Echinosporins as new cell cycle inhibitors and apoptosis inducers from marine-derived Streptomyces albogriseolus.

Bioassay-guided fractionation of the ethyl acetate extract from the fermentation broth of marine-derived Streptomyces albogriseolus A2002 led to the isolation of echinosporin (1) and 7-deoxyechinosporin (2). Compound 1 inhibited the proliferation of tsFT210, K562 and HCT-15 cancer cells (IC(50) 91.5 microM, 25.1 microM and 247 microM respectively) and 2 showed the same effect on K562 cells (IC(50) 143 microM). Flow cytometric analysis suggested that 1 and 2 exert their anti-proliferative effects on those cells through inhibiting cell cycle at the G(2)/M phase and inducing apoptosis.

A new phenanthraquinone from the stems of Bauhinia variegata L.

A new phenanthraquinone, named bauhinione (1), has been isolated from Bauhinia variegata L., and its structure has been elucidated as 2,7-dimethoxy-3-methyl-9,10-dihydrophenanthrene-1,4-dione on the basis of spectroscopic analysis.

Bioactive saponins from Dioscorea futschauensis.

A new anti-neoplastic spirostanol saponin, (25S)-spirost-5-en-3 beta, 27-diol-3O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl (1-->3)]-beta-D-glucopyranoside and three known compounds viz. prosapogenin A of dioscin, dioscin and gracilin were isolated from Dioscorea futschauensis by bioactivity-guided fractionation. Their structures were elucidated mainly by means of spectroscopic analysis. Their bioactivity against Pyricularia oryzae and cytotoxic activity on ts-FT210 cell line was evaluated.

Tryprostatin A, a specific and novel inhibitor of microtubule assembly.

We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 microM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 microM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase.

Screening of cell cycle inhibitors from microbial metabolites by a bioassay using a mouse cdc2 mutant cell line, tsFT210.

We have established a bioassay system using a mouse cdc2 mutant cell line, tsFT210, to detect inhibitors of the mammalian cell cycle. When cultured at the high temperature, restrictive temperature at 39.4 degrees C, tsFT210 cells can be arrested at G2 phase and are large in size. Four hours after release from G2 arrest, the cells entered into the G1 phase. At this time, G1 phase cells were easily discriminated from the G2/M-cells by their size under microscopic observation. The cell-morphology-based bioassay utilizing tsFT210 cells is very simple and sensitive for detecting cdc2 kinase inhibitors and also G2/M-phase inhibitors of the mammalian cell cycle. To demonstrate the merits of this bioassay, the effects of protein kinase inhibitors isolated from actinomycetes were investigated. RK-286C and RK-1409, which are structurally related to staurosporine, inhibited cell cycle progression at the G2 phase in both G2-synchronized and nonsynchronized cultures of tsFT210 cells. Another kinase inhibitor, sangivamycin, inhibited cell cycle progression at the G2 phase of cells released from temperature arrest but did not inhibit that of the exponentially growing cells. Using the bioassay system, we carried out screening of the cell cycle inhibitors from the microbial metabolites and have discovered several new inhibitors, including novel compounds such as tryprostatins A, B and acetophthalidin. Thus, this bioassay allowed for the detection of cell cycle inhibitors and provided a convenient and useful method for the screening of new inhibitors from the microbial metabolites.

Expression of transforming growth factor beta in radiation interstitial pneumonitis.

We investigated the changes in transforming growth factor beta 1 (TGF-beta 1) mRNA and TGF-beta 3 protein expression that occur in radiation interstitial pneumonitis. We used TGF-beta 1-cDNA probe in situ hybridization and TGF-beta 3 polyclonal antibody in immunohistochemical techniques. Our results showed that the distribution of TGF-beta 1 mRNA and TGF-beta 3 protein basically coincided in blood vessels, airways, lung parenchyma, and alveolar macrophages. However, bronchial epithelial cells expressed only TGF-beta 3 proteins and no TGF-beta 1 mRNA. We found an increased expression of TGF-beta 1 mRNA and TGF-beta 3 proteins in radiation interstitial pneumonitis.

Novel mammalian cell cycle inhibitors, tryprostatins A, B and other diketopiperazines produced by Aspergillus fumigatus. I. Taxonomy, fermentation, isolation and biological properties.

Two novel diketopiperazines named tryprostatins A (1) and B (2) and a new natural product belonging to the diketopiperazine series, designated as demethoxyfumitremorgin C (3), together with four known diketopiperazines, fumitremorgin C (4), 12,13-dihydroxyfumitremorgin C (5), fumitremorgin B (6) and verruculogen (7), were isolated from the fermentation broth of Aspergillus fumigatus BM939 by the combined use of solvent extraction, silica gel column chromatography, preparative TLC and repeated-preparative HPLC. The diketopiperazines showed an inhibitory activity on the cell cycle progression of mouse tsFT210 cells in the M phase with the MIC values of 16.4 microM (1), 4.4 microM (2), 0.45 microM (3), 4.1 microM (4), 60.8 microM (5), 26.1 microM (6) and 12.2 microM (7), respectively.

