Onion (Allium cepa L.) and garlic (Allium sativum L. forma pekinense Makino) are known to exhibit anticarcinogenic activities via enzymatic inhibition, enzymatic induction, and apoptosis. In addition, they possess the following properties: antiinflammatory, antioxidant, antimicrobial, antifungal, and antiparasitic properties. Further, they play a role in the prevention of cardiovascular diseases. In orter to develop natural healthy foods that can prevent and combat the above diseases, we have tried to isolate a stable substance from the acetone extract of onion and garlic. Onions and garlics were separately chopped, homomenated and soaked in acetone for 3 days at room temperature. The respective filtrate was evaporated at 40℃ in vacuo to yield a residue, which was subjected to Diaion HP-20 and then repeatedly chromatographed on silica gel to give novel compounds named onionin A (1) from onion, and garlicnin A (2) and garlicnin B (3) from garlic. Onion A (1): The polirive HR-FAB-MS of 1 showed peaks due to [M+Na]^+ and [C_6H_<11>OS]^+ at m/z 243.0489 (Calcd for C_9H_<16>O_2S_2Na, 243.0489), 70%, and at m/z 131.0525 [Calcd for C_6H_<11>OS, 131.0531], base peak, respectively. The ^1H- and ^<13>C-NMR spectra and various 2D-NMR techniques revealed the structure of 1 to be 3,4-dimethyl-5-(1'Z-propenyl-tetrahydrothiophen-2-sulfoxide-S-oxide. Garlicnin A (2): The positive HR-FAB-MS of 2 showed peaks due to [M+Na]^+ at m/z 243.0490 (Calcd for C_9H_<16>O_2S_2Na, 243.0490). The structure of 2 was determined by the ^1H- and ^<13>C-NMR spectra and various 2D-NMR techniques to be 3,4-dimethyl-5-(2'-propenyl)-tetrahydrothiophen-2-sulfoxide-S-oxide. Garlicnin B (3): The positive HR-FAB-MS of 3 showed peaks due to [M+Na]^+ and [C_<12>H_<20>O_2S_4Na]^+ at m/z 347.0244 (Calcd for C_<12>H_<20>O_2S_4Na, 347.0244). The structure of 3 was deduced by the ^1H- and ^<13>C-NMR spectra and various 2D-NMR techniques to be 3,4-dimethyl-5-(4',5'-dithia-1'E,7'-octadiene)-tetrahydrothiophen-2-sulfoxide-S-oxide. It is known that inhibition of M2 macrophage polarization suppresses tumor cell proliferation. We have then examined the inhibitofy effect of 1, a new compound isolated from onion, on CD163 expression, M2 macrophage marker, by a Cell noncompetitive enzyme-linked immunosorbent assay (Cell-ELISA). As a result, 1 significantly inhibited CD163 expression. This result suggests that onionin A (1) has a potential to suppress tumor cell proliferation by inhibition of M2 macrophage polarization.

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Microorganisms have an amazing capability to produce various secondary metabolites with unique structures and various biological activities. The microbial metabolites have been major sources of pharmaceutical leads and therapeutic agents, and also they have a great potential as the bioprobes for chemical biology study. We therefore have constructed a fraction library of microbial metabolites by a systematic separation method to discover and isolate useful and/or novel metabolites. The frations may contain valuable compounds with novel structures, activities, or key metabolites of a particular biosynthetic pathway. Each fraction is analyzed on an LC/MS with phogodiode-array-detector to speculate a structures of each metabolite by UV and mass spectra within the fractions. The spectral data were stored into the spectral database to discover novel metabolites. We have also constructed an LC/MS data plot, which is 2D data plot with retention time for X-axis and m/z value for Y-axis on the LC/MS analysis, to find particular metabolites group for each strain. In this time, we found an unidentified peak with UV maxima of 280 nm and m/z value of 428 [M+H]^+ in the fraction library of Streptomyces spirovercillatus JC-8444 through the spectral database search. It was isolated and the structure was determined to be verticilactam (1). 1 had a unique 16-membered macrolactam skeleton including β-keto-amide moiety, which was the first example as a natural product and even in synthetic products in our best knowledge. Based on the structure of verticilactam (1), type I polyketide synthases (PKSs) were speculated for the biosynthesis. A 24-membered lactam might be derived through polyketide chain extension, then post-PKS modification processes involving Diels-Alder reaction and tetrahydrofuran formation might generate 1. These results suggest that the construction of the microbial metabolite fraction library is useful to discover a novel compound. Also, we have already found a analog of 1 on the LC/MS data plot by the similarity of m/z value.

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Okadaic acid (OA) as well as dinophysistoxin-1 (DTX1) has been causative for diarrheic shellfish poisoning. Biochemical experiments were carried out to investigate interactions of the two toxins with novel okadaic acid binding proteins 2.1 (OABP2.1) and 2.3 (OABP2.3), originally isolated from the marine sponge Halichondria okadai. Firstly, the recombinant proteins for OABPs 2.1 and 2.3 were expressed in Escherichia coli BL21 (DE3) cells. Binding assays using [24-^3H]OA demonstrated that the dissociation constants K_d for the recombinant OABPs 2.1 and 2.3 were determined as 1.30±0.56 nM and 1.54±0.35 nM, respectively. Binding of [24-^3H]OA to recombinant OABP2.1 was almost equally replaced with OA and DTX1. OA-induced cytotoxicity to mouse leukemia P388 cells was inhibited by the recombinant OABPs 2.1 and 2.3 in a comparable manner. These results suggest that regulation of OA-induced toxocity by OABPs 2.1 and 2.3 may be important for symbiotic relationship present in the sponge H. okadai.

