Phytochemical and mycological investigation ofArtemisia monosperma

Both polar and nonpolar fractions ofArtemisia monosperma were found to contain taraxasterol, taraxasterol acetate, pseudotaraxasterol acetate, lupeol, β-sitosterol and 3′,5-dihydroxy-4′,6,7-trimethoxyflavone. None of the isolated compounds except 3′,5-dihydroxy-4′,6,7-trimethoxyflavone provided successful control againstRhizoctonia damping-off of cotton in a greenhouse experiment when used as seed treatment. However, on evaluation ofA. monosperma dried shoot as amendment on controlling soil-borne plant pathogens, it was effective in decreasing the damping-off disease of cotton caused byRhizoctonia solani and provided a firmer plant stand. Higher doses of amendment (2 and 4%) caused a significant drop in the number of propagules ofR. solani in soil when incorporated 3 and 6 weeks before planting. In most cases incorporation ofA. monosperma in the soil caused a distinct increase in the total fungal population.In vitro studies showed toxicity of diffused or extracted substances fromA. monosperma for growth and pectolytic and cellulolytic enzyme activities of the target pathogen.     

Biotransformation of soybean isoflavones by a marine <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 060524 and cytotoxicity of the products

A marine Streptomyces sp. 060524 capable of hydrolyzing the glycosidic bond of isoflavone glycosides, was isolated by detecting its β-glucosidase activity. 5 isoflavone aglycones were isolated from culture filtrates in soybean meal glucose medium. They were identified as genistein (1), glycitein (2), daidzein (3), 3′,4′,5,7-tetrahydroxyisoflavone (4), and 3′,4′,7-trihydroxyisoflavone (5), based on UV, NMR and mass spectral analysis. The Streptomyces can selectively hydroxylate at the 3′-position in the daidzein and genistein to generate 3′-hydroxydaidzein and 3′-hydroxygenistein, respectively. The Strain biotransformed more than 90% of soybean isoflavone glycosides into their aglycones within 108 h. 3′-hydroxydaidzein and 3′-hydroxygenistein exhibited stronger cytotoxicity against K562 human chronic leukemia than daidzein and genistein.     

Cloning,expression, isolation,and properties of thymidine kinase from herpes simplex virus type 1, strain L2

Thymidine kinase UL23 gene (EC 2.7.1.145) from the L2 acyclovir-sensitive strain of herpes simplex virus type 1 was cloned and expressed in E. coli. The enzyme was purified by chromatography to the purity of 90% according to PAG electrophoresis data. The Michaelis constants for the reactions with thymidine and acyclovir were determined. The enzyme was found to phosphorylate modified nucleosides, particularly 3′-deoxythymidine, 3′-deoxy-2′,3′-didehydrothymidine, 2′,3′-dideoxycytidine, 9-[(hydroxyethyl)methyl]guanine, E-5-(2-bromovinyl-2′-deoxyuridine, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, 2′,3′-dideoxydehydrothymidine, β-L-2′,3′-dideoxy-3′-thiacytidine, and 3′-fluoro-3′-deoxythymidine. Some properties of the purified enzyme were compared with those of thymidine kinases of other herpes simplex virus strains. It was shown that acyclovir H-phosphonate inhibited the enzyme.     

The leaves of the common box,Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid,anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,¹H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.     

Synthesis and biological properties of pyrimidine 4′-fluoronucleosides and 4′-fluorouridine 5′-<Emphasis Type="Italic">O</Emphasis>-triphosphate

4′-Fluoro-2′,3′-O-isopropylidenecytidine was synthesized by the treatment of 5′-O-acetyl-4′-fluoro-2′,3′-O-isopropylideneuridine with triazole and 4-chlorophenyl dichlorophosphate followed by ammonolysis. The interaction of 4′-fluoro-2′,3′-O-isopropylidenecytidine with hydroxylamine resulted in 4′-fluoro-2′,3′-O-isopropylidene-5′-O-acetyl-N ⁴-hydroxycytidine. The removal of the 2′,3′-O-isopropylidene groups led to acetyl derivatives of 4′-fluorouridine, 4′-fluorocytidine, and 4′-fluoro-N ⁴-hydroxycytidine. 4′-Fluorouridine 5′-O-triphosphate was obtained in three steps starting from 4′-fluoro-2′,3′-O-isopropylideneuridine. 4′-Fluorouridine 5′-O-triphosphate was shown to be an effective inhibitor of HCV RNA-dependent RNA polymerase and a substrate for the NTPase reaction catalyzed by the HCV NS3 protein, the hydrolysis rate being similar to that of ATP. It could also activate a helicase reaction with an efficacy of only threefold lower than that for ATP.     

