The potential of specific ubiquitin-proteasome inhibitors, which act as anti-cancer agents, has intensively been investigated. In 2003, a proteasome inhibitor Velcade^[○!R] was approved by FDA for the treatment of patients with multiple myeloma. On the other hand, polyubiquitination of proteins requires the sequential action of three enzymes, E1, E2, and E3, which leads to the idea that inhibitors against E1, E2, and E3 are also drug candidates for the treatments of diseases related to ubiquitin-dependent proteolysis. In this study, we succeeded in isolating a new E2 inhibitor, leuettamol A, which inhibits the formation of the Ubc13-Uev1A complex, from the sponge Leucetta microphaphis. This is the first report of the isolation of the E2 inhibitor. In addition, we isolated aaptamine, isoaaptamine, and demethylaaptamine from the marine sponge Aaptos suberitoides as proteasome inhibitors. Interstingly, they showed different inhibior specificities against chymotrypsin-like, trypsin -like, and caspase-like activities of the proteasome. Although proteasome inhibitors so far reported sow the most potent inhibitory abilities against the chymotrypsin-like activity among three activities, aaptamine congeners showed the same levels of inhibitory potencies against caspase-like and chymotrypsin-like activities. The designing of proteasome inibitors with different inhibitory mechanisms and/or different susceptibilities to three different active sites of the proteasome will provide lead compounds with reduced toxicity. It has been reported that combination of two proteasome inhibitors. Velcade^[○!R] and salinosporamide A, with different inhibitory specificities triggers in vivo synergistic cytotoxicity in multiple myeloma. Combination of aaptamine congeners with known proteasome inhibitors would produce a synergistic cytotoxic effect on cancer cells.

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Based on the fact that some of the bioactive mateials 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 cytotoxic azaphilones, chaetomugilin A-H, were produced by Chaetomium globosum 106B-6, which was originally separated from marine fish Mugil cephalus. Our continuing search for cytotoxic metabolites from this fungal strain has led to the isolation of five new azaphilones designated as chaetomugilins I (1), J (2), 11-epichaetomugilin A (3), 4'-epichaetomugilin A (4) and 106B-6 XXVIII (5). These metabolites isolated in this investigaton exhibited significant cytotoxic activity against the murine P388 leukemia cell line, the human HL-60 leukemia cell line, the murine L1210 leukemia cell line, and the human KB epidermoid carcinoma cell line. In addition, chaetomugilin I (1) was proved to show selective cytotoxicity to a disease-orinted panel of 39 human cancer cell lines. The absolute sterostructures of 1-5 have been elucidated on the bsis of spectroscopic analyses using 1D and 2D NMR techniques, the modified Mosher's method and some chemical transformatons. Compounds 1-5 were exzmined together with 5-FU as standard sample in the cytotoxic assay using the murine P388 leukemia cell line, the human HL-60 leukemia cell line, the murine L1210 leukemia cell line, and the human KB epidermoid carcinoma cell line. Compounds 1 and 2 exhibited significant cytotoxic activity against all cell lines equal to 5-FU. In addition, 1 was examined using a disease-oriented panel of 39 human cell lines. The effective concentration (MG-MID), the delta and range values of 1 (-5.15, 0.63 and 1.21, respectively) disclosed that compound 1 showed potent and selective ctotoxic activity (effective value: MG-MID≤-5, delta>0.5 and range>1.0). Furthermore, evaluation of the pattern of differential cytotoxicity using the COMPARE program suggested that the mode of action of 1 might be different from that shown by any other anticancer drugs developed to date.

