Increased metabolite production by deletion of an HDA1-type histone deacetylase in the phytopathogenic fungi, Magnaporthe oryzae (Pyricularia oryzae) and Fusarium asiaticum.

Histone deacetylases (HDACs) play an important role in the regulation of chromatin structure and gene expression. We found that dark pigmentation of Magnaporthe oryzae (anamorph Pyricularia oryzae) ΔMohda1, a mutant strain in which an orthologue of the yeast HDA1 was disrupted by double cross-over homologous recombination, was significantly stimulated in liquid culture. Analysis of metabolites in a ΔMohda1 mutant culture revealed that the accumulation of shunt products of the 1,8-dihydroxynaphthalene melanin and ergosterol pathways were significantly enhanced compared to the wild-type strain. Northern blot analysis of the ΔMohda1 mutant revealed transcriptional activation of three melanin genes that are dispersed throughout the genome of M. oryzae. The effect of deletion of the yeast HDA1 orthologue was also observed in Fusarium asiaticum from the Fusarium graminearum species complex; the HDF2 deletion mutant produced increased levels of nivalenol-type trichothecenes. These results suggest that histone modification via HDA1-type HDAC regulates the production of natural products in filamentous fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural products of fungi have significant impacts on human welfare, in both detrimental and beneficial ways. Although HDA1-type histone deacetylase is not essential for vegetative growth, deletion of the gene affects the expression of clustered secondary metabolite genes in some fungi. Here, we report that such phenomena are also observed in physically unlinked genes required for melanin biosynthesis in the rice blast fungus. In addition, production of Fusarium trichothecenes, previously reported to be unaffected by HDA1 deletion, was significantly upregulated in another Fusarium species. Thus, the HDA1-inactivation strategy may be regarded as a general approach for overproduction and/or discovery of fungal metabolites.

4-Hydroxy-17-methylincisterol, an inhibitor of DNA polymerase-alpha activity and the growth of human cancer cells in vitro.

An ergosterol derivative, 4-hydroxy-17-methylincisterol (HMI), was found to be an inhibitor of mammalian DNA polymerases in vitro. HMI inhibited the activity of calf thymus DNA polymerase alpha (pol. alpha). Among the polymerases tested, pol. alpha was the most sensitive to inhibition by HMI, and the inhibition was concentration dependent. The inhibitory effect of HMI on pol. alpha was almost the same as that shown by aphidicolin, a well-known potent pol. alpha inhibitor. HMI had relatively less effect on rat DNA pol. beta, human immunodeficiency virus type 1 reverse transcriptase (HIV-RT), and calf thymus terminal deoxynucleotidyl transferase (TdT) in vitro, and did not influence the activities of prokaryotic DNA polymerases such as Klenow Fragment of DNA polymerase I, or the DNA-metabolic enzyme DNase I. HMI was found to be able to prevent the growth of human cancer cell lines originating from patients with leukemia or various solid tumors; its IC50 values ranged from 7.5 to 12 microM. We also synthesized other ergosterol derivatives and tested them, and found that two compounds, 17-methylincisterol and 4-acetyl-17-methylincisterol, have similar inhibitory effects.

The cyanogenic glucoside, prunasin (D-mandelonitrile-beta-D-glucoside), is a novel inhibitor of DNA polymerase beta.

A DNA polymerase beta (pol. beta) inhibitor has been isolated independently from two organisms; a red perilla, Perilla frutescens, and a mugwort, Artemisia vulgaris. These molecules were determined by spectroscopic analyses to be the cyanogenic glucoside, D-mandelonitrile-beta-D-glucoside, prunasin. The compound inhibited the activity of rat pol. beta at 150 microM, but did not influence the activities of calf DNA polymerase alpha and plant DNA polymerases, human immunodefficiency virus type 1 reverse transcriptase, calf terminal deoxynucleotidyl transferase, or any prokaryotic DNA polymerases, or DNA and RNA metabolic enzymes examined. The compound dose-dependently inhibited pol. beta activity, the IC(50) value being 98 microM with poly dA/oligo dT(12-18) and dTTP as the DNA template and substrate, respectively. Inhibition of pol. beta by the compound was competitive with the substrate, dTTP. The inhibition was enhanced in the presence of fatty acid, and the IC(50) value decreased to approximately 40 microM. In the presence of C(10)-decanoic acid, the K(i) value for substrate dTTP decreased by 28-fold, suggesting that the fatty acid allowed easier access of the compound to the substrate-binding site.

