Iodination of Ricin D

Approximately five tyrosine residues of ricin D were iodinated preferentially under appropriate conditions probably forming diiodotyrosine. Iodination of this toxin carried out in 0.1 m phosphate buffer at pH 7.0 and 0°C for 60 min with a 20 fold molar excess of iodine per mole of protein, yielded a main component which appeared as a single band on polyacrylamide gel disc electrophoresis. Analysis of protein-bound radioactivity and the content of diiodotyrosine of ¹⁸¹I-labeled ricin D revealed that two tyrosine residues in the isoleucyl chain and three in the alanyl chain were substituted. The toxicity of iodinated ricin D decreased to one hundredth of that of native protein, However, the hemagglutinating activity of this protein was not affected by the iodination reaction.     

Anti-prion activity of an RNA aptamer and its structural basis

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP^C) of PrP into an abnormal form (PrP^Sc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP^C is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP^Sc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP^C, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.     

Molecular Characterization of a Novel Armadillo Repeat-Like Protein Gene Differentially Induced by High-Salt Stress and Dehydration from the Model Legume Lotus Japonicus

Armadillo (ARM) repeat proteins have tandem repeats of a degenerate sequence motif required for protein–protein interaction and are distributed widely in eukaryotes. In this study, we isolated and characterized a novel ARM repeat-like protein gene from the model legume Lotus japonicus that is differentially induced by abiotic stress. The gene, LjTDF-5, encodes a hypothetical protein of 369 aa with a protein signature "ARM-type fold". Three-dimensional protein structure was predicted to consist of 23 α-helices and no β-sheets by homology modeling. The LjTDF-5-homologous genes were distributed broadly in the plant kingdom and the C-terminal region, around 60 amino acids in length, was highly conserved in all of the homologs examined although any known functional domains or protein signatures in this region were not detected in silico analyses. Subcellular localization assays revealed that the sGFP-fused LjTDF-5 protein localized to the nuclei of onion epidermis cells, despite the protein not containing a typical nuclear localization signal. In quantitative real-time RT-PCR, the expression of LjTDF-5 was highly induced by 100 mM NaCl in the roots and by dehydration in the shoot, but not by abscisic acid (ABA, 10 μM). These results suggest that the ARM repeat-like protein LjTDF-5 functions in or around the nucleus in response to high-salt stress and dehydration in L. japonicus.     

Semisynthesis and biological evaluation of a cotylenin A mimic derived from fusicoccin A

In an effort to overcome the unavailability of cotylenin A (CN A), an anticancer agent and a stabilizer of protein–protein interactions (PPIs) mediated by 14-3-3 proteins, ISIR-050 was designed as a CN A mimic. The synthesis was accomplished via a semisynthetic approach starting from fusicoccin A. ISIR-050 showed interferon-α (IFNα)-dependent growth inhibitory activity and a PPI stabilization effect similar to those of CN A. The biochemical analysis suggested that ISIR-050 and CN A induce the same pharmacological response to IFNα-treated cancer cells and that 14-3-3 proteins play a role in the mode of action.     

Design, synthesis and biological activity of novel non-peptidyl endothelin converting enzyme inhibitors, 1-phenyl-tetrazole-formazan analogues

A novel non-peptidyl endothelin converting enzyme inhibitor was obtained through a pharmacophore analysis of known inhibitors and three-dimensional structure database search. Analogues of the new inhibitor were designed using the structure-activity relationship of known inhibitors and synthesized. In anesthetized rats, intraperitoneal administration of the analogues suppressed the pressor responses induced by big endothelin-1.     

Secreted Frizzled-related Protein 5 Diminishes Cardiac Inflammation and Protects the Heart from Ischemia/Reperfusion Injury

Wnt signaling has diverse actions in cardiovascular development and disease processes. Secreted frizzled-related protein 5 (Sfrp5) has been shown to function as an extracellular inhibitor of non-canonical Wnt signaling that is expressed at relatively high levels in white adipose tissue. The aim of this study was to investigate the role of Sfrp5 in the heart under ischemic stress. Sfrp5 KO and WT mice were subjected to ischemia/reperfusion (I/R). Although Sfrp5-KO mice exhibited no detectable phenotype when compared with WT control at baseline, they displayed larger infarct sizes, enhanced cardiac myocyte apoptosis, and diminished cardiac function following I/R. The ischemic lesions of Sfrp5-KO mice had greater infiltration of Wnt5a-positive macrophages and greater inflammatory cytokine and chemokine gene expression when compared with WT mice. In bone marrow-derived macrophages, Wnt5a promoted JNK activation and increased inflammatory gene expression, whereas treatment with Sfrp5 blocked these effects. These results indicate that Sfrp5 functions to antagonize inflammatory responses after I/R in the heart, possibly through a mechanism involving non-canonical Wnt5a/JNK signaling.     

Ghrelin Augments the Expressions and Secretions of Proinflammatory Adipokines,VEGF120 and MCP‐1, in Differentiated 3T3‐L1 Adipocytes

Ghrelin is a physiological‐active peptide with growth hormone‐releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin‐direct‐effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3‐L1 adipocytes stimulated with ghrelin in vitro . The stimulation with 50 nmol/L ghrelin for 24 h resulted in the significant 1.9‐fold increase on vascular endothelial growth factor‐120 (VEGF₁₂₀) releases (p < 0.01) and the 1.7‐fold on monocyte chemoattractant protein‐1 (MCP‐1) (p < 0.01) from 3T3‐L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, IL‐10 and adiponectin secretions. In addition, Akt phosphorylation on Ser473 and c‐Jun NH₂‐terminal protein kinase (JNK) phosphorylation on Thr183/Tyr185 were markedly enhanced 1.4‐fold (p < 0.01) and 1.6‐fold (p < 0.01) in the ghrelin‐stimulated adipocytes, respectively. Furthermore, the treatment with LY294002 (50 μmol/L) and Wortmannin (10nmol/L), inhibitors of phosphatidylinositol 3‐kinase (PI3K), significantly decreased the amplified VEGF₁₂₀ secretion by 29% (p < 0.01) and 28% (p < 0.01) relative to the cells stimulated by ghrelin alone, respectively, whereas these inhibitors had no effects on increased MCP‐1 release. On the other hand, JNK inhibitor SP600125 (10 μmol/L) clearly reduced the increased MCP‐1, but not VEGF₁₂₀, release by 35% relative to the only ghrelin‐stimulated cells (p < 0.01). In conclusion, ghrelin can enhance the secretions of proinflammatory adipokines, VEGF₁₂₀ and MCP‐1, but fails to affect IL‐10 and adiponectin which are considered to be anti‐inflammatory adipokines. Moreover, this augmented VEGF₁₂₀ release is invited through the activation of PI3K pathways and the MCP‐1 is through JNK pathways. Consequently, our results strongly suggest that ghrelin can induce the development of diabetes via its direct‐action in peripheral tissues as well as via in CNS. J. Cell. Physiol. 230: 199–209, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.