Novel mammalian cell cycle inhibitors, tryprostatins A, B and other diketopiperazines produced by Aspergillus fumigatus. II. Physico-chemical properties and structures.

Two novel diketopiperazines named tryprostatins A and B and a new natural product belonging to the diketopiperazine series, designated as demethoxyfumitremorgin C, together with four known diketopiperazines, fumitremorgin C, 12,13-dihydroxyfumitremorgin C, fumitremorgin B and verruculogen, are new M phase inhibitors of the mammalian cell cycle, which were isolated from the secondary metabolites of Aspergillus fumigatus. The structures of tryprostatins A, B and demethoxyfumitremorgin C were determined mainly by the use of spectroscopic methods especially by detailed analyses of their 1H and 13C NMR spectra with the aid of 2D NMR techniques including pulse field gradient heteronuclear multiple-bond correlation (PFG-HMBC) spectroscopy. Their absolute configurations were determined on the basis of the optical rotational values and CD spectra.

Collagen gene expression in radiation interstitial pneumonitis.

By using type I and type III collagen cDNA probe and cDNA-mRNA in situ hybridization, we observed the changes of rat lung alpha 1(I) and alpha 1(III) collagen gene expression in radiation interstitial pneumonitis. The results showed that the expressed cell of type I and type III collagen were scattered within the fibroblasts in the thickened interalveolar walls. The type I and type III collagen mRNA content in irradiated animals were higher than those in the controls at 0.5, 1, 2, 3, 6, and 12 months.

Dauricoside, a new glycosidal alkaloid having an inhibitory activity against blood-platelet aggregation.

Dauricoside (1), a new glycosidal alkaloid, was isolated from the rhizomes of Menispermum dauricum DC. along with dauricine (2), daurisoline (3), dauriporphine (4), menisporphine (5), and 6-O-demethylmenisporphine (6), and its structure was determined by means of spectroscopic methods. Compounds 1, 2, and 3 inhibited blood-platelet aggregation induced by adenosine 5'-diphosphate (ADP).

Constituents of a fern, Davallia mariesii Moore. II. Identification and 1H- and 13C-nuclear magnetic resonance spectra of procyanidin B-5, epicatechin-(4 beta----8)-epicatechin-(4 beta----6)-epicatechin, and epicatechin-(4 beta----6)-epicatechin-(4 beta----8)-epicatechin-(4 beta----6)-epicatechin.

Procyanidin B-5 (1), epicatechin-(4 beta----8)-epicatechin-(4 beta----6)-epicatechin (2), and epicatechin-(4 beta----6)-epicatechin-(4 beta----8)-epicatechin-(4 beta----6)-epicatechin (3), which showed an inhibitory effect toward protein kinase C, were isolated from the rhizomes of Davallia mariesii Moore. Detailed analyses of their 1H- and 13C-nuclear magnetic resonance spectra were carried out by the use of two-dimensional nuclear magnetic resonance techniques.

Constituents of a fern, Davallia mariesii Moore. I. Isolation and structures of davallialactone and a new flavanone glucuronide.

A new gamma-lactone derivative named davallialactone (4) and the 7-O-beta-D-glucuronide of (+/-)-eriodictyol (5a) have been isolated from Davallia mariessi Moore along with caffeic acid (1), 4-beta-D-glucopyranosylcaffeic acid (2) and 4-O-beta-D-glucopyranosyl-p-coumaric acid (3). The structures of the new compounds were determined by chemical and spectroscopic methods including two-dimensional nuclear magnetic resonance (2D NMR) techniques, especially 1H-detected heteronuclear multiple-bond multiple-quantum coherence and long-range C-H J-resolved 2D NMR techniques.

[Isolation and elucidation of a new minor saponin from the leaves of Panax ginseng C.A. Meyer].

The structure of a new minor saponin isolated from the leaves of Panax ginseng C. A. Meyer was elucidated on the basis of chemical evidences and spectral data. The saponin was named as ginsenoside-La.

[A new minor saponin from the leaves of Panax ginseng C. A. Meyer].

Two minor compounds isolated from the leaves of Panax ginseng C. A. Meyer were characterized as 20(R)-protopanaxadiol (I) and 3 beta, 6 alpha, 12 beta-trihydroxydammar-20 (22), 24-diene-6-O-alpha-L-rhamnosyl-(1----2)-beta-D-glucopyranoside (II) on the basis of spectral analysis and chemical evidences. I was isolated for the first time from the leaves; II was shown to be a new saponin and was named as ginsenoside-Rg4.