Agonist-induced internalization plays a key role in the tight regulation of the extent and duration of G protein-coupled receptor signaling. Previously, we have shown that the Bombyx corazonin receptor (BmCrzR) activates both Gαq- and Gαs-dependent signaling cascades. However, the molecular mechanisms involved in the regulation of the internalization and desensitization of BmCrzR remain to be elucidated. Here, vectors for expressing BmCrzR fused with enhanced green fluorescent protein (EGFP) at the C-terminal end were used to further characterize BmCrzR internalization. We found that the BmCrzR heterologously expressed in HEK-293 and BmN cells was rapidly internalized from the plasma membrane into the cytoplasm in a concentration- and time-dependent manner via a β-arrestin (Kurtz)-dependent and clathrin-independent pathway in response to agonist challenge. While most of the internalized receptors were recycled to the cell surface via early endosomes, some others were transported to lysosomes for degradation. Assays using RNA interference revealed that both GRK2 and GRK5 were essentially involved in the regulation of BmCrzR phosphorylation and internalization. Further investigations indicated that the identified cluster of Ser/Thr residues (411TSS413) was responsible for GRK-mediated phosphorylation and internalization. This is the first detailed investigation of the internalization and trafficking of Bombyx corazonin receptors.

•A novel oligosaccharide BBPW-2, was isolated and purified from Bombyx batryticatus.•The chemical structure characterization of BBPW-2 was elucidated.•The in vitro inhibitory activity was measured by MTT assay and crystal violet assay.•The growth inhibition mechanism was explored through apoptosis and cell-cycle analysis.An oligosaccharide BBPW-2 was isolated and purified from Bombyx batryticatus, its molecular weight was 2.0 × 103 Da, and its structure was elucidated by compositional, methylation and NMR analysis. Our results showed that BBPW-2 consisted of β-d-(1 → 2,6)-glucopyranose and β-d-(1 → 2,6)-mannosyl units serving as the backbone, α-d-(1 → 2)-galactopyranose and α-d-(1 → 3)-mannosyl units as branches, and α-d-Manp and β-d-Glcp as terminals. The in vitro inhibitory activity of BBPW-2 was measured using MTT and and crystal violet assays, which suggested that BBPW-2 had direct cytotoxic effects on the cancer cell lines HeLa and HepG2 (particularly HeLa cells), and had a long-term antiproliferative effect on MCF-7 cells, respectively. Apoptosis and cellcycle analysis of HeLa cells showed that BBPW-2 induced cellcycle disruption in the G0/G1 and G2/M phases accompanied by an impressive increment of early apoptotic cells and late apoptotic and necrotic cells. These results suggest that BBPW-2 could be a potential chemotherapeutic drug and its antitumor effects deserve further study.

Recent work has demonstrated that hydrogen peroxide functions as a signaling molecule controlling different essential processes in plants and mammals, which can be produced by superoxide dismutase (SOD) and xanthine oxidase (XO) and decomposed by catalase (CAT), respectively. Progeny diapause of the silkworm, Bombyx mori, is induced by diapause hormone (DH) and the expression of DH gene in the maternal generation has been determined. In order to investigate the relationship between the metabolism of H2O2 and the expression of DH gene, level of H2O2 and activities of SOD, XO and CAT between univoltine and polyvoltine strains, which can produce diapause and non-diapause eggs, respectively, at embryonic and pupal stages were measured. Our results showed that there were significant differences in the metabolism of hydrogen peroxide between two strains and between embryonic and pupal stages. Compared to polyvoltine strain, level of hydrogen peroxide in univoltine strain was significantly higher from stage 19 to stage 21 but lower from stage 24 to stage 29 and the whole pupal stage (Fig. 1). Variations of hydrogen peroxide indicated that hydrogen peroxide may be involved in the active release of DH and the progeny diapause decision by DH rather than the expression of DH gene.