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Gyobu N.,Biomedicinal Information Research Center
Methods in molecular biology (Clifton, N.J.)

Once 2D crystals suitable for electron crystallography have been obtained, grid preparation for cryo-EM is a critical step in obtaining high-resolution structural information. Specimens have to be prepared in a manner that prevents dehydration and disruption of the crystals in the vacuum of the electron microscope. Sugar embedding is an effective way to preserve specimens in the native and hydrated state. Preparation of almost perfectly flat specimens is another prerequisite. Imperfect specimen flatness is a crucial problem in the recording of images and diffraction patterns at higher tilt angles because it causes the blurring of spots perpendicular to the tilt axis. In this chapter, we describe the protocols of preparing 2D crystal specimen for electron crystallographical data collection. These protocols cover preparation of a flat carbon support film by sparkless carbon evaporation, sugar embedding using back injection, and the recently developed carbon sandwich technique. Source

Hwang J.-H.,Japan National Institute of Advanced Industrial Science and Technology | Takagi M.,Biomedicinal Information Research Center | Murakami H.,Aichi Cancer Center Research Institute | Sekido Y.,Aichi Cancer Center Research Institute | And 2 more authors.
Cancer Letters

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with a poor prognosis. Thus, novel therapeutic agents need to be developed for treating it. We recently reported the isolation of the novel anti-MPM compound designated as JBIR-23 from Streptomyces sp. AK-AB27. In this study, JBIR-23 exerted its cytotoxic effect on MPM cells by promotion of tubulin polymerization and G 2/M arrest, which was followed by apoptosis induction via the caspase pathway through phosphorylation of p38 mitogen-activated protein kinase and c-jun N-terminal kinase. Furthermore, in vivo analysis demonstrated that JBIR-23 prevented tumor growth in tumor-bearing nude mice without evident side effects. © 2010 Elsevier Ireland Ltd. Source

Izumikawa M.,Biomedicinal Information Research Center | Takagi M.,Biomedicinal Information Research Center | Shin-Ya K.,Japan National Institute of Advanced Industrial Science and Technology
Journal of Natural Products

The search for metabolites of Kibdelosporangium sp. AK-AA56 resulted in the discovery of novel N-phenylacetylated peptides, JBIR-78 (1) and JBIR-95 (2). Compounds 1 and 2 were established to be N-phenylacetylated heptapeptides by extensive NMR and HRESIMS analyses. The absolute configuration of the standard amino acids including a cysteic acid moiety was determined using Marfey's method on the acid hydrolysates of 1 and 2. The relative and absolute configurations of a nonstandard amino acid, β-hydroxyleucine, were elucidated using the J-based and modified Mosher's methods, respectively. In an antimicrobial test, 1 showed antibacterial activity against Micrococcus luteus. © 2012 The American Chemical Society and American Society of Pharmacognosy. Source

Khan S.T.,Biomedicinal Information Research Center | Khan S.T.,King Saud University | Takagi M.,Biomedicinal Information Research Center | Shin-Ya K.,Japan National Institute of Advanced Industrial Science and Technology
Microbes and Environments

Actinobacteria associated with 3 marine sponges, Cinachyra sp., Petrosia sp., and Ulosa sp., were investigated. Analyses of 16S rRNA gene clone libraries revealed that actinobacterial diversity varied greatly and that Ulosa sp. was most diverse, while Cinachyra sp. was least diverse. Culture-based approaches failed to isolate actinobacteria from Petrosia sp. or Ulosa sp., but strains belonging to 10 different genera and 3 novel species were isolated from Cinachyra sp. Source

Khan S. T.,Biomedicinal Information Research Center | Komaki H.,Biomedicinal Information Research Center | Komaki H.,Chiba Institute of Technology | Motohashi K.,Biomedicinal Information Research Center | And 4 more authors.
Environmental Microbiology

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture-dependent and -independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR-43 exhibited cytotoxic activity against cancer cell lines. JBIR-34 and -35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd. Source

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