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Fuscoporia torulosa (Pers.) (Hymenochaetaceae) is a mushroom forming woody fruiting body on living or dead trees. Two curious pentacyclic triterpenoids, fuscotorunones A and B, each of which has a unique ε-caprolactone in ring E, were isolated from the fruiting bodies of F. torulosa. The structures of fuscotorunones A and B were elucidated using MS analyses, IR spectrum and extensive 2D-homo and heteronuclear NMR data interpretation. Furthermore, a predicted biosynthetic pathway from 2,3-oxidosqualene to fuscotorunones A and B in F. torulosa is proposed.

During the search for new antitrypanosomal drug leads, four antitrypanosomal compounds, of three depsipeptides and one nortriterpenoid, were isolated from cultures of the mutant strain IU-3 of the insect pathogenic fungus Ophiocordyceps coccidiicola NBRC 100683. Their structures were identified by the analysis of high resolution-electron ionization (HR-EI)-MS and HR-FAB-MS, and (1)H- and (13)C-NMR spectra, including extensive two dimensional (2D)-heteronuclear NMR experiments, and comparison with literature data for destruxin A (1), destruxin B (2), destruxin E chlorohydrin (3) and helvolic acid (4). Compounds 1-4 showed in vitro antitrypanosomal activity against Trypanosoma brucei brucei GUTat3.1 with IC50 values of 0.33, 0.16, 0.061 and 5.08 µg/mL, respectively.

Over several hundred new compounds have been isolated from the bryophytes and more than 40 new carbon skeletal terpenoids and aromatic compounds found in this class. Most of liverworts elaborate characteristic odiferous, pungent and bitter tasting compounds many of which show, antimicrobial, antifungal, antiviral, allergenic contact dermatitis, cytotoxic, insecticidal, anti-HIV, superoxide anion radical release, plant growth regulatory, neurotrophic, NO production inhibitory, muscle relaxing, antiobesity, piscicidal and nematocidal activity. Several inedible mushrooms produce spider female pheromones, strong antioxidant, or cytotoxic compounds. The present paper concerns with the isolation of terpenoids, aromatic compounds and acetogenins from several bryophytes and inedible fungi and their cytotoxic and antiviral activity.

In this study, we describe a rational design strategy to develop a latent ratiometric fluorescent pH probe from self-assembled seminaphthorhodafluor (SNARF) derivatives in aqueous conditions. Analysis of the characteristics of SNARF derivatives with protected phenolic groups allowed classification of the threshold between the assembled and monomeric states according to the Hansch–Fujita hydrophobic parameters for a substituent inserted as a SNARF-OH phenolic-protecting group. The esterase-activated latent ratiometric fluorescent pH probe SNARF-OBn(OAc) was characterized in the application of tuneable small-molecule aggregation and disaggregation. Before esterase treatment, SNARF-OBn(OAc) formed self-assembled clusters, emitting no fluorescence. The significant fluorescence was observed after esterase treatment because the self-assembled clusters of SNARF-OBn(OAc) diffuse as SNARF-OH monomers, demonstrating the intracellular application of SNARF-OBn(OAc) as a latent ratiometric fluorescent pH probe for floating cells. Furthermore, to directly visualize the state of self-assembled SNARF derivatives for investigating the mechanism of their cellular uptake, SNARF-Dan was rationally designed and applied for cellular imaging. Through the cellular application of SNARF-Dan, it was suggested that the self-assembled SNARF derivatives were introduced into the cell via macropinocytosis.

During the search for new antitrypanosomal drug leads, three new antitrypanosomal compounds, cardinalisamides A-C (1-3), were isolated from cultures of the insect pathogenic fungus Cordyceps cardinalis NBRC 103832. Their structures were elucidated using MS analyses and extensive 2D-heteronuclear NMR. The absolute configurations of 1-3 were addressed by chemical degradation and Marfey's analysis. 1-3 showed in vitro antitrypanosomal activity against Trypanosoma brucei brucei with IC50 values of 8.56, 8.65 and 8.63 μg ml(-1), respectively.

Yellow pigments, fomitellanols A (1a) and B (2a), and drimane-type sesquiterpenoid ethers of isocitric acid, cryptoporic acids P (3) and Q (4), have been isolated from the fruiting bodies of Fomitella fraxinea (Polyporaceae). Their structures were established by a combination of extensive NMR spectroscopy and/or X-ray crystallographic analyses, and their biological activity against COX-1, COX-2, and 5-LO was investigated.

