Occurrence and diversity of mesophilic Shewanella strains isolated from the North-West Pacific Ocean

Although bacteria of the genus Shewanella belong to one of the readily cultivable groups of "Gammaproteobacteria", little is known about the occurrence and abundance of these microorganisms in the marine ecosystem. Studies revealed that of 654 isolates obtained from marine invertebrates (ophiuroid Amphiopholis kochii, sipuncula Phascolosoma japonicum, and holothurian Apostichopus japonicus, Cucumaria japonica), seawater and sediments of the North-West Pacific Ocean (i.e. the Sea of Japan and Iturup Is, Kurile Islands), 10.7% belonged to the genus Shewanella. The proportion of viable Shewanella species varied from 4% to 20% depending on the source of isolation. From the isolation study, representative strains of different phenotypes (from seventy presumptive Shewanella strains) were selected for detailed characterization using phenotypic, chemotaxonomic, and phylogenetic testing. 16S rDNA sequence-based phylogenetic analysis confirmed the results of tentative identification and placed the majority of these strains within only a few species of the genus Shewanella with 98-99% of 16S rDNA sequences identity mainly with S. japonica and S. colwelliana, suggesting that the strains studied might belong to these species. Numerically dominant strains of S. japonica were metabolically active and produced proteinases (gelatinases, caseinases), lipases, amylases, agarases, and alginases. Shewanella strains studied demonstrated weak antimicrobial and antifungal activities that might be an indication of their passive role in the colonization on living and non-living surfaces.     

Excitation and deexcitation processes of Eu(III) benzo-15-crown-5 complex studied on comparing with its first coordination sphere

The coordination sphere and the deexcitation mechanism of the Eu(III) benzo-15-crown-5 complex, Eu(B15C5), were studied with references of the Eu(III) complexes with a similar coordination sphere; the dibenzo-18-crown-6 complex, Eu₃(B₂18C6)₂, and the cryptand[2.2.1] complex, Eu([2.2.1]). NMR spectroscopy reveals that the Eu(B15C5) complex is quite stable in acetonitrile solution whereas only 40% of the Eu(III) ion forms the complex in the equimolar Eu(NO₃)₃ and B₂18C6 acetonitrile solution. The coordination sphere of the Eu(III) complexes in acetonitrile solutions were also discussed by the degenerate ⁷F₀→⁵D₀ transition energy levels. The Eu(B15C5) have a negative shift compared with the europium(III) nitrate in acetonitrile and it is explained by the coordination of both nitrate ions and the crown ether ligand. Energy transfer from the n–π* excited state located in the catechol structure to the central europium ion was first observed as the sensitized luminescence of ⁵D₀→⁷F_J. The excited state lifetime of the Eu(B15C5) complex was first determined as 201 μs in the present study.     

Camptothecin-somatostatin conjugates inhibit the growth of small cell lung cancer cells

The effects of camptothecin-somatostatin (CPT-SS) conjugates were investigated on small cell lung cancer (SCLC) cells. CPT was coupled to a SS agonist (SSA), c(Cys-Phe-DTrp-Lys-Thr-Cys)Thr-NH2 using the built in nucleophile assisted-releasing group (L1) N-methyl-aminoethyl-Gly-Dser-Nle-Dtyr-Dser or (L2) aminoethyl-Gly-Dser-Nle-Dtyr-Dser. The resulting CPT-L1-SSA and CPT-L2-SSA inhibited the specific binding of [125I-Tyr11]SS to NCI-H69 cell membranes with IC50 values of 0.2 and 2.1 nM, respectively. [125I]CPT-L1-SSA was internalized by SCLC cells at 37 degrees C but not at 4 degrees C. CPT-L1-SSA and CPT-L2-SSA inhibited in a dose-dependent manner the increase in adenylylcyclase activity caused by 25 microM forskolin. In vitro, 0.3 microM CPT-L1-SSA half-maximally inhibited the clonal growth of SCLC cells and 1 microM CPT-L1-SSA strongly inhibited 3H-thymidine incorporation into DNA and trypan-blue exclusion. These results suggest that CPT conjugated peptides such as CPT-L1-SSA may prove useful for exploring the efficacy of receptor-directed cytotoxicity to inhibit the proliferation of SCLC cells.     