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Metagenomics is a resesarch field based on the methodology extracting genomic DNA directly from environmental samples without any microbial culture or isolation steps. Utilizing this technique, it is expected to be possible to obtain biosynthetic genes of bioactive natural products from 99% of unculturable bacteria in the nature. In order to clone biosynthetic tene cluster of clinically potential marine natural products and produce by heterologous expression, we constructed about 200 thousands member of marine metagenomic library from tidal flat sediments and marine invertebrates collected off Ariake Sea, Kumamoto. Constructed marine metagenomic library was screened for siderophore production to obtain several active clones which were producting something ferric ion chelator. Nucleotide sequence of the zctive clone revealed that there were clustered 5 open reading frames, and homology search supported by BLAST suggested that they were highly similar to biosynthetic gene cluster of vibrioferrin, a siderophore reported from marine bacterium Vibrio parahaemolyticus. Active compound was purified from the culture broth of the clone by several chromatography steps to afford single isolated compound. All spectral data including NMR, MS, and UV indicated that the compound was identical to vibrioferrin. As dexcribed above, we demonstrated cloning of whole biosynthetic tene cluster of functional natural products by metagenomic approach, and fully produced the encoded compound by heterologous expression in Escherichia coli. Details of metagenomic library construction, siderophore screening, DNA analysis, compound production and identification, as well as metalloproteinase inhibitory activity involved in cancer metastasis will be presented.

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Immunosuppressants are used for the prevention of transplantation of organs and tissues, and treatment of allergic and autoimmune diseases. The high-potency imunosuppressants, cyclosporin A and FK506, are applied in a clinical setting. The studies of working mechanism of cyclosporin A and FK506 revealed the immune activation system via carcineurin and NF-AT. In the course of our screening for novel immunosuppressants, we already reported to isolate new immunosuppressants, ushikulides A (2) and B (3) and their biological activities. A new immunosuppressant, ushikulide C (1), was isolated from the culture broth of Streptomyces sp. IUK-2. From EtOAc extracts of 5 day-old culture broth of the strain, compound 1 was purified by a combination of SiO_2 gel chromatography and reverse-phase HPLC. The molecular formula of 1 was established as C_<40>H_<70>O_<11> by HRFAB-MS. The ^1H and ^<13>C NMR spectral data of 1 were similar to those of 2. The planer structure of 1 was determined by various NMR spectral data. The relative stereochemistry of 1 was estimated by J-based configuration analysis. Most relative stereochemistry of 1 was same as that of 2. IC_<50> value of 1 was calculated to be 70nM against concanavalin A-induced proliferation of murine splenocytes. Compound 1 did not show significant cytotoxicity for KB cells at a concentration 10μM.

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Seven oligosacchrides containing fructopyranosyl (Fp) residue have been isolated from fermented beverage of plant extract. Their NMR spectra showed difficulties in analyzing because of the signal overlapping of one of the methylene proton H-6, and methine protons H-3, H-4, and H-5 in ^1H-NMR and methine carbons C-3, C-4 and C-5 in ^<13>C-NMR. Additional NMR signals from other residues cause further complexity. We use two NMR techniques; HR-HMBC and H2BC in addition to the conventional 2D NMR methods. The key HMBC correlation peak inducating the linkage between fructofuranose (Ff) and Fp (Fp-C-2/Ff-H-6) of 4 was hidden by intra-residual HMBC correlation signal (Fp-C-2/Fp-H-1). These two correlation signals could be discriminated by the fine structure of each proton signal of Fp-H-1 (3.80ppm, d, 12.3Hz) and Ff-H-6 (3.80ppm, dd, 10.8, 6.2) using HR-HMBC. The fine structure of both protons in the HR-HMBC spectrum revealed the Fp2→6Fflinkage of 4. The C-4 and C-5 in Fp could not be assigned by HMBC, becaluse HMBC can not discriminate the ^2J_<CH> and ^3J_<CH> correlations. This problem could be solved by using H2BC method, which detects the former correlation selectively by the two step magnetization transfer ^1J_<CH>→^3J_<CH>. One of the H-6 signals of Fp in 3 was separated from other signals, which showed correlation signal to one carbon. This could be assigned as C-5.

