Dioncophylline E from Dioncophyllum thollonii,the first 7,3'-coupled dioncophyllaceous naphthylisoquinoline alkaloid

The isolation and structural elucidation of dioncophylline E, a novel naphthylisoquinoline alkaloid from the rare West African liana Dioncophyllum thollonii, is described. The alkaloid displays an unusual 7,3'-linkage between the naphthalene and the isoquinoline portions. Due to the resulting medium degree of steric hindrance exerted by the ortho-substituents next to the biaryl axis, the compound undergoes slow rotation about the axis at room temperature and thus is the first such alkaloid that occurs as a mixture of two configurationally semi-stable atropo-diastereomers. Dioncophylline E exhibits good antimalarial activity against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum while its antitrypanosomal activity against Trypanosoma cruzi is weak and that against T. brucei rhodesiense is moderate. Furthermore, four additional naphthylisoquinoline alkaloids previously known from the related plant species Triphyophyllum peltatum, have been identified in D. thollonii.     

In vivo localization and identification of the antiplasmodial alkaloid dioncophylline A in the tropical liana Triphyophyllum peltatum by a combination of fluorescence, near infrared Fourier transform Raman microscopy, and density functional theory calculations

Near infrared Fourier transform (NIR FT) micro Raman spectroscopy in combination with density functional theory (DFT) calculations has been applied for an in vivo localization of the antiplasmodial naphthylisoquinoline alkaloid dioncophylline A (1) in the tropical liana Triphyophyllum peltatum. Fluorescence microscopy images suggest finding this active agent in 10 mum big inclusions located in the cortex of the stem or the beginning of the leaves. By means of spatially resolved FT Raman micro spectroscopy, we could detect dioncophylline A (1) in these inclusions. FT Raman spectroscopy is an extremely selective tool capable of differentiating between various structurally similar naphthylisoquinoline alkaloids. With the help of DFT calculations, we succeeded in assigning the differences found in the FT Raman spectra of the various naphthylisoquinolines to nuC=C vibrations of the naphthyl ring. The presented results are of relevance for the investigation and extraction of new antimalarial active agents.     

Extract screening by HPLC coupled to MS-MS,NMR, and CD: a dimeric and three monomeric naphthylisoquinoline alkaloids from Ancistrocladus griffithii

Three new monomeric naphthylisoquinoline alkaloids, ancistrogriffines A, B, and C, and the first dimer of a 7,8'-coupled naphthylisoquinoline, ancistrogriffithine A, have been detected by phytochemical online screening of plant extracts of Ancistrocladus griffithii, using the analytical 'triad' HPLC-MS/MS, HPLC-NMR, and HPLC-CD. Ancistrogriffithine A, as well as ancistrogriffines A and C, were structurally completely assigned (including the absolute configuration) right from the extract, without previous isolation. Furthermore, two related, but known alkaloids, ancistrocladine and hamatine, were identified. Except for ancistrogriffine B, which occurs in trace quantities only, all new alkaloids were then isolated preparatively and the initial assignments were fully confirmed by conventional offline methods. Of particular interest is the constitutionally and configurationally unprecedented structure of ancistrogriffithine A, which is simultaneously the first dimeric naphthylisoquinoline alkaloid from an Asian Ancistrocladus species. Ancistrogriffithine A and ancistrogriffine A are active against Plasmodium falciparum. Furthermore, the latter compound shows good activity against Leishmania donovani. The results demonstrate the ability of modern online methods like HPLC-NMR, -MS/MS, and -CD to serve as powerful tools for the reliable structural elucidation of even complex structures of trace compounds in crude biological matrices.     

