Jannaschia cystaugens sp. nov., an Alexandrium (Dinophyceae) cyst formation-promoting bacterium from Hiroshima Bay, Japan.

Heterotrophic bacteria isolated from water samples taken from Hiroshima Bay, Japan, and referred to as Alexandrium (Dinophyceae) cyst formation-promoting bacteria, were assigned to the Roseobacter-Sulfitobacter-Silicibacter group within the alpha-Proteobacteria on the basis of nearly complete 16S rRNA gene sequences. Phylogenetic analyses showed that two strains, CFPB-A9T and CFPB-A5, are closely related to each other and that their closest relative was Jannaschia helgolandensis (95.9 % sequence similarity). These strains were Gram-negative, motile, obligately aerobic rods that required sodium ions and 2-7 % sea salts for growth and did not produce bacteriochlorophyll a. Their optimal growth temperature was 25-30 degrees C. The strains had Q-10 as the dominant respiratory quinone. Primary cellular fatty acid in both strains was 18 : 1omega7c. The DNA G + C contents of strains CFPB-A9T and CFPB-A5 were 59.1 and 59.2 mol%, respectively. Based on physiological, biological, chemotaxonomic and phylogenetic data, the strains are considered to represent a novel species, Jannaschia cystaugens sp. nov., with type strain CFPB-A9T (= LMG 22015T = NBRC 100362T).

Antitumor activity of eosinophils activated by IL-5 and eotaxin against hepatocellular carcinoma.

We examined the antitumor effects of eosinophils to explore the potential of eosinophils as effector cells in tumor cytotoxicity. We expressed eotaxin in hepatocellular carcinoma cells, MH134, and injected them into either normal or IL-5 TG mice intradermally and monitored cell growth. In normal mice, growth of MH134 cells containing the expression plasmid pCXN2-eotaxin was similar to that of vector-transfected MH134 cells for a period of 2 weeks, suggesting that expression of eotaxin does not change the growth rate of tumor cells. In IL-5 TG mice, however, the growth of eotaxin expressing MH134 cells was significantly suppressed. LPS induced eosinophils to produce TNF-alpha to kill MH134 cells in vitro. Intratumor injection of LPS is effective to kill MH134-pCXN2 and MH134-pCXN2-eotaxin only in normal mice. Administration of anti-CD4 or anti-CD8 antibodies suppressed growth of MH134-pCXN2-eotaxin cells compared with control antibodies, suggesting that T cells may interfere with immunity against MH134. Administration of anti-IL-5Ralpha and anti-asialo GM1 antibodies enhanced growth of MH134-pCXN2-eotaxin cells, suggesting involvement of eosinophils and NK cells in suppression of tumor cell growth. Although we cannot exclude the possibility that NK cells participate in tumor cell killing in vivo, the presence of NK markers such as DX5, asialo GM1, Ly49, and CD94, and NKG2D on large numbers of eosinophils activated by eotaxin suggests that eosinophils function in such suppression of tumor cell growth. Furthermore, we showed that anti-NKG2D antibodies could significantly inhibit the LPS-induced cytotoxicity against MH134 by highly enriched fraction of eosinophils.

Development and functional analysis of eosinophils from murine embryonic stem cells.

We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony-stimulating factor-deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)-5 with either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) for 20 d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL-5, IL-3, GM-CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL-3 or IL-5. Levels of GATA-1, Friend of GATA (FOG)-1, PU.1, CCAAT/enhancer binding protein (C/EBP)alpha, C/EBPbeta, IL-3 receptor alpha (IL-3Ralpha), GM-CSF receptor alpha (GM-CSFRalpha), and MBP mRNAs were increased in ES cells 10 d after transfer onto OP9 cells. In contrast, C/EBPepsilon, IL-5Ralpha, and eosinophil peroxidase mRNAs were induced in response to IL-3 and IL-5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr-1, F4/80, B220, CCR3, IL-3Ralpha, IL-5Ralpha, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils.

m-Diethynylbenzene macrocycles: syntheses and self-association behavior in solution.

m-Diethynylbenzene macrocycles (DBMs), buta-1,3-diyne-bridged [4(n)]metacyclophanes, have been synthesized and their self-association behaviors in solution were investigated. Cyclic tetramers, hexamers, and octamers of DBMs having exo-annular octyl, hexadecyl, and 3,6,9-trioxadecyl ester groups were prepared by intermolecular oxidative coupling of dimer units or intramolecular cyclization of the corresponding open-chain oligomers. The aggregation properties were investigated by two methods, the (1)H NMR spectra and the vapor pressure osmometry (VPO). Although some discrepancies were observed between the association constants obtained from the two methods, the qualitative view was consistent with each other. The analysis of self-aggregation by VPO revealed unique aggregation behavior of DBMs in acetone and toluene, which was not elucidated by the NMR method. Namely, the association constants for infinite association are several times larger than the dimerization constant, suggesting that the aggregation is enhanced by the formation of dimers (a nucleation mechanism). In polar solvents, DBMs aggregate more strongly than in chloroform due to the solvophobic interactions between the macrocyclic framework and the solvents. Moreover, DBMs self-associate in aromatic solvents such as toluene and o-xylene more readily than in chloroform. In particular, the hexameric DBM having a large macrocyclic cavity exhibits extremely large association constants in aromatic solvents. By comparing the aggregation properties of DBMs with the corresponding acyclic oligomers, the effect of the macrocyclic structure on the aggregation propensity was clarified. Finally, it turned out that DBMs tend to aggregate more readily than the corresponding phenylacetylene macrocycles, acetylene-bridged [2(n)]metacyclophanes, owing to the withdrawal of the electron density from the aromatic rings by the butadiyne linkages which facilitates pi-pi stacking interactions.