A Dipterocarpaceae plant Shorea roxburghll G. DON is widely distributed in Thailand and the neighboring countries, Cambodia, India, Laos, Malaysia, Myanmar, and Vietnam, etc. The plant is locally called "Phayom", and its bark has been used as an astringent or a preservative for traditional beverages in Thailand. In addition, the bark has also been used for treatments of dysentery, diarrhea, and cholera in Indian folk medicine. In the present study, isolation and structure elucidation of five new 3-ethyl-4-phenyl-3,4-dihydroisocoumarins, phayomphenols (1-5), are presented. Several oligostilbenoids isolated concurrently from this medicinal plant were found to show preventive effects from metabolic syndrome, i.e. inhibiting effects against plasma glucose elevation in sucrose-loaded and triglyceride elevation in olive oil-loaded mice. To clarify the mode of action of the antihyperglycemic and antihyperlipidemic activities, effects of the active oligostilbenoids on gastric emptying in mice, glucose uptake in isolated intestinal tissues as well as inhibitory activities against intestinal a-glucosidase were examined and discussed. In addition, the protective effects of these stilbenoids against liver injury and fatty liver revealed in the present study will also be presented.

抄録全体を表示

Marine dinoflagellates of the genus Amphidinium are well-known as a producer of unique cytotoxic metabolites. We have isolated two new macrolides, iriomoteolides-13a (1) and -10a (2), from the benthic dinoflagellate Amphidinium species collected off Iriomote Island, Japan. In this symposium, we will discuss the isolation and structural elucidation of these new macrolides. The dinoflagellate Amphidinium sp. (strain KCA09053) was cultivated in 50 L of seawater medium, 16 h light and 8 h dark. The algal cells obtained from 350 L of the medium were extracted with MeOH/toluene (3:1). The toluene-soluble materials of the extract were subjected to a SiO_2 gel and C_<18> columns, and one of a macrolide containing fractions were separated by C_<18> HPLC to afford new compounds, iriomoteolides-13a (1) and -10a (2) Structure elucidation of iriomoteolides-13a (1) and -10a (2) were carried out by detailed analyses of 2D NMR data and MS spectra. Iriomoteolide-13a (1) is a novel 22-membered macrolide with a tetrahydrofuro[3,2-b]furan ring, a tetrahydropyran ring, three one-carbon branches, a ketone carbonyl, and three hydroxyl groups. On the other hand, iriomoteolide-10a (2) is a new 21-membered macrolide with a tetrahydropyran ring, six one-carbon branches, two ketone carbonyls, and two hydroxyl groups. Compounds 1 and 2 exhibited cytotoxic activity against human epidermoid carcinoma KB cells (IC_<50>: 0.5 and 0.9 μg/mL, respectively).

抄録全体を表示

Reveromycin A (RM-A) is a polyketide compound with a distinctive spiroacetal core structure, has isolated as an inhibitor of mitogenic activity induced by epidermal growth factor from Streptomyces reveromyceticus SN-593. RM-A shows various biological activities and one of them is a strong anti-osteoclastic activity, which is the most prominent feature of RM-A as a drug lead. We recently have succeeded in the identification of the biosynthetic gene cluster of RM-A by gene disruption and complementation analyses. We also identified reveromycin T (RM-T) as the biosynthetic precursor of RM-A by feeding experiments. During such experiments, we found that unexpected compounds were produced by the addition of small amount of alcohol to S. reveromyceticus SN-593 during fermentation. They were speculated to be novel reveromycin derivatives by a photodiode array detector attached LC/MS analysis. It is important to expand structural variety of RMs for structure-activity relationship study for a feature drug development. Therefore, we tested some culture conditions, and found that the addition of alcohol to a culture broth during the fermentation led to the production of new RM derivatives. This method was applied to a large-scale fermentation, and two new RMs, RM-T mono methyl and mono ethyl esters were produced and isolated by the addition of methanol and ethanol, respectively. Their structures were elucidated by spectroscopic and chemical methods. Moreover, their cytotoxic activities against human cancer cell lines, HL-60 and K562, and mouse cell lines FT210, and antibacterial activity against Escherichia coli HO-141 were evaluated. The cytotoxic activities against human cancer cell lines of the new RMs were improved.

