Diapause is a developmental transition in insects based on seasonal adaptation to adversity; it is regulated by a circadian clock system and the endocrine system. However, the molecular node and its mechanism underlying the effects of these systems are still unclear. Here, a mutant of Bombyx mori with the circadian clock gene Period (Per) knocked out was constructed, which dramatically changed the classic diapause-destined pathway. Per-knockout silkworms powerfully attenuated, but could not completely block, the predetermined effects of temperature and photoperiod on diapause determination, and this effect depended on the diapause hormone (DH) pathway. The impaired transcription-translation feedback loop of the circadian clock system lacking the Per gene caused direct up-regulation of the expression of GRD, a receptor of γ-aminobutyric acid (GABA), by changing expression level of Cycle. The synthesis of GABA in the tissue complex of brain-suboesophageal ganglion then increased and restricted the decomposition, which continuously promoted the GABAergic signal to play a role, and finally inhibiting (delaying) the release of DH to the hemolymph, and reducing the diapause-inducing effect of DH. The results provided an example to explain the regulatory mechanism of the circadian clock on endocrine hormones in the silkworm.
Vitamin C (VC) is an essential nutrient for many animals. However, whether insects, including Bombyx mori, can synthesize VC remains unclear. In this article, the optimized HPLC method was used to determine the content of l‐ascorbic acid (AsA) in silkworm eggs, larvae and pupae, and the activity of l‐gulono‐1,4‐lactone oxidase (GULO), a key enzyme in VC synthesis. The RNA interference method was used to determine the effect of the BmGulo‐like gene on embryonic development and GULO activity in the pupal fat body. The AsA content increased significantly during E144 h–E168 h in the late embryonic stage and P48 h–P144 h in the middle‐late pupal stage, in which exogenous VC was not ingested. Furthermore, the body AsA content in larvae fed VC‐free feed also increased with larval stage. The GULO enzymatic activity was present in eggs and the fat bodies of larvae and pupae, even when the larvae were reared with fresh mulberry leaves. Moreover, the activity was higher in the later embryonic stages (E144 h–E168 h) and the early pupal stage (before P24 h). The GULO activity in the pupal fat body dramatically decreased when the screened BmGulo‐like gene (BGIBMGA005735) was knocked down with small interfering RNA; in addition, the survival rate and hatching rate of eggs significantly decreased 21% and 44%, respectively, and embryonic development was delayed. Thus, Bombyx mori can synthesize AsA through the l‐gulose pathway, albeit with low activity, and this synthesis ability varies with developmental stages.
OBJECTIVE To investigate the significance and relationship between matrix metalloproteinase 9 (MMP-9) and infiltration of macrophages in the process of invasion and metastasis in oral squamous cell carcinoma (OSCC). METHODS The immunohistochemical SABC method was used to detect the expression of MMP-9 and CD68 (for labeling macrophages) in 42 cases of OSCC and in 10 normal tissues. RESULTS The expression of MMP-9 and macrophage counts in the OSCC cases were significantly higher compared to normal tissues (P<0.05). The expression of MMP-9 and macrophage counts were related to lymphnode metastasis and the TNM stage (P<0.05), showing that there was a positive correlation among these parameters (γ = 0.443, P<0.01). CONCLUSION Both MMP-9 and macrophages may play an important role in the process of invasion and metastasis in OSCC, and this cellular activity may relate to the macrophages which affect the tumor cells and upregulate the expression of MMP-9.
Circadian clock system disorders can lead to uncontrolled cell proliferation, but the molecular mechanism remains unknown. We used a Bombyx mori animal model of single Period gene ( BmPer ) expression to investigate this mechanism. A slow growing developmental cell model (Per-KD) was isolated from a B. mori ovarian cell line (BmN) by continuous knock down of BmPer expression. The effects of BmPer expression knockdown (Per-KD) on cell proliferation and apoptosis were opposite to those of m/hPer1 and m/hPer2 in mammals. The knockdown of BmPer expression led to cell cycle deceleration with shrinking of the BmN cell nucleus, and significant inhibition of nuclear DNA synthesis and cell proliferation. It also promoted autophagy via the lysosomal pathway, and accelerated apoptosis via the caspase pathway.
The circadian clock plays an integral role in hormone biosynthesis and secretion. However, how the circadian clock precisely coordinates hormonal homeostasis to maintain normal animal development remains unclear. Here, we show that knocking out the core clock gene Cryptochrome 1 (Cry1) significantly delays the developmental time in Bombyx mori. This study focuses on the ecdysone and juvenile hormone signalling pathways of fifth instar larvae with the longest developmental time delay. We found that the mutant reduced prothoracicotropic hormone synthesis in the brain, and could not produce sufficient ecdysone in the prothoracic gland, resulting in a delayed peak of 20‐hydroxyecdysone titre in the hemolymph of fifth instar larvae, prolonging developmental time. Moreover, further investigation revealed that the mutant enhanced juvenile hormone biosynthesis and signalling pathway and that this higher juvenile hormone titre also resulted in prolonged developmental time in fifth instar larvae. Our results provide insights into the molecular mechanisms by which the circadian clock regulates animal development by maintaining hormonal homeostasis.
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