Small-molecular inhibitors of Ca2+-induced mitochondrial permeability transition (MPT) derived from muscle relaxant dantrolene

A structure consisting of substituted hydantoin linked to a 5-(halophenyl)furan-2-yl group via an amide bond was identified as a promising scaffold for development of low-molecular-weight therapeutic agents to treat vascular dysfunction, including ischemia/reperfusion injury. Among the compounds synthesized, 5-(3,5-dichlorophenyl)-N-{2,4-dioxo-3-[(pyridin-3-yl)methyl]imidazolidin-1-yl}-2-furamide (17) possessed the most potent inhibitory activity against Ca²⁺-induced mitochondrial swelling. The structural development, synthesis and structure–activity relationship of these compounds are described.     

CRTH2 plays an essential role in the pathophysiology of Cry j 1-induced pollinosis in mice

PGD(2) is the major prostanoid produced during the acute phase of allergic reactions. Two PGD(2) receptors have been isolated, DP and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), but whether they participate in the pathophysiology of allergic diseases remains unclear. We investigated the role of CRTH2 in the initiation of allergic rhinitis in mice. First, we developed a novel murine model of pollinosis, a type of seasonal allergic rhinitis. Additionally, pathophysiological differences in the pollinosis were compared between wild-type and CRTH2 gene-deficient mice. An effect of treatment with ramatroban, a CRTH2/T-prostanoid receptor dual antagonist, was also determined. Repeated intranasal sensitization with Cry j 1, the major allergen of Cryptomeria japonica pollen, in the absence of adjuvants significantly exacerbated nasal hyperresponsive symptoms, Cry j 1-specific IgE and IgG1 production, nasal eosinophilia, and Cry j 1-induced in vitro production of IL-4 and IL-5 by submandibular lymph node cells. Additionally, CRTH2 mRNA in nasal mucosa was significantly elevated in Cry j 1-sensitized mice. Following repeated intranasal sensitization with Cry j 1, CRTH2 gene-deficient mice had significantly weaker Cry j 1-specific IgE/IgG1 production, nasal eosinophilia, and IL-4 production by submandibular lymph node cells than did wild-type mice. Similar results were found in mice treated with ramatroban. These results suggest that the PGD(2)-CRTH2 interaction is elevated following sensitization and plays a proinflammatory role in the pathophysiology of allergic rhinitis, especially pollinosis in mice.     

WGEF activates Rho in the Wnt-PCP pathway and controls convergent extension in Xenopus gastrulation

The Wnt-PCP (planar cell polarity, PCP) pathway regulates cell polarity and convergent extension movements during axis formation in vertebrates by activation of Rho and Rac, leading to the re-organization of the actin cytoskeleton. Rho and Rac activation require guanine nucleotide-exchange factors (GEFs), but the identity of the GEF involved in Wnt-PCP-mediated convergent extension is unknown. Here we report the identification of the weak-similarity GEF (WGEF) gene by a microarray-based screen for notochord enriched genes, and show that WGEF is involved in Wnt-regulated convergent extension. Overexpression of WGEF activated RhoA and rescued the suppression of convergent extension by dominant-negative Wnt-11, whereas depletion of WGEF led to suppression of convergent extension that could be rescued by RhoA or Rho-associated kinase activation. WGEF protein preferentially localized at the plasma membrane, and Frizzled-7 induced colocalization of Dishevelled and WGEF. WGEF protein can bind to Dishevelled and Daam-1, and deletion of the Dishevelled-binding domain generates a hyperactive from of WGEF. These results indicate that WGEF is a component of the Wnt-PCP pathway that connects Dishevelled to Rho activation.     

Hypoxia induces expression of a GPI-anchorless splice variant of the prion protein

The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI-anchorless PrP (GPI(-) PrPSV), was unglycosylated and soluble in non-ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non-neuronal tissues. When long-term passaged T98G cells were placed in a low-oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI(-) PrPSV expression in T98G cells.     