Irradiation of mammalian cultured cells with a collimated heavy-ion microbeam

As the first step for the analysis of the biological effect of heavy charged-particle radiation, we established a method for the irradiation of individual cells with a heavy-ion microbeam apparatus at JAERI-Takasaki. CHO-K1 cells attached on a thin film of an ion track detector, CR-39, were automatically detected under a fluorescence microscope and irradiated individually with an 40Ar13+ ion (11.5 MeV/nucleon, LET 1260 keV/microm) microbeam. Without killing the irradiated cells, trajectories of irradiated ions were visualized as etch pits by treatment of the CR-39 with an alkaline-ethanol solution at 37 degrees C. The exact positions of ion hits were determined by overlaying images of both cells and etch pits. The cells that were irradiated with argon ions showed a reduced growth in postirradiation observations. Moreover, a single hit of an argon ion to the cell nucleus resulted in strong growth inhibition. These results tell us that our verified irradiation method enables us to start a precise study of the effects of high-LET radiation on cells.     

Comparative radiation tolerance based on the induction of DNA double-strand breaks in tobacco BY-2 cells and CHO-K1 cells irradiated with gamma rays

Higher plants are generally more tolerant to ionizing radiation than mammals. To explore the radiation tolerance of higher plants, the induction of DNA double-strand breaks (DSBs) by gamma rays was investigated in tobacco BY-2 cells and compared with that in Chinese hamster ovary (CHO)-K1 cells as a reference. This is the first examination of radiation-induced DSBs in a higher plant cell. The resulting DNA fragments were separated by pulsed-field gel electrophoresis and stained with SYBR Green I. The initial yield of DSBs was then quantified from the fraction of DNA fragments shorter than 1.6 Mbp based on the assumption of random distribution of DSBs. The DSB yield in tobacco BY-2 cells (2.0 +/- 0.1 DSBs Gbp(-1) Gy(-1)) was only one-third of that in CHO-K1 cells. Furthermore, the calculated number of DSBs per diploid cell irradiated with gamma rays at the mean lethal dose was five times greater in BY-2 cells (263 +/- 13) than in CHO-K1 cells. These results suggest that the radiation tolerance of BY-2 cells appears to be due not only to a lower induction of DNA damage but also to a more efficient repair of the induced DNA damage.     

Identification of an allele of VAM3/SYP22 that confers a semi-dwarf phenotype in Arabidopsis thaliana

The short stem and midrib (ssm) mutants of Arabidopsis thaliana show both semi-dwarf and wavy leaf phenotypes due to defects in the elongation of the stem internodes and leaves. Moreover, these abnormalities cannot be recovered by exogenous phytohormones. ssm was originally identified as a single recessive mutant of the ecotype Columbia (Col-0), but genetic crossing experiments have revealed that this mutant phenotype is restored by another gene that is functional in the ecotype Landsberg erecta (Ler) and not in Col-0. Map-based cloning of the gene that is defective in ssm mutants has uncovered a small deletion in the sixth intron of a gene encoding a syntaxin, VAM3/SYP22, which has been implicated in vesicle transport to the vacuole. This mutation appears to cause a peptide insertion in the deduced VAM3/SYP22 polypeptide sequence due to defective splicing of the shortened sixth intron. Significantly, when compared with the wild-type Ler genome, the wild-type Col-0 genome has a single base pair deletion causing a frameshift mutation in SYP23, a gene with the highest known homology to VAM3/SYP22. These findings suggest that VAM3/SYP22 and SYP23 have overlapping functions and that the vesicle transport mediated by these syntaxins is important for shoot morphogenesis.     

KATAMARI1/MURUS3 Is a novel golgi membrane protein that is required for endomembrane organization in Arabidopsis

In plant cells, unlike animal and yeast cells, endomembrane dynamics appear to depend more on actin filaments than on microtubules. However, the molecular mechanisms of endomembrane-actin filament interactions are unknown. In this study, we isolated and characterized an Arabidopsis thaliana mutant, katamari1 (kam1), which has a defect in the organization of endomembranes and actin filaments. The kam1 plants form abnormally large aggregates that consist of endoplasmic reticulum with actin filaments in the perinuclear region within the cells and are defective in normal cell elongation. Map-based cloning revealed that the KAM1 gene is allelic to the MUR3 gene. We demonstrate that the KAM1/MUR3 protein is a type II membrane protein composed of a short cytosolic N-terminal domain and a transmembrane domain followed by a large lumenal domain and is localized specifically on Golgi membranes. We further show that actin filaments interact with Golgi stacks via KAM1/MUR3 to maintain the proper organization of endomembranes. Our results provide functional evidence that KAM1/MUR3 is a novel component of the Golgi-mediated organization of actin functioning in proper endomembrane organization and cell elongation.