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Ophiocoma scolopendrina is a tropical and subtropical ophiuroid widely distributed in the Indo-Pacific. O. scolopendrina is microphsgous feeder inhibiting in the intertidal zones. Despite its wide occurrence, this echinoderm has been largely overlooded by marine natural pruduct chemists. In the couse of our search for cytotoxic compounds from marine organisms, we found activity in the crude extract of O. scolopenfrina, from which we have isolate ophiodilactones A and B (1 and 2, respectively). The organic layer of the extract of the ophiuroid was subjected to a modification of Kupchan's solvent partitioning scheme to yield 60% MeOH, CHCl_3, and n-hexane layers. The CHCl_3 layer was separated by ODS flash chromatography, and the cytotoxic fraction was purified by reversed-phase HPLC to give ophiodilactones A (1.4mg) and B (0.9mg). Their structures were elucidated by interpretation of spectroscopic data. Planer structures of ophiodilactones were deduced from COSY, HSQC, and HMBC spectra (Figure 1 and 6a). To assign the location of the hydroxyl group, deuterium-induced shifts were measured in the ^<13>C NMR spectrum (Figure 2). The sizes of the lactone rings were assigned by IR spectrum. The relative stereochemistry was elucidated on the basis of NOESY data (Figure 3 and 6b), and the absolute stereochemistry was determined by the CD spectrum (Figure 5). Ophiodilactones A and B exhibited moderate cytotoxic activity against P388 murine leukemia cells with IC_<50> values of 5.0 and 2.2μg/mL, respectively. Ophiodilactones are new members of phenylalanine-derived γ-lactones, which have been isolated from fungi, tunicates, cyanobacteria, and plants. The carbon skeleton of ophiodilactone A is idiintical with that of maculalactone M. from the cyanobacterium, whereas the carbon skeleton of ophiodilactone B is new. Because O. scolopendrina inhibits the rockey shore, ophiodilactones are suspected to originate in the dietary cyanobacteria colonizing on the surface of rocks.

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Actinobacteria primarily isolated from terrestrial habitats have served as one of the main sources of bioactive compounds. A sharp decrease in the discovery of new compounds and increase in the re-isolation of known compounds require new approaches. One approach is the screening of actinobacteria from marine habitats. Actinobacteria from a marine sponge Haliclona sp. were isolated using four different medium including two mewly designed medium, jewfish and mussel extract agars. Strains belonging to seven different genera of actinobacteria including six novel species belonging to genus Streptomyces were isolated from these medium. These phylogenetically new strains were screened for the production of noel compoundsd and for the presence of genes for secondary metabolites (including genes for PKS, NRPS and HMGR). Unique gene sequences from these strains predicted the production of novel compounds. Interstingly a total of 7 novel compounds (JBIR-29, -30, -34, -35, -39, -40, and -43) together with 10 known compounds were isolated from these strains. JBIR-34 and -35 were of special interest, with respect to their novel chemical structures. This rate of novel compounds discovery from marine sponge-derived Streptomyces is remarkable compared to that of terrestrial Streptomyces. Our studies strongly suggest the presence of a number of yet unexplored marine actinobacterial diversity, a promising source of novel compounds for therapeutic use.

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Manzamine alkdloids are well known to be a group of marine natural products, which have a β-carboline moiety and a unique heterocyclic ring system. Since manzamine A had been isolated from a marine sponge Haliclona sp. in 1986, approximately 100 manzamine alkdloids containing biogenetic precursors have been idolated so far. A great number of studies on them, such as isolation and structural analysis, biosynthesis, and total synthesis, have been actively performed due to their intriguing structures and intersting biological activities. We have also isolated s seris of manzamine alkaloids from several genera and their structures have been elucidated by means of NMR spectra, chemical correlations, X-ray crystallography, and CD spectra. During our continuous search for structurally interesting metabolites from marine sponges, new manzamine alkdloids, zamamidines A-C (1-3), have been isolated, and their structures were elucidated by spectroscopic data. 1-3 are new manzamine alkdloids possessing a second β-carboline ring via an ethylene chain at N-2 of manzamine H, 1,2,3,4-tetrahydromanzamine B, and manzamine D, respectively. Since no manzamine alkdloids possessing any side chain at N-2 other than a methyl group have been reported to date, 1&#12316;3 are the first examples of it. Zamamidines A-C (1-3) showed inhibitory activities against Plasmodium falciparum (Kl strain) and Tripanosoma brucei brucei (GUTat 3.1) in vitro.