Synthesis and biological activity of 26-norbrassinolide, 26-norcastasterone and 26-nor-6-deoxocastasterone.

26-Norbrassinolide, identified as a metabolite of brassinolide in cultured cells of the liverwort, Marchantia polymorpha, as well as 26-norcastasterone and 26-nor-6-deoxocastasterone were synthesized. Synthesis of these new brassinosteroids was conducted by employing the orthoester Claisen rearrangement and asymmetric dihydroxylation as key reactions. The modified rice lamina inclination test indicated that these three 26-norbrassinosteroids were less active than their corresponding C28 brassinosteroids. Growth-promoting activities were also examined by using the brassinosteroid-deficient, dwarf mutant lkb of garden pea (Pisum sativum L.). In this assay, 26-norbrassinolide was as effective as brassinolide and 26-norcastasterone was more effective than castasterone although 26-nor-6-deoxocastasterone was much less effective than 6-deoxocastasterone. Therefore, removal of C-26 of brassinosteroids does not necessarily reduce the biological activity. The role of C-26 removal in Marchantia cells remains unclear.

Sesquiterpene O-naphthoquinones from the root bark of Ulmus davidiana.

Three new sesquiterpene ortho-naphthoquinones, davidianones A, B and C, together with four known compounds, mansonones E, F, H and I, were isolated from the root bark of Ulmus davidiana. On the basis of spectral data including pulse field gradient two-dimensional NMR spectroscopy, the structures of new compounds were established as 3-hydroxymethyl-6,9-dimethylnaphtho(1,8-b,c)pyran-7,8-dione, 6-methoxycarbonyl-3,9-dimethylnaphtho(1,8-b,c)pyran-7,8-dione, 6-dimethoxymethyl-3,9-dimethylnaphtho(1.8-b,c)pyran-7,8-d ion e, respectively. Their antioxidative activities were evaluated by a thiobarbituric acid method using rat liver microsomes, with mansonone F showing the greatest activity.

Ellagic acid rhamnosides from the stem bark of Eucalyptus globulus.

Four ellagic acid rhamnosides were isolated from the stem bark of Eucalyptus globulus. Their structures have been established on the basis of the analysis of their 1H NMR, 13C NMR, HMBC, IR and MS spectral data. The HMBC data of these compounds were most useful for their structure determinations, with these bring determined to be 3-O-methylellagic acid 3'-O-alpha-rhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-3''-O-acetylrhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-2''-O-acetylrhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-4''-O-acetylrhamnopyranoside, respectively. Their antioxidant activities were evaluated by measuring the inhibition of lipid peroxidation using rat liver microsomes, with IC50 values of 10.0-14.0 microg/ml.

Cytotoxic benzyl dihydroflavonols from Cudrania tricuspidata.

Three new dihydroflavonols, gericudranins A-C were isolated from the stem bark of Cudrania tricuspidata. They were identified as 6,8-di-p-hydroxybenzyltaxifolin, 8-p-hydroxybenzyltaxifolin and 6-p-hydroxybenzyltaxifolin, respectively, by means of spectral studies. These compounds were cytotoxic to human tumor cell lines, such as CRL 1579 (skin), LOX-IMVI (skin), MOLT-4F (leukemia), KM12 (colon) and UO-31 (renal) in culture, with ED50 values of 2.7-31.3 micrograms ml-1.

Synthesis of 6-oxy functionalized campest-4-en-3-ones: efficient hydroperoxidation at C-6 of campest-5-en-3-one with molecular oxygen and silica gel.

As a reference compound library for the investigation of biosynthesis of brassinosteroids, focused on a pathway from campesterol (1) to campestanol (2), 6-oxy functionalized campest-4-en-3-ones as well as campest-5-en-3-one (7) and campestane-3,6-dione were prepared from 1. Oxidation of 1 with pyridinium chlorochromate buffered by calcium carbonate gave 5-en-3-one (7) in 76% yield. Treatment of 7 with silica gel under an oxygen atmosphere in ethyl ether at room temperature produced efficient hydroperoxidation at the C-6 position to give 6alpha-hydroperoxycampest-4-en-3-one and 6beta-hydroperoxycampest-4-en-3-one in 34% and 49% yields, respectively. These compounds were converted to 6alpha-hydroxycampest-4-en-3-one and 6beta-hydroxycampest-4-en-3-one by reduction with triethyl phosphite. This provided the first example of the practical use of hydroperoxidation at C-6 of a Delta(5(6))-unsaturated 3-oxo-steroid with molecular oxygen and silica gel. On the other hand, oxidation of 1 with pyridinium chlorochromate in the absence of calcium carbonate gave campest-4-ene-3,6-dione in 64% yield. This compound was then converted in a highly stereoselective manner to campestane-3,6-dione with A/B trans ring junction by reduction with titanium (III) chloride in 85% yield.