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Based on the fact that some of the bioactive materials isolated marine animals have been produced by bacteria, we have focused our attention on new antitumour materials from microorganisms inhabiting the marine environment. As part of this endeavor, we have reported that cephalimysin A were produced by Aspergillus fumigatus, which was originally separated from marine fish Mugil ephalus. Our continuing search for cytotoxic metabolites from this fungal strain has led to the isolation of three new metabolites having a spiro-heterocyclic γ-lactam core cephalimysind B-D (1-3), as well as FD-838 (4). Compounds 1-3 are the diastereomers of 4. Compounds 2 and 3 exhibit an opposite absolute configuration at a spiro carbon to that of other known naturally occurring spiro-heterocyclic γ-lactams. In addition, we succeeded in the chemical transformation of the four natural procudts (1-4) into their epimers (1'-4') at C-8 to afford all the stereoisomers of FD-838 (4) with three stereogenic centers. Consequently, the relationship between the absolute configuration at stereogenic centers and the CD Cotton effects for these compounds could be unambiguously established. Split Cotton effects (□_<max>: around 350 and 320 nm) were observed for the 5S,8S isomer, ie., positive and negative Cotton effects. The 5R,8S isomer showed negative nad positive Cotton effects. While the 5R,8S isomer and the 5S,8R isomer showed a negatibe Cotton effect and a positive Cotton effect (λ_<max>: around 345 nm), respectively. In addition, we found that the coupling constant of H-9 and 9-OH in the ^1H NMR spectra show the orientations of 9-OH and 8-OCH_3, i.e., 9-OH oriented cis to 8-OCH_3 for a large coupling constant (J=12Hz), and trans to 8-OCH_3 for a small coupling constant (J=4Hz). This would be occured by a hydrogen-bond, which held the conformation of 9-hydroxy. Such findings are useful for exzmining the stereochemistry of spirofuranone-lactams. As a primary screeing for antitumor activity, cancer cell growth inhibitory properties of the natural products 1-4 and their epimers 1'-4' were examined using the murine P388 leukemia, the human HL-50 leukemia, the murine L1210 leukemia, and the human KB epidermoid carcinoma cell lines. All the compounds except 1 exhibited moderate activity against the P388 and HL-60 cell lines. The cancer cell growth inhibitory properties did not imply a structure-activity relatonship, and so molecular target screening are in progress.

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KODA (Figure 1) exhibits flower-inducing activity toward Lemna paucicostata after reacting with norepinephrine in vitro. FN1 and FN2 are the major active compounds produced by the chemical reaction (Figure 1). Until now these compounds have not yet been detected even in a trace amounts in L. paucicostata exposed to drought stress. We have found that flower-inducing activity of Lemna extracts increased when the plants were exposed to drought stress (Figure 2). Therefore we tried to isolate and characterize compounds involved in the flower-inducing activity from L. aucicostata exposed to drought stress. During exposing to the drought stress for 120 min, Lemna plants produced several compounds, designated as LDS1, LDS2 and LDS3 (Figure 3). The MeOH extracts from 1,500g fw of Lemna plants was separated into several fractions by preparative HPLC and subjected to bioassay for detecting flower-inducing activity toward L. paucicostata. The flower-inducing activity was found in a fraction where LDS1 was detected. Guided by the flower-inducing activity, the active compound (2.4mg) was isolated. As the retention time and UV, and MS of flower-inducing compound were identical to those of LDS1, the desired compound is identified to be LDS1. Therefore LDS1 must be a principle flower-inducing compound production in L. paucicostata exposed to drought stress. We have also isolated LDS2 (2.0mg) and LDS3 (2.4mg) from the extracts without monitoring the flower-inducing activity. The planar structure of LDS1 was characterized based on the NMR and the high-resolution mass spectroscopic analysis (Figure 5) to be (11E,15Z)-9,13-dihydroxy-10-oxo-octadeca-11, 15-dienoic acid, although the configurations at C9 and C13 were not deduced. Structures of LDS2 and LDS3 were also characterized except their stereo chemistry as shown in Figure 5. LDS1 exhibited significant flower-inducing activity at the concentration of 10 nM and the activity was comparable to FN1, 2 at the concentration of more than 10nM. In contrast to LDS1, the activity of LDS2 and LDS3 was much less than LDS1 (Figure 6). The levels of endogenous LDS1-3 increased in parallel with the flowering activity found in the extracts of L. paucicostata when the plants were exposed to drought stress (Figure 7).

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Monpanoki (Argusia (or Messerschmidia or Tournefortia) argentea (Boraginaceae)) is locally used in Okinawa in Japan as an antidote for poisoning from snakes venoms, Trimeresurus flavoviridis (habu). A methanolic extract of the plant significantly inhibited hemorrhage induced by crude venom of Trimeresurus flavoviridis. The extract was then separated according to antivenom activity by using silica gel (ODS) column to afford rosmarinic acid (RA) (1) as an active principle. RA (1) significantly inhibited the hemorrhagic effect of crude venoms of T. flavoviridis, Crotalus atrox, Gloydius blomhoffii, Bitis arietans as well as snake venom metalloproteinases, HT-b (C. atrox), bilitoxin 2 (Agkistrodon bilineatus), HF (B. arietans), and Acl-proteinase (Deinagkistrodon acutus). This is the first report of the antihemorrhage activity of RA (1) and RA (1) greatly contributes to the antihemorrhagic efficiency of this plant against crude snake venoms and hemorrhagic toxins. Furthermore, mechanistic evidence of RA's neutralization of the hemorrhagic effects of snake venom was invesitigated. Inhibitions against fibrinogen hydreolytic and collagen hydrolytic activities of T. flavoviridis venom were examined by SDS-PAGE. Histopathological study was done by using microscope afer administration of venom with or without RA. RA was found to markedly neutralize venom-induced hemorrhage, fibrinogenolysis, cytotoxicity and digestion of type IV collagen activity. Moreover, both hemorrhage and neutrophil infiltrations were inhibited by RA, although both were observed in pathology sections administered only T. flavoviridis venom. These results demonstrate that RA inhibited most of the hemorrhage effects of venom. These findings indicate that rosmarinic acid can be expected to provide therapeutic benefits in neutralization of snake venom accompanied by heat stability.