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We studied on two kinds of antioxidants produced by dinoflagellate Aphidinium sp. The aqueous ethanos extract of the dinoflagellate Amphidinium sp. showed the activation of the Nrf2-ARE pathway of SH-SY5Y cell. NF-E2 related factor 2 (Nrf2) is a transcription factor, regulates the gene expression of teh antioxidant proteins with binding to the antioxidant response element (ARE). The activation of the Nrf2-ARE pathway gives the oxidative stress resistance to cell. We tried to isolate the activator of the Nrf2-ARE pathway. The continuous chromatography of the extract gave one fraction that shows the biological activity. This fraction contains three compounds, and we are now checking that which compound is the activator. The aqueous ethanol extract of another dinoflagellate Amphidinium sp. showed the inhibition of the neruonal cell death caused by 6-hydroxydopamine. 6-Hydroxydopamine is often used for inducing Parkinsonism, the inhibitor is useful to study Parkinson's disease. The reverse phase chromatography of the extract gave three compounds. One of them is a carotenoid, peridinin. Now, we tried to isolate other compounds and check which compound is the inhibitor.

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ABCG2 is a member of the ATP-binding cassette (ATP) family of transporters, which transport certain chemicals out of cells using the energy of ATP hydrolysis. Of the 48 human ABC transporter genes, three are associated with multidrug resistance in caner - P-glycoprotein (P-gp), the multidrug associated protein 1 (MRP1), and the breast cancer resistance protein (BCRP or ABCG2). The overexpression of ABCG2 in cancer cells plays an important mediator of exporting chemotherapeutic agents, mitoxantrone, topotecan, irinotecan, doxorubicin, SN-38, and flavopiridol, resulting in cellular resistance to them. Therefore, compounds inhibiting the ABCG2 could be used to develop therapeutic agents that reduce multidrug resistance in cancer cells. To identify lead compounds for development as ABCG2 inhibitors, the AGCG2 high through-put assay was applied to a large repository of natural pruduct extracts maintained by National Cancer Institue (NCI). Bioassay-guided fractionation of the extract of the marine ascidian Botryllus tyreus resulted in purification of a seris of botryllamides as AGCG2 inhibitors. We determined their structures by the combination of NMR analyses and ESI-MS analyses. The inhibitory activities of botryllamides on ABCG2 and other ABC multi-drug transporters were evaluated by the flow-cytometry based substrate efflux assay. Furthermore, botryllamide F and G were synthesized, followed by the analysis of the structure-activity relationship with synthetic botryllamide analogues.

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In our quest for neurotoxic copounds in marine benthic organisms, we discovered novel functionalized peptide toxin aculeine A (Acu-A) from a sponge Axinyssa aculeata. Acu-A is a 45 amino acid-residue peptide with novel post translational modification with long chain polypropanamine attached to the N-terminus. Aculeines were proconvulsant in mice after contral administration. Acu-A was shown to induce calcium influx in cultures HEK293 cells and cultured rat hippocampal cells in an external Ca^<2+> dependent manner suggesting membrane disrupting function of the molecule. Partial amino acid sequence of Acu-A was determined by combination of Edman detradation and MADLDI-TOFMS analysis of the enzyme digests. The whole amino acid sequence for Acu A was deduced from nucleic acid sequence determined by 3' and 5'-RACE agreeing well with the above amino acid sequence where six cysteine residues were arranged like typical cystine knots such as conotoxin. These data suggested that Acu-A was a novel class of polyamine-modified cystine knots.

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During the course of screening for antioxidants from microorganic metabolites, our research group has so far isolated several interesting 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging compounds. We recently found that an actinomycete USF-TC31 strain separated from a marine algae collected in Izu Peninsula, Japan, produced new compounds which had a DPPH radical-scavenging activity. Six compounds were isolated as radical scavengers from the culture broth of a marine-derived actinomycete USF-TC31 strain. Two compounds were novel compounds, and their structures were determined to be N-carbamoyl-2,3-dihydroxybenzamide (5) and 2-acetamido-3-(2,3-dihydroxybenzoylthio)propanoid acid (6) on the basis of spectroscopic data. Four compounds were known compounds, and they were identified to be anthranilic acid (1), 2,3-dihydroxybenzoic acid (2), 2,3-dihydroxybenzamide (3) and benadrostin (4). In addition, compound 6 was characterized to be a racemate by a specific rotation. Each of obtained compound was evaluated for DPPH radical scavenging actibity, and compounds 2, 3, 5 and 6 exhibited potent activities in somparison with the positive control BHT (butylhydroxytoluene).