抄録全体を表示

Metastasis is the major cause of cancer mortality. During the process of metastasis, invasion and migration are critical steps for cancer cells to spread from the primary tumor and establish the secondary tumor. Therefore, suppression of these steps is considered as a key approach to control tumor progression. In our continuing search for novel inhibitors of tumor cell migration and invasion from microorganisms, we found pterocidin, a polyketide with an α,β-unsaturated-δ-lactone from the culture extract of sediment-derived Streptomyces, which was previously isolated from another Streptomyces as a cytotoxic compound by our group. In this study, we elucidated all stereocenters present in this molecule by a series of J-based configuration analyses and NOESY analysis, coupled with chemical derivatization and chiral anisotropy analysis. In addition, we discovered two novel migration inhibitors, ulbactins F and G from sponge-derived Brevibacillus. These compounds belong to a class of secondary metabolites biosynthesized from salicylic acid and cysteins. Their most intriguing structural feature is a heterocyclic ring system consisting of two nitrogen- and sulfur-containing five-membered rings fused into a tricyclic system. Absolute configurations of these compounds were established by X-ray crystallographic analysis.

抄録全体を表示

The ocean is a rich source of natural products with novel structural features and biological activities. We have investigated bioactive compounds from marine organisms such as sponges, ascidians, and marine-derived microorganisms. In the course of our search program for novel and useful compounds, three new sesquiterpenes (1-3), designated euryspongin A-C, were isolated from a marine sponge Euryspongia sp. collected at Iriomote Island. In this presentation, we described the isolation and structure elucidation including the stereochemistry of euryspongins A-C. From the EtOH extract of the sponge, compounds 1-3 were purified by ODS column chromatography and HPLC. The structure of 1 was assigned on the basis of spectroscopic data including various NMR experiments. Compound 1 had a unique bicyclic furanosesquiterpene structure with six- and eight-membered rings. The stereochemistry of 1 was assigned by ^1H-^1H coupling constants, NOESY spectrum, and a comparison of the specific rotation of 1 with that of the related compound. ^1H-NMR spectra of 2 and 3 were very similar to that of 1. Comparison of NMR data among compounds 1-3 indicated that a furan ring in 1 was replaced by an α,β-unsaturated-γ-lactone ring in 2 and 3. The stereochemistry of compounds 2 and 3 were deduced to be the same as that of 1 by the analysis of ^1H-^1H coupling constants and NOESY data. Sesquiterpenes having a six- and eight-membered bicyclic structure are quite rare in the nature. To our knowledge, only four compounds have been reported as natural products in this class. Investigation on the biological activity of 1-3 is now in progress.

抄録全体を表示

Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic of members of the galectin family of oligosaccharide-binding proteins. The lectin activity was remarkably stable, with only a modest loss in hemagglutination after exposure of the protein to 100℃ for 1 h, and showed little sensitivity to calcium concentration. CchG-1 and -2 appeared as 16 and l8kDa in SDS-PAGE, respectively, whereas MALDI-TOF MS indicated broad ion clusters centered at 16,216 and 16,423 respectively. The amino acid sequences of the CchGs were deduced using a combination of Edman degradation and cDNA cloning and revealed that the proteins were distant orthologues of animal prototype galectins and that multiple isolectins comprised the CchGs. The biochemical properties of the CchGs as well as their unexpected activity on mammalian excitatory amino acid receptors suggest that further analysis of these new members of the galectin family will yield further glycobiological and neurophysiological insights.

抄録全体を表示

Among the Hymenopteran insect venoms, those having a large nest and social life such as honeybee, hornets and paper wasps have been well documented. Their venoms are composed of a number of peptides and proteins, which is used for defending their nests and themselves. In contrast, the venoms of solitary wasps and bees, those having solitary life, have not been studied well. We have surveyed solitary wasp venoms inhabiting in Japan and found neurotoxins and antimicrobial peptides. However, solitary bee venoms have never been studied until quite recently. The first study of solitary bee venoms has reported only in 2008 about the European solitary bee, Melecta albifrons. A novel peptide, melectin, was isolated and characterized. Melectin has similar characteristics to those of melittin and mastoparan from the honeybee and hornet venom, respectively: rich in hydrophobic and basic amino acids, amphipathic, and showing antimicrobial, mast cell degranulating and hemolytic activities. Accordingly, this peptide belongs to linear cationic α-helical peptides. In 2010, another solitary bee venom peptide, osmin, was found from the European solitary bee Osmia rufa. Chemical and biological characteristics of osmin are basically similar to those of melectin. We have been studying solitary bee venom inhabiting in Japan and found two major peptide components, xylopin from Xylocopa appendiculata circumvolans and sculpin from Megachile sculpturalis, belonging to linear cationic α-helical peptides. Reported herein is the chemical and biological characterization of these novel venom peptides.