Clavatol and patulin formation as the antagonistic principle of <Emphasis Type="Italic">Aspergillus clavatonanicus</Emphasis>, an endophytic fungus of <Emphasis Type="Italic">Taxus mairei</Emphasis>

Many endophytic fungi are known to protect plants from plant pathogens, but the antagonistic mechanism has rarely been revealed. In this study, we wished to learn whether an endophytic Aspergillus sp., isolated from Taxus mairei, would indeed produce bioactive components, and if so whether (a) they would antagonize plant pathogenic fungi; and (b) whether this Aspergillus sp. would produce the compound also under conditions of confrontation with these fungi. The endophytic fungal strain from T. mairei was identified as Aspergillus clavatonanicus by analysis of morphological characteristics and the sequence of the internal transcribed spacers (ITS rDNA) of rDNA. When grown in surface culture, the fungus produced clavatol (2′,4′-dihydroxy-3′,5′-dimethylacetophenone) and patulin (2-hydroxy-3,7-dioxabicyclo [4.3.0]nona-5,9-dien-8-one), as shown by shown by NMR, MS, X-ray, and EI-MS analysis. Both exhibited inhibitory activity in vitro against several plant pathogenic fungi, i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, and Pythium ultimum. During confrontation with P. ultimum, A. clavatonanicus antagonized its growth of P. ultimum, and both clavatol as well as patulin were formed as the only bioactive components, albeit with different kinetics. We conclude that A. clavatonanicus produces clavatol and patulin, and that these two polyketides may be involved in the protection of T. mairei against attack by plant pathogens by this Aspergillus sp.     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

Algal Carotenoids 51. Secondary carotenoids 2.Haematococcus pluvialis aplanospores as a source of (3S, 3′S)-astaxanthin esters

Aplanospores ofHaematococcus pluvialis MUR 145 contained 0.7% carotenoids (dry wt. basis) consisting of β,β-carotene (5% of total carotenoid), echinenone (4%), canthaxanthin (4%), (3S,3′S)-astaxanthin diester (34%), (3S,3′S)-astaxanthin monoester (46%), (3S,3′S)-astaxanthin (1%) and (3R,3′R,6′R)-lutein (6%). The astaxanthin esters were examined by TLC and HPLC and VIS,¹H NMR and mass spectra recorded. Their chirality was determined by the camphanate method (Vecchi & Müller, 1979) after anaerobic hydrolysis. The tough cell wall of the aplanospores required enzymatic treatment prior to pigment extraction. The potential use of this microalga as a feed ingredient in aquaculture is discussed briefly.     

Quinoline alkaloids as intercalative topoisomerase inhibitors

Quinoline alkaloids are abundant in the Rutaceae, and many have exhibited cytotoxic activity. Because structurally related antitumor alkaloids such as camptothecin and fagaronine are known to function as intercalative topoisomerase poisons, it is hypothesized that cytotoxic Stauranthus alkaloids may also serve as intercalative topoisomerase inhibitors. To test this hypothesis theoretically, ten Stauranthus quinoline alkaloids were examined for potential intercalation into DNA using a molecular docking approach. Four of the alkaloids (stauranthine, skimmianine, 3′,6′-dihydroxy-3′,6′-dihydrostauranthine, and trans-3′,4′-dihydroxy-3′,4′-dihydrostauranthine) were able to intercalatively dock consistently into DNA. In order to probe the intermolecular interactions that may be responsible for intercalation of these quinoline alkaloids, density functional calculations have been carried out using both the B3LYP and M06 functionals. M06 calculations indicated favorable π–π interactions between either skimmianine or stauranthine and the guanine–cytosine base pair. Furthermore, the lowest-energy face-to-face orientation of stauranthine with guanine is consistent with favorable dipole–dipole orientations, favorable electrostatic interactions, and favorable frontier molecular orbital interactions. Likewise, the lowest-energy face-to-face orientation of stauranthine with the guanine–cytosine base pair reveals favorable electrostatic interactions as well as frontier molecular orbital interactions. Thus, not only can quinoline alkaloids dock intercalatively into DNA, but the docked orientations are also electronically favorable.      

<Emphasis Type="Italic">SpnH</Emphasis> from <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> encodes a rhamnosyl 4′-<Emphasis Type="Italic">O</Emphasis>-methyltransferase for biosynthesis of the insecticidal macrolide,spinosyn A

Deoxysugar, 2′, 3′, 4′-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4′-O-methylation of 2′, 3′-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4′-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4′-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4′-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.     

cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from <Emphasis Type="Italic">Coleus blumei</Emphasis> involved in rosmarinic acid biosynthesis

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K _m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and 40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.     

Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

Cis-2′, 3′-Dihydrodiol production on flavone B-Ring by Biphenyl Dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli

Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments. The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2′ and C3′ on the B-ring of flavone, resulting in the formation of flavone cis-2′, 3′-dihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since this product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Study of soybean and lentil root exudates

Summary Some polyphenolic compounds have been identified in root exudates of two species of legumes (both cultivated in sterile conditions): for lentil, three desoxy-5 flavones (4′,7-dihydroxy, 3′,4′,7-trihydroxy and 4′,7-dihydroxy, 3′-methoxyflavones) and for soybean: two isoflavonoids, coumestrol and daidzein (or 4′,7-dihydroxyisoflavone). Identified compounds are polyphenolic aglycones whereas roots contain the corresponding glycosides, showing that a hydrolysis takes place at the time of exudation. Compounds are observed in plantlet exudates as soon as the first day after planting. Their daily quantitative variations parallel those of total phenolic compounds. Exudated coumestrol and 4′,7-dihydroxyflavone amounts are estimated at about 20% of root content in these molecules.     