Ancistrolikokine D,a 5,8'-coupled naphthylisoquinoline alkaloid,and related natural products from Ancistrocladus likoko

A new naphthylisoquinoline alkaloid, ancistrolikokine D, and the likewise 5,8'-coupled alkaloid ancistroealaine A, as well as two further, biosynthetically related, but nitrogen-free natural products, ancistronaphthoic acid B and cis-isoshinanolone, have been isolated from Ancistrocladus likoko J. LEACUTE;ONARD (Ancistrocladaceae). The 5,8'-coupling of the new alkaloids and of the alkaloids isolated earlier hints at a close phylogenetic relationship of A. likoko to other Central African Ancistrocladus species. The compounds show moderate activities against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei rhodesiense.     

Ancistroheynine B and two further 7,3'-coupled naphthylisoquinoline alkaloids from Ancistrocladus heyneanus Wall

A new axially chiral naphthylisoquinoline alkaloid, ancistroheynine B (7), has been isolated from the leaves of the Indian liana Ancistrocladus heyneanus Wall., along with two known related alkaloids, ancistrocladidine (3) and ancistrotanzanine C (6), which are 7,3'-coupled, too. The structural elucidation was achieved by chemical, spectroscopic, and chiroptical methods. Biological activities of ancistroheynine B against the pathogens of malaria, leishmaniasis, Chagas' disease, and African sleeping sickness were evaluated.     

Stress-related polyketide metabolism of Dioncophyllaceae and Ancistrocladaceae

The discovery of a novel biosynthetic pathway to isoquinoline alkaloids is described. The naphthylisoquinoline alkaloid dioncophylline A, one of the most prominent representatives of a new class of structurally and pharmacologically intriguing secondary metabolites, is shown to originate from acetate units, both molecular halves, the isoquinoline part and the naphthalene portion, being formed from identical polyketide precursors. All other tetrahydroisoquinoline alkaloids previously investigated, ultimately originate from aromatic amino acids. The novel pathway to isoquinoline alkaloids (hence acetogenic) was proved by feeding experiments with (13)C-labelled precursors administered to callus cultures of Triphyophyllum peltatum (Dioncophyllaceae), followed by NMR investigations using the potent cryoprobe methodology. The new pathway is largely stress-sensitive: upon exposure to chemical, biotic or physical stress, T. peltatum stops producing the isoquinoline part, so that the naphthalene moiety accumulates in the chemical form of naphthoquinones like plumbagin and droserone and the chiral tetralone isoshinanolone.     

Austrocolorins A1 and B1: atropisomeric 10,10'-linked dihydroanthracenones from an Australian Dermocybe sp

The atropisomeric austrocolorins A(1) (7) and B(1) (8), new members of the rare tricolorin class of 10,10'-coupled dihydroanthracenones, are isolated from an indigenous Australian toadstool belonging to the subgenus Dermocybe of Cortinarius, and their structure and absolute central and axial configuration is deduced from the spectroscopic data, and confirmed by chemical degradation and chiral HPLC analysis.     

Convergence in the biosynthesis of acetogenic natural products from plants,fungi, and bacteria

This review deals with polyketides to which nature has developed different biosynthetic pathways in the course of evolution. The anthraquinone chrysophanol is the first example of an acetogenic natural product that is, in an organism-specific manner, formed via more than one polyketide folding mode: In eukaryotes, like e.g., in fungi, in higher plants, and in insects, it is synthesized via folding mode F, while in prokaryotes it originates through mode S. It has, more recently, even been found to be synthesized by a third pathway, named mode S′. Thus, chrysophanol is the first polyketide synthase product that originates through a divergent–convergent biosynthesis (depending on the respective producing organisms). A second example of a striking biosynthetic convergence is the isoquinoline alkaloids. While all as yet investigated representatives of this large family of plant-derived metabolites (more than 2500 known representatives!) are formed from aromatic amino acids, the biosynthetic origin of naphthylisoquinoline alkaloids like dioncophylline A is unprecedented in following a route to isoquinolines in plants: we have shown that such naphthylisoquinolines represent the as yet only known polyketidic di- and tetrahydroisoquinolines, originating from acetate and malonate units, exclusively. Both molecular halves, the isoquinoline part and the naphthalene portion, are even synthesized from a joint polyketide precursor, the first proven case of the F-type folding mode in higher plants. The biosynthetic origins of the natural products presented in this paper were elucidated by feeding ¹³C₂-labeled acetate (or advanced precursors) to the respective producing organisms, with subsequent NMR analysis of their ¹³C₂ incorporation patterns using the potent cryoprobe methodology, thus making the full polyketide folding pattern visible.     