抄録全体を表示

Gram-negative bacteria communicate with one another using N-acylhomoserine lactones (AHLs) as signaling molecules. This mechanism, known as quorum sensing (QS), is needed to develop pathogenic and symbiotic interactions with eukaryotic hosts such as animals and plants. Increasing evidence indicates that certain bacteria, namely endobacteria, inhabit fungal cells and establish symbiotic relationships with hosts. However, it has not been clear whether bacterial QS acts in developing the relationships. Here we describe the isolation and identification of N-heptanoylhomoserine lactone and N-octanoylhomoserine lactone from the culture broth of the zygomycete fungus Mortierella alpina A-178. This suggested the presence of endobacteria in the fungus, which was confirmed by PCR, fluorescence in situ hybridization, and transmission electron microscopy. Two major bands detected by PCR-denaturing gradient gel electrophoresis showed sequence identity with the 13-Proteobacterium Castellaniella defragrans (100%) and Gram-positive bacterium Cryobacterium sp. (99.8%). The production of AHLs depended on the presence of endobacteria and was induced in response to the increase in the concentration of AHLs, suggesting that the bacterium conducts AHL-mediated QS in the fungus. This research is the first to report the production of AHLs by endofungal bacteria and raises the possibility that QS plays roles in the development of fungus-endobacterium symbiosis.

抄録全体を表示

The joro spider venom is a mixture of acyl-polyamine analogs whose chemical structures consist of 4 building blocks (Fig.1). It is difficult to isolate each constituent of venom even by HPLC because those compounds are very similar. Therefore those compounds were analyzed by mass spectrometry to get the structural information. In order to obtain structural information of the venom, especially that of polyamine backbone, high-energy collision-induced dissociation (HE-CID), which can cleave C-C bonds, is necessary. These have been analyzed by combination of solid-phase extraction and 4-sector tandem MS. We reported that the product ion spectra obtained by the JMS-S3000 SpiralTOF were very similar to those obtained by the 4-sector tandem MS. In this report, we analyzed known acyl-polyamines in joro spider venom using the combination of easy sample preparation and the SpiralTOF. The 14 peaks corresponding to the sodium ion adduct of known acyl-polyamine molecules were observed by the SpiralTOF (Fig.4). The Charge-Remote Fragmentations (CRF) by means of HE-CID were observed from all 14 components by the SpiralTOF-TOF. In addition, obtained product ion spectra pattern from the precursor ions which have same structural groups were very similar each other (Fig.6). It was suggested that these compounds could be easily identified if the database of the product ion spectra by HE-CID would be available.

抄録全体を表示

Ants are social insects, which means they live in large colonies or groups. An ant has eyes that allow them to see extremely well because of the many lenses. Ants also use chemicals called pheromones to leave scent trails for other ants to follow. When the visual information and the chemical information are contradictory, to which information does an ant give priority? In the present study, we study on the selection of the foraging path of garden ant, genus Lasius, which can recognize and response to both visual information and chemical information. Specifically, by setting un-optimized initial pheromone path that connects the nest to a feeding site with one corner of finite angles along the path, we made situations such that the optimal foraging path and along-pheromone path are separated from each other. Hence with varying the corner angle and the total path length, we observed whether ants keep initial path or newly develop the optimal path. It was found that for homing ants that got food, a sharp transition from the regime of keeping the initial pheromone path to the regime of developing the optimal path takes place as the relative angle between the direction assingned by the chemical information and that assingned by the visual information exceeds a threshold. Next, it is believed that the trail pheromone has a species-specific structure. F. Kern et al. identified Mellein as a trail pheromone of the ant (Lasius niger) which widely inhabits Europe. Until 2005, the similar kind of ant which inhabits Japan was named Lasius niger. However, recently, the ant has been called Lasius japonicus from the behavioral and the physiological difference. This fact suggests that a new trail pheromone exists to Lasius japonicus. We now wish to report the identification of the multicomponent trail pheromone of Lasius japonicus, which consists of a synergistic mixture of three alkyl ketones. 2-Tridecanone, 2-undecanone and 2-dodecanone are components of the trail pheromone of the garden ant, Lasius japonicus. These ketones were extracted from the rectal fluid of dissected worker ants, and identified by the mass spectra and gas-chromatographic retention times. The same ketones could also be detected in the material excreted by the ants on their foraging path. We believe this identification to be carried out at the lowest level of detection yet achieved for a pheromone, and for it to be the most complex mixture yet identified as an insect trail pheromone.

