Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine

Hangzhou, China

Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine

Hangzhou, China
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Chen Y.-N.,Zhejiang Sci-Tech University | Chen Y.-N.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Gu X.,Van Andel Research Institute | Zhou X.E.,Van Andel Research Institute | And 12 more authors.
Protein Science | Year: 2017

TBC1D15 belongs to the TBC (Tre-2/Bub2/Cdc16) domain family and functions as a GTPase-activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark-TBC1D15 and Sus-TBC1D15 belong to the same subfamily of TBC domain-containing proteins, and their GAP-domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost. © 2017 The Protein Society


Si H.,Zhejiang Sci-Tech University | Si H.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Cao Y.,Zhejiang Sci-Tech University | Cao Y.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 10 more authors.
BMC Genomics | Year: 2017

Background: A transposable element (TE) is a DNA fragment that can change its position within a genome. Transposable elements play important roles in maintaining the stability and diversity of organisms by transposition. Recent studies have shown that approximately half of the genes in Bombyx mori are TEs. Results: We systematically identified and analyzed the BmAGO2-associated TEs, which exceed 100 in the B. mori genome. Additionally, we also mapped the small RNAs associated with BmAGO2 in B.mori. The transposon Bm1645 is the most abundant TE associated with BmAGO2, and Bm1645-derived small RNAs represent a small RNA pool. We determined the expression patterns of several Bm1645-derived small RNAs by northern blotting, and the results showed there was differential expression of multiple small RNAs in normal and BmNPV-infected BmN cells and silkworms from various developmental stages. We confirmed that four TE-siRNAs could bind to BmAGO2 using EMSA and also validated the recognition sites of these four TE-siRNAs in Bm1645 by dual-luciferase reporter assays. Furthermore, qRT-PCR analysis revealed the overexpression of the four TE-siRNAs could downregulate the expression of Bm1645 in BmN cells, and the transcription of Bm1645 was upregulated by the downregulation of BmAGO2. Conclusions: Our results suggest Bm1645 functions as a source of small RNAs pool and this pool can produce many BmAGO2-associated small RNAs that regulate TE's expression. © 2017 The Author(s).


Lv Z.,Zhejiang Sci-Tech University | Lv Z.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Wang T.,Zhejiang Sci-Tech University | Wang T.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 20 more authors.
International Journal of Genomics | Year: 2013

The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412 bp. The open reading frame contains 603 bp, which encodes 200 amino acid residues. This recombinant BmBras2 protein was subsequently used as an antigen to raise a rabbit polyclonal antibody. Western blotting and real-time PCR analyses showed that BmBras2 was expressed during four developmental stages. The BmBras2 expression level was the highest in the pupae and was low in other life cycle stages. BmBras2 was expressed in all eight tested tissues, and it was highly expressed in the head, intestine, and epidermis. Subcellular localization studies indicated that BmBras2 was predominantly localized in the nuclei of Bm5 cells, although cytoplasmic staining was also observed to a lesser extent. A cell proliferation assay showed that rBmBras2 could stimulate the proliferation of hepatoma cells. The higher BmBras2 expression levels in the pupal stage, tissue expression patterns, and a cell proliferation assay indicated that BmBras2 promotes cell division and proliferation, most likely by influencing cell signal transduction. © 2013 Zhengbing Lv et al.


Lv Z.,Zhejiang Sci-Tech University | Lv Z.,Zhejiang provincial key laboratory of silkworm bioreactor and biomedicine | Zhang X.,Zhejiang Sci-Tech University | Zhang X.,Zhejiang provincial key laboratory of silkworm bioreactor and biomedicine | And 22 more authors.
Gene | Year: 2012

Background: Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. However, its molecular roles are largely unknown. Methods: To better understand the function of prohibitin protein in silkworm (BmPHB), its coding sequence was isolated from a cDNA library of silkworm pupae. An His-tagged BmPHB fusion protein was expressed in . Escherichia coli Rosetta (DE3) and purified with affinity and reversed-phase chromatography. Purified rBmPHB was used to generate anti-BmPHB polyclonal antibody. The subcellular localization of BmPHB was analysed by immunohistochemistry. Results: BmPHB gene has an ORF of 825. bp, encoding a predicted peptide with 274 amino acid residues. Immunostaining indicate that prohibitin is expressed in nucleus and predominately in cytoplasm. Western blot analyses indicated that, in the fifth instar larva, BmPHB was expressed descendingly in gonad, malpighian tubule, trachea, fatty body, intestine, and head. However, no expression was detected in larva's silk gland and epidermis. In addition, BmPHB was expressed in the nascent egg, larva and pupa, but not in the moth. Conclusions: The expression of . BmPHB gene presents differential characteristic in different stage and tissues. It may play important roles in the development of silkworm. General significance: Studies on . prohibitin have been still restricted to a few specific insects and insect cell lines such as . Drosophila, . Acyrthosiphon pisum and mosquito cell lines, not yet in silkworm. This is a first characterization of prohibitin in silkworm, . B. mori. © 2012 Elsevier B.V.


Zhang J.,Zhejiang Sci-Tech University | Zhang J.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Liu Y.,Zhejiang Sci-Tech University | Liu Y.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 23 more authors.
Gene | Year: 2013

MicroRNAs are indispensable players in the regulation of a broad range of biological processes. Here, we report the first deep sequencing of the whitespotted bamboo shark (Chiloscyllium plagiosum) liver. We mapped 91 miRNAs in the Callorhinchus milii genome that have previously been described in the Danio rerio, Fugu rubripes, Oryzias latipes, Xenopus laevis, Xenopus tropicalis, Homo sapiens, and Mus musculus. In addition, 156 new putative candidate (PC) C. plagiosum miRNAs were identified. From these 247 miRNAs, 39 miRNA clusters were identified, and the expression of these clustered miRNAs was observed to vary significantly. A total of 7 candidate miRNAs were selected for expression confirmation by stem-loop RT-PCR. This study resulted in the addition of a significant number of novel miRNA sequences to GenBank and laid the foundation for further understanding of the function of miRNAs in the regulation of C. plagiosum liver development. © 2013 Elsevier B.V.