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In the course of our screening program for microbial anti-infectives such as anti-microbial antibiotics and portentiators of exisiting antibiotic sctivity against methicillin-resistant Staphylococcus aureus (MRSA) or Candida albicans, eight active compounds (1-8) including five new ones (xanthoradones A (1) and B (2), rugulosins B (4) and C (5) and 6'-hydroxy-3'-methoxy-mitorubrin (6)) were isolated from the culutures broth of Penicillium radicum FKI-3765-2, which was isolated from a soil sample collected at Hilo, Hawaii, USA. From EtOAc extracts of the 13 day-old culture broth of the strain, all the active compounds (1-8) were purified by ODS column chromatography and HPLC. The structures were culcidated by various NMR experiments. Compounds 1 and 2 have an asymmetric biaryl skeleton, which contains a dihydronaphthopyranone and a naphthoquinone moiety. Compound 4 is a hetero-dimer of anthraquinones, whereas 5 is a homo-dimer. Compound 6 is a new member of azaphilone family. Compounds 1 and 2 potentiated imipenem activity against MRSA by decreasing MIC value of imipenem from 16 to 0.060 and 0.030 μf/ml, respectively. Compounds 4 and 5 showed moderate ajtimicrobial activity against MRSA. Compound 6 moderately potintiated miconazole activity against C. albicans.

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The glutamic acid shows an extremely important function in the basal metabolism as a supply source of nitrogen which is necessary for the protein synthesis. The Hemerocallis fulva var. sempervirens (Liliaceae), contains few amino acids including glutamic acid, but it contains a large amount of oxypinnatanine (OPT) which is a particular glutamic acid deriative with furfurly group. OPT has been isolated from Euscaphis japonica (Staphyleaceae), Honkenya peploides (Caryophyllaceae) besides the Hemerocallis genus, but no research has been carried out concerning the physiological role of OPT. In thid study, to clarify the physiology function of OPT in Hemerocallis plants, the following two points were examined. (1) Isolation of OPT related compounds, to clarify the biosynthetic pathway of OPT We isolated two novel amino acid amides connected with the fructopyranose, kwansonines A (1) and B (2), together with three known amino acid amides, longitubanines A (3), B (4) and pinnatanine (5), from H. fulva L. var. sempervirens. This is the first report on the isolation of amino acid amide N-furctoside from Hemerocallis genus plant. (2) Participation of OPT in the growth processes of Hemerocallis plants Seasonal variations of OPT in the roots, leaves and flowers of five Hemerocallis plants (Hemerocallis fulva var. semervirens, H. fulva var. kwanso, H. fulva var. disticha, H. dumortieri var. esculenta, and H. fulva var. fulva) were investigated. The contents of OPT were highest at the early growth stage and decreased with growing up except for H. dumortieri var. esculenta. This suggested that OPT was produced and stored at the early growth stage and used at the growth process. It was considered that OPT participated in the growth of the plants instead of glutamic acid.

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In the course of our research on the active compounds of tropical and subtropical medicinal plants, we investigated the constituents of the seeds of Erythrina velutina (Leguminosae), commonly known as "Mulungu" in Brazil. This plant is widely used as a remedy for insomnia and inflammatory conditions, and for its anticancer effects. OUr phytochemical investigations of the seeds of E. velutina resulted in the isolation of six new Erythrinan alkaloids (1-6), seven known alkaloids (7-13), and an indole alkaloid (14). These compounds were elucidated by using HRMS and ^1H, and ^<13>C NMR techniques. We also studied the biological activities regarding the sleep promoting and anticancer effects by inestigating the isolated compounds from this plant. Hypaphorine (14) was investigated reagrding sleep-promoting effects in normal mice, and the results showed that it significantly increased the non-rapid eye movement (NREM) sleep time during the first hour after its administration. The NREM dleep time was enhanced by 33% in experimental mice when compared to that of controls. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an important member of the TNF super family. However, the clinical application of TRAIL in cancer therapy is limited because many cnacer cells have been idintified as resistant to its cytotoxicity. We examined the enhancing effects of the various isolated compounds on the cytotoxicity of TRAIL against Jurcat cells. Erysotrine (9) showed superior when combined with TRAIL among the samples tested, and exhibited no cytotoxicity (>50μg/mL) when administered alone.