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Investigation of the tannin constituents of Tamarix nilotica (Tamaricaceae) has resulted in the isolation of one new (1) and the three known trimeric ellagitannins hirrtellins T1 (4), T2 (2) and T3 (3). On the basis of spectroscopic and chemical experiments, the new tannin (1) was assigned a unique macrocyclic structure consisting of three tatally acylated glucose cores connected together by a p-dehydrodigalloyl (p-GOG) and two m-dehydrodigalloyl (m-GOG) moieties in the mode shown by the formula 1. Major T. nilotica dimers, hirtellins A (5) and B (6) and tamarixinin A (7), together with some related tannins, remurins A (8) and B (9), milotinins M4 (10) and D8 (11), with good abundance in the plant, were tested for their possible direct cytotoxic activity on human oral squamous cell carcinoma (HSC-2, HSC-3, anc HSC-4) and promyelocytic leukemia (HL-60) cell lines compared with their effects on human oral normal cells (HGF, HPC and HPLF), and all of the tested tannins were potently cytotoxic against HL-60 cells. Among all of the tested tannins, hirtellin A (5) and nilotinin D8 (11) showed potent cytotoxic effects and elevated tumor specificity (TS) values against all tested tumor cell lines. Upon oujr effort to find an alternative source for the stable production of these tannins through a biotechnological approach we established shoot cultures of T. tetrandra capable of producting ellagitannins. Chromatographic separations of an extract of cultured tissues, afforded 14 known monomeric-tetrameric together with new monomeric (12) and trimeric (13) ellagitannins, belonging to the different type of the tamaricaceous tannins. Known tannins were identified as 4 monomers, tellimagrandins I (14) and II (15), nilotinin M2 (16), remurin A (8), 8 dimers, hirtellins A (5), B (6) and C (19), isohirtellin C (20), milotinin D3 (17), tamarixinins A (7), B (21) and C (18), a trimer, hirtellin T1 (4) and a tetramer, hirtellin Q1 (22). The structure where O-1 of a glucose core of hirtellin T1 (4) was deacylated was assigned for the new trimer 13. The new monomer (12) was determined as methyl (2,3-di-O-galloyl-4,6-hexahydroxydiphenoyl)-β-D-glucoside. The influences of light, and manipulating nutrients in the culture medium on the growth and tannin production in cultured shoots of T. tetrandra were examined. Tamaricaceous plants produce unique class of hydrolysable tannins, which could be candidates for developing low-toxocity antitumor agents. T. tetrandra cultured tissues produce monomeric-tetrameric ellagitannins common to tamaricaceous plants. Although the intact plant produce high amoung of hirtellin B (6), the cultured tissues of T. tetrandra accumulalte high amoung of hirtellin A (5), which has been shown to have significant host-mediated antitumor activity in a previous study, and prominent selective cytotosic effects against broad specrum of tumor cell lines in our present investigation.

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A cytotoxic monoterpene, a conjugated unsaturated γ-lactone (YS-Lactone), was isolated from the leaf and stem of Aruncus dioicus var. tenuifolius, and the structure including relative and absolute stereochemistry was determined. The elucidated structure was identical to the aglycon of Kodemariosides, the monoterpene acylglucosides isolate dfrom other plant, Spiraea cantoniensis. However, an unglycosylated aglycon of these compounds has not been reported from S. cantoniensis, or the cytotoxicity of this aglycon has not been studied. We clarified that YS-Lactone shows relatively high cytotoxicity to Jurkat-T cell (IC_<50> 1.3μM) and P388 cell (IC_<50> 2.4μM). Moreover, induction of apoptosis in HL-60 cell by YS-Lactone at 6.0μM was suggested by morphological change of nublear and DNA ladder.

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Marine benthi organisms have yielded a variety of natural products with neuropharmacological applications. Here we describe the isolation and pharmacological characterization of four novel, neurologically active 8-oxoderivatives of purine, 1-4, isolated from Haplosclerida sponges collected in the Republic of Palau. The structure of 1 was determined based on spectral data and was confirmed by X-ray crystallography. The structures of 2-4 were determined similarly using spectral data by analogy of that of 1. Compound 1 induced convulsant behavior upon intracerebroventricular injection into mice, with a CD_<50> value of 2,4 nmol/mouse. Purines 2-4 were active in mouse bioassays at higher doses. The seizurogenic activity observed with 1 was correlated with inhibition of neruonal GABAergic transmission, with only a modest mpact of excitatory signaling, in electrophysiological recordings from hippoampal neurons in cultured and acute brain slice preparations. Despite having a purine template structure, the inhibitory activity of 1 was not prevented by a nonselective adenosine receptor antagonist. The natural product 1 therefore represents a novel substituted purine that elicits convulsions through its actions on inhibitory neurotransmission. The four 8-oxoisoguanine analogs comprise a new family of compounds closely related in structure to both important endogenous neurosignaling molecules and commonly used CNS stimulants.