Farnesyl caffeate, synthesis of propolis and polar bud excretion, has been reported to exhibit anti-allergic effects in mice. However, little is known about anti-tumor effects. In this study, we investigated the effect of Farnesyl caffeate in cell proliferation of lung carcinoma cell line (H157). Antiproliferative effect and apoptosis on H157 cell were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT) and DNA fragmentation assay, respectively. Farnesyl caffeate inhibited the growth of H157 cell in dose-dependent manner. Morphological changes of nuclei by staining Hoechst 33258 and DNA fragmentation suggested that Farnesyl caffeate induced death involved in a mechanism of apoptosis. Moreover, caspase-3, caspase-7 and caspase-9 were activated by Farnesyl caffeate on H157 cell. The protein expressions of Bax and Bcl-2 were down-regulated by Farnesyl caffeate, resulting in cytochrome c release from the mitochondria. Farnesyl caffeate significantly increased the expression of p53 proteins which indicates that p53 plays a pivotal role in the initiation phase of Farnesyl caffeate-induced HepG2 cell apoptosis. These results demonstrated Farnesyl caffeate-induced apoptosis in human lung carcinoma cell line. More detailed mechanism of Farnesyl caffeate-induced H157 apoptosis remains to be elucidated.

Growth of neurite processes is a critical step in neuronal development, regeneration, differentiation, and response to injury. The discovery of compounds that can stimulate neurite formation would be important for developing new therapeutics against both neurodegenerative disorders and trauma-induced neuronal injuries. Semisynthetic derivatives of artemisinin, an active compound in Artemisia annua, have been effectively used in malaria treatment, but they have been shown to possess neurotoxic potential. In this study, we found unexpectedly that artemisinin and its derivatives induced neurite outgrowth of PC12 cells. Artemisinins containing an endoperoxide bridge such as artemisinin and dihydroartemisinin induced growth of neurite processes at concentrations that were slightly cytotoxic, artemisinin having the most potent maximal effect among them. Deoxyartemisinin, which lacks the endoperoxide bridge, was ineffective. Artemisinin-treated cells expressed increased levels of the neuronal marker βIII-tubulin. Artemisinin upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), critical signaling molecules in neuronal differentiation. Consistent with activation of the two MAPKs, neurite outgrowth induced by artemisinin was inhibited by the MAPK/ERK kinase inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Artemisinin also induced phosphorylation of cyclic AMP response element-binding protein (CREB) that was almost completely attenuated by PD98059 but not by SB203580. Taken together, our results indicate that artemisinin and its derivatives containing the endoperoxide bridge induced differentiation of PC12 cells toward a neuronal phenotype and suggest that both activation of ERK signaling pathway, which leads to CREB phosphorylation, and activation of p38 MAPK signaling pathway are involved in this process.

Obesity is a major public health problem. An in-depth knowledge of the molecular mechanisms of oro-sensory detection of dietary lipids may help fight it. Humans and rodents can detect fatty acids via lipido-receptors, such as CD36 and GPR120. We studied the implication of the MAPK pathways, in particular, ERK1/2, in the gustatory detection of fatty acids. Linoleic acid, a dietary fatty acid, induced via CD36 the phosphorylation of MEK1/2-ERK1/2-ETS-like transcription factor-1 cascade, which requires Fyn-Src kinase and lipid rafts in human taste bud cells (TBCs). ERK1/2 cascade was activated by Ca2+ signaling via opening of the calcium-homeostasis modulator-1 (CALHM1) channel. Furthermore, fatty acid–evoked Ca2+ signaling and ERK1/2 phosphorylation were decreased in both human TBCs after small interfering RNA knockdown of CALHM1 channel and in TBCs from Calhm1−/− mice. Targeted knockdown of ERK1/2 by small interfering RNA or PD0325901 (MEK1/2 inhibitor) in the tongue and genetic ablation of Erk1 or Calhm1 genes impaired preference for dietary fat in mice. Lingual inhibition of ERK1/2 in healthy volunteers also decreased orogustatory sensitivity for linoleic acid. Our data demonstrate that ERK1/2-MAPK cascade is regulated by the opening of CALHM1 Ca2+ channel in TBCs to modulate orogustatory detection of dietary lipids in mice and humans.—Subramaniam, S., Ozdener, M. H., Abdoul-Azize, S., Saito, K., Malik, B., Maquart, G., Hashimoto, T., Marambaud, P., Aribi, M., Tordoff, M. G., Besnard, P., Khan, N. A. ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans.