Activation and inhibition of stimulus features in conjunction search.

Two visual search experiments were conducted to examine how activation of target-relevant features and inhibition of target-irrelevant features are involved in conjunction search. The hypothesis was that if an excitatory mechanism is involved, it should be revealed as facilitation when a target and distractors are repeated in two successive displays. If an inhibitory mechanism is involved, suppression should be obtained when distractor features from one display determine the target in the following display. The results of Experiment 1 showed that facilitation was observed consistently across two set sizes (5 and 9), whereas suppression was obtained only with larger set size (9). This pattern of results was replicated and extended in Experiment 2 with different set size. It seems that both excitatory and inhibitory mechanisms are involved in conjunction search. The excitatory mechanism seems to play a role in conjunction search regardless of search difficulty, whereas the inhibitory mechanism might play a role only when set size is larger.

Absolute Configuration of Kelsoene and Prespatane.

The absolute configurations of the rare sesquiterpenes, kelsoene and prespatane, were determined to be (1S, 2R,5S, 6S, 7R, 8R)-2,8-dimethyl-6-(1-methylethenyl)tricyclo[5.3.0.0(2,5)] decane (IUPAC name) and (1R, 2S, 5R, 6R, 7R, 8S)-1,5-dimethyl-8-(1-methylethenyl) tricyclo-[5.3.0.0(2,6)] decane, respectively, on the basis of the observed chemical shifts and NOEs in NOESY and NOEDS experiments after conversion with the chiral reagent, 2'-methoxy-1,1'-binaphthalene-2-carbohydroximoyl chloride (MBCC). The absolute configurations of kelsoene and prespatane thus determined suggest that initial cyclization of FPP from the si-face at C-10 to form a 10R-germacradienyl cation leads to kelsoene, while that from the re-face leads to prespatane via the 10S-germacradienyl cation.

Enantiomeric purity and odor characteristics of 2- and 3-acetoxy-1, 8-cineoles in the rhizomes of Alpinia galanga willd.

(S)-(+)-O-methylmandelate esters of trans- and cis-1,3, 3-trimethyl-2-oxabicyclo[2.2.2]octan-5- and 6-ols (2- and 3-hydroxy-1,8-cineoles) were prepared, and eight diastereomers were separated. The absolute configuration of the asymmetric carbons of the cineole moiety of each diastereomer was determined by (1)H NMR data according to the Mosher theory. Each mandelate was reduced with LiAlH(4) to obtain optically pure hydroxy-1,8-cineoles, this being followed by acetylation to afford optically pure acetoxy-1, 8-cineoles. These acetates were subjected to chiral GC, using a cyclodextrin column, and the enantiomeric purity of trans- and cis-1, 3,3-trimethyl-2-oxabicyclo[2.2.2]octan-5- and 6-yl acetates in the aroma concentrate from the rhizomes of Alpinia galanga was determined as 93.9 (5S), 19.4 (5R), 63.5 (6R), and 100 (6R) % ee, respectively. The aroma character of each enantiomer was also evaluated by GC-sniffing.

A new inhibitor of protein kinase C, RK-1409 (7-oxostaurosporine). II. Fermentation, isolation, physico-chemical properties and structure.

RK-1409, a new inhibitor of protein kinase C, was isolated from the culture broth of Streptomyces platensis subsp. malvinus RK-1409. The structure was elucidated as 7-oxostaurosporine on the basis of spectroscopic analyses and oxidation of staurosporine.

Reveromycins, new inhibitors of eukaryotic cell growth. III. Structures of reveromycins A, B, C and D.

Reveromycins A, B, C and D are new group inhibitors of the mitogenic activity of epidermal growth factor (EGF), produced by Streptomyces sp. Reveromycins are novel polyketide type antibiotics which have two terminal carboxylic groups, a spiroketal, a succinate and a varied side chain in the molecule. Determination of their structures by chemical and spectroscopic methods, in particular NMR studies, is described.