Co-expression of<Emphasis Type="Italic">flavonoid 3′ 5′-hydroxylase</Emphasis> and<Emphasis Type="Italic">flavonoid 3′-hydroxylase</Emphasis> Accelerates Decolorization in Transgenic Chrysanthemum Petals

Theflavonoid 3′,5′-hydroxylase (F3′,5′H) gene, derived from petunia, was introduced into chrysanthemum tissues by Agrobacterium-mediated genetic transformation. Cotyledon expiants were co-cultured withA. tumefaciens LBA 4404 harboring the vector pMBP that carriesF3′,5′H under the control of the CaMV 35S promoter andnptll as a selectable marker gene. After 72 h of co-cultivation, the expiants were placed on an MS medium supplemented with 4 mg L^-1 BA, 0.1 mg L^-1 NAA, 400 mg L^-1 carbenicillin, and 100 mg L^-1; kanamycin. After 4 weeks, kanamycin-resistant adventitious shoots had developed at a frequency of 6.3%. These shoots were then rooted and acclimatized in potting soil. Integration ofF3′,5′H into the plant genome was confirmed by Southern blot analysis. Flower buds that had red petals did not differ between the transgenic and the wild-type plants. However, petal color did change from red to bright orange to yellow when the buds developed into fully opened flowers on the transgenics. Spectrometric analysis revealed that the content of flavonoid compounds was more rapidly reduced in the transgenic petals as floral development proceeded. RT-PCR analysis showed thatF3′,5′H andflavonoid 3′hydroxylase (F3′H) were expressed simultaneously in the transgenic plants. Therefore, we suggest that this more rapid change in petal color results from 1) competition between levels of transgenicF3′,5′H and endogenousF3′H, each of which uses the same substrate in the flavonoid biosynthetic pathway and 2) the intrinsic substrate specificity of chrysanthemumDFR (dihydroflavonol 4-reductase).     

Facile synthesis of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N 6-ethenoadenosine nucleosides

Facile synthetic methods of 2′,5′-dideoxy-, 2′,3′-dideoxy- and 3′-deoxy-1,N ⁶-ethenoadenosine nucleosides by either an enzymatic dideoxyribosyl transfer reaction or a simple chemical reaction were proposed. The synthetic products were isolated and purified by preparative HPLC and their structures were confirmed by¹H NMR (500 MHz) and FAB-MS including high resolution mass measurement. These modified nucleoside analogs have not been reported yet. Therefore, these modified nucleoside analogs are of potential value to be studied further for biological activity such as anticancer or antiviral.     

Theoretical study of spin-spin coupling across the hydrogen (O-H⋯N) bond in adenosine derivatives

The study of spin-spin coupling constants across hydrogen bond provides useful information about configuration of complexes. The interesting case of such interactions was observed as a coupling across an intramolecular hydrogen bond in 8-bromo-2′,3′-O-isopropylideneadenosine between the -CH₂OH (at 5″ proton) group and the nitrogen atom of adenine. In this paper we report theoretical investigations on the ^4h J _NH coupling across the H″-C-O-H···N hydrogen bond in adenosine derivatives in various solvent models. Figure Coupling constants in 8-bromo-2′,3′-O-isopropylideneadenosine Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

The degradation of α-quaternary nonylphenol isomers by Sphingomonas sp. strain TTNP3 involves a type II ipso-substitution mechanism

The degradation of radiolabeled 4(3′,5′-dimethyl-3′-heptyl)-phenol [nonylphenol (NP)] was tested with resting cells of Sphingomonas sp. strain TTNP3. Concomitantly to the degradation of NP, a metabolite identified as hydroquinone transiently accumulated and short-chain organic acids were then produced at the expense of hydroquinone. Two other radiolabeled isomers of NP, 4(2′,6′-dimethyl-2′-heptyl)-phenol and 4(3′,6′-dimethyl-3′-heptyl)-phenol, were synthesized. In parallel experiments, the 4(2′,6′-dimethyl-2′-heptyl)-phenol was degraded more slowly than the other isomers of NP by strain TTNP3, possibly because of effects of the side-chain structure on the kinetics of degradation. Alkylbenzenediol and alkoxyphenol derivatives identified as metabolites during previous studies were synthesized and tested as substrates. The derivatives were not degraded, which indicated that the mineralization of NP does not proceed via alkoxyphenol as the principal intermediate. The results obtained led to the elucidation of the degradation pathway of NP isomers with a quaternary α-carbon. The proposed mechanism is a type II ipso substitution, leading to hydroquinone and nonanol as the main metabolites and to the dead-end metabolites alkylbenzenediol or alkoxyphenol, depending on the substitution at the α-carbon of the carbocationic intermediate formed.