A novel phenolic metabolite, ornatipolide, was isolated from the Boletus ornatipes fungus. Its structure was established by a combination of spectroscopic and chemical methods, and by an X-ray crystallographic analysis.

During the search for new antitrypanosomal drug leads, four antitrypanosomal compounds, of three depsipeptides and one nortriterpenoid, were isolated from cultures of the mutant strain IU-3 of the insect pathogenic fungus Ophiocordyceps coccidiicola NBRC 100683. Their structures were identified by the analysis of high resolution-electron ionization (HR-EI)-MS and HR-FAB-MS, and (1)H- and (13)C-NMR spectra, including extensive two dimensional (2D)-heteronuclear NMR experiments, and comparison with literature data for destruxin A (1), destruxin B (2), destruxin E chlorohydrin (3) and helvolic acid (4). Compounds 1-4 showed in vitro antitrypanosomal activity against Trypanosoma brucei brucei GUTat3.1 with IC50 values of 0.33, 0.16, 0.061 and 5.08 µg/mL, respectively.

During the search for secondary metabolites with antiproliferative activity, six new lanostane triterpenoids, tyrosamic acids A-F (1-6) together with ten known compounds (7-16), were isolated from the fruiting body of Tyromyces sambuceus. Their structures were elucidated using MS analyses, extensive 2D-heteronuclear NMR data interpretation and the structure of 3 was further confirmed by single-crystal X-ray data analyses. All lanostane triterpenoids (1-16) possesses a carboxy group at C-20 position and their strength of antiproliferative activity was affected by the presence or absence of a hydroxy group at C-15 position and at the side chain. Four of the compounds (1, 6, 10, 14) showed antiproliferative activities against human cancer cell lines with IC₅₀ values of 16.8-48.3 µM (HL-60).

During our search to discover new antitrypanosomal compounds, eight known plant compounds (three phenolic compounds and five kawa lactones) were evaluated for in vitro activity against Trypanosoma brucei brucei. Among them, we found two phenolic compounds and three kawa lactones possessing an α-pyrone influenced antitrypanosomal property. In particular, β-phenethyl caffeate, farnesyl caffeate and dihydrokawain exhibited high or moderate selective and potent antitrypanosomal activity in vitro. We detail here the antitrypanosomal activity and cytotoxicities of the compounds, in comparison with two commonly used antitrypanosomal drugs (eflornithine and suramin). Our findings represent the first report of the promising trypanocidal activity of these compounds.

During the course of our screening program to discover new antitrypanosomal compounds, 17 known plant aromatic compounds [12 bis(bibenzyls)s and 5 bibenzyls] were evaluated for in vitro activity against Trypanosoma brucei brucei. Sixteen compounds were found to exhibit antitrypanosomal activity. In particular, three compounds, marchantin A (1), plagiochin A (5) and 2(R)-2-isopropenyl-6,7-dihydroxy-4-(2-phenylethyl)dihydrobenzofuran (16) demonstrated moderate selective and potent antitrypanosomal activities in vitro. We detail here the antitrypanosomal properties and cytotoxicities of the compounds in comparison with two commonly used therapeutic drugs, eflornithine and suramin. Our finding represents the first report of the promising trypanocidal activity of these compounds. The research also provides valuable insight into structure-activity relationships and the possible mode of action of the compounds.

Vasorelaxant effects of a series of bis(bibenzyls) from liverworts such as Marchantia polymorpha and Marchantia paleacea on rat aorta demonstrated that they relaxed phenylephrine (PE)-induced contractions, which may be mediated through the increased release of NO from endothelial cells as well as opening of K+ channels, and inhibition of Ca2+ influx through voltage-dependent Ca2+ channels (VDCs) and/or receptor-operated Ca2+ channels (ROCs). Structure–activity relationship based on their structures was discussed. The presence of two aromatic rings which can be connected through two atoms bridge spacer may play an important role for vasorelaxant effect.

Opaliferin, a polyketide with a unique partial structure in which a cyclopentanone and tetrahydrofuran were connected with an external double bond, was isolated from the insect pathogenic fungus Cordyceps sp. NBRC 106954. The structure and relative configuration of opaliferin were determined by spectroscopic analysis and X-ray crystallography. The absolute configuration was established by anomalous dispersion effects in X-ray diffraction measurements on the crystal of di(p-bromobenzoyl) ester of opaliferin. A plausible biosynthetic pathway for opaliferin is proposed.