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Pleurocybella porrigens (Angel's wings in English; Sugihiratake in Japanese) is widespread and common throughout temperature regions of the world. It has been eaten for a long time all over the world. However, in 2004, 55 people got poisoned by eating this mushroom, and 17 people among them died of aute encephalopaty. In order to elucidate the molecular mechanism of the disease, we examined toxicity of extract of the mushroom and found cytotoxicity of the extract against mouse cerebrum glial cells. Therefore, we tried to isolate the toxic principle(s) from the extracts. Dried fruiting bodies of P. porrigens were extracted with hexane, followed ayb EtOAc, EtOH, H_2O, and boiled H_2O. The EtOH fraction which showed cytotoxicity to mouse glial cells was subjected to open silica gel column chromatography with stepwise elution by acetone and 90% MeOH. The 90% MeOH fraction was separated with repeated HPLC to afford compounds 1-6. The structures of 1-6 were determined by the interpretation of spectroscopic data. All the compounds except for 6 had β-hydroxy valine moieties, and compounds 1 to 3 were novel. Compounds 1, 3, 4, and 5 showed toxicity against mouse cerebrum glial cells at 10μg/mL but 6 exhibited no activity. This result indicates that the 3-hydroxyvaline moiety is indispensable for the cytotoxicity. The relation between these compounds and acute encephalophathy is now being elucidated.

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Filopodia, the spike-like cell membrane projections, are known to contribute to cell motility. However, the molecular mechanism for filopodia formation still remains obscure. Therefore, the inhibitor against filopodia formation would be a useful tool to elucidate the mechanisms for filopodia formation. In the course of the screening for the inhibitor against the filopodia formation, we found that one cultured broth of Streptomyces sp. #1869-19 showed the strong inhibition. Isolation of the active substances from the cultured broth of this strain showed that two substances, glucopiericidin A (GPA) and piericidin A (PA) synergistically inhibited the filopodia when they were co-treated (Fig. 1). So far, the mode of action of GPA has not yet been clarified. However, because we found that glycolysis inhibitor 2-deoxglucose as well as GPA could synergistically inhibit the filopodia formation with PA (Fig. 2), GPA would be an inhibitor of glycolysis. Indeed, metabolome analysis revealed that GPA decreased the celllular level of final products of glycolysis (Fig. 2) by inhibiting either the step of glucose uptake into cells or the subsequent step of glucose phosphorylation. Because GPA inhibited Glucose Transporter-mediated uptake of ^3H-labeled 2-deoxyglucose (Fig. 3), it was indicated that GPA suppressed glycolysis via inhibition of of Glucose Transporter. On the other hand, PA is known as the inhibitor of mitochondrial respiratory chain complex 1, and co-treatment of GPA and PA decreased cellular ATP levels synergistically (Fig. 4). Because the filopodia formation is the ATP-energy dependent, depletion of cellular ATP induced by GPA and PA might be a cause of inhibition of filopodia formation (Fig. 5).

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The Type III secretion system (T3SS) is the most importan pathogenic mechanism, which causes infection among many Gram-nagative pathogenic bacteria by injection of effector proteins into host cells. Since T3SS is not essential for the survival of these bacteria, T3SS inhibitors may attenuate not to cause resistances and affect normal bacterial flora. Thus, T3SS is expected to be a good therapeutic target. AS a part of screening by the assay utilizing T3SS-induced hemolysis in enteropathogenic Escherichia coli (EPEC), we have screened 13,300 microbial extracts and found that the extracts of the strain Streptomyces sp. K06-0806 and K07-0034 showed potent T3SS inhibitory activity. After the fermentation and purification of the active compound, linear polyketides, aurodox and factumycin (Fig. 2), were isolated as T3SS inhibitors from the culture broths of K06-0806 and K07-0034, respectively. The strain K07-0034 also produced new factumycin analogs, 28β-factumycin and 8,9-dihydrofactumycin. Aurodox and factumycin are known as protein biosynthesis inhibitors as well as tetracyline. The inhibition of protein biosynthesis by aurodox is caused by its binding to the bacterial elongation factor Tu (EF-Tu). However, aurodox inhibited the hemolytic activity caused ty T3SS of EPEC in the 30 times lower concentration (IC_<50>: 1.2μg/mL). On the contary, some antibiotics (tetarcycline, colismycin, pactamycin etc.) inhibited both the hemolytic activity and the anti-EPEC activity at the same concentration. These results indicated aurodox could inhibit T3SS by unknown mechanism. The SDS-PAGE study showed that aurodox inhibited the expression of T3SS-related proteins, Tir, EspB, Map and EspF without the inhibition of EPEC viability and the expression of the house-keeping protein, GroEL. The RT-PCR study of the genus of LEE locus of enterocyte effacement) showed that aurodox reduced the transcription of many T3SS-related genes such as ler (LEE=encoded regulator),eae (LEE2: Type III apparatus), espA, espD, espB, one of 16S rRNA gene. Aurodox significantly reduced the transcription of ler, a gene of positive regulator of LEE, even at 0.5μg/mL. This result suggests the specific inhibition of aurodox against the expression of T3SS-related proteins is caused by the inhibition of teranscripton of ler. In vivo study was carried out using C3H/HeJ mie infected by Citrobacter rodentium. Orally administrated aurodox caused murine survival with the repression of bacterial colonization in the intestinal mucosa. Thererfore, aurodox is suggested to reduce the bacterial pathogenesia by the inhibition of T3SS.

