3-O-ß-D-Glucopyranosyltheobroxide from Aerial Parts of Cowpea (Vigna unguiculata).

Theobroxide has been isolated from culture filtrates of Lasiodiplodia theobromae as a potato tuber-inducing compound. In this study, the metabolism of theobroxide was investigated using cowpea as an experimental model and [2H3-7]theobroxide as a substrate for analyzing a metabolite, which revealed that theobroxide applied exogenously to the roots was converted into 3-O-ß-D-glucopyranosyltheobroxide.

Chemical Promotion of Endogenous Amounts of ABA in Arabidopsis thaliana by a Natural Product, Theobroxide.

Plant hormones are a group of structurally diverse small compounds that orchestrate the cellular processes governing proper plant growth and environmental adaptation. To understand the details of hormonal activity, we must study not only their inherent activities but also the cross-talk among plant hormones. In addition to their use in agriculture, plant chemical activators, such as probenazole and uniconazole, have made great contributions to understand hormonal cross-talk. However, the use of plant chemical activators is limited due to the lack of activators for certain hormones. For example, to the best of our knowledge, there are only a few chemical activators previously known to stimulate the accumulation of ABA in plants, such as absinazoles and proanthocyanidins. In many cases, antagonistic effects have been examined in experiments using exogenously applied ABA, although these studies did not account for biologically relevant concentrations. In this report, it was found that a natural product, theobroxide, had potential as a plant chemical activator for stimulating the accumulation of ABA. Using theobroxide, the antagonistic effect of ABA against GAs was proved without exogenously applying ABA or using mutant plants. Our results suggest that ABA levels could be chemically controlled to elicit ABA-dependent biological phenomena.

Interactions among LOX metabolites regulate temperature-mediated flower bud formation in morning glory (Pharbitis nil).

We examined the relationship between temperature (15-35°C) and flower induction as it is influenced by linolenic acid (LA) cascade products, lipoxygenase (LOX; EC 1.13.11.12), allene oxide synthase (AOS; EC 4.2.1.92), and allene oxide cyclase (AOC; EC 5.3.99.6) generated in morning glory (Pharbitis nil Choisy). The maximum amount of LOX protein was detected when plants were grown at 30°C, whereas endogenous AOS and AOC proteins were markedly accumulated at 15°C. Although both test levels of 9(S)- and 13(S)-hydroperoxy linolenic acid (HPOT) showed similar temperature dependencies, reflecting the profile of LOX, the relative amount of 13(S)-HPOT was much higher than that of 9(S)-HPOT, regardless of temperature regime. This implied a faster reaction pathway to 9,10-α-ketol octadecadienoic acid (KODA) in the LA cascade. In the 13(S)-HPOT pathway, the highest level of endogenous jasmonic acid (JA) was observed at 15°C. Our results suggest that at a high temperature (30°C), 9(S)-HPOT may be readily metabolized into KODA to promote flower bud formation. By contrast, at a low temperature, high levels of AOS and AOC result in an accumulation of JA that inhibits this developmental process. Accordingly, depending on the growing temperature, flower bud formation in P. nil is possibly regulated by the interactions among LOX metabolites, with KODA serving as a promoter and JA as an inhibitor.

Bioconversion of proposed precursors into theobroxide and related compounds.

We have previously reported a tetraketide origin for theobroxide and its related compound. In the present study, bioconversion of natural and deuterium-labeled precursors of this proposed biosynthetic pathway by Lasiodipoldia theobromae was investigated. Theobroxide was quantified after bioconversion from each proposed precursor. The transformation of the isotopically labeled precursor to products was tracked by 2H NMR measurement.

Expression of allene oxide cyclase from Pharbitis nil upon theobroxide treatment.