Ursolic acid is triterpene isolated from Uncaria rhynchophylla and is a pharmacologically active substance. The induction of dendritic cell maturation is critical for the induction of Ag-specific T-lymphocyte response and may be essential for the development of human vaccine relying on T cell immunity. In this study, we investigated that the effect of Ursolic acid on the phenotypic and functional maturation of human monocyte-derived dendritic cells in vitro. Dendritic cells harvested on day 8 were examined using functional assay. The expression levels of CD1a, CD80, CD83, CD86, HLA-DR and CCR7 on Ursolic acid-primed dendritic cells was slightly enhanced. Ursolic acid dose-dependently enhanced the T cell stimulatory capacity in an allogeneic mixed lymphocyte reaction, as measured by T cell proliferation. The production of IL-12p70 induced by Ursolic acid-primed dendritic cells was inhibited by the anti-Toll-like receptor-2 (TLR2) mAb and anti-TLR4 mAb. Moreover, Ursolic acid-primed dendritic cells expressed levels of mRNA coding for both TLR2 and TLR4. The majority of cells produced considerable interferon-gamma (IFN-gamma), but also small amounts of interleukin (IL-4)-4. Ursolic acid-primed dendritic cells have an intermediate migratory capacity towards CCL19 and CCL21. These results suggest that Ursolic acid modulates human dendritic cells function in a fashion that favors Th1 polarization via the activation of IL-12p70 dependent on TLR2 and/or TLR4, and may be used on dendritic cells-based vaccines for cancer immunotherapy.

Microbial biotransformation of naturally occurring pungent sesquiterpene 1,2-dials, polygodial and cinnamodial, and a diterpene 1,2-dial, sacculatal as well as their tetrahydro derivatives was carried out by using Aspergillus niger. The pungent polygodial and sacculatal are toxic against A. niger not to produce any metabolites while A. niger biotransformed cinnamodial to the lactonized products in small amount. On the other hands, the dihydroxy derivatives of the former two dialdehydes were bioconverted by the same fungus to give hydroxy-, oxo-, carboxylic- and epoxy-products. The stereostructures of each metabolite and their metabolic pathways were described.

Dendritic cells (DC) are key antigen-presenting cells that link innate and adaptive immunity and ultimately activate antigen-specific T cells. In the current study, we demonstrated that two triterpene esters, uncarinic acid C (1) and uncarinic acid D (2), which are isolated from the hooks of Uncaria rhynchophylla, activate phenotypic and cytokine production alterations in DC. We also show that 1 and 2 modulate human DC function in a fashion that favors Th1 cell polarization. The effect of 1 (E configuration at the 2' position) was approximately 20 times more potent than that of 2 (Z configuration at 2'). These results indicated that the configuration of the 2' double bond greatly effects activity. Thus, 1 and 2 may prove useful as DC-based vaccines for cancer immunotherapy.

The biotransformation of (+)-isopinocampheol and (−)-isopinocampheol and (6R)-(−)-caryophyllene oxide was carried out by Aspergillus niger TBUYN-2. A. niger converted (+)-isopinocampheol to give (+)-5-hydroxyisopinocampheol and (+)-1-hydroxyisopinocampheol. On the other hand, (−)-isopinocampheol was converted to (−)-5-hydroxyisopinocampheol, (−)-1-hydroxyisopinocampheol and (+)-2-hydroxyisopinocampheol. In the case of (6R)-(−)-caryophyllene oxide, (−)-caryophyllene-12-oic acid and (−)-caryophyllene-6,7,12-triol were obtained as new compounds. Clovandiol, which is a component of Salvia canariensis, was also obtained from (6R)-(−)-caryophyllene oxide following incubation for 4 days with shaking in medium without microorganisms. Copyright © 2010 John Wiley & Sons, Ltd.

The biotransformation of (+)-isopinocampheol and (−)-isopinocampheol and (6R)-(−)-caryophyllene oxide was carried out by Aspergillus niger TBUYN-2. A. niger converted (+)-isopinocampheol to give (+)-5-hydroxyisopinocampheol and (+)-1-hydroxyisopinocampheol. On the other hand, (−)-isopinocampheol was converted to (−)-5-hydroxyisopinocampheol, (−)-1-hydroxyisopinocampheol and (+)-2-hydroxyisopinocampheol. In the case of (6R)-(−)-caryophyllene oxide, (−)-caryophyllene-12-oic acid and (−)-caryophyllene-6,7,12-triol were obtained as new compounds. Clovandiol, which is a component of Salvia canariensis, was also obtained from (6R)-(−)-caryophyllene oxide following incubation for 4 days with shaking in medium without microorganisms. Copyright © 2010 John Wiley & Sons, Ltd.

