Bufadienolides from bulbs of Urginea lydenburgensis (Hyacinthaceae: Urgineoideae)

Bulbs of the ethnomedicinal hyacinthac Urginea lydenburgensis have yielded two bufadienolides, 16beta-acetoxy-3beta,14beta-dihydroxy-19-formyl-bufa-4,20,22-trienolide (scillicyanosidin) and 4beta,8beta,11alpha,14beta-tetrahydroxybufa-5,20,22-trienolide-12-one, 2alpha,3beta-O-4,6-dideoxy-L-glucose (lydenburgenin).     

Chemotaxonomic relevance of cardenolides in Urginea fugax

In a chemotaxonomic approach the investigation of a methanolic extract of bulbs of Urginea fugax (MORIS) STEINH. resulted in the detection of several cardenolides. The structure of a novel compound, named fugaxin (1), was established as 12alpha,14beta-dihydroxy-2alpha,3beta-(tetrahydro-3',5'-dihydroxy-4'-methoxy-6'-methyl-2H-pyran-2',4'-diylbisoxy)-card-4,20-dienolide by extensive NMR spectroscopic studies including 2D-NMR techniques (COSY, HSQC, HMQC) and selective 1D experiments (NOE, TOCSY) as well as HR-ESI-MS. The chemotaxonomic relevance of the occurrence of cardenolides in the genus Urginea is discussed.     

Bufadienolides and phytoecdystones from the rhizomes of Helleborus thibetanus (Ranunculaceae)

Two new bufadienolide glycosides with an A/B trans ring structure, 14β,16β-dihydroxy-3β-(β-d-glucopyranosyloxy)-5α-bufa-20,22-dienolide (1), and 14β,16β-dihydroxy-3β-[β-d-glucopyranosyl-(1→4)-(β-d-glucopyranosyloxy)]-5α-bufa-20,22-dienolide (2), two known ecdysteroids (polypodine B and 20-hydroxyecdysone) (3-4), and six known bufadienolide and its glycosides with 5β-OH (hellebrigenin, 16β-hydroxyhellebrigenin-3-O-α-l-rhamnoside, hellebrigenin 3-O-β-d-glucoside, hellebrin, 16β-hydroxyhellebrigenin-3-O-β-d-glucoside, and deglucohellebrin) (5-10) were isolated from the rhizomes of Helleborus thibetanus. The structures of compounds 1 and 2 were elucidated using various spectroscopic methods. All compounds were reported for the first time from the title plant and their chemotaxonomic significance for the genus Helleborus was discussed.     

Steryl esters and phenylethanol esters from Syringa komarowii

Three new steryl esters and a new phenylethanol ester, together with 22 known compounds were isolated from the aqueous ethanolic extract of the whole plants of Syringa komarowii. The new compounds were elucidated as stigmastane-3beta,6alpha-diol 3-O-tetradecanoate (1), stigmastane-3beta,6alpha-diol 3-O-palmitate (2), stigmastane-3beta,6alpha-diol 3-O-stearate (3), and 2-(4-hydroxyphenyl)-ethyl dotriacontanoate (4) on the basis of extensive spectral data and chemical evidences.     

Differential amplification of satellite PaB6 in chromosomally hypervariable Prospero autumnale complex (Hyacinthaceae)

MethodsA new satellite repeat PaB6 has previously been identified, and monomers were reconstructed from next-generation sequencing (NGS) data of P. autumnale cytotype B⁶B⁶ (2n = 12). Monomers of all other Prospero cytotypes and species were sequenced to check for lineage-specific mutations. Copy number, restriction patterns and methylation levels of PaB6 were analysed using Southern blotting. PaB6 was localized on chromosomes using fluorescence in situ hybridization (FISH).ConclusionsAlthough present in all Prospero species, PaB6 has undergone differential amplification only in chromosomally variable P. autumnale, particularly in cytotypes B⁶B⁶ and B⁵B⁵. These arose via independent chromosomal fusions from x = 7 to x = 6 and 5, respectively, accompanied by genome size increases. The copy numbers of satellite DNA PaB6 are among the highest in angiosperms, and changes of PaB6 are exceptionally dynamic in this group of closely related cytotypes of a single species. The evolution of the PaB6 copy numbers is discussed, and it is suggested that PaB6 represents a recent and highly dynamic system originating from a small pool of ancestral repeats.     

