Optimization of a coagulation factor VIIa inhibitor found in factor Xa inhibitor library

An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a, was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in the TF-dependent extrinsic pathway.     

Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated or UV-damaged DNA. The activated form of Claspin contains two repeated phosphopeptide motifs that mediate its binding to Chk1. We show that these phosphopeptide motifs bind to Chk1 by means of its N-terminal kinase domain. The binding site on Chk1 involves a positively charged cluster of amino acids that contains lysine 54, arginine 129, threonine 153, and arginine 162. Mutagenesis of these residues strongly compromises the ability of Chk1 to interact with Claspin. These amino acids lie within regions of Chk1 that are involved in various aspects of its catalytic function. The predicted position on Chk1 of the phosphate group from Claspin corresponds to the location of activation-loop phosphorylation in various kinases. In addition, we have obtained evidence that the C-terminal regulatory domain of Chk1, which does not form a stable complex with Claspin under our assay conditions, nonetheless has some role in Claspin-dependent activation. Overall, these results indicate that Claspin docks with a phosphate-binding site in the catalytic domain of Chk1 during activation by ATR. Phosphorylated Claspin may mimic an activating phosphorylation of Chk1 during this process.     

Studies on the endogenous L-selectin ligands: systematic and highly efficient total synthetic routes to lactamized-sialyl 6-O-sulfo Lewis X and other novel gangliosides containing lactamized neuraminic acid

Systematic syntheses of lactamized neuraminic acid-containing gangliosides GM4, sulfated sialylparagloboside, and sulfated/nonsulfated sialyl Lewis X are described. The highly efficient, one-step lactamization of neuraminic acid was accomplished by treatment of the N-deacetylated sialic acid (neuraminic acid)-containing gangliosides with HBTU and HOBt in DMF at 65 degrees C. Both the lactamized neuraminic acid residue and the sulfate group at O-6 of the GlcNAc residue were found to be involved in the antigenic determinant defined by G159 monoclonal antibody, while the fucose residue may not be critical for the recognition by G159 mAb.     

Gene Silencing Mediated by Endogenous MicroRNAs under Heat Stress Conditions in Mammalian Cells

Heat shock, sudden change in temperature, triggers various responses in cells for protecting the cells from such a severe circumstance. Here we investigated gene silencing mediated by endogenous microRNAs (miRNAs) in mammalian cells exposed to a mild hyperthermia, by means of miRNA activity assay using a luciferase reporter gene as well as miRNA expression analysis using a DNA microarray. Our findings indicated that the gene silencing activities involving miRNAs were enhanced without increasing in their expression levels under heat-stress conditions. Additionally, the gene silencing activity appeared to be independent of the cytoprotective action involving heat shock proteins that are immediately activated in heat-shocked cells and that function as molecular chaperons for restoring heat-denatured proteins to normal proteins. Our current findings suggested the possibility that gene silencing involving endogenous miRNAs might play a subsidiary role in heat-shocked cells for an aggressive inhibition of the expression of heat-denatured proteins.     

Haemolysin produced by Vibrio mimicus activates two Cl- secretory pathways in cultured intestinal-like Caco-2 cells

Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH-induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl- by activating both Ca2+ -dependent and cAMP-dependent Cl- secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl- secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl- secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.     

Requirement of tryptophan hydroxylase during development for maturation of sensorimotor gating

Deficits in sensorimotor gating, a function to focus on the most salient stimulus, could lead to a breakdown of cognitive integrity, and could reflect the "flooding" by sensory overload and cognitive fragmentation seen in schizophrenia. Sensorimotor gating emerges at infancy, and matures during childhood. The mechanisms that underlie its development are largely unclear. Here, we screened the mouse genome, and found that tryptophan hydroxylase (TPH) is implicated in the maturation of sensorimotor gating. TPH, an enzyme involved in the biosynthesis of serotonin, proved to be required only during the weaning period for maturation of sensorimotor gating, but was dispensable for its emergence. Proper serotonin levels during development underlie the mature functional architecture for sensorimotor gating via appropriate actin polymerization. Thus, maintaining proper serotonin levels during childhood may be important for mature sensorimotor gating in adulthood.     

Isolation and characterization of a new Clostridium sp. that performs effective cellulosic waste digestion in a thermophilic methanogenic bioreactor

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55 degrees C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.