Habropetaline A,an antimalarial naphthylisoquinoline alkaloid from Triphyophyllum peltatum

The isolation, structural elucidation, and antiprotozoal activities of habropetaline A, a novel naphthylisoquinoline alkaloid from Triphyophyllum peltatum, are described. This alkaloid had previously only been identified on line, by the LC-MS/MS-NMR-CD triad, in the crude extract of the rare and difficult-to-provide related plant species Habropetalum dawei, whose small quantities available had not permitted to isolate the compound. As predicted by quantitative structure-activity relationship (QSAR) investigations, habropetaline A exhibits strong antimalarial activity against Plasmodium falciparum, while it is inactive against other protozoal pathogens (Trypanosoma brucei rhodesience, T. cruzi, and Leishmania donovani).     

Structural and biochemical characterization of the vaccinia virus envelope protein D8 and its recognition by the antibody LA5

Smallpox vaccine is considered a gold standard of vaccines, as it is the only one that has led to the complete eradication of an infectious disease from the human population. B cell responses are critical for the protective immunity induced by the vaccine, yet their targeted epitopes recognized in humans remain poorly described. Here we describe the biochemical and structural characterization of one of the immunodominant vaccinia virus (VACV) antigens, D8, and its binding to the monoclonal antibody LA5, which is capable of neutralizing VACV in the presence of complement. The full-length D8 ectodomain was found to form a tetramer. We determined the crystal structure of the LA5 Fab-monomeric D8 complex at a resolution of 2.1 Å, as well as the unliganded structures of D8 and LA5-Fab at resolutions of 1.42 Å and 1.6 Å, respectively. D8 features a carbonic anhydrase (CAH) fold that has evolved to bind to the glycosaminoglycan (GAG) chondroitin sulfate (CS) on host cells. The central positively charged crevice of D8 was predicted to be the CS binding site by automated docking experiments. Furthermore, sequence alignment of various poxvirus D8 orthologs revealed that this crevice is structurally conserved. The D8 epitope is formed by 23 discontinuous residues that are spread across 80% of the D8 protein sequence. Interestingly, LA5 binds with a high-affinity lock-and-key mechanism above this crevice with an unusually large antibody-antigen interface, burying 2,434 Ų of protein surface.     

Analytical characterisation of crude extracts from an African Ancistrocladus species using high-performance liquid chromatography and capillary electrophoresis coupled to ion trap mass spectrometry

The analysis by HPLC, CE and CE-MS/MS of root bark extracts of a, so far undescribed, Central-African Ancistrocladus species (family Ancistrocladaceae) is described. Owing to the complexity of the extract, the application of reversed-phase HPLC resulted in a partially incomplete separation of the naphthylisoquinoline alkaloids, whilst CE using a non-aqueous buffer proved to be a very valuable complementary method for a first characterisation of the crude extract. By performing additional CE-MS/MS experiments, in combination with parallel isolation studies and structural elucidation using conventional methods, six alkaloidal substances present in the plant could be identified.     

中国角麦蛾属分类研究(鳞翅目:麦蛾科)

角麦蛾属Deltophora Janse昆虫广布全世界,已知有21种,但过去在中国没有记录,本文报道2新种和中国1新纪录种：指角麦蛾D.digitiformis sp.nov。分布河南（登封、济源）;方瓣角麦蛾D.quadrativalvata sp.nov。分布河北（涞源、井陉、内丘）和河南（登封）;中国新纪录种远东角麦蛾D.korbi(Caradja),分布河北（蔚县）,国外分布朝鲜和日本,文中给出了中国角麦蛾属分种检索表和雌雄外生殖器特征图。     