抄録全体を表示

(+)-Epirhododendrin (1), a rhododendrol glycoside, was isolated from the stem bark of Acer nikoense. This compound has a characteristic oxymethine at C-2 and its configuration has been verified as S. In contrast, (-)-rhododendrin (2) as C-2 epimer of 1 was frequently found in number of plant species. Biosynthetic pathway and stereocontrolled synthesis of 1 and 2 were well studied by several research groups. However, molecular design of novel biologically active compounds on the basis of the rhododendrol skeleton has been rarely investigated. Rhododendrol glycoside 1 and 2 contain a monophenol moiety. When a hydroxyl group introduces into C-2' position of 1 and 2, they change into alkyl resorcinol 3 and 4. Alkyl resorcinol has been known as a potent and lipophilic tyrosinase inhibitor, which can be useful for the development of antioxidant, insecticide and functional cosmetics. Therefore, we performed concise synthesis of 1-4 and their evaluation of tyrosinase inhibitory activity in this study. Synthesis of 1 and 2 from raspberry ketone (5) was achieved in 36% yield via 5 steps containing trichloroacetimidate glycosylation as a key reaction. According to the similar synthetic route, 3 and 4 were furnished from 2,4-dihydroxybenzaldehyde (9) in 22% yield via 7 steps. Although separable condition of 1 and 2 could not be optimized, 3 and 4 were isolated with the aid of RP-HPLC using 12% MeCN in H_2O as a mobile phase. On the stage of the biological evaluation, 3 and 4 acted as potent tyrosinase inhibitors. It should be noted that IC_<50> of 4 is about twofold more potent than that of 3. This significant activity indicates that 4 might possess a unique property of stereochemical recognition against tyrosinase.

抄録全体を表示

Long-chain fatty acids are indispensable precursors in biosyntheses of phospholipids and signal transducers. Because of their low solubility in aqueous media, fatty acids are in need of binding proteins to increase their concentration in biological fluid. Fatty acid-binding proteins (FABPs) bind free fatty acids with high affinity to provide their intracellular solubilization and trafficking. The molecular recognition of fatty acid by FABPs effects both preference to long chain fatty acids and tolerance to variation in chain length and unsaturation. We were particularly interested in the molecular mechanism of this fuzzy recognition, and launched structural and mechanistic investigations on human heart FABP (hFABP3)-fatty acid complexes. In this study, we would like to report (i) X-ray crystal structures of hFABP3 with/without fatty acid, (ii) hFABP3-fatty acid interaction analysis by isothermal titration calorimetry (ITC), and (iii) dynamic structural analyses of hFABP3-fatty acid complexes by molecular dynamics (MD) simulations. Present results showed that (i) the binding of fatty acids to hFABP3 were accompanied by fixation of water molecules inside the binding pocket, (ii) the reordering of water molecules is important for hFABP3-fatty acids interaction, and (iii) hFABP3-bound fatty acids undergo conformational changes inside the binding pocket. A detailed study on the correlation between the dynamic structure of bound fatty acids and the reordering of water molecules inside the binding pocket is currently underway.

