Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (T-m) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthostericbinding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested Lamino acids, but no D-amino acid, induced T-m shifts of T1r2a/T1r3LBD, indicating the broad L-amino acid specificities of the proteins probably with several different manners of recognition. The T-m shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF

Functional characterisation of a nicotinic acetylcholine receptor alpha subunit from the brown dog tick, Rhipicephalus sanguineus

Open Access funded by Biotechnology and Biological Sciences Research Council Under a Creative Commons license This work was supported by a Biotechnology and Biological Sciences Research Council (UK) Industrial CASE studentship award (BBSSM200411428) to K.L. with co-funding from Pfizer Animal Health, UK. We thank Dr. Andy Ball for help with rearing of ticks.Peer reviewedPublisher PD

The Epistatic Relationship between BRCA2 and the Other RAD51 Mediators in Homologous Recombination

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA–damage site

Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR–neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH–π interactions in the Ls-AChBP–CTD complex than in the Ls-AChBP–IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs

オリゴペプチドの疎水性と構造との相関関係に対する定量的解析

京都大学0048新制・論文博士農学博士乙第7322号論農博第1606号新制||農||580(附属図書館)学位論文||H2||N2235(農学部図書室)UT51-90-V142(主査)教授 藤田 稔夫, 教授 駒野 徹, 教授 松野 隆一学位規則第5条第2項該当Kyoto UniversityDFA

Automatic detection of fish sounds based on multi-stage classification including logistic regression via adaptive feature weighting

This paper presents a method for automatic detection of fish sounds in an underwater environment. There exist two difficulties: (i) features and classifiers that provide good detection results differ depending on the underwater environment and (ii) there are cases where a large amount of training data that is necessary for supervised machine learning cannot be prepared. A method presented in this paper (the proposed hybrid method) overcomes these difficulties as follows. First, novel logistic regression (NLR) is derived via adaptive feature weighting by focusing on the accuracy of classification results by multiple classifiers, support vector machine (SVM), and k-nearest neighbors (kNN). Although there are cases where SVM or k-NN cannot work well due to divergence of useful features, NLR can produce complementary results. Second, the proposed hybrid method performs multi-stage classification with consideration of the accuracy of SVM, k-NN, and NLR. The multistage acquisition of reliable results works adaptively according to the underwater environment to reduce performance degradation due to diversity of useful classifiers even if abundant training data cannot be prepared. Experiments on underwater recordings including sounds of Sciaenidae such as silver croakers (Pennahia argentata) and blue drums (Nibea mitsukurii) show the effectiveness of the proposed hybrid method

Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-π bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-α nAChR subunits