Coincident isolation of a novel homoisoflavonoid from Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae)

We report on the first phytochemical investigation of a member of the African genus Resnova (Hyacinthoideae: Hyacinthaceae). From the dichloromethane extract of the bulbs of both Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae) a novel 3-benzyl-4-chromanone homoisoflavonoid, 5,6-dimethoxy-7-hydroxy-3-(4′-hydroxybenzyl)-4-chromanone, was isolated. A further 11 known homoisoflavonoids were also identified, the 12 in total presenting a clear biosynthetic sequence. Eight of the 12 compounds found were common to both species.     

Strict consistency between genetic and topographic landscapes of the brown tree frog (Buergeria robusta) in Taiwan

Taiwan presents an excellent opportunity to build a phylogeographic paradigm for fine-scaled differentiation occurring within short distances on an single island. Due to the limitation of habitat availability on the island, demographic histories of species in Taiwan were strongly influenced by glacial-interglacial cycles. Nevertheless, there are relatively few studies demonstrating such phylogeographic patterns for islands, especially in subtropical Asia. In this study, we aim to construct the genetic landscape of a philopatric stream frog Buergeria robusta by an intense and fine-scaled collection throughout the island. The deduced genetic landscape of B. robusta presented extremely high congruence with the actual topography of Taiwan. Two major lineages were found on the eastern and the western sides of Taiwan with a non-overlapping distribution, indicating the importance of the Central Mountain Range as the major biogeographic barrier. Both clades showed a strong and congruent tendency of demographic or distributional expansion in recent history based on different analyses. Population expansion of such a subtropical lowland species might be a result from a release of available habitat in post-glacial periods.     

New bile alcohols - synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols.

The synthesis of (22R)- and (22S)-5beta-cholestane-3alpha,7alpha,12alpha,22,25-pentols is described. Bisnorcholyl aldehyde was prepared from cholic acid and converted into the cholestane-pentols by a Grignard reaction with 3-methyl-3-(tetrahydropyran-2-yloxy)-butynylmagnesium bromide followed by hydrogenation and acid hydrolysis. One of the synthetic pentols, the 22R-isomer was identical with a metabolite of 5beta-cholestane-3alpha,7alpha,25-triol formed in the rabbit.     

Taxoid metabolism: Taxoid 14beta-hydroxylase is a cytochrome P450-dependent monooxygenase

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.     

The absolute stereochemistry of a diterpene from Ballota aucheri

The semi-synthetic transformation of hispanolone, isolated from Ballota africana, into 6beta-hydroxy-15,16-epoxylabda-8,13(16),14-trien-7-one has established an ent-labdane absolute stereochemistry for a diterpene metabolite originally isolated from B. aucheri.     

Constituents from Miliusa balansae (Annonaceae)

The styryl derivatives 3,4-dimethoxy-6-styryl-pyran-2-one and (2E,5E)-2-methoxy-4-oxo-6-phenyl-hexa-2,5-dienoic acid methyl ester were isolated from leaves and branches of Miliusa balansae (Annonaceae). In addition, the geranylated homogentisic acid derivative miliusate, four flavanones and two dihydrochalcones were identified. Their structures were elucidated by spectroscopic means.     

Cloning and functional characterisation of a cis-muuroladiene synthase from black peppermint (Menthaxpiperita) and direct evidence for a chemotype unable to synthesise farnesene