Sequence evidence in the archaeal genomes that tRNAs emerged through the combination of ancestral genes as 5' and 3' tRNA halves

The discovery of separate 5' and 3' halves of transfer RNA (tRNA) molecules-so-called split tRNA-in the archaeal parasite Nanoarchaeum equitans made us wonder whether ancestral tRNA was encoded on 1 or 2 genes. We performed a comprehensive phylogenetic analysis of tRNAs in 45 archaeal species to explore the relationship between the three types of tRNAs (nonintronic, intronic and split). We classified 1953 mature tRNA sequences into 22 clusters. All split tRNAs have shown phylogenetic relationships with other tRNAs possessing the same anticodon. We also mimicked split tRNA by artificially separating the tRNA sequences of 7 primitive archaeal species at the anticodon and analyzed the sequence similarity and diversity of the 5' and 3' tRNA halves. Network analysis revealed specific characteristics of and topological differences between the 5' and 3' tRNA halves: the 5' half sequences were categorized into 6 distinct groups with a sequence similarity of >80%, while the 3' half sequences were categorized into 9 groups with a higher sequence similarity of >88%, suggesting different evolutionary backgrounds of the 2 halves. Furthermore, the combinations of 5' and 3' halves corresponded with the variation of amino acids in the codon table. We found not only universally conserved combinations of 5'-3' tRNA halves in tRNA(iMet), tRNA(Thr), tRNA(Ile), tRNA(Gly), tRNA(Gln), tRNA(Glu), tRNA(Asp), tRNA(Lys), tRNA(Arg) and tRNA(Leu) but also phylum-specific combinations in tRNA(Pro), tRNA(Ala), and tRNA(Trp). Our results support the idea that tRNA emerged through the combination of separate genes and explain the sequence diversity that arose during archaeal tRNA evolution.     

Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.     

Glycoform analysis of Japanese cypress pollen allergen, Cha o 1: a comparison of the glycoforms of cedar and cypress pollen allergens

A Japanese cypress (Chamaecyparis obtusa) pollen allergen, Cha o 1, is one of the major allergens that cause allergic pollinosis in Japan. Although it has been found that Cha o 1 is glycosylated and that the amino acid sequence is highly homologous with that of Japanese cedar pollen allergen (Cry j 1), the structure of N-glycans linked to Cha o 1 remains to be determined. In this study, therefore, we analyzed the structures of the N-glycans of Cha o1. The N-glycans were liberated by hydrazinolysis from purified Cha o 1, and the resulting sugar chains were N-acetylated and pyridylaminated. The structures of pyridylaminated N-glycans were analyzed by a combination of exoglycosidase digestion, two dimensional (2D-) sugar chain mapping, and electrospray ionization mass spectrometry analysis. Structural analysis indicated that the major N-glycan structure of Cha o1 is GlcNAc2Man3Xyl1Fuc1GlcNAc2 (89%), and that high-mannose type structures (Man9GlcNAc2, Man7GlcNAc2) occur as minor components (11%).     

Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB

Elastin is a crosslinked hydrophobic protein found in abundance in vertebrate tissue and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this study, the central residues of the (LGGVG)(6) elastin mimetic are targeted. Using a combination of a statistical analysis based on structures in the Brookhaven Protein Data Bank (PDB), 1D cross-polarization magic-angle-spinning (CPMAS) NMR spectroscopy, and 2D off-magic-angle-spinning (OMAS) spin-diffusion experiments, it is determined that none of the residues are found in a singular regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, there are multiple conformations and significant disorder. Furthermore, the conformational ensembles are not reflective of proteins generally, as in the PDB, suggesting that the structure distributions in elastin mimetics are unique to these peptides and are a salient feature of the functional model of the native protein.     

Preparation of human immune effector T cells containing iron-oxide nanoparticles

Preparation of human immune T cells containing iron-oxide nanoparticles was carried out for the development of magnetically mediated immunotherapy. Peripheral blood lymphocytes (PBLs) after the incubation with magnetite nanoparticles were found to contain measurable ferric ions, which suggested the incorporation of magnetite nanoparticles. Transmission electron microscopic (TEM) study indicated that the incorporation of magnetite nanoparticles was mediated by endocytosis of PBLs. Furthermore, the effects of dosages and diameter of magnetite nanoparticles on the magnetite incorporation were investigated, and it was demonstrated that the increase in dosage promoted the incorporation of nanoparticles and the uptake into PBLs was more effective for magnetite nanoparticles, which formed smaller aggregations in medium. Finally, the demonstration of magnetite incorporation into enriched T cells and tumor antigen-specific cytotoxic T lymphocyte (CTL) line promises the achievement of magnetically mediated immunotherapy with tumor-specific CTLs containing magnetic nanoparticles.