Sparassis crispa (SC), known as Hanabiratake in Japanese, is an edible mushroom with various medicinal properties. We isolated 3 novel phthalides, designated hanabiratakelide A (1), B (2), and C (3), from the SC fruit body. In this investigation, 3 known phthalides (4—6), ubiquinone-9, and 2 known unsaturated fatty acids were also isolated. Their structures were elucidated primarily through extensive NMR experiments. The isolated compounds 1—6 were tested for their anti-oxidant activity. The in vitro superoxide dismutase-like activity of the 3 hanabiratakelides was stronger than that of vitamin C. The compounds also exerted inhibitory effects on lipopolysaccharide-stimulated nitric oxide and prostaglandin E2 production by a murine macrophage cell line, RAW264. In addition, the growth of the colon cancer cell lines Caco-2 and colon-26 was significantly inhibited by treatment with the 3 hanabiratakelides. In vivo, the frequency of azoxymethane-induced aberrant crypt foci was reduced in SC-fed F344/N rats compared to rats fed a standard diet. In conclusion, 3 novel phthalides, hanabiratakelides, derived from SC were shown to possess anti-oxidant, anti-inflammatory, and anti-tumor activity.

Monocytes give rise to macrophages, osteoclasts (OCs), and dendritic cells (DCs). Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB (RANK) ligand induce OC differentiation from monocytes, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) trigger monocytic differentiation into DCs. However, regulatory mechanisms for the polarization of monocytic differentiation are still unclear. The present study was undertaken to clarify the mechanism of triggering the deflection of OC and DC differentiation from monocytes. GM-CSF and IL-4 abolished monocytic differentiation into OCs while inducing DC differentiation even in the presence of M-CSF and RANK ligand. GM-CSF and IL-4 in combination potently up-regulate tumor necrosis factor-α (TNF-α) converting enzyme (TACE) and activity in monocytes, causing ectodomain shedding of M-CSF receptor, resulting in the disruption of its phosphorylation by M-CSF as well as the induction of osteoclastogenesis from monocytes by M-CSF and RANK ligand. Interestingly, TACE inhibition robustly causes the resumption of the surface expression of M-CSF receptor on monocytes, facilitating M-CSF–mediated phosphorylation of M-CSF receptor and macrophage/OC differentiation while impairing GM-CSF– and IL-4–mediated DC differentiation from monocytes. These results reveal a novel proteolytic regulation of M-CSF receptor expression in monocytes to control M-CSF signaling and monocytic differentiation into macrophage/OC-lineage cells or DCs.

A screening of 30 crude extracts of marine sponges against human promyelocytic leukemia cells (HL-60) yielded an EtOAc extract of the sponge Callyspongia sp. (Callyspongiidae) with significant activity. Further bioassay-guided fractionation of the EtOAc extract led to the isolation of three polyacetylene metabolites: a new polyacetylene diol, callyspongidiol (1), along with two known compounds, siphonodiol (2) and 14,15-dihydrosiphonodiol (3). Their structures were determined by a combination of spectroscopic analyses. Compounds 1-3 exhibited antiproliferative activity against HL-60 with IC(50) values of 6.5, 2.8, and 6.5 microg/ml, respectively. These metabolites induce apoptosis in HL-60 cells. Dendritic cells (DC) differentiated with 1-3 enhance the differentiation of naïve T cells towards the Th1 type.

We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging. UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated by nitro reduction, directly linked to SNARF. Although UTX-12 itself is colorless and non-fluorescent in aqueous solution, nitro reduction triggers the release of SNARF which has well-characterized long wavelength absorption and fluorescence that is sensitive to pH. The resultant SNARF, released intracellularly by enzymatic reduction of UTX-12, allows measurement of pH by pH-dependent dual emission shifts. UTX-12 showed clear differences in fluorescence behavior between hypoxic and aerobic conditions in liver microsomes and inside V79 cells. These data are confirmation that UTX-12 is biologically reduced inside tumor cells and the released SNARF should monitor intracellular pH of tumor cells selectively with reduced background signal.