Using oligonucleotide primers designed to the known gene sequence of an (E)-beta-farnesene (EbetaF) synthase, two cDNA sequences (MxpSS1 and MxpSS2) were cloned from a black peppermint (Menthaxpiperita) plant. MxpSS1 encoded a protein with 96% overall amino acid sequence identity with the EbetaF synthase. Recombinant MxpSS1 produced in Escherichia coli, after removal of an N-terminal thioredoxin fusion, had a K(m) for FPP of 1.91+/-0.1 microM and k(cat) of 0.18 s(-1), and converted farnesyl diphosphate (FPP) into four products, the major two being cis-muurola-3,5-diene (45%) and cis-muurola-4(14),5-diene (43%). This is the first cis-muuroladiene synthase, to be characterised. MxpSS2 encoded a protein with only two amino acids differing from EbetaF synthase. Recombinant MxpSS2 protein showed no activity towards FPP. One of the two mutations, at position 531 (leucine in MxpSS2 and serine in EbetaF synthase) was shown, by structural modelling to occur in the J-K loop, an element of the structure of sesquiterpene synthases known to be important in the reaction mechanism. Reintroduction of the serine at position 531 into MxpSS2 by site-directed mutagenesis restored EbetaF synthase activity (K(m) for FPP 0.98+/-0.12 microM, k(cat) 0.1 s(-1)), demonstrating the crucial role of this residue in the enzyme activity. Analysis, by GC-MS, of the sesquiterpene profile of the plant used for the cloning, revealed that EbetaF was not present, confirming that this particular mint chemotype had lost EbetaF synthase activity due to the observed mutations.     

Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from homo sapiens

Human quinolinate phosphoribosyltransferase (EC 2.4.2.19) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.     

The chemotaxonomic and medicinal significance of phenolic acids in Arctopus and Alepidea (Apiaceae subfamily Saniculoideae)

The occurrence of (R)-3′-O-β-d-glucopyranosylrosmarinic acid, rosmarinic acid and caffeic acid in two important South African medicinal plants is reported for the first time. (R)-3′-O-β-d-Glucopyranosylrosmarinic acid and rosmarinic acid were isolated and identified in several samples from three species of the genus Arctopus L. (sieketroos) and three species of the genus Alepidea F. Delaroche (ikhathazo), both recently shown to be members of the subfamily Saniculoideae of the family Apiaceae. The compounds occur in high concentrations (up to 15.3 mg of (R)-3′-O-β-d-glucopyranosylrosmarinic acid per g dry wt) in roots of Arctopus. Our results provide a rationale for the traditional uses of these plants, as the identified compounds are all known for their antioxidant activity, with rosmarinic acid further contributing to a wide range of biological activities. Furthermore, we confirm the idea that (R)-3′-O-β-d-glucopyranosylrosmarinic acid is a useful chemotaxonomic marker for the subfamily Saniculoideae.     

3-Methylbutanoyl and 3-methylbut-2-enoyl disaccharides from green coffee beans (Coffea arabica)

The aerial parts of Teucrium oliverianum yielded two neo-clerodane diterpenoids, teucrolin F and G, together with the known teucrolin E. The previously proposed structure for teucrolin E was revised so that it contains a tetrahydrofuran ring instead of an oxetane ring. This was based on analysis of the NMR spectroscopic data of its diacetate, including its NOE spectra. In addition, the structural assignments of the new diterpenoids were based on 1H and 13C NMR spectroscopic studies, mainly 2D NMR experiments, including homonuclear and heteronuclear correlations.     

An unusual homoisoflavanone and a structurally-related dihydrochalcone from Polygonum ferrugineum (Polygonaceae)

The homoisoflavanone 5,7-dihydroxy-6-methoxy-3-(9-hydroxy-phenylmethyl)-chroman-4-one (1) and its structurally related 2',4',6'-trihydroxy-3'-methoxy-alpha-hydroxymethyl-beta-hydroxy-dihydrochalcone (2) along with the known pashanone (3), flavokawin B (4) and cardamonin or alpinetin chalcone (5) pinostrobin (6) and 5,8-dimethoxy-7-hydroxychroman-4-one (7) were isolated from dry leaves of Polygonum ferrugineum (Polygonaceae). To our knowledge, this is the first report of the isolation of a homoisoflavanone from the Polygonum genus and the Polygonaceae family, and could be an important chemotaxonomic finding. In addition, the pattern of substitution of this homoisoflavanone is different from others previously reported.     

Mulinane-type diterpenoids from Mulinum spinosum

In addition to the known mulinolic and mulinenic acids, two diterpenoids, mulin-11-ene-13-alpha,14-alpha-dihydroxy-20-oic and mulin-12-ene-14-one-20-oic acids, were isolated from the aerial parts of Mulinum spinosum (Cav.) Pers. (Apiaceae). Their structures were determined based on spectroscopic studies.     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.