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Hypoxic enrvironment on tumor is now recognized to be an important factor for tumor growth, angiogenesis, metastasis and response to chemotherapy and irradiation. Then, the compounds, which exhibit growh inhibitory activity against tumor cells under hypoxic environment slelctively, are expected to be promising nes lead for anti-cancer drugs. Furthermore it is an important to find new responsible molecules for the adaptation of hypoxia in tumor cells through analysis of action-mechanism of the compound, because the adaption of hypoxia in tumor cells is not completely clarified. In the course of our search for hypoxia-selective growth inhibitors, a furanosesterterpene, furospinosulin-1 (1), was isolates from an Indonesia marine sponge of Suberea sp. The compound 1 showed selective growth inhibitory activity against human prostate cancer cells DU145 under 1% or low oxygen atmosphere dose-dependently, while the compound 1 did not inhibit production of Hypoxia Inducible Factor-1α (HIF-1α), which is considered to be a major transcription factor to adapt hypoxic environment in tumor. Moreover, furospinosulin-1 (1) suppressed tumor growth in the mice s.c.-inoculated mouse sarcoma S180 cells. Then, the Pathway-Specific Oligo GEArray analysis was carried out to elucidate action-mechanism of compound 1. The data suggested hat furospinosulin-1 (1) might inhibit signaling of insulin growth factor 1 receptor (IGF-1R) via suppression of insulin-like growth factor-2 (IGF-2).

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Protein conjugates and biotin conjugates of tetrodotoxin (TTX) were prepared by reductive amination between 11-oxoTTX and bydrazides. The mixture of 11-oxoTTX and its 4,9-anhydro derivative (ratio 2:5) was obtained from TTX by Pfitzner-Moffatt oxidation as previously reported by Lazdunski et al. We optimized this reaction, and obtained them reproductively in approximately 60% yield. By analysis using MALDI-TOF MS, four molecules of TTX were suggested to be conjugated to one molecule of BSA (bovine serum albumin). TTX was biotylated by using biotin PEG_4 hydrazide and another biotin hydrazide reagent which has an alkyl linker between biotin and hydrazide. The inhibitory activity of this TTX-biotin conjugate to sodium channels in Neuro2A cell line was estimated approximately 1/100 of that of TTX by the preliminary result.

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Protein phosphatase type1 (PP1), together with protein phosphatase 2A (PP2A), is a major serine/threonine protein phosphatase (PP). Preiously, we reported that tautomycetin (TC) is a specific inhibitor of PP1 using cell extracts and purified enzymes. Then we demonstrated that treatment of COS-7 cells with 5μM TC for 5h inhibited endogenous PP1 by more than 90% without affecting PP2A activity, showing that TC preferentially inhibits PP1 in the cells. Using of tautomycetin, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK without affecting those of JNK and p38. The TC-mediated inhibition of PP1 also suppressed activation of Raf-1 and B-Raf. Furthermore, a physical interaction between PP1C and Raf-1 was observed. We were also interested in role of PP1 in TNF-α-induced signaling. We show that TC specifically inhibits activation of NF-κB among the three TNF-α effectors (NF-κB, JNK and caspase). Specifically, TC-treatment suppressed activation of IKK. Co-immunoprecipitation experiment showed that PP1C physically interacted with IKK complex. Pull-down analysis using recombinant GST-TNF-α, showed that PP1C was recruited to TNFR1 together with IKK complex, RIP and TAK1 upon stimulus. Ours is the first demonstration that PP1 positively regulates the TNF-α-induced NF-κB pathway at the level of IKK activation. Constitutively activation of Raf/ERK and/or NF-κB pathway was observed in several tumor cells, and it is thought EGF/Raf inhibitor and NF-κB inhibitor could be used for cancer therapy. Thus, TC might be a potential therapeutic reagent for cancer.