GPR120 and GPR40 are G-protein-coupled receptors whose endogenous ligands are medium- and long-chain free fatty acids, and they are thought to play an important physiological role in insulin release. Despite recent progress in understanding their roles, much still remains unclear about their pharmacology, and few specific ligands for GPR120 and GPR40 besides medium- to long-chain fatty acids have been reported so far. To identify new selective ligands for these receptors, more than 80 natural compounds were screened, together with a reference compound MEDICA16, which is known to activate GPR40, by monitoring the extracellular regulated kinase (ERK) and [Ca(2+)](i) responses in inducible and stable expression cell lines for GPR40 and GPR120, respectively. MEDICA16 selectively activated [Ca(2+)](i) response in GPR40-expressing cells but not in GPR120-expressing cells. Among the natural compounds tested, grifolin derivatives, grifolic acid and grifolic acid methyl ether, promoted ERK and [Ca(2+)](i) responses in GPR120-expressing cells, but not in GPR40-expressing cells, and inhibited the alpha-linolenic acid (LA)-induced ERK and [Ca(2+)](i) responses in GPR120-expressing cells. Interestingly, in accordance with the pharmacological profiles of these compounds, similar profiles of glucagon-like peptide-1 secretion were seen for mouse enteroendocrine cell line, STC-1 cells, which express GPR120 endogenously. Taken together, these studies identified a selective GPR40 agonist and several GPR120 partial agonists. These compounds would be useful probes to further investigate the physiological and pharmacological functions of GPR40 and GPR120.

Riccardin C, a nuclear receptor LXRalpha selective agonist, is an 18-membered macrocyclic bisbibenzyl isolated from several liverworts. Synthesis of riccardin C and its seven O-methylated derivatives was accomplished. The synthetic sequence highlights an intramolecular Suzuki-Miyaura coupling in the formation of the 18-membered biaryl linkage present in riccardin C. The structure-activity relationship of these compounds suggests that all of the phenolic hydroxy groups present in riccardin C are essential for the activation of LXRalpha.

We used functional magnetic resonance imaging (fMRI) to identify brain areas involved in auditory rhythm perception. Participants listened to three rhythm sequences that varied in temporal predictability. The most predictable sequence was an isochronous rhythm sequence of a single interval (ISO). The other two sequences had nine intervals with unequal durations. One of these had interval durations of integer ratios relative to the shortest interval (METRIC). The other had interval durations of non-integer ratios relative to the shortest interval (NON-METRIC), and was thus perceptually more complex than the other two. In addition, we presented unpredictable sequences with randomly distributed intervals (RAN). We tested two hypotheses. Firstly, that areas involved in motor timing control would also process the temporal predictability of sensory cues. Therefore, there was no active task included in the experiment that could influence the participant perception or induce motor preparation. We found that dorsal premotor cortex (PMD), SMA, preSMA, and lateral cerebellum were more active when participants listen to rhythm sequences compared to random sequences. The activity pattern in supplementary motor area (SMA) and preSMA suggested a modulation dependent on sequence predictability, strongly suggesting a role in temporal sensory prediction. Secondly, we hypothesized that the more complex the rhythm sequence, the more it would engage short-term memory processes of the prefrontal cortex. We found that the superior prefrontal cortex was more active when listening to METRIC and NON-METRIC compared to ISO. We argue that the complexity of rhythm sequences is an important factor in modulating activity in many of the rhythm areas. However, the difference in complexity of our stimuli should be regarded as continuous.

Multiple myeloma is still an incurable disease, most commonly occurring in the elderly. The myeloma-induced bone marrow microenvironment protects myeloma cells from drug-induced apoptosis. Therefore, the development of novel and tolerable therapeutic alternatives to overcome the drug resistance is an important clinical issue. Valproic acid (VPA), a safe and widely used anti-epileptic agent, is revisited as a class I- and IIa-specific histone deacetylase inhibitor. In the present study, we evaluated the effect as well as a mechanism of actions of VPA on myeloma cell growth and survival, with special reference to the myeloma-induced bone marrow microenvironment. VPA at therapeutic concentrations for epilepsy induced cell death in primary CD138-positive myeloma cells as well as myeloma cell lines, but not in CD138-negative bone marrow cells. VPA suppressed osteoclastogenesis as well as osteoclast-mediated myeloma cell growth. VPA also inhibited vascular tubule formation enhanced by co-cultures of myeloma cells and osteoclasts in concert with thalidomide. In addition, VPA induced both caspase-dependent and -independent cell death in myeloma cells, and potentiated the anti-myeloma effects of melphalan and dexamethasone. Collectively, VPA is suggested to exert multi-factorial anti-myeloma actions, and may serve as a safe adjuvant to be included in conventional chemotherapies against myeloma.