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In the ocean, many structurally unique compounds that have significant biological activities have been isolated from various marine invertebrates. Especially, sponges, belonging to the porifera, are also isolated many natural products. Recent research suggests that many marine sponges harbor various microbial symbionts, by which many bioactive compounds are produced, and that the number of cultivable bacteria represents 1% or fewer of the total environmental bacteria. So, to take advantage of sponge symbionts efficiently the metagenomic analysis is optimal as culture independent analysis technique. In this study, we isolate and analyze metagenomic DNA from bacterial symbionts of Japanese marine sponge, H. okadai, and obtained genomic information for bacterial symbionts. Marine sponge, H. okadai, was crushed with buffer and separated by centrifuge and the genomi DNA was extracted from bacteria fraction. Firstly, isolated genomic DNA was sequenced on a Roche FLX pyrosequencer (Roche, Mannheim, Germany). Pyrosequencing of this DNA yielded 230,000 reads (50 Mbp). Then, metagenomic DNA was ligated into fosmid vector pCC1FOS vector (Epicentre) and the ligated vectors were transformed into E. coli EPI300 (Epicentre). The transformants were spread onto LB medim. A total of 150000 fosmid clones metagenomic library were constructed.

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Caffeoylquinic acid (CQA) is one of the phenylpropanoids found in a variety of natural resources and foods, such as sweetpotato, propolis, and coffee. Previously, we found that 3,5-di-O-caffeoylquinic acid (3,5-di-CQA, 1) and 3,4,5-tri-O-caffeoylquinic acid (3,4,5-tri-CQA, 2) possesed neuroprotective effect against amyloid-β_<1-42> (Aβ)-induced cell death. Proteomics analysis of CQA-treated cells has shown that 1 and 2 induced the oerexpression of glycolysis enzymes (phosphoglycerate kinase-1; PGK1, phosphoglycerate mutase 1; PGAM1). In this study, we investigated structure activity relationships of CQAscontaining 1 and 2 on energy generation promotion activity. Moreover, we carried out the measurement of escape latency time to find the hidden platform in Morris water maze (MWM) using senescence-accelerated-prone mice (SAMP8). To investigate the relationship between the upregulated glycolytic enzyme's effects on energy generation and the neuroprotective activity, the level of ATP on cells treated with 1 and 2 were evaluated. Intracellullar ATP level in the neuronal cells treated with compounds 1 and 2 was higher than in control cells. Intracellular ATP level in 2 treated cell was higher activity than 1 treated ell. And we checked the mRNA expression of glycolysis enzymes on treated cells with 1 and 2, respectively. As a result, the mRNA expression level of glycolysis enzyme, PGAM1, PGK1, and G3PDH, were increased by compounds 1 and 2. Additionally, we carried out the measurement of escape latency time to find the hidden platform in Morris water maze. Consequently, 1 and 2 administration induced the improvement of spatial learning and memory on SAMP8 mice, and the overexpression of PGK1 mRNA in SAMP8 brain. In this session, we also describe the recent findings about mechanism of neuroprotective activity and activation on energy metabolism by CQAs.

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Streptomyces sp. 2238-SVT4 produces hatomarubigins A, B, C and D, with belong to the angucycline family and reverse colchicine-resistance in multidrug-resistant tumor cells. Among them, hatomarubigin D has a unique dimeric structure with a methylene linkage and little is known about the mechanism of the methylene bridge formation between two aromatic rings. In this study, hatomarubigin biosynthesis genes were obtained and analyzed to determine the mechanism of hatomarubigin D biosynthesis. PCR using aromatase and cyclase gene-specific primers identified the hrb gene cluster for angucycline biosynthesis in Streptomyces sp. 2238-SVT4. The cluster consisted of 39 open reading frames including minimal polyketide synthase, ketoreducrtase, aromatase, cyclase, O-methyltransferase, oxidoreductase and oxygenase genes. Expression of a part of the gene cluster containig hrbR1 to hrbX in Streptomyces lividans TK23 resulted in the production of hatomarubigins A, B and C. Hatomarubigin D was obtained from the conversion of hatomarubigin C by a purified enzyme encoded by hrbY in the presence of methylcobalamin and NADPH. To analyze hrb gene functions, we constructed an expression plasmid containing the hrb cluster without hrbU, a putative O-methyltransferase gene. Sl lividans harboring this plasmid produced a new metabolite, hatomarubigin E. The molecular formula of hatomarubigin E was established as C_<19>H_<16>O_5 by high-resolution FAB-MS. The structure was elucidated to be 8-O-demethylhatomarubigin C by NMR spectroscopic analysis. A purified enzyme encoded by hrbU ocnverted hatomarubigin E into hatomarubigin C in the presence of S-adenosylmethionine, indication that hatomarubigin E is a substrate of HrbU O-methyltransferase and a biosynthetic intermediate of hatomarubigin C.