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We previously confirmed that Alternaria brassicicola ATCC96836 producdd brassicicene (BC) C and that Orf8 and Orf6, which were identified in the BC C biosynthetic gene cluster in this strain, were fusicoccadiene (FD) synthase and 16-O-methyltransferase, respectively. In the present study, the early steps of BC C biosynthesis after the formation of FD were investigated. Plasmids carrying the FD synthase gene (orf8), one (or two) of five P450 genes (orf1, orf2, orf5, orf7 and orf11) identified in the cluster and a P450 reductase gene cloned from strain ATCC96836 were constructed and introduced into Sacchromyces cerevisiae. Based on the structures of the compounds produced by the transformants, Orf1 is suggested to be an 8β-hydroxylation enzyme that yield FD 8β-ol, followed by 16-hydroxylation by Orf7 to produce FD 8β,16-diol. Functional analyses of the other genes in the cluster are now in progress.

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Gardenia jasminoides produces geniposide, a well known iridoid glucoside. In the iridoid biosynthetic pathway, cyclopenta-[c]-pyran skeleton derived from geranyl diphosphate is glucosylated. However, this glucosylation step and iridoid 1-O-glucosyltransferase are still unclear. We attempted to characterize glucosylation activity toward iridoids and isolate a cDNA encoding a iridoid glucosyltransferase from G. jasminoides. G. jasminoides cell suspension cultures converted exogenously supplid genipin to geniposide, and also geniposide to gardenoside. The glucosylation activity was dependent on culture stage of cells. The glucosylation activity was not affected by addition of methyl jasmonate to the cell cultures. In Catharanthus reseus cell cultures, which also produce many iridoids and terpenoid indole alkaloids derived from iridoids, genipin was conerted to geniposide, but the activity was much lower than that of G. jasminoides. Thirteen cDNAs encoding plant secondary product glycosyltransferases (PSPGs) were cloned from cultured G. jasminoides cells, using a RACE-PCR method based on the highly conserved C-terminal region of PSPGs. We prepared the recombinant enzymes expressed in E. coli and analyzed their genipin glucosylation activities. GjUGT2 catalyzed the formation of geniposide from genipin, and also exhibited an 1-O-glucosylation activity toward 7-deoxyloganetin and produced 7-deoxyloganin, whereas hardly of did not accept other phenolic compound such as p-nitrophenol, quercetin, esucletin, and trans-zeatin as sugar-acceptor substrates. To our knowledge this is the first report describing cDNA cloning of the iridoid-specific glucosyltransferase, and provide useful information on the iridoid biosynthetic pathway.

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The pathway of leucine to mevalonate has been attractive attention in the biosynthesis of isoprenoid in parasitic protozoa and myxobacterium recently. This "leucine-mevalonate" pathway was also effectively observed in the biosynthesis of characteristic lipid core in halophilic archaea Halobacterium salinarium JCM 9120. The labeling experiment with L-[5,5-^2H_3]leucine strongly suggested the intact incorporation of the terminal methyl group of leucine to the branched methyl group of isoprenoidal lipd core which is characteristic for halophilci archaea. Further, the substrate stereochemistry of isovaleryl-CoA dehydrogense at diastereotopic methyl group was determined by the incorporation experiments of the corresponding two isotopically-labeled leucine. At first, improved isotopic labeling at the diastereotopic methyl group of leucine ((4S)- and (4R)-[5-^2H_1]leuine) was deeloped. The C-5 deuterium of the (4R) isomer was effectively incorporated (20% incorporation) and the incorporation of deuterium of the (4S) isomer was low (-5% incorporation). Thus, (4R)-[5-^2H]leucine was converted to (3Z)-[3-^2H]isovaleryl-CoA. The substrate stereochemistry of the enzyme was same as the corresponding enzyme derived from rat liver. These results will open the vision to determine the enzymes concerned about amino acid metabolism from the unknown genetic resource of halophilic archaea.