Vege and Mar: two novel hAT MITE families from Drosophila willistoni

Two novel families of miniature inverted repeat transposable elements (MITEs), Vege and Mar, are described from Drosophila willistoni. Based on their structures, both element families are hypothesized to belong to the hAT superfamily of transposable elements. Both elements have perfect, inverted terminal repeats and 8-bp target site duplications and were found to have inserted within fixed copies of nonautonomous P elements. Vege is present in all studied D. willistoni populations and appears to have a relatively low copy number. Mar was identified in only a single D. willistoni population, and its copy number is presently unknown. Although MITEs occupy relatively large proportions of the genomes of a broad range of organisms, this may be their first unambiguous identification in any species of the genus Drosophila.     

Functional classification of protein 3D structures from predicted local interaction sites

A new approach to the functional classification of protein 3D structures is described with application to some examples from structural genomics. This approach is based on functional site prediction with THEMATICS and POOL. THEMATICS employs calculated electrostatic potentials of the query structure. POOL is a machine learning method that utilizes THEMATICS features and has been shown to predict accurate, precise, highly localized interaction sites. Extension to the functional classification of structural genomics proteins is now described. Predicted functionally important residues are structurally aligned with those of proteins with previously characterized biochemical functions. A 3D structure match at the predicted local functional site then serves as a more reliable predictor of biochemical function than an overall structure match. Annotation is confirmed for a structural genomics protein with the ribulose phosphate binding barrel (RPBB) fold. A putative glucoamylase from Bacteroides fragilis (PDB ID 3eu8) is shown to be in fact probably not a glucoamylase. Finally a structural genomics protein from Streptomyces coelicolor annotated as an enoyl-CoA hydratase (PDB ID 3g64) is shown to be misannotated. Its predicted active site does not match the well-characterized enoyl-CoA hydratases of similar structure but rather bears closer resemblance to those of a dehalogenase with similar fold.     

Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography

We provide a catalog of 3D cryo soft X-ray tomography (cryo-SXT) images obtained from ～6 to 12μm thick mouse adenocarcinoma cells. Included are multiple representative images of nuclei, nucleoli, nuclear membrane, nuclear membrane channels, mitochondria, lysosomes, endoplasmic reticulum, filaments and plasma membrane, plus three structures not previously described by cryo-SXT, namely Golgi, microvilli and nuclear-membrane blebs. Sections from the 3D cryo-SXT tomograms for all the preceding structures closely resemble those seen by thin-section transmission electron microscopy (TEM). Some structures such as nuclear-membrane channels and nuclear-membrane blebs are more easily detected by cryo-SXT than TEM most likely due to their better contrast and cellular preservation in cryo-SXT combined with the ability to rapidly locate these structures within a full 3D image. We identify and discuss two current limitations in cryo-SXT: variability in image quality and difficulties in detecting weaker contrast structures such as chromatin and various nuclear bodies. Progress on these points is likely to come from the solution of several technical problems in image acquisition, plus the implementation of advanced cryo soft X-ray microscopy approaches such as phase contrast or optical sectioning.     

苔藓动物主要类群28S rDNA多变区的分子形态分析及其系统学意义

测定了苔藓动物主要代表类群3种苔虫的28S rDNA全序列(3 459～3 613 bp),结合已公布的相关类群的28S rDNA全序列数据,分析了其一级结构特征,并分别绘制了D2、D3和D8区段的二级结构.比较分析结果显示:各类群的全序列总长均超过3 300 bp,GC含量为41.8%～57.7%;D3和D8区段的二级结构保守性较高,D2区差异显著;各区段的长度变化较大,但是,大多都具有相似的主干结构.基于二级结构多变区分子形态学特征的比较基本和基于一级结构的分子系统学结论相吻合,提示它们同样包含着重要的系统发育信息.     