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The mushroom Pleurocybela porrigens (Angel's wings in English; Sugihiratake in Japanese) is widespread and common throughout temperate regions of the world. It has been eaten for a lng time all over the world. However, in autumn 2004 in Japan, fifty-five people got poisoned by eating this mushroom, and seventeen peoplel among them died of acute encephalopaty. There had been no report regarding toxocity of the fruiting bodies until the incident. Under these circumstances, we tried to isolate teh principle(s) of the disease. Purification of a glycoprotein showing lethal activity against mouse The mushroom was extracted with water and boiling water. After repeated chromatography of the water-soluble fractions, a glycoprotein was purified. The substance showed lethal toxocity toward mice at a dose of 24mg/kg (i.p.). Purification, characterization, and cDNA cloning of a lectin (PPL) PPL was purified from this mushroom. The results of SDS-PAGE gel filtration and MALDI-TOF-mass of PPL indicated that its molecular mass was 56kDa, and it was composed of four 14kDa subunits with no disulfide bonds. The complete amino acid sequence was determined by amino acid sequencing. The cDNA of PPL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA of the protein consisted of 411 bp encoding 137 amino acids. Intravenous (i.v.) (50mg/kg) or intraperitoneal (i.p.) (150mg/kg) administration of PPL to mice did not show any toxocity. However, i.v. (9mg/kg) administration of the protein to rats exhibited lethal toxocity. Purification of unusual amino acids showing cytotoxicity against mouse cerebrum glial cells Six amino acid derivatives (1-6) including three novel ones (1-3) were isolated from the mushroom. These compounds were cytotoxic to mouse glial cells. The structural novelty and analogy of the amino acids is such that each acid has the β-hydroxyvaline unit adducted to endogenous molecules, which inspired us to conclude the occurrence of an aziridine-amino acid (7) as the common precursor of the six compounds. We synthesized this compound and proved its occurrence in this mushroom. Compound 7 showed toxocity against rat CG4-16 oligodendrocyte cells; 7 significantly reduced the cell viability at concentrations up to 10μg/ml (87μM). Mechanism of the acute encephalopathy We found that a mixture of the letnal glycoprotein and PPL showed protease activity and disrupted the blood-brain barrier (BBB) in mice. We speculated that compound 7 or its derivatives caused demyelinating symptom after disruption of BBB. Verfication of the hypothesis is now on progress.

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Aaptamine (6) was a characteristeic marine natural product possessing 1H^benzo[de][1,6]-naphthyridine ring, which was isolated from the Okinawan marine sponge Aaptos aaptos. Approximately 20 related alkaloids have been isolated from several species of marine sponges so far. A lot of interstint researches have been performed since the isolation of 6 in 1982 due to its unique structures and various biological activities. During our continuous search for structurally interesting metabolites from marine sponges, a new bromoindole alkaloid, 6-bromoconicamin (1), a hybrid compound of 1 and bisdemethylaaptamne (5), nakijinamine A (2), and its derivatives, nakijinamines B (3) and C (4), have been isolated from two collections of Suberites sp. (SS-1083 and SS-1084). Their structuers were elucidated from spectroscopic data and chemical conversions. 6-Bromoconicamin (1) is an indole alkaloid having bromine atom at C-6 in conicamin, which was isolated from the Mediterranean marine tunicate Aplidium conicum. Nakijinamine A (2) is the hybrid compound of 1 and bisdemethylaaptamine (5), while nakijinamines B (3) and C (4) are related alkaloids of 2. Although the elucidatio of the gross structure of 2 from its spectroscopic data was very difficult due to broadening signals in ^1H NMR spectrum of 2, the structure of 2 was able to be a ssigned from chemical conversions of 2. The structure of 3 was assigned as the debromo analog of 2 by the comparison of NMR data with 2. Although 4 had a lot of quaternary carbons and hetero atoms in its structure, its gross structure was elucidated from careful analysis of spectroscopic data and comparison of NMR data with known compounds. The confirmation of the structure of 4 and biological activities of 1-4 are in progress.

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Streptothricins (STs) are broad-spectrum antibiotics that were first isolated from Streptomyces lavendulae in 1943. However STs are not currently used therapeutically due to their nephrotoxicity. All STs consist of a carbamoylated D-gulosamine to which a homopolymer of 1 to 7 β-lysine residues and a streptolidine lactam, and amide form of the unusual amino acid streptolidine, are attached. Recently, we have idintified a novel enzyme, ST hydorolase (SttH). SttH converted ST-F (with one β-lysine residue) and ST-D (with 3 β-lysine residue) to ST-F-acid and ST-D-acid, respectively, by the hydrolysis of streptolidine lactam. Interestingly, the selective toxocity of ST-D was altered from broad-spectrum to bacterial-specific, although ST-F-acid was detoxified in both prokaryotes and eukaryotes, demonstrating that the moiety of β-lysine homopolymer plays a crucial role in antibiotic activity. In fact, it is known that ST-D is a more effective antibiotic than ST-F. The biosynthetic mechanism of β-lysine homopolymer is thus intriguing in the ST biosynthesis. This presentation will focus on non-ribosomal peptide synthetase (NRPS) genes involved in the ST biosynthesis. In addition, the biosynthetic mechanism of β-lysine homopolymer will be discussed.

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Obesity is recognized as an energy balance disorder; energy input exceeds energy output. Recent medications approved for trhe treatment of obesity attempt to restore energy balance by reducing energy input by suppressing appetite or inhibiting lipid lipase to interfere with lipid absorption from the small intestine. Another potential strategy for the treatment is to block the synthesis of triacylglyerol (TG), the final storage form of free long-chain fatty acid. Therefore, inhibitors of TG synthesis are expected as therapeutic agents for obesity. We established a high content assay to observe the TG biosynthetic pathway using intact animal cells. During our screening for microbial inhibitors of TG synthesis in the cell-based assay utilizing Chinese hamster ovary (CHO-K1) cells, new biaryl dihydronaphthopyranones with a 2,4-dihydroxyoctan side chain, named dinapinones A1 (1) and A2 (2), were isolated from the culture broth of Penicillium pinophilum FKI-3864 by solvent extraction, silica gel and ODS column chromatographies and prepartive HPLC using a reverse phase (ODS and C30) column. Furthermore, monapinone A (3), the common monomer part of 1 and 2, was found to be produced by fermentation of the strain in seawater-containing medium. The planner and relative stereochemistries of 1, 2 and 3 were elucidated by various NMR experiments, suggesting to be 3S^*, 13R^* and 15R^*. Further, circular dichroism (CD) was studied to confirm that the absolute axial (C8-C8') configurations of 1 and 2 were comparable to the values reported for (P)- and (M)-vioxanthin (4). As a result, 1 had the (M)-configuration, accordingly 2 has the (P)-configuration. In the similar fashion, comparison of data with CD spectrum reported for S- and R-semivioxanthis (5) indicted that the absolute stereochemistry at C-3 in was elucidated to be 3S. Accordingly, the total absolute stereochemistry of 3 was concluded to be 3S13R15R. Furthermore, the absolute stereochemistry was elucidated by in vitro biosynthesis from the structurally well-defined monomer 3 to 1 and 2, resulting in (M)-3S13R15R-1 and (P)-3S13R15R-2, respectively. Compound 2 strongly inhibited TG synthesis IC_<50>; 0.65μM), while 1 and 3 did not inhibit the synthesis even at 12μM. Interestingly, a mixture of isolated 1 and 2 (1:1) showed the most potent TG inhibitory activity with an IC_<50> of 0.054μM.