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Sugar-sugar glycosyltransferases attach additional sugar groups to an existing sugar moiety of small molecule glycosides. Although they play an important role in producing structural diversity of secondary products in higher plants, few studies have centered on them. We recently isolated a cDNA clone encoding a sugar-sugar glycosyltransferase (CaUGT3) catalyzing 1,6-glucosylation of curcumin glucoside from Catharantus roseus. CaUGT3 exhibited a unique glucosylchain elongation activity forming not only gentiobioside but also gentiotrioside and gentiotetroside in a sequential manner. In this study, we examined the catalytic function and substrate specificity of CaUGT3 and found that CaUGT3 exhibited glucosyl transfer activity toward various flavonol- and flavone glucosides, with the highest catalytic specificity toward kaempferol 3-O-glucoside. Furthermore, we also investigated the functional propertied of CaUGT3 using homology modeling and site-directed mutagenesis, and identified amino acids positioned in the acceptor binding pocket as crucial for providing enough space to accommodate flavonoid glucosides instead of flavonoid aglycones. These results provide basic information for understanding and engineering the catalytic functions of sugar-sugar glycosyltransferases involved in biosynthesis of plant glycosides.

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Flavonoids are most commonnly conjugated with various sugar moieties by UDP-sugar:glycosyltransferases (UGTs) in a lineage-specific manner. Generally, the phylogenetics and regiospecificity of flavonoid UGTs are correlated, indicating that the regiospecificity of UGT differentiated prior to speciation. By contrast, it is unclear how the sugar donor specificity of UGTs evolved. Here, we report the biochemical, homology-modeled, and phylogenetic characterization of flavonoid 7-O-glucuronosyltransferases (F7GAT), which is responsible for producing specialized metabolites in Lamiales plants. All of the Lamiales F7GATs were found to be members of the UGT88-related cluster and specifically used UDP-glucuronic acid (UDPGA). We identified an Arg residue that is specifially conserved in the PSPG box in the Lamiales F7GATs. Substitution of this Arg with Trp was sufficient ot conver the sugar donor specificity of the Lamiales F7GATs from UDPGA to UDP-glucose. Homology modeling of the Lamiales F7GAT suggested that the Arg residue plays a critical role in the specific recognition of anionic carboxylate of the glucuronic acid moiety of UDPGA with its cationic guanidinium moiety. These results support the hypothesis that differentiation of sugar donor specificity of UGTs occurred locally, in specific plant lineages, after establishment of general regiospecificity for the sugar acceptor. Thus, the plasticity of sugar donor specificity explains, in part, the extraordinary structural diversification of phytochemicals.

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An industrial fungus Aspergillus oryzae and a toxigenic fungus A. Flavus are close relatives and some strains of both species posses a biosynthetic gene (cpa) cluster for mycotoxin cyclopiazonic acid (CPA). Compared with the cpa cluster of A. flavus, 2 extra genes, cpaD and cpaE, exist in the cluster of A. oryzae. It is noteworthy that the 2 genes, which have been lost in a detrimental fungus, A. flavus, are apparently retained in a domesticated fungus A. oryzae. We, therefore, addressed the physiological role of the A. oryzae-specific genes. A compound that was not produced in the cpaD knockout mutant was isolated from the culture broth of the wild-type strain (CPA producer NBRC4177). Its structure was determined as a 2-oxocyclopiazonic acid, suggesting that cpaD is responsible for the conversion fo CPA to 2-oxocyclopiazonic acid in A. oryzae. The toxicity of CPA as a mycotoxin is considered to be attributed to the inhibition of sarcoplasmic reticulum Ca^<2+>-ATPase. The inhibitory aactivity of 2-oxocyclopiazonic acid against Ca^<2+>-ATPase was 5-fold lower than that of CPA. Furthermore, the comparison of the nucleotide sequence of cpa cluster between A. oryzae and A. flavus implies that the common ancestor of the two species had the cpa cluster that consists of 7 genes, and the 2 genes, which migt be disadvantageous to survive in the wild, could have been lost from the A. flavus genome during evolution. We propose that the presence of the favorable gene for human consumption in the mcotoxin gene cluster of A. oryzae, but not A. flavus, is evidence for the domestication of A. oryzae.

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