In previous reports we have reported that theobroxide induces characteristic accumulation of allene oxide cyclase (AOC; EC 5.3.99.6) protein and jasmonic acid (JA) in Pharbitis nil. In the present study, PnAOC, an AOC gene from Pharbitis nil was cloned. Immunofluorescence assays indicated that the AOC protein is located in the chloroplast of vascular bundles in Pharbitis nil leaves. The PnAOC cDNA sequence lacking the chloroplast signal peptide was successfully expressed in Escherichia coli, and a gas chromatography-mass spectrum assay suggested the relative AOC activity of the recombinant PnAOC protein in comparison with Arabidopsis AOC2. Interestingly, a biphasic expression of PnAOC was induced by theobroxide, which is consistent with the accumulation patterns of AOC protein and JA. All these results indicate that AOC is the primary target of theobroxide regulation and suggest that feedback regulation of PnAOC by JA occurs upon theobroxide treatment in Pharbitis nil.

Temperature regulates tuber-inducing lipoxygenase-derived metabolites in potato (Solanum tuberosum).

Temperature is one of the major environmental factors affecting potato tuberization. It has been suggested that lipoxygenase (LOX) mediates between temperature and tuber induction. In this study, the contents of the LOX-derived metabolites hydroperoxylinolenic acid (HPOT), jasmonic acid (JA), tuberonic acid (TA) and tuberonic acid glucoside (TAG) were analyzed in leaves of potatoes growing at different temperatures. At low, tuber-inducing temperature, endogenous levels of JA, TA and TAG rise, indicating their crucial role in tuber induction. The concentration of 13(S)-HPOT seems not to be directly affected by temperature. Instead, the molecule has only a short half-life in leaves and is readily metabolized.

Novel cyclic peptide, epichlicin, from the endophytic fungus, Epichloe typhina.

The novel cyclic peptide, epichlicin, was isolated from Epichloe typhina, an endophytic fungus of the timothy plant (Phleum pretense L.). Its structure was determined by NMR studies and by mass spectrometry. Enantiomers of 3-amino tetradecanoic acid, a constituent amino acid of epichlicin, were synthesized as authentic standards. The stereochemistry of each amino acid was elucidated through a combination of the advanced Marfey method and chemical manipulation. Epichlicin showed inhibitory activity toward the spore germination of Cladosporium phlei, a pathogenic fungus of the timothy plant at an IC50 value of 22 nM.

Anti-babesial compounds from Curcuma xanthorrhiza.

Anti-babesial ingredients, (12R)- and (12S)-12,13-dihydro-12,13-dihydroxyxanthorrhizols, were isolated from Curcuma xanthorrhiza. The structures were established by the extensive NMR techniques. The assignments of (1)H NMR data of (12R)-12,13-dihydro-12,13-dihydroxyxanthorrhizol was revised, and (12S)-12,13-dihydro-12,13-dihydroxyxanthorrhizol was isolated as a pure form for the first time. The IC(50) of the active compounds were compared with that of commercial drug, diminazene aceturate (Ganaseg). IC(50) values of Ganaseg, (12R)-, and (12S)-12,13-dihydro-12,13-dihydroxyxanthorrhizols were 0.6 microg mL(-1), 8.3 microg mL(-1) and 11.6 microg mL(-1), respectively.

Inhibition of stem elongation in spinach by theobroxide.

In the current study, we investigated the influences of theobroxide on stem elongation in spinach (Spinacia oleracea). Our results showed that stem elongation and flower formation were inhibited by spraying spinach plants with theobroxide under inductive, long day conditions (16 h light/8 h dark), while application of exogenous applied GA3 prevented the effect of theobroxide. Quantitative analysis showed that theobroxide suppressed GA1 biosynthesis, whereas the endogenous content of jasmonic acid was unchanged. However, under short day conditions (10 h light/14 h dark), there were no differences in stem length between treated and untreated plants. These results suggest that the inhibition of stem elongation by theobroxide is probably due to the suppression of gibberellin biosynthesis.

Inhibitory role of gibberellins in theobroxide-induced flowering of Pharbitis nil.

Theobroxide, a novel active compound isolated from a fungus, has been reported previously to induce potato tuberization and flower bud formation in Pharbitis nil under non-inductive long-day conditions. Up to date, the action mechanism of theobroxide on flower-bud induction of P. nil, however, is still unknown. In the present study, we observed a reduction of the stem length, along with the induction of flower buds, in theobroxide-treated and short-day-grown P. nil plants. Also, the results showed that flower bud formation was delayed markedly in P. nil seedlings with removal of cotyledons or exposure to night break. The suppression effect of night-break and cotyledon-removal, however, was abolished completely by spraying theobroxide. Endogenous gibberellin(1/3) contents in P. nil plants treated with theobroxide or grown under short-day conditions were relatively lower, suggesting that gibberellins probably are negatively involved in theobroxide- and short-day-induced flower-bud formation of P. nil.