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The naturally occurring isopentenyl derivatives of purines presently known consist of triacanthine and isoprenoid cytokinins such as N^6-isopentenyladenine, trans-zeatin, cis-zeatin, dihydrozeatin, and their sugar conjugates. In the course of our continuing search for biologically active compound from plants, we found that the MeOH extrace of the seeds of Japanese honey locust, Gleditsia japonica Miquel (Leguminosae), which has been used as a diuretic and an expectorant in oriental traditional medicine, accelerated acetylcholinesterase (AChE) activity. The MeOH extract was suspended in water and partitioned successively with hexane and EtOAc. The aqueous layer was lyophilized to yield syrup. The water-soluble fraction was separated repeatedly on an ODS column employing MeOH in H_2O gradient mixtures to afford five new purine alkaloids designated saikachinosides A (1), B (2),C (3), and locustosides A (4), B (5), as well as triacanthine. The structures of 1-5 were determined to be new 3-prenylated isoguanine glycosides by interpretation of ESIMS, IR, UV, and 1D and 2D NMR spectral data. Of these, the structures of 1 and 4 were confirmed by X-ray crystalographic analyses. Absolute configuration of glucose and apiose obtained after acid hydroysis of these compounds was determined as all D by GC-MS analyses of the trimethylsilylated thiazolidine derivatives. Although several naturall occurring prenylated purines such as isoprenoid cytokinins have been reported, most of them are N^6-prenylated adenine derivatives. There are very few, if any, compounds having a 3-prenylated purine skeleton. Compounds 1-5 were evaluated using electric eel AChE. All compounds isolated accelerated AChE activity.

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Acetoacetyl-coenzyme A (CoA) is the precursor of 3-hydroxy-3-methylglutaryl (HMG)-CoA in the mevalonate pathway, which is essential for terpenoid backbone biosynthesis. It is also the precursor of poly-β-hydroxybutyrate, a polymer belonging to the polyester class that is procuced by microorganisms. The de novo synthesis of acetoacetyl-CoA is usually catalyzed by acetoacetyl-CoA thiolase via a thioester-dependent Claisen-condensation reaction between two molecules of acetyl-CoA. Here we report that the nphT7 gene found in the mevalonate pathway gene cluster from a soil-isolated Streptomyces sp. strain encodes an unusual acetoacetyl-CoA synthesizing enzyme. The recombinant enzyme overexpressed in Escherichia coli catalyzes a single condensation of acetyl-CoA and malonyl-CoA to give acetoacetyl-CoA and CoA. Replacement of malonyl-CoA with malonyl-[acyl carrier protein] resulted in loss of the condensation activity. No acetoacetyl-CoA synthesizing activity was detected through the condensation of two molecules of acecyl-CoA. The site directed mutagenesis experiments suggested that the NphT7-catalyzed reaction proceeds through almost identical reaction mehanisms to that of KAS III and Type III PKS, where the Cys-His-Asn catalytic triad is involved.Based on these properites of NphT7, we propose to name this novel enzyme of the thiolase superfamily acetoacetyl-CoA synthase. Co-expression of the nphT7 gene with the HMG-CoA synthase gene and the HMG-CoA reductase gene in a heterologous host allowed 3.5-fold higher production of mevalonate than when only the HMG-CoA synthase gene and the HMG-CoA reductase gene were expressed. This result suggests that the nphT7 gene can be used to significantly increase the concentration of acetoacetyl-CoA in cells, eventually leadin to the production of useful terponoids and poly-β-hydroxybutyrate.

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Marinc dinoflagelletes are known to produce bioactive secondary metabolites. Members of Amphidinium are among the most abundant and diverse sand-dwelling benthic dinoflagellates world-wide, and have been proven to be important sources of structurally unique polyketides. we have isolated a series of macrolides, iriomoteolides, from a marine benthic Amphidinium sp. collected off Iriomote Island. In our continuint investigation for new anticancer drug leads from the dinoflagellate Amphidinium sp., we ave resulted in the isolation of a novel cytotoxic polyketide, amphirionin-2 (1) from a amrine dinoflagellate Amphidiniium sp. (strain KCA09051) collected off Iriomote Island. The coutured algal cells of the Amphidinium KCA09051 strain were extracted with MeOH/toluene. The toluene-soluble fractions of the extract were subjected to SiO_2 gel, C_<18>, and then NH_2-SiO_2 columns. One of cytotoxic fractions was separated by C_<18> HPLC to afford a new cytotoxic metabolite, amphirionin-2 (1). Structure elucidatio of was carried out using detailed analyses of 2D NMR spectra. The relative stereochemistry for 1 were assigned by combination of conformational analyses using NMR. Amphirionin-2 (1) is a novel polyketide with two hexahydrofuro[3,2-b]furan ring moieties, twi branched methyls, and a bydroxyl group. Amphirionine-2 (1) exhibited cytotoxocity against murine hepatic cancer cells MH134 (IC_<50>: 0.1μg/mL).

