2'-Deoxymugineic acid (DMA), one of mugineic acid-family phytosiderophores (MAs), was synthesized in vitro both from Lmethionine and from nicotianamine (NA) with a cell-free system derived from root tips of iron-deficient barley (Hordeum vulgare L.). The reactions producing DMA from NA needed an amino group acceptor (i.e. 2-oxoglutarate, pyruvate, or oxalacetic acid) and a reductant (i.e. NADH or NADPH). The activity of the enzymes to produce NA from L-methionine was the highest at about pH 9.This biosynthetic activity was markedly induced by iron-deficiency stress. The synthesis of NA from S-adenosyl-L-methionine was more efficient than from L-methionine. From the results with the cell-free system reported here, we propose a revised biosynthetic pathway of MAs.Plants with 'Strategy 2' for iron acquisition (16) excrete iron-chelators, called phytosiderophores, from the roots to solubilize the external insoluble Fe3", and the amount of the secreted phytosiderophores increases under iron deficiency stress. MAs2 are the only examples of the phytosiderophores known so far (21). Their chemical properties (9,14,20,23) and their physiological significance (4,7,8,11,12,15,22) have already been established.The biosynthetic pathway of MAs is presently considered to be that three molecules of L-methionine are combined to form NA, which is then converted to DMA by deamination and hydroxylation at the 3"-carbon, and subsequent series of hydroxylation of DMA lead to other members of MAs. This pathway has been supported by the incorporation of L-['4C] methionine or L-['3C]methionine into MAs in vivo (5, 10) and the inhibition of deamination in vivo (6, 18). To characterize the biosynthesis furthermore, we recently developed a cellfree system for the biosynthesis of NA from L-methionine, using crude protein fraction from the roots of iron-deficient barley (18). For a larger scale preparation of the sample, we 'Partly supported by Grant-in-Aid (No. 62219004 and No. 62303014) from the Ministry of Education, Science and Culture, Japan 2Abbreviations: MAs, mugineic acid-family phytosiderophores; DMA, 2'-deoxymugineic acid; NA, nicotianamine; SAM, S-adenosyl-L-methionine; Taps, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid also reported the cell-free system from suspension culture of Nicotiana megalosiphon (19).On the above-mentioned hypothetical pathway of the biosynthesis of MAs, NA is a key substance, not only because its structure is much similar to MAs but also because the biosynthetic steps from NA to MAs can be thought to be evolutionally acquired by the 'strategy 2' plants, or lost by the 'strategy 1' plants: NA is widely distributed among the plants of both strategy 1 and 2, but MAs can be found only in some graminaceous plants (1,3,17). Nevertheless, there has been no direct evidence that NA is converted to DMA. The principal reason for this lack of data may be that there have been no systems to produce DMA in vitro.We thus improved our previous cell-free system in the following order. First, the cel...

Kaempferol belongs to the flavonoid family and has been used in traditional folk medicine. Here, we investigated the antitumor effects of kaempferol on cell cycle arrest and autophagic cell death in SK-HEP-1 human hepatic cancer cells. Kaempferol decreased cell viability as determined by MTT assays and induced a G2/M phase cell cycle arrest in a concentration-dependent manner. Kaempferol did not induce DNA fragmentation, apoptotic bodies or caspase-3 activity in SK-HEP-1 cells as determined by DNA gel electrophoresis, DAPI staining and caspase-3 activity assays, respectively. In contrast, kaempferol is involved in the autophagic process. Double-membrane vacuoles, lysosomal compartments, acidic vesicular organelles and cleavage of microtubule-associated protein 1 light chain 3 (LC3) were observed by transmission electron microscopy, LysoΤracker red staining, GFP-fluorescent LC3 assays and acridine orange staining, respectively. In SK-HEP-1 cells, kaempferol increased the protein levels of p-AMPK, LC3-II, Atg 5, Atg 7, Atg 12 and beclin 1 as well as inhibited the protein levels of CDK1, cyclin B, p-AKT and p-mTOR. Taken together, CDK1/cyclin B expression and the AMPK and AKT signaling pathways contributed to kaempferol-induced G2/M cell cycle arrest and autophagic cell death in SK-HEP-1 human hepatic cancer cells. These results suggest that kaempferol may be useful for long-term cancer prevention.

Eleven new triterpenoid saponins, ardisianosides A (1), B (2), C (4), D (5), E (6), F (7), G (15), H (16), I (17), J (18), and K (19), together with 10 known saponins, were isolated from the whole plants of Ardisia japonica. The structures of the new saponins were established on the basis of extensive 1D and 2D NMR and MS studies coupled with chemical degradations. The cytotoxic activities of saponins 1-21 are reported against three human cancer cell lines, namely, HL-60 myeloid leukemia, KATO-III stomach adenocarcinoma, and A549 lung adenocarcinoma cells.

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