Ushikulides A and B, immunosuppressants produced by a strain of Streptomyces sp.

Novel immunosuppressants, ushikulides A and B, were isolated from the culture broth of Streptomyces sp. IUK-102. Ushikulides A and B both have the same molecular formula, determined as C40H68O10. The structures of both compounds were elucidated to be novel 22-membered macrolides. Both compounds showed immunosuppressive activity for murine splenocyte proliferation in vitro.

Allene oxide cyclase is essential for theobroxide-induced jasmonic acid biosynthesis in Pharbitis nil.

Theobroxide, a natural product, strongly stimulates the biosynthesis of jasmonic acid (JA) in Pharbitis nil. In this study, we investigated the accumulation of protein by the immunoblot analysis of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC), key enzymes in JA biosynthesis, and how the endogenous levels of JA in P. nil are affected by theobroxide. The effect of JA on the accumulations of these proteins was monitored simultaneously. The results show that theobroxide treatment led to a high level accumulation of JA, which is due to high accumulations of LOX, AOS, and AOC proteins induced by theobroxide treatment both under short day (SD) and long day (LD) conditions. However, under SD conditions AOS and AOC proteins are not enhanced by JA treatment. Kinetic analysis of protein levels shows that a biphasic activation of AOC protein by theobroxide is displayed and the first activation of AOC protein together with elevated JA levels is observed within 30min after treatment. Meanwhile, AOS and LOX proteins are activated by theobroxide later than AOC protein, suggesting that AOC plays an essential role in the initial JA formation induced by theobroxide. Since theobroxide-increased JA levels also show a biphasic manner similar to AOC activation and AOS, LOX proteins are activated later than AOC, and thus we propose a positive JA feedback regulation. Interestingly, AOS protein, which is also the enzyme for the biosynthesis of 9,10-ketol-octadecadienoic acid (KODA, a flowering inducing factor), accumulates markedly due to the simultaneous involvement of theobroxide and SD conditions, suggesting that AOS probably plays a role in flower bud formation in P. nil.

Evaluation of the inhibitory activities of the extracts of Indonesian traditional medicinal plants against Plasmodium falciparum and Babesia gibsoni.

Twenty-four kinds of water extracts derived from 22 plants that are traditionally used for the treatment of malaria on Java Island, Indonesia, were screened for their antibabesial and antimalarial activities. Among the extracts, 8 extracts displayed strong antimalarial activity, with an inhibition range from 89.6 to 100%, and 15 showed strong antibabesial activity, with an inhibition range from 84.2 to 98.1%. The extracts of Achillea millefolium, Baeckea frutenscens, Brucea javanica, Curcuma xanthorrhiza, Strychnos lucida and Swietenia macrophylla showed both strong antibabesial and antimalarial activities. The antimalarial activities paralleled the antibabesial activities, but the converse was not true.

Production of phleichrome by Cladosporium phlei as stimulated by diketopiperadines of Epichloe typhina.

Epichloe typhina is an endophytic fungus, while Cladosporium phlei is a pathogenic fungus of the timothy plant (Phleum pretense L.). We found two activities in the culture filtrate of E. typhina: one stimulated the pathogenic fungus, C. phlei, to produce phleichrome and the other inhibited its growth. The active ingredients that stimulated the production of phleichrome and inhibited the growth of C. phlei were isolated and characterized. The isolated compounds were identified as cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were stimulatory compounds, and p-hydroxybenzaldehyde, which was the growth inhibitory compound, based on an analysis of their spectral data. Of the two stimulatory compounds, cyclo-(L-Pro-L-Phe) showed higher activity. However, when 500 microg of cyclo-(L-Pro-L-Phe) was spotted on the TLC plate for bio-autography, a growth inhibitory zone was identified in the central red region, which contained phleichrome. On the other hand, phleichrome showed antifungal activity against E. typhina in the light, so it is assumed that there might be antagonism between the endophytic fungus, E. typhina, and the pathogenic fungus, C. phlei.