Sandaracopimaric acid and Sandaracopimaradiene-3beta-ol are diterpenes isolated from the heatwood of Cryptomeria japonica and are pharmacologically active substances. Dendritic cells (DC) are key antigen presenting cells (APC), which link innate and adaptive immunity, ultimately activating antigen-specific T cells. We demonstrate that Sandaracopimaric acid and Sandaracopimaradiene-3beta-ol activate humans DC as documented by phenotypic and functional maturation and altered cytokine production. The expression of the co-stimulatory molecules such as CD83, CD86 and HLA-DR on Sandaracopimaric acid- and Sandaracopimaradiene-3beta-ol-primed DC was enhanced. Sandaracopimaric acid- and Sandaracopimaradiene-3beta-ol-primed DC also enhanced the T cell stimulatory capacity in an allo MLR. Naive T cells co-cultured with Sandaracopimaric acid- or Sandaracopimaradiene-3beta-ol-primed DC turned into typical Th1 cells, which produced large quantities of IFN-gamma and released small amounts of IL-4 depending on IL-12 secretion. Sandaracopimaric acid- or Sandaracopimaradiene-3beta-ol-primed DC had a high migration to macrophage inflammatory protein (MIP)-3beta. These results suggest that some diterpenes modulate human DC function in a fashion that favors Th1 cell polarization and may be used on DC-based vaccines for cancer immunotherapy.

We have designed and synthesized new hypoxic-neoplastic cells-targeted indoleamine 2,3-dioxygenase (IDO) inhibitors. 1-Methyl-tryptophan (1MT)-tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-dioxide) hybrid inhibitors including 1 (TX-2236), 2 (TX-2235), 3 (TX-2228), and 4 (TX-2234) were prepared. All of these compounds were uncompetitive IDO inhibitors. TPZ-monoxide hybrids 1 and 3 showed higher IDO inhibitory activities than TPZ hybrids 2 and 4. Among these hybrids, hybrid 1 was the most potent IDO inhibitor. TPZ hybrids 2 and 4 showed stronger hypoxia-selective cytotoxicity than TPZ to EMT6/KU cells. These data suggest that TPZ hybrids 2 and 4 may act through their dual biological functions: first, they function as hypoxic cytotoxins in hypoxic cells, and then are metabolized to their TPZ-monoxide (3-amino-1,2,4-benzotriazine 1-oxide) hybrids, which function as IDO inhibitors.

Siphonodiol is a polyacetylene diol isolated from marine sponges Callyspongia sp. We demonstrate that the effect of Siphonodiol on the phenotypic and functional maturation of human monocyte derived DC in vitro. Human monocytes were exposed to Siphonodiol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR on LPS-primed DC were partially enhanced by Siphonodiol. Siphonodiol augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC. Siphonodiol dose-dependently enhanced the production of IL-12p70 by LPS-primed DC and this cytokine production was inhibited by anti-TLR4 mAb. IFN-gamma secretion from naive T cells co-cultured with DC differentiated with LPS was augmented by Siphonodiol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by Siphonodiol depends on TLR4 and via the activation of IL-12p70.

Farnesoid X receptor (FXR) controls the expression of critical genes in bile acid and cholesterol homeostasis. To study FXR and to develop a regulator of cholesterol, some nonsteroidal and steroidal ligands have been found in addition to endogenous ligands for FXR. In this study, we discovered five bile acid derivatives (methyl cholate, methyl deoxycholate, 5β-cholanic acid, 5β-cholanic acid-7α,12α-diol, and NIHS700) and two natural products (marchantin A and marchantin E) that activated FXR in the reporter assay. These compounds activated FXR to a high level comparable to the most potent endogenous bile acid, chenodeoxycholic acid, although it was not predicted from their structures; five of them were similar to the lower potency bile acids, and two were structurally much different from bile acids. The elevation levels of reporter gene expression by some of the screened compounds were varied in Cos-7, HepG2, HuH-7, and Caco-2 cells. These compounds also controlled the expression of genes regulated by FXR, and some of the compounds regulated these genes in a cell-type–specific and/or gene-selective fashion. Therefore, molecular design of the compounds can cause selective modulation of the expression of FXR target genes. Keywords:: farnesoid X receptor (FXR), reporter assay, ginkgolic acid, marchantin, cell-type–specific modulation

The inhibition of lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) by herbertenoids and cuparenoids isolated from liverworts in RAW 264.7 macrophages was evaluated. Among compounds tested, herbertenediol, cuparenediol, 1,2-diacetoxyherbertene and 2-hydroxy-4-methoxycuparene exhibited significant activity. For 2-hydroxy-4-methoxycuparene, chosen as representative compound, the strong inhibitory activity was related to the inhibition on LPS-induced iNOS mRNA. The structure-activity relationship will be discussed.