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Recently, we have developed a unique new MALDI-SpiralTOF-TOF tandem mass spectrometer utilizing a spiral ion trajectory TOFMS as the 1^<st> MS and an offset parabolic reflectron as the 2^<nd> MS(Fig. 1). We've found that the system can produce informative product ion mass spectra suitable for structural determination of nantural products such as polyethers and polyamines. This new system can produce high-energy collision induced dissociation (HE-CID) product ion spectra that are nearly identical to those produced by 4-sector tandem mass spectrometer systems because (1) monoisotopic precursor ions are selected by the long flight lengh 1^<st> MS and (2) product ions due to metastable fragmentation (a.k.a., PSD) are eliminated by the electric sectors used in the spiral ion optics of the 1^<st> MS. We have examined the applicability of the MALDI-SpiralTOF-TOF system to the structural elucidation of Joro Spider Toxin JSTX-3 and Yessotoxin (YTX), and we compared the data acquired on the MALDI-SpiralTOF-TOF tandem MS to those previously acquired on 4-sector tandem MS system (Fig. 5), (Fig. 6). In spite of the different mass analyzers and different ionization methods used in the two systems, the two systems produced virtually identical HE-CID product ion spectra from both analytes.

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Entomopathogenic fungi infect insects through the cuticle, grow as hyphal bodies or hyphae in the hemocoel, and cause host death by nutritional destruction of tissues, and producint toxic metabolites and pathogenic enzymes. To date, entomopathogenic fungi have not been extensively studied by natural product chemists. We focused on entomopathogenic fungi as new resources for biologically active compounds, and studied the diversity of their secondary metabolites. In this study, we show the isolation and structure elucidation of novel pyrone diterpene-type compounds, metarhizin A-H (1-8) from Metarhizium flavoviride, and their antiproliferative activities on human cancer cell lines. Additionally, we synthesized many metarhizin derivatives, evaluated their antiproliferative activities, and discuss the structural requirements for this activity. A novel pyrone diterpene-type compound, Nf-1 (9), was also isolated from Nectria flammea. Oomycetes from fungus-like mycelium and were calssified as fungi in the past. However, the cell walls of them are c omposed of cellulose rather than chitin, as in the fungi. In the present, the ultrastructure, biochemistry and molecular sequences of them indicate that the oomycetes belong to the Kingdom Chromista, not the Kingdom Fungi. We also focused on oomycetes as new resources for natural product chemistry. A new oxidized lanostane-type compound, St-1 (23), was isolated from the mycelium of oomycete Saprolegnia terrestris. The structure of 23 was determined by spectral means including EIMS, and ^1H and ^<13>C NMR.

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In recent research for bioactive substances of Arabidopsis thaliana, seven new oxylipins named Arabidopsides A-G were isolated from the aerial parts of this plant. Arabidopsides A-G are monogalactosyl diacylglycerides or digalactosyl diacylglycerides containing 12-oxophytodienoic acid (OPDA) and/or dinor-oxophytodienoc acid (dn-OPDA) which are known as precursors of jasmonic acid (JA) and have received much attention because they play important roles in regulation of developmental and defense gene expression of plants as JA and methyl jasmonate (MeJA). On the other hand, it is known that green leaf volatiles (GLVs) and heavy metals induce OPDA and JA in various plants. In this research, we investigated the effect of volatile compounds and heavy metals as potent inducers for triggering accumulations of Arabidopsides in mature leaves of A. thaliana. Leaves were exposed to GLVs and heavy metals solution. The stress-exposed leaf tissues were submerged and extracted in methanol. The extracted samples were analyzed by ODS-HPLC. In these analyses, GLVs- and heave metals-treatment induced strong increase of Arabidopsides. In addition, GLVs- and heavy metals-induced Arabidopsides were not able to detected inthe coi 1-1 mutant. These results indicate that abiotic stress- induced Arabidopsides are involved in the mechanism of stress response through COI 1 signaling in A. thaliana.

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Ciguatera fish poisoning (CFP) is an ichthyosarcotoxism caused by the consumption of tropical and subtropical coral fish contaminated with toxic polyther compounds, ciguatoxins (CTXs) and maitotoxin (MTX). CTX is known to activate the voltage-gated sodium channel in the nerves of intact mammals, leading to an influx of intra-cellular sodium and depolarization of the nerve fibre. Garrec et al. have reported that the extracts of some southern medicinal plants, traditionally used to treat CFP, to prevent invitro neurotoxicity produced by CTX or brevetoxin (BTX). Here, we studied new CFP antitoxic compounds isolated from two southern medicinal plants, Argusia Argentea and Vitex rotundifolia Linne fil. by using in vitro neurotoxocity assay.

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