Novel potato micro-tuber-inducing compound, (3R,6S)-6-hydroxylasiodiplodin, from a strain of Lasiodiplodia theobromae.

A novel potato micro-tuber-inducing compound was isolated from the culture broth of Lasiodiplodia theobromae Shimokita 2. The structure of the isolated compound was determined as (3R,6S)-6-hydroxylasiodiplodin by means of spectroscopic analyses, the modified Mosher method, and chemical conversion. The compound showed potato micro-tuber-inducing activity at a concentration of 10(-4) M, using the culture of single-node segments of potato stems in vitro.

Anti-babesial compounds from Curcuma zedoaria.

Anti-babesial activity was confirmed in an extract of the bark of Curcuma zedoaria. The active ingredients were isolated, and their chemical structures were determined to be zedoalactones A, B, and C based on spectral data. Zedoalactone C is a hitherto unreported compound. The IC50 vales of these active compounds against Babesia gibsoni were compared with a standard drug, diminazene aceturate. The IC50 value of diminazene aceturate was 0.6 microg/mL, while those of zedoalactones A, B, and C were 16.5, 1.6 and 4.2 microg/mL, respectively.

Anti-babesial activity of some central kalimantan plant extracts and active oligostilbenoids from Shorea balangeran.

Bark extracts from a total of 22 species of Central Kalimantan plants were evaluated for their anti-babesial activity against Babesia gibsoni in vitro. Of these plant species, extracts of Calophyllum tetrapterum, Garcinia rigida, Lithocarpus sp., Sandoricum emarginatum, and Shorea balangeran showed more than 90% inhibition of the parasite growth at a test concentration of 1000 microg/mL. Activity-guided fractionation of the bark of S. balangeran (Dipterocarpaceae) led to the reisolation of oligostilbenoids, vaticanol A(1), B(2), and G(3). The structures were determined on the basis of spectral evidence. Compounds 1 and 3 showed complete inhibition on the growth of Babesia gibsoni in vitro at a concentration of 25 microg/mL, and compound 2 at concentration of 50 microg/mL.

Novel antifungal compounds produced by Sterile Dark, an unidentified wheat rhizosphere fungus.

Two novel antifungal compounds were isolated from a culture broth of Sterile Dark, an unidentified fungus isolated from the rhizosphere of wheat grown in a continuous cropping field. These compounds were elucidated to be phthalide-based compounds by spectroscopic analyses.

The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain.

To clarify the processes involved in plant immunity, we have isolated and characterized a single recessive Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response (HR). This mutant shows spontaneously activated expression of pathogenesis-related (PR) genes, and leading to a 32-fold increase in salicylic acid (SA). Inoculation of cad1 mutant plants with Pseudomonas syringae pv tomato DC3000 shows that the cad1 mutation results in the restriction of bacterial growth. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (membrane attack complex and perforin) domain of complement components and perforin proteins that are involved in innate immunity in animals. Furthermore, cell death is suppressed in transgenic cad1 plants expressing nahG, which encodes an SA-degrading enzyme. We therefore conclude that the CAD1 protein negatively controls the SA-mediated pathway of programmed cell death in plant immunity.

Anti-babesial and anti-plasmodial compounds from Phyllanthus niruri.

Bioassay-guided fractionation of boiled aqueous extracts from the whole plant of Phyllanthus niruri led to the isolation of 1-O-galloyl-6-O-luteoyl-alpha-d-glucose (1), with IC(50) values of 4.7 microg/mL against Babesia gibsoni and 1.4 microg/mL against Plasmodium falciparum in vitro. The known compounds beta-glucogallin (2), quercetin 3-O-beta-d-glucopyranosyl-(2-->1)-O-beta-d-xylopyranoside (3), beta-sitosterol, and gallic acid were also isolated. Structures of these compounds were elucidated on the basis of their chemical and spectroscopic data.