The binding of okadaic acid analogs to recombinant OABP2.1 originally isolated from the marine sponge Halichondria okadai

The binding between [24-³H]okadaic acid (OA) and a recombinant OA binding protein OABP2.1 was examined using various OA analog, including methyl okadaate, norokadanone, 7-deoxy OA, and 14,15-dihydro OA, 7-O-palmitoyl DTX1, to investigate the structure activity relationship. Among them, 7-O-palmitoyl DTX1, which is one of the diarrhetic shellfish poisoning (DSP) toxins identified in shellfish, displayed an IC₅₀ for [24-³H]OA binding at 51 ± 6.3 nM (Mean ± SD). In addition, a synthetic compound, N-pyrenylmethyl okadamide, exhibited its IC₅₀ at 10 ± 2.9 nM (Mean ± SD). These results suggested that the recombinant OABP2.1 and the N-pyrenylmethyl okadamide might be core substances in a novel assay for the DSP toxins.     

Structure-based design and synthesis of a bivalent iminobiotin analog showing strong affinity toward a low immunogenic streptavidin mutant

The streptavidin/biotin interaction has been widely used as a useful tool in research fields. For application to a pre-targeting system, we previously developed a streptavidin mutant that binds to an iminobiotin analog while abolishing affinity for natural biocytin. Here, we design a bivalent iminobiotin analog that shows 1000-fold higher affinity than before, and determine its crystal structure complexed with the mutant protein.     

Decrease in Hydraulic Conductivity of a Paddy Field using Biocalcification in situ

The hydraulic conductivity of a paddy field (Anthraquic Dystrustept), a silty clay soil containing more than 29% (w/w) of gravel, in Nagoya University Farm was reduced by in situ treatment of subsurface soil using bentonite and biocalcification (microbial calcium carbonate precipitation) through the addition of CaCl₂, urea, and corn steep liquor (CSL). The treatment decreased the hydraulic conductivity of the field from an average of 10^?3 cm/s to a range of 10^?5 to 10^?7 cm/s during 69 days, with reducing the proportion of pores of subsurface soil larger than 75 µm in diameter. The biocalcification effect was observed at 10-cm thickness from the treated subsurface. Laboratory soil core experiments demonstrated that the decrease in the hydraulic conductivity was not attributed to the effect of bentonite but mainly to the effect of biocalcification. The addition of CSL enhanced the urease activity of soil required for biocalcification, even at 4°C, as indicated by a decrease in urease activation energy temperature sensitivity. These experimental results agreed with the gradual decrease in hydraulic conductivity observed in the field when the average daily temperature was 7°C (days 24–69). It was suggested that the biocalcification is a potential technique to reduce the hydraulic conductivity of paddy field.     

Indirect reciprocity can overcome free-rider problems on costly moral assessment

Indirect reciprocity is one of the major mechanisms of the evolution of cooperation. Because constant monitoring and accurate evaluation in moral assessments tend to be costly, indirect reciprocity can be exploited by cost evaders. A recent study crucially showed that a cooperative state achieved by indirect reciprocators is easily destabilized by cost evaders in the case with no supportive mechanism. Here, we present a simple and widely applicable solution that considers pre-assessment of cost evaders. In the pre-assessment, those who fail to pay for costly assessment systems are assigned a nasty image that leads to them being rejected by discriminators. We demonstrate that considering the pre-assessment can crucially stabilize reciprocal cooperation for a broad range of indirect reciprocity models. In particular for the most leading social norms, we analyse the conditions under which a prosocial state becomes locally stable.     

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.     

Functional involvement of TMF/ARA160 in Rab6-dependent retrograde membrane traffic

The small GTPase Rab6 regulates retrograde membrane traffic from endosomes to the Golgi apparatus and from the Golgi to the endoplasmic reticulum (ER). We examined the role of a Rab6-binding protein, TMF/ARA160 (TATA element modulatory factor/androgen receptor-coactivator of 160 kDa), in this process. High-resolution immunofluorescence imaging revealed that TMF signal surrounded Rab6-positive Golgi structures and immunoelectron microscopy revealed that TMF is concentrated at the budding structures localized at the tips of cisternae. The knockdown of either TMF or Rab6 by RNA interference blocked retrograde transport of endocytosed Shiga toxin from early/recycling endosomes to the trans-Golgi network, causing missorting of the toxin to late endosomes/lysosomes. However, the TMF knockdown caused Rab6-dependent displacement of N-acetylgalactosaminyltransferase-2 (GalNAc-T2), but not beta1,4-galactosyltransferase (GalT), from the Golgi. Analyses using chimeric proteins, in which the cytoplasmic regions of GalNAc-T2 and GalT were exchanged, revealed that the cytoplasmic region of GalNAc-T2 plays a crucial role in its TMF-dependent Golgi retention. These observations suggest critical roles for TMF in two Rab6-dependent retrograde transport processes: one from endosomes to the Golgi and the other from the Golgi to the ER.