Six naphthylisoquinoline alkaloids and a related benzopyranone from a Congolese Ancistrocladus species related to Ancistrocladus congolensis

From the roots of a recently discovered Ancistrocladus taxon, with close affinities to Ancistrocladus congolensis regarding molecular ITS sequence data, six naphthylisoquinoline alkaloids, 5'-O-demethylhamatine (2), 5'-O-demethylhamatinine (3), 6-O-demethylancistroealaine A (4), 6,5'-O,O-didemethylancistroealaine A (5), 5-epi-6-O-methylancistrobertsonine A (6), and 5-epi-4'-O-demethylancistrobertsonine C (7), have been isolated, along with a likewise benzopyranone carboxylic acid, 8. The structural elucidation succeeded by chemical, spectroscopic, and chiroptical methods. Their bioactivities were tested against protozoan parasites causing severe tropical diseases. Furthermore, eight known related alkaloids were identified.     

Three-dimensional modelling of the catalytic domain of Streptococcus mutans glucosyltransferase GtfB

Glucosyltransferases (GtfB/C/D) of Streptococcus mutans, a pathogen for human dental caries, synthesize water-insoluble glucan through the hydrolysis of sucrose. Genetic and biochemical approaches have identified several active sites of these enzymes, but no three-dimensional (3D) structural evidence is yet available to elucidate the subdomain arrangement and molecular mechanism of catalysis. Based on a combined sequence and secondary structure alignment against known crystal structures of segments from closely related proteins, we propose here the 3D model of an N-terminal domain essential for the sucrose binding and splitting in GtfB. A Tim-barrel of (alpha/beta)(8) structural characteristics is revealed and the structural correlation for two peptides is described.     

A Method to Fabricate Disconnected Silver Nanostructures in 3D

The standard nanofabrication toolkit includes techniques primarily aimed at creating 2D patterns in dielectric media. Creating metal patterns on a submicron scale requires a combination of nanofabrication tools and several material processing steps. For example, steps to create planar metal structures using ultraviolet photolithography and electron-beam lithography can include sample exposure, sample development, metal deposition, and metal liftoff. To create 3D metal structures, the sequence is repeated multiple times. The complexity and difficulty of stacking and aligning multiple layers limits practical implementations of 3D metal structuring using standard nanofabrication tools. Femtosecond-laser direct-writing has emerged as a pre-eminent technique for 3D nanofabrication.^1,2 Femtosecond lasers are frequently used to create 3D patterns in polymers and glasses.^3-7 However, 3D metal direct-writing remains a challenge. Here, we describe a method to fabricate silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm. The method enables the fabrication of patterns not feasible using other techniques, such as 3D arrays of disconnected silver voxels.⁸ Disconnected 3D metal patterns are useful for metamaterials where unit cells are not in contact with each other,⁹ such as coupled metal dot^10,11or coupled metal rod^12,13 resonators. Potential applications include negative index metamaterials, invisibility cloaks, and perfect lenses.In femtosecond-laser direct-writing, the laser wavelength is chosen such that photons are not linearly absorbed in the target medium. When the laser pulse duration is compressed to the femtosecond time scale and the radiation is tightly focused inside the target, the extremely high intensity induces nonlinear absorption. Multiple photons are absorbed simultaneously to cause electronic transitions that lead to material modification within the focused region. Using this approach, one can form structures in the bulk of a material rather than on its surface.Most work on 3D direct metal writing has focused on creating self-supported metal structures.^14-16 The method described here yields sub-micrometer silver structures that do not need to be self-supported because they are embedded inside a matrix. A doped polymer matrix is prepared using a mixture of silver nitrate (AgNO₃), polyvinylpyrrolidone (PVP) and water (H₂O). Samples are then patterned by irradiation with an 11-MHz femtosecond laser producing 50-fs pulses. During irradiation, photoreduction of silver ions is induced through nonlinear absorption, creating an aggregate of silver nanoparticles in the focal region. Using this approach we create silver patterns embedded in a doped PVP matrix. Adding 3D translation of the sample extends the patterning to three dimensions.     

Reconstitution of water channel function and 2D-crystallization of human aquaporin 8

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3?.