A new inhibitor of protein kinase C, RK-1409B (4'-demethylamino-4'-hydroxy-3'-epistaurosporine).

RK-1409B, a new inhibitor of protein kinase C, was isolated from the culture broth of Streptomyces platensis subsp. malvinus RK-1409. The structure was elucidated on the basis of spectroscopic analyses. RK-1409B inhibited protein kinase C in vitro and the morphological change of a human chronic leukemia cell line, K-562, induced by phorbol 12,13-dibutyrate with IC50 value of 0.4 microM.

A new inhibitor of protein kinase C, RK-1409 (7-oxostaurosporine). I. Taxonomy and biological activity.

A novel inhibitor of protein kinase C was found in the fermentation of a soil actinomycete, strain RK-1409. According to the taxonomic studies, the producing strain was designated as Streptomyces platensis subsp. malvinus RK-1409. The protein kinase C inhibitor, RK-1409 (7-oxostaurosporine) inhibited the morphological change of a human chronic erythroleukemia cell, K-562, induced by phorbol 12,13-dibutyrate (PDBu) at the concentration of 10 ng/ml. The concentration of 3 ng/ml inhibited the activity of protein kinase C in vitro. RK-1409 inhibited the cell cycle progression at G2 phase of K-562 cells.

F13459, a new derivative of mycophenolic acid. II. Physico-chemical properties and structural elucidation.

F13459 is a new inhibitor of synthesis and trafficking of virus glycoprotein isolated from the culture broth of a Penicillium sp. The molecular formula of F13459 was determined to be C27H28O11 by HRFAB-MS and NMR spectral analyses. The structure of F13459 was elucidated to be 3,4-dihydro-3,4,6,8-tetrahydroxy-3-methyl-1H-2-benzopyran-1-one 4-O-mycophenolate, an ester derivative of mycophenolic acid. F13459 was isolated as the optically inactive form. F13459 exists in epimeric mixtures at C-3' through relatively fast hemiacetal-ketone tautomerism and at C-4' through slow keto-enol tautomerism. Those epimerizations were confirmed by NOE differential experiments for fast chemical exchange and equilibrium and by deuteration experiments in NMR for slow chemical exchange.

SW-163C and E, novel antitumor depsipeptides produced by Streptomyces sp. II. Structure elucidation.

SW-163C and E are novel antitumor antibiotics, which belong to quinomycin family, isolated from the culture broth of Streptomyces sp. SNA15896. These compounds were determined to be cyclic depsipeptides having 3-hydroxyquinaldic acid as a chromophore and a sulfur-containing intramolecular cross linkage through various spectroscopic analyses.

SNA-8073-B, a new isotetracenone antibiotic inhibits prolyl endopeptidase. I. Fermentation, isolation and biological properties.

SNA-8073-B, an inhibitor of prolyl endopeptidase isolated from the broth filtrate of Streptomyces sp. SNA-8073, is a new isotetracenone antibiotic. It was purified by ethyl acetate extraction, silica gel column chromatography and high performance liquid chromatography on ODS column. SNA-8073-B has the molecular formula of C20H16O5 and is a stereoisomer of SNA-8073-A (fujianmycin B, rubiginone A2). SNA-8073-B inhibited prolyl endopeptidase of Flavobacterium non-competitively (IC50 = 8.9 microM) when Z-Gly-Pro-pNA was used as a substrate, but SNA-8073-A did not show any inhibition even at 60 microM.

Reveromycins, new inhibitors of eukaryotic cell growth. I. Producing organism, fermentation, isolation and physico-chemical properties.

New antibiotics named reveromycins A, B, C and D were isolated as inhibitors of mitogenic activity induced by epidermal growth factor (EGF) in a mouse epidermal keratinocyte. Reveromycins were produced by a soil actinomycete (strain SN-593) which belongs to the genus Streptomyces.

Reveromycins, new inhibitors of eukaryotic cell growth. II. Biological activities.

Reveromycins A, B, C and D showed inhibitory activity against EGF-stimulated mitogen response in Balb/MK cells. Furthermore reveromycins A, C and D exhibited morphological reversion of srcts-NRK cells, antiproliferative activity against human tumor cell lines and antifungal activity. The effects of reveromycins A, C and D on eukaryotic cells were closely similar to each other, but those of reveromycin B were very weak. In vitro studies revealed that reveromycin A is a selective inhibitor of protein synthesis in eukaryotic cells.