Search

It is well known that phosphorylation of extracellular signal-regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signaling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage- and dose-dependent. The absence of Ca2+ reduced PTTH-stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH-stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH-regulated RSK phosphorylation, indicating its redox regulation. A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, decreased PTTH-stimulated RSK phosphorylation, indicating that ERK is an upstream signaling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH-stimulated RSK phosphorylation, and a PKC activator, phorbol 12-myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI-D1870, a specific RSK inhibitor, partly prevented PTTH-stimulated RSK phosphorylation and significantly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH-stimulated ecdysteroidogenesis. This article is protected by copyright. All rights reserved.

Aims This study aimed to investigate the potential of Bacillus methylotrophicus as a probiotic. Methods and Results A Bacillus isolate designated strain C14 was isolated from Korean traditional fermented soybean paste (doenjang). The strain was identified, and its physiological and biochemical properties were characterized. The gastrointestinal tolerance and immunomodulatory function of strain C14 were also investigated. Strain C14 was identified as B. methylotrophicus by analysis of its biochemical properties using the API 50CHB system and by phylogenetic analysis of the 16S rDNA sequence. Strain C14 showed >80% and >75% of survival for artificial gastric juices (pH 2·5 and 1% pepsin) and 0·5% (w/v) bile salt, respectively. Heat-killed B. methylotrophicus C14 inhibited the adhesion of various pathogens and enhanced the adhesion of probiotic bacteria to Caco-2 cells. The heat-killed cells also induced high levels of immune cell proliferation compared with the control and stimulated interleukin-6 and tumour necrosis factor-α production in mouse macrophages. Conclusions Bacillus methylotrophicus C14 could be used as a probiotic. Significance and Impact of the Study Recently identified B. methylotrophicus is a new potential probiotic with high gastrointestinal tolerance.

Odorant-binding proteins (OBPs) are crucial in the olfactory pathway of insects. In the present study, the antenna-enriched OBP AlinOBP13 was investigated because of its potential contribution to the peripheral olfactory perception in the alfalfa plant bug Adelphocoris lineolatus. The results of quantitative reverse transcriptase-PCR showed that the transcript level of AlinOBP13 was higher in the adult stage than in the nymph stages. The transcript levels of AlinOBP13 in the male and female antennae significantly increased after 4 and 8 h of starvation, respectively. Fine ultrastructures of different types of chemosensilla in both female and male antennae were investigated using transmission electron microscopy and immunocytochemical labelling. The results revealed that the anti-AlinOBP13 antiserum strongly and specifically labelled short basiconic sensilla; this antiserum was restricted to the inner lumen and the cavities below the sensillum base of the sensilla. By contrast, multiporous sensilla trichodea, medium long sensilla basiconica, and aporous sensilla chaetica were not labelled. The present study is the first to report an OBP showing specific expression in the short basiconic sensilla of a member of the Hemipteran species. The results of a fluorescence displacement binding assay indicated that recombinant AlinOBP13 showed a more specific binding preference to terpenoids than to sex pheromones and other classes of chemicals. This binding ability was dramatically affected by pH; higher binding affinities were displayed at pH 10.0 than at pH 7.4 and 5.0. In addition, the results of dose-dependent electroantennogram recordings from the antennae showed that both female and male adult bugs responded to the terpenoids tested, suggesting an apparent physiological relevance of AlinOBP13 in A. lineolatus chemoreception. The results of this study suggest that AlinOBP13 functions as a specific carrier of terpenoids and provide insights into the mechanism of A. lineolatus in response to green volatiles.

Olfaction plays an important role in insects' survival and reproduction. Odorant-binding proteins (OBPs) are considered to be one of the crucial proteins in the insect olfactory pathway. In this study, an antenna-specific OBP of the alfalfa plant bug, Adelphocoris lineolatus AlinOBP5, was expressed and purified in vitro. The binding affinities of AlinOBP5 with sex pheromone analogues of the Miridae and cotton volatiles were investigated by fluorescence competitive binding assays. The binding sites of AlinOBP5 were predicted by three-dimensional structure modelling and molecular docking, and site-directed mutagenesis. AlinOBP5 could not effectively bind with sex pheromone analogues of Miridae but showed high binding abilities with specific cotton volatiles, such as cis-nerolidol, ethyl laurate, β-ionone, β-caryophyllene, 2,3-dimethylbenzoic acid and (E)-farnesol. The strongest binding affinity was to cis-nerolidol, suggesting a role of AlinOBP5 in general odorant chemoreception. Based on the relatively strong binding affinity and the reported physiological activity of cis-nerolidol in other insects, we chose cis-nerolidol for further homology modelling and ligand docking. The results of molecular simulation and site-directed mutagenesis indicated that two amino acids, Lys74 and Pro121, in the protein binding pocket are the key amino acids involved in the binding of cis-nerolidol. The Lys74 residue may participate in specific recognition of ligands, and the Pro121 residue plays a crucial role in ligand binding and release by changing the binding pocket environment and stabilizing the conformation of the C-terminus of AlinOBP5.

The purpose of this study was to examine the mitogenic effects of 20-hydroxyecdysone on neurogenesis in mushroom bodies of the adult cockroach, Diploptera punctata. The occurrence of neurogenesis was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU). The number of BrdU-labeled cells in the mushroom bodies was high shortly after adult ecdysis, then gradually decreased, and proliferation ceased on day 8. 20-Hydroxyecdysone injection during the early adult stages significantly delayed the decrease in mitotic activity. Moreover, 20-hydroxyecdysone injection during the late stage stimulated quiescent mushroom body neuroblasts to initiate their mitotic activity in a dose-dependent manner. These results indicated that the mushroom body neuroblasts of this insect become quiescent in the maturing central nervous system, but retain the capacity for proliferation if exposed to appropriate environmental signals. We conclude that 20-hydroxyecdysone has a mitogenic effect on neurogenesis in mushroom bodies of this insect.