抄録全体を表示

Exposure of UVB causes production and secretion of several major melanogenic cytokines such as endothelin-1 (ET-1) and stem cell factor (SCF) via autocirne of interleukin (IL)-1α in keratinocyte. Those melanogenic cytokines stimulate melanocytes, leading to increase of epidermal pigmentation via activation of intracellular signaling cascades and up-regulation of several melanocyte-specific proteins. The inhibitor of UVB-induced melanocyte activation can contribute to improvement or prevention of epidermal pigmentation disorders. In this study, we isolated three new cembrene compounds (1〜3) and (+)-nephthenol from an Okinawan soft coral by a bioassay of the inhibitory effect of melanogenesis on B16 melanoma cells. The isolated compounds significantly decreased melanin contents at 30 μM. In addition, new cemberene compound 1 attenuated tyroinase activity of B16 melanoma cells without cytotoxicity. Furthermore, we investigated whether compound 1 inhibited UVB-induced melanocytes activation using cell-insert method (melanocytes co-cultured with UVB-exposed keratinocytes). Compound 1 decreased tyrosinase activity of melanocytes without cytotoxicity. Western blotting analysis revealed that tyrosinase expression was increased by co-culturing with UVB exposed-keratinocytes, whereas treatment with compound 1 suppressed its expression. These results suggest that new cembrene compounds inhibit melanogenesis by interrupting tyrosinase expression.

抄録全体を表示

Gambierol (1), a polycyclic ether natural neurotoxin isolated from the ciguatera causative dinoflagellate, Gambierdiscus toxicus has been reported to be a potent and subtype-selective blocker of voltage-gated potassium channels (VGPCs). Previous studies of marine polycyclic ether molecules have suggested the importance of the whole skeleton of polycyclic ether for potent biological activity. We have previously investigated the peripheral structure-activity relationships of Gambierol, however, it remained unclear whether the full length of polycyclic ether skeleton is essential for its toxicity. In this presentation, we designed and synthesized two structurally simplified analogues of Gambierol comprising BCDEFGH- and EFGH- rings of the parent compound (2 and 3). Surprisingly, both analogues showed comparable potency to Gambierol on VGPCs inhibition in cerebellar granule cells of mice. These results indicated that we obtained the easily synthesized analogues of Gambierol that have potent biological activity. Moreover, to investigate the additional functions of truncated analogues, we examined the effect of these compounds in a model of Alzheimer's disease (AD) obtained from triple transgenic mice, which expresses amyloid beta (An) accumulation and tau hyperphosphorylation. In vitro preincubation of the neurons of these mice with Gambierol or analogues decreased steady-state level of the NMDA receptor subunit 2A without affecting the 2B subunit. In addition, treatment of these compounds reduced the intra- and extracellular levels of Aβ and the levels of hyperphosphorylated tau. This study constitutes the first discovery of designed structurally simplified analogues of polycyclic ether compound possessing potent biological activity. Furthermore, it suggested the practicality of gambierol and analogues as chemical probes for understanding the function of VGPCs and the mechanism of modulation of the accumulation of Aβ and hyperphosphorylated tau by NMDA receptors.

抄録全体を表示

It is now generally accepted that the requirement for minimum 6 months treatment for Tuberculosis is due to the difficulty in eradicating dormant states of M. tuberculosis. Although physiology of the latent M. tuberculosis infections is still unclear, hypoxic condition was found to induce a dormant state of Mycobacterium sp., which has a drug susceptibility profile resembling that of the latent M. tuberculosis infection. Based on these findings, we have established a screening system for anti-dormant mycobacterial substances. As a result of screening, we isolated three new aminolipopeptides, named trichoderins A (1), A1 (2), and B (3) from a marine sponge-derived fungus of Trichoderma sp. Trichonderins showed potent anti-mycobacterial activity against M. smegmatis and M. bovis BCG under standard aerobic growth condition as well as dormancy-inducing hypoxic condition, with MICs in the range of 0.02-1.56 μg/mL. In addition, we also isolated the marine spongian diterpene alkaloids, agelasines B (4), C (5), and D (6), as anti-dormant mycobacterial substances with MICs in the range of 0.8-12.5 μg/mL. On the other hand, it is known that the transformant, which over-expresses the target molecule of anti-microbial compound, becomes resistant to the compound. In order to identify the gene that could confer a resistance to trichoderin A (1) or agelasine D (6), the transformants of M. smegmatis, which over-expressed the genes of M. bovis BCG randomly, were prepared by geomic DNA library of M. bovis BCG. Then, the transformant of M. smegmatis, which over-expressed a part of genes that coded mycobacterial ATP synthase, was found to exhibit a resistance to trichoderin A (1). In addition, trichoderin A (1) reduced ATP contents in M. bovis BCG. These findings suggested that the anti-mycobacterial activity of trichoderin A (1) might come from the inhibition of ATP synthesis. In the case of agelasine D (6), the four genes, BCG3184c, 3185c, 3186c and 3187c, were identified as candidates for target molecule of agelasine D (6) according to the analysis of cosmids in the agelasine D (6)-resistant transformants.

