Diversity and composition of flower-visiting insects and related factors in three fruit tree species.

Animal-mediated pollination is an essential ecosystem service for the production of many fruit trees. To reveal the community composition of flower-visiting wild insects which potentially contribute to fruit production and to examine the effects of geographic location, local meteorological conditions and locally introduced domesticated pollinators on them, we investigated the community composition of insects visiting the flowers (hereafter, "visitors") of apple, Japanese pear and Oriental persimmon for 1‒3 years at 20 sites around Japan. While most of the variation (82%) of the community composition was explained by tree species with a slight contribution by geographic distance (2%), maximum temperature and tree species contributed 62% and 41% of the variation in total abundance of the visitors, respectively. Though the dominant families of the visitors varied spatiotemporally, the community composition of the visitors of apple and Japanese pear clearly differed from that of Oriental persimmon. While Andrenidae and Syrphidae together accounted for 46%‒64% of the visitors of apple and Japanese pear, Apidae represented 57% of the visitors of Oriental persimmon. The taxonomic richness, diversity and evenness of the visitors were best predicted by locally introduced domesticated pollinators and local meteorological conditions of wind speed and maximum temperature. Amongst these selected factors, locally introduced domesticated pollinators could have the largest impact. It seemed to be strongly related to the reduction of taxonomic richness, diversity and evenness of the visitors, accounting for 41‒89% of the variation. Results suggested that the community composition and total abundance of potential pollinators were predominantly determined by tree species and temperature, but locally introduced domesticated pollinators could have a determinantal pressure on the taxonomic diversity of the community.

A quantitative affinity-based technique for the identification of potential lead compounds.

Here, we describe the development and validation of a quantitative analytical method for rapid evaluation of protein-compound interactions. The method uses size-exclusion chromatography in a 96-well format with liquid chromatography/mass spectrometry (qSEC-LC/MS) by which the amount of a compound that was originally in complex with a target protein is determined as an indicator of the binding affinity. Proof of concept of this new analytical approach was performed using a thrombin-inhibitor model. The results showed that the qSEC-LC/MS could be developed into an effective affinity-based analytical technique, despite a few limitations such as difficulty in determining the K(d) value accurately.

A quantitative analysis to unveil specific binding proteins for bioactive compounds.

The mechanisms of the action of bioactive compounds discovered via 'black box' assays using phenotypic indicators generally remain unknown, with the major challenges being the identification of target proteins. In this study, we aimed to develop an efficient methodology to unveil target proteins that are rarely characterised. Proof-of-concept experiments were performed using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a well-known calmodulin (CaM) antagonist, as a bait compound. The results showed that in our methodology CaM was identified as a W-7-specific binding protein in the cytosolic fraction of a rat brain extract, whereas other proteins acquired in the same experiment were recognised as non-specific binding proteins. The binding affinity of CaM to W-7 is not very high (dissociation constant: 1.6 × 10(-5) M), showing that the recognition specificity is applicable to compounds with very low binding affinities.

Rapid Detection of Infestation of Apple Fruits by the Peach Fruit Moth, Carposina sasakii Matsumura, Larvae Using a 0.2-T Dedicated Magnetic Resonance Imaging Apparatus.

Infestation of harvested apple fruits by the peach fruit moth (Carposina sasakii Matsumura) was studied using a dedicated magnetic resonance imaging (MRI) apparatus equipped with a 0.2-T permanent magnet. Infested holes on the three-dimensional (3-D) images tracked ecological movements of peach fruit moth larvae within the food fruits, and thus in their natural habitat. Sensitive short solenoid coil and surface coil detectors were devised to shorten measurement times. The short solenoid coil detected infestation holes at a rate of 6.4 s per image by the single-slice 2-D measurement. The multi-slice 2-D measurement provided six slice images of a fruit within 2 min taken by the two detectors. These results indicate that the 0.2-T MRI apparatus allows one to distinguish sound fruits from infested ones, and also as a means for plant protection and the preservation of natural ecological systems in foreign trade.

A fluorescence polarization-based assay for the identification and evaluation of calmodulin antagonists.

A fluorescence polarization (FP) assay was developed to identify calmodulin (CaM) antagonists. A fluorescent tracer was newly designed by covalently labeling N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which is a well-known CaM antagonist, with the Cy5 dye. In the FP assay, the tracer (Cy5-W-7) was bound to CaM with a dissociation constant (K(d)) of 6.5 microM and demonstrated efficient competitive activity with other CaM antagonists, including W-7, chlorpromazine, trifluoperazine, W-5, and clozapine, indicating that Cy5-W-7 binds to the ligand-binding site of CaM in a specific manner. The inhibitory activities of Cy5-W-7 and CaM antagonists were subsequently measured by the CaM-dependent calcineurin phosphatase assay, and the results were confirmed with those of the FP assays. In addition, assay optimization for high-throughput screening was performed, and a Z' factor of 0.7 was achieved in a 1536-well format. The FP assay was found to be a simple and reliable alternative to conventional assays for evaluating CaM antagonists.

The design of a new potent and selective ligand for the orphan bombesin receptor subtype 3 (BRS3).

Extensive SAR studies on the unselective BRS3 agonist, [H-D-Phe6,beta-Ala11,Phe13,Nle14]-bombesin-(6-14)-nonapeptide amide, have highlighted structural features important for BRS3 activity and have provided guidance as to the design of selective agonists. A radically modified heptapeptide agonist, maintaining only the Trp-Ala moiety of the parent [H-D-Phe6,betaAla11,Phe13,Nle14]-peptide amide, and with a very different carboxyl terminal region, has been produced which was potent at BRS3 and essentially had no NMB or GRP receptor activity. Its structure is Ac-Phe-Trp-Ala-His(tauBzl)-Nip-Gly-Arg-NH2.

Identification of a sex pheromone component of Pseudococcus cryptus.

A sex pheromone component of Pseudococcus cryptus has been isolated and identified. The crude pheromone extract obtained by airborne collection was fractionated by liquid chromatography (LC) on Florisil, and further purified by high performance liquid chromatography and preparative Gas Chromatography (GC). The pheromone component was shown to be an ester, the alcohol part of which was identical to the known alcohol moiety of the pheromone of Planococcus citri. The chemical structure was determined to be 3-isopropenyl-2,2-dimethylcyclobutylmethyl 3-methyl-3-butenoate by MS and 1H NMR analyses. The absolute configuration of the pheromone was assigned as (1R,3R) by comparison of the retention time of the alcohol derived from the P. cryptus pheromone with those of the alcohol derived from P. citri pheromone, and a synthetic sample of alcohol enriched in the (1R,3R)-enantiomer, using a chiral GC stationary phase. The structure of the pheromone was confirmed by synthesis, and by bioassays in a glasshouse.

Synthesis of the sex pheromone of the citrus mealybug, Pseudococcus cryptus.

The sex pheromone of the citrus mealybug (Pseudococcus cryptus), [(1R,3R)-3-isopropenyl-2,2-dimethylcyclobutyl]methyl 3-methyl-3-butenoate, was synthesized from (+)-alpha-pinene in five operational steps in a 43% overall yield. The synthetic pheromone was identical with the natural pheromone in (1)H-NMR and mass spectroscopic properties, and showed almost the same pheromonal activity as the natural pheromone.