Purpose: Similar to osteoclastogenesis, angiogenesis is enhanced in the bone marrow in myeloma in parallel with tumor progression. We showed previously that myeloma cells and osteoclasts are mutually stimulated to form a vicious cycle to lead to enhance both osteoclastogenesis and tumor growth. The present study was undertaken to clarify whether myeloma cell-osteoclast interaction enhances angiogenesis and whether there is any mutual stimulation between osteoclastogenesis and angiogenesis. Experimental Design: Myeloma cells and monocyte-derived osteoclasts were cocultured, and angiogenic activity produced by the cocultures was assessed with in vitro vascular tubule formation assays and human umbilical vascular endothelial cell (HUVEC) migration and survival. Osteoclastogenic activity was determined with rabbit bone cell cultures on dentine slices. Results: Myeloma cells and osteoclasts constitutively secrete proangiogenic factors, vascular endothelial growth factor (VEGF) and osteopontin, respectively. A cell-to-cell interaction between myeloma cells and osteoclasts potently enhanced vascular tubule formation. Blockade of both VEGF and osteopontin actions almost completely abrogated such vascular tubule formation as well as migration and survival of HUVECs enhanced by conditioned medium from cocultures of myeloma cells and osteoclasts. Furthermore, these factors in combination triggered the production of osteoclastogenic activity by HUVEC. Conclusions: Osteoclast-derived osteopontin and VEGF from myeloma cells cooperatively enhance angiogenesis and also induce osteoclastogenic activity by vascular endothelial cells. These observations suggest the presence of a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion.

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However, clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody, we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the α2 domain of HLA-A. Here, we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells, lymphocytes, or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors, suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely, 2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs, melphalan, or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore, administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I–targeting therapy against multiple myeloma. [Cancer Res 2007;67(3):1184–92]

Two novel red pigments, uroleuconaphins A1 (1) and B1 (2) were isolated from the aphid Uroleucon nigrotuberculatum (Olive). The structures and the absolute configurations of 1 and 2 were determined by single-crystal X-ray analysis of their derivatives. These structures were constituted as dimeric compounds of two molecules of quinone A (3), which were linked via a dihydrofuran ring system.

Three benzophenone derivatives, daldinals A–C, from the Japanese fungus Daldinia childiae were evaluated for their inhibitory activity of nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) in RAW 264.7 cells. They strongly suppressed the LPS-induced production of NO with IC50 values of 15.2, 4.6 and 6.4 μM, respectively. To clarify the mechanism involved, total RNA extraction, followed by reverse-transcribed, polymerase chain reaction (PCR) for inducible nitric oxide synthase (iNOS) mRNA and finally electrophoresis on agarose gel were performed. These experimental results suggest that the inhibition of the LPS-induced NO production of daldinal B is due to the inhibition of iNOS mRNA synthesis. Further, their biological activities were compared with those of other metabolites obtained during our studies on biologically active substances from inedible fungi in recent years.

Stromata of Hypoxylon fragiforme were studied during the vegetation period by hplc profiling, revealing changes in the composition during stromatal development. Cytochalasin H and two new cytochalasins named fragiformins A-B were identified as major constituents of the young, maturing stromata, whereas mature, ascogenous material yielded large amounts of mitorubrin-type azaphilones. The above compounds, further cytochalasins from Xylariaceae and other fungi, and additional azaphilones of the mitorubrin type were assayed for their nematicidal effects against Caenorhabditis elegans and their antimicrobial activities against Bacillus subtilis, Yarrowia lipolytica, and various filamentous fungi. The results confirmed data in the literature on broad-spectrum non-selective activities of azaphilones and cytochalasins in biological systems. Most interestingly, laboratory cultures of the above Hypoxylon spp. mainly produced dihydroisocoumarin derivatives and were found devoid of mitorubrins and cytochalasins. These rather drastic changes in the secondary metabolism of H. fragiforme and the above biological activities are discussed in relation to the possible biological functions of secondary metabolites (extrolites) in the Hypoxyloideae.