dependent kinases.

[The inhibitory effect of a third generation retinoid, R8605, on protein kinase C activity in vitro].

R8605 is a newly synthesized compound of the third generation retinoids produced by the Institute of Materia Medica. It exhibited in vitro inhibitory effects on the diolein and TPA (12-O-tetradecanoylphorbol-13-acetate) induced activation of Protein Kinase C (PKC) from rat brain and also showed inhibitory effects on the calcium/phospholipid-independent phosphorylation of protamine by PKC. The IC50 values were 65, 70, 100/mumol/L respectively. It is suggested that the inhibition of PKC activity by R8605 is related to the inhibition of tumor promotion.