Yasuyuki Yagi

Induction of neurite outgrowth in PC12 cells by artemisinin through activation of ERK and p38 MAPK signaling pathways.

Growth of neurite processes is a critical step in neuronal development, regeneration, differentiation, and response to injury. The discovery of compounds that can stimulate neurite formation would be important for developing new therapeutics against both neurodegenerative disorders and trauma-induced neuronal injuries. Semisynthetic derivatives of artemisinin, an active compound in Artemisia annua, have been effectively used in malaria treatment, but they have been shown to possess neurotoxic potential. In this study, we found unexpectedly that artemisinin and its derivatives induced neurite outgrowth of PC12 cells. Artemisinins containing an endoperoxide bridge such as artemisinin and dihydroartemisinin induced growth of neurite processes at concentrations that were slightly cytotoxic, artemisinin having the most potent maximal effect among them. Deoxyartemisinin, which lacks the endoperoxide bridge, was ineffective. Artemisinin-treated cells expressed increased levels of the neuronal marker β(III)-tubulin. Artemisinin upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), critical signaling molecules in neuronal differentiation. Consistent with activation of the two MAPKs, neurite outgrowth induced by artemisinin was inhibited by the MAPK/ERK kinase inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Artemisinin also induced phosphorylation of cyclic AMP response element-binding protein (CREB) that was almost completely attenuated by PD98059 but not by SB203580. Taken together, our results indicate that artemisinin and its derivatives containing the endoperoxide bridge induced differentiation of PC12 cells toward a neuronal phenotype and suggest that both activation of ERK signaling pathway, which leads to CREB phosphorylation, and activation of p38 MAPK signaling pathway are involved in this process.

Phenanthrene derivatives from Cymbidium Great Flower Marie Laurencin and their biological activities.

A new phenanthrendione, ephemeranthoquinone B (1), two phenanthrenes, marylaurencinols A (2) and B (3), and a phenanthrene glucoside, marylaurencinoside A (4), were isolated from the roots of Cymbidium Great Flower Marie Laurencin, along with six known phenanthrenes, 5-10. The structures of these compounds were established by a combination of extensive NMR spectroscopy and/or X-ray crystallographic analysis and chemical degradation. The compounds were tested for antibacterial activities against Bacillus subtilis and Klebsiella pneumoniae and for cytotoxic activity against the human promyelocytic leukemia (HL-60) cell line. Compounds 1, 3, and 6 showed antibacterial activities with minimum inhibitory concentration (MIC) values in the range of 4.88 to 65.10 μM. Notably, ephemeranthoquinone B (1) had a strong antibacterial effect on B. subtilis. Furthermore, 1 exhibited moderate cytotoxic activity (IC(50) 2.8 μM) against HL-60 cells. Compounds 4-9 also showed weak cytotoxic activity against the HL-60 cell line with IC(50) values of 19.3-52.4 μM.

Zierane sesquiterpene lactone, cembrane and fusicoccane diterpenoids, from the Tahitian liverwort Chandonanthus hirtellus.

Cembrane-type diterpenoids, 13,18,20-epi-iso-chandonanthone (1) and (8E)-4alpha-acetoxy-12alpha,13alpha-epoxycembra-1(15),8-diene (2), two fusicoccane-type diterpenoids, fusicoauritone 6alpha-methyl ether (3) and 6beta,10beta-epoxy-5beta-hydroxyfusicocc-2-ene (4) and a zierane sesquiterpene gamma-lactone, chandolide (5) were isolated from the Tahitian liverwort Chandonanthus hirtellus (Web.) Mitt., together with eight known diterpenoids, chandonanthine (6), fusicogigantone A (7), fusicogigantone B (8), fusicogigantepoxide (9), anadensin (10), fusicoauritone (11), ent-verticillol (12) and ent-epi-verticillol (13). Their structures were established by a combination of extensive NMR spectroscopy and/or X-ray crystallographic analyses. Compounds 1, 5 and 10 showed weak cytotoxic activity against HL-60. Compound 3 also indicated weak cytotoxic activity against KB cell lines.

Cytotoxic, radical scavenging and antimicrobial activities of sesquiterpenoids from the Tahitian liverwort Mastigophora diclados (Brid.) Nees (Mastigophoraceae)

A drimane, (+)-drimenol (1), five known herbertanes, (−)-α-herbertenol (2), (−)-herbertenediol (3), mastigophorene A (4), (−)-mastigophorene C (5) and (−)-mastigophorene D (6), a pimarane, (−)-ent-pimara-8(14),15-dien-19-oic acid (7), and two eudesmanolides, (−)-diplophyllolide A (8) and (−)-diplophyllin (9) were isolated from the Tahitian Mastigophora diclados (Brid.) Nees. Herbertane sesquiterpenes (2, 3, 5 and 6) showed cytotoxicity against HL-60 and KB cell lines, radical scavenging activity and antimicrobial activity against Bacillus subtilis. (−)-Diplophyllolide A (8) also exhibited cytotoxicity against HL-60 and KB cell lines.

Antioxidant Properties of 2-O-β- D -Glucopyranosyl- L -ascorbic Acid

The antioxidant activity of a provitamin C agent, 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), was compared to that of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) and ascorbic acid (AA) using four in vitro methods, 1,1-diphenyl-picrylhydrazyl (DPPH) radical-scavenging assay, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+)-scavenging assay, oxygen radical absorbance capacity (ORAC) assay, and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis inhibition assay. AA-2βG slowly and continuously scavenged DPPH radicals and ABTS•+ in roughly the same reaction profiles as AA-2G, whereas AA quenched these radicals immediately. In the ORAC assay and the hemolysis inhibition assay, AA-2βG showed similar overall activities to AA-2G and to AA, although the reactivity of AA-2βG against the peroxyl radical generated in both assays was lower than that of AA-2G and AA. These data indicate that AA-2βG had roughly the same radical-scavenging properties as AA-2G, and a comprehensive in vitro antioxidant activity of AA-2βG appeared to be comparable not only to that of AA-2G but also to that of AA.

Generation of Mast Cells from Mouse Fetus: Analysis of Differentiation and Functionality, and Transcriptome Profiling Using Next Generation Sequencer

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy.