Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.     

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) > D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).     

Distribution of proglucagon mRNA and GLP-1 in the brainstem of chicks

Glucagon-like peptide-1 (GLP-1), structurally similar to glucagon, synthesized from the precursor proglucagon, is a well known anorexigenic peptide in the brain of several animal species. However, there are no previous reports concerning GLP-1-containing neurons in the chick brain. The aim of the present study was to investigate the distribution of proglucagon mRNA and GLP-1-immunoreactive (GLI) perikarya in various regions of the chick brain. We detected proglucagon mRNA in the brainstem, and to a lesser extent in the telencephalon. In the brainstem, a study using immunohistochemistry revealed that GLI perikarya were present in the nucleus motorius nervi facialis pars dosalis, nucleus motoris dorsalis nervi vagi and nucleus tractus solitarii. Furthermore, we found that proglucagon mRNA expression in the brainstem decreased after 24 h fasting. The present findings support the idea that endogenous GLP-1 is involved in feeding behavior of chicks.     

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.     

Prostaglandin E2 production in ovarian cancer cell lines is regulated by cyclooxygenase-1, not cyclooxygenase-2

The significance of cyclooxygenase-2 (COX-2) expression in ovarian cancer has been discussed. In this study, we found increased expression of COX-1 mRNA and protein in three out of 10 ovarian cancer cell lines. Prostaglandin E 2 (PGE2) production was elevated in these three cell lines, but not in other seven cell lines. COX-2 protein was not detected in any of the cell lines. Cytosolic prostaglandin E synthase (cPGES) mRNA and protein were detected in all 10 cell lines. Membrane-associated PGES-1 (mPGES-1) was detected in some of the ovarian cell lines, but its presence did not correspond with PGE2 production. In contrast, mPGES-2 mRNA and protein were detected in all 10 cell lines. A nonselective COX inhibitor (indometacin) and a selective COX-1 inhibitor (SC-560) strongly inhibited PGE2 production by the three cell lines, while selective COX-2 inhibitors (NS-398 and rofecoxib) did not inhibit PGE2 production. In addition, increased expression of COX-1, not COX-2 protein was observed in the mass of ovarian cancer tissues from 22 patients when compared with that in normal tissue. These findings suggest that COX-1 might be a major enzyme regulating PGE2 production in ovarian cancer cells.     

Purification and characterization of a novel aminoacylase from Streptomyces mobaraensis

A novel aminoacylase was purified to homogeneity from culture broth of Streptomyces mobaraensis, as evidenced by SDS-polyacrylamide gel electrophoresis (PAGE). The enzyme was a monomer with an approximate molecular mass of 100 kDa. The purified enzyme was inhibited by the presence of 1,10-phenanthroline and activated by the addition of Co2+. It was stable at temperatures of up to 60 degrees C for 1 h at pH 7.2. It showed broad substrate specificity to N-acetylated L-amino acids. It catalyzed the hydrolysis of the amide bonds of various N-acetylated L-amino acids, except for Nepsilon-acetyl-L-lysine and N-acetyl-L-proline. Hydrolysis of N-acetyl-L-methionine and N-acetyl-L-histidine followed Michaelis-Menten kinetics with K(m) values of 1.3+/-0.1 mM and 2.7+/-0.1 mM respectively. The enzyme also catalyzed the deacetylation of 7-aminocephalosporanic acid (7-ACA) and cephalosporin C. Moreover, feruloylamino acids and L-lysine derivatives of ferulic acid derivatives were synthesized in an aqueous buffer using the enzyme.     

Growth inhibition by tungsten in the sulfur-oxidizing bacterium Acidithiobacillus thiooxidans

Growth of five strains of sulfur-oxidizing bacteria Acidithiobacillus thiooxidans, including strain NB1-3, was inhibited completely by 50 microM of sodium tungstate (Na(2)WO(4)). When the cells of NB1-3 were incubated in 0.1 M beta-alanine-SO(4)(2-) buffer (pH 3.0) with 100 microM Na(2)WO(4) for 1 h, the amount of tungsten bound to the cells was 33 microg/mg protein. Approximately 10 times more tungsten was bound to the cells at pH 3.0 than at pH 7.0. The tungsten binding to NB1-3 cells was inhibited by oxyanions such as sodium molybdenum and ammonium vanadate. The activities of enzymes involved in elemental sulfur oxidation of NB1-3 cells such as sulfur oxidase, sulfur dioxygenase, and sulfite oxidase were strongly inhibited by Na(2)WO(4). These results indicate that tungsten binds to NB1-3 cells and inhibits the sulfur oxidation enzyme system of the cells, and as a result, inhibits cell growth. When portland cement bars supplemented with 0.075% metal nickel and with 0.075% metal nickel and 0.075% calcium tungstate were exposed to the atmosphere of a sewage treatment plant containing 28 ppm of H(2)S for 2 years, the weight loss of the portland cement bar with metal nickel and calcium tungstate was much lower than the cement bar containing 0.075% metal nickel.     

Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi

A resynthesis study was conducted to clarify the relationship between the root endophyte, Heteroconium chaetospira and the ericaceous plant, Rhododendron obtusum var. kaempferi. The host plant roots were recovered 2 months after inoculation, and the infection process and colonization pattern of the fungus were observed under a microscope. The hyphae of H. chaetospira developed structures resembling ericoid mycorrhizas, such as hyphal coils within the host epidermal cells. These structures were morphologically the same as previously reported ericoid mycorrhizal structures. The frequencies of hyphal coils within the epidermal cells of host roots ranged from 13 to 20%. H. chaetospira did not promote or reduce host plant growth. This is the first reported study that H. chaetospira is able to form structures resembling mycorrhizas within the roots of ericaceous plants.     

A novel eukaryotic selenoprotein in the haptophyte alga Emiliania huxleyi

The diversity of selenoproteins raises the question of why many life forms require selenium. Especially in photosynthetic organisms, the biochemical basis for the requirement for selenium is unclear because there is little information on selenoproteins. We found six selenium-containing proteins in a haptophyte alga, Emiliania huxleyi, which requires selenium for growth. The 27-kDa protein EhSEP2 was isolated, and its cDNA was cloned. The deduced amino acid sequence revealed that EhSEP2 is homologous to protein disulfide isomerase (PDI) and contains a highly conserved thioredoxin domain. The nucleotide sequence contains an in-frame TGA codon encoding selenocysteine at the position corresponding to the cysteine residue in the reaction center of known PDIs. However, no typical selenocysteine insertion sequence was found in the EhSEP2 cDNA. The EhSEP2 mRNA level was related to the abundance of selenium. E. huxleyi possesses a novel PDI-like selenoprotein and may have a novel type of selenocysteine insertion machinery.     

Suppression of adriamycin-induced apoptosis by sustained activation of the phosphatidylinositol-3'-OH kinase-Akt pathway

The mechanisms by which growth factors trigger signal transduction pathways leading to protection against apoptosis are of great interest. In this study, we investigated the effect of hepatocyte growth factor (HGF/SF) and epidermal growth factor (EGF) on adriamycin (ADR)-induced apoptosis. Treatment of human epithelial MKN74 cells with ADR, a DNA topoisomerase IIalpha inhibitor, caused apoptosis. However, cells pretreated with HGF/SF, but not those pretreated with EGF, were resistant to this apoptosis. The protective effect of HGF/SF against the ADR-induced apoptosis was abolished in the presence of either LY294002, an inhibitor of phosphatidylinositol-3'-OH kinase (PI3-K) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, an inhibitor of Akt, thus implicating the activation of PI3-K-Akt signaling in the antiapoptotic action of HGF/SF. Immunoblotting analysis revealed that HGF/SF stimulated the sustained phosphorylation of Akt for several hours but that EGF stimulated the phosphorylation only transiently. Furthermore, ADR-induced activation of caspase-9, a downstream molecule of Akt, was inhibited for at least 24 h after HGF/SF stimulation, but it was not affected by EGF stimulation. Cell-surface biotin-labeling analysis showed that the HGF/SF receptor remained on the cell surface until at least 30 min after HGF/SF addition but that the EGF receptor level on the cell surface was attenuated at an earlier time after EGF addition. These results indicate that HGF/SF, but not EGF, transmitted protective signals against ADR-induced apoptosis by causing sustained activation of the PI3-K-Akt signaling pathway. Furthermore, the difference in antiapoptotic capacity between HGF/SF and EGF is explained, at least in part, by the delayed down-regulation of the HGF/SF receptor.