抄録全体を表示

Ladder-shaped polycyclic ethers (LSPs) exhibit potent toxicity due to their strong interactions to channel proteins. For example, brevetoxin binds sodium channels, and gambierol binds potassium channels. However the detail of this interaction has not been clarified yet. We estimated the hypothesis that LSPs interact with alpha helices of channel proteins through the hydrogen bond as figure 1. To prove this hypothesis, we attempt to prepare a double transmembrane peptide (A). This peptide was designed to have amphiphilic character, which may cause this peptide making a four alpha helix bundle like channel proteins. N-terminal peptide thioester and C-terminal cysteine peptide were synthesized by solid phase peptide synthesis. These peptides were connected by native chemical ligation subsequently to obtain peptide (A). Next, the optimal method of reconstitution in liposomes was investigated. As the result, the detergent removal method by dialysis using CHAPS was the best suited. In order to analyze the interaction between the peptide and lipid bilayer, gel filtration, digestion by a protease, and FRET experiments were conducted. For the positive and negative controls, peptide (B) and (C) were prepared by chemical synthesis. Peptide (B) is known as a transmembrane peptide, and peptide (C) is known to bind the surface of lipid bilayer. In the gel filtration, peptide (A) and LUV were eluted at the same time, indicating peptides to associate with liposomes. In the digestion by a protease, thermolysin, was used as a protease that cut the N side of hydrophobic residues. After treatment of peptide (A) reconstituted in liposome with thermolysin for 1 hour and 3 hours, the peptide MS peak was detected by LC-ESI MS. In contrast, the peptide not reconstituted in liposome was not detected after 1 hour digestion. However, any digested fragments of the peptide were not detected. A FRET experiment was planned, in addition. When FRET between fluorescent labeled phospholipids and the fluorescent group of peptide (F) is observed, it shows that the center of the peptide is located at the center of the lipid bilayer. As the result, the FRET induced fluorescence, though in a low intensity, was observed in the fluorescent labeled peptide (F) and peptide (B). FRET between the double transmembrane peptide and LSP (yessotoxin) will be similarly attempted to observe.

抄録全体を表示

Imaging of biological components is a fundamental and important technology in life science. Bioluminescent imaging enables high-sensitive and non-invasive detection of biological components. Green fluorescent protein (GFP) emits light under irradiation of UV light. Static imaging of reporter genes in living cells has been achieved by using GFP. Luciferases and photoproteins require biological components (such as reactive oxygen species (ROS), calcium ion, and ATP) for light emission. These proteins enable dynamic imaging of biological components in living cells. Pholasin is a photoprotein of a common piddock, Pholas dactylus, which emits light in the presence of ROS. Pholasin is commercially available as a ROS indicator. Pholasin has a chromophore for emission of light and its structure has not been determined. Here we report that the chromophore is constituted from apo-Pholasin and dehydrocoelenterazine (DCL). DCL is the organic substance of symplectin, a photoprotein of a luminescent squid (Symplectoteuthis oualaniensis), and is a component of the chromophore of symplectin. Luminescence of symplectin is also initiated by ROS. The similar involvement of ROS in the luminescence of Pholasin and symplectin formulated a hypothesis that DCL might be the organic substance of Pholasin. Actually, addition of DCL resulted in enhancing the activity of Pholasin. The existence of DCL in Pholasin was evidenced by isolating DCL as dithiothreitol adduct from Pholasin. The structure was identified by LC-MS comparing with the synthetic compound. From these results, we concluded that DCL is the organic substance of Pholasin and forms the chromophore for emission of light.

抄録全体を表示