Shi X.,Zhejiang Sci-Tech University | Fang Q.,Zhejiang Sci-Tech University | Ding M.,Zhejiang Sci-Tech University | Wu J.,Zhejiang Sci-Tech University | And 6 more authors.
Journal of Biomaterials Applications | Year: 2016

To develop biocompatible composite microspheres for novel hemostatic use, we designed and prepared a novel biomaterial, composite microspheres consisting of carboxymethyl chitosan, sodium alginate, and collagen (CSCM). The ultra-structure of CSCM was investigated by scanning electron microscopy assay. In hemostatic function experiment, it was found that CSCM could facilitate platelet adherence, platelet aggregation, and platelet activation in vitro. Besides, the maximum swelling of CSCM submerged in PBS for 50 min was over 300% of that exhibited by commercial hemostatic compound microporous polysaccharide haemostatic powder (CMPHP). In addition, CSCM exhibited good biodegradability and non-cytotoxicity. These results demonstrated that CSCM may be useful in platelet plug formation, and this study would provide important information for further research on hemostasis experiment in vivo. © 2015 The Author(s).


Yu W.,Zhejiang Sci-Tech University | Yu W.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Wang M.,Zhejiang Sci-Tech University | Wang M.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 6 more authors.
International Journal of Genomics | Year: 2013

Storage protein 2 (SP2) not only is an important source of energy for the growth and development of silkworm but also has inhibitory effects on cell apoptosis. Endothelial cell (EC) apoptosis is an important contributing factor in the development of atherosclerosis; therefore, study of the antiapoptotic activity of SP2 on ECs provides information related to the treatment of atherosclerosis and other cardiovascular diseases. In this study, the sp2 gene was cloned and expressed in Escherichia coli to produce a 6xHis-tagged fusion protein, which was then used to generate a polyclonal antibody. Western blot results revealed that SP2 levels were higher in the pupal stage and hemolymph of fifth-instar larvae but low in the egg and adult stages. Subcellular localization results showed that SP2 is located mainly on the cell membrane. In addition, a Bac-to-Bac system was used to construct a recombinant baculovirus for SP2 expression. The purified SP2 was then added to a culture medium for human umbilical vein ECs (HUVECs), which were exposed to staurosporine. A cell viability assay demonstrated that SP2 could significantly enhance the viability of HUVEC. Furthermore, both ELISA and flow cytometry results indicated that SP2 has anti-apoptotic effects on staurosporine-induced HUVEC apoptosis. © 2013 Wei Yu et al.


Li B.,Zhejiang Sci-Tech University | Li B.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Cheng X.,Zhejiang Sci-Tech University | Cheng X.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 8 more authors.
Mycoscience | Year: 2016

Ganoderma lingzhi is a kind of precious medicinal mushroom. MicroRNAs (miRNAs) play an important role in the regulation of a broad range of biological processes. In this study, we first report on the miRNAs of the G. lingzhi sporocarp. In total, 132 known miRNAs and 34 putative candidate (PC) miRNAs were identified for the first time in G. lingzhi sporocarp. Through target gene prediction, 111 of 132 known miRNAs had targeted genes, 58 of them had more than one, and most predicted targeted genes might be regulated by more than one miRNA; functional enrichment by KEGG analysis demonstrated that target genes related to cancer and signal transduction occupied the largest proportion. A total of seven miRNAs (four known miRNAs and three PC miRNAs) that had high abundance were selected for expression confirmation by quantitative real-time PCR (qRT-PCR) and northern blotting. This is the first comprehensive study of the miRNA composition in G. lingzhi, which resulted in the addition of a significant number of novel miRNA sequences to miRBase and laid the foundation for further understanding of the function of miRNAs in the regulation of G. lingzhi. © 2016 Published by Elsevier B.V.


Yu W.,Zhejiang Sci-Tech University | Yu W.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Li J.,Zhejiang Sci-Tech University | Li J.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 12 more authors.
Virology Journal | Year: 2012

Background: The polyhedrin gene promoter has an essential role in regulating foreign gene expression in baculovirus expression vector systems (BEVS); however, the high-level transcription mechanism is still unknown. One-hybrid screening in yeast is a powerful way of identifying rapidly heterologous transcription factors that can interact with the polyhedrin promoter DNA sequence. In the current study, total RNA was extracted from the fat bodies of fifth-instar silkworm larvae that had been infected with Bombyx mori nuclear polyhedrosis virus (BmNPV) for 5 days; complementary DNA (cDNA) was then generated using reverse-transcription (RT)-PCR to construct a silkworm gene expression library. Key polyhedrin promoter bait sequences were synthesized to generate a bait yeast strain, which was used to screen the one-hybrid cDNA library. Results: In total, 12 positive yeast colonies were obtained from the SD/-Leu/AbA plates; sequencing analysis showed that they belong to two different protein cDNA colonies. Positive colonies underwent bioinformatics analysis, which revealed one colony to be ribosomal proteins [B. mori ribosomal protein SA (BmRPSA)] and the other to be NPV DNA-binding proteins (DBP). To further verify the regulatory function of these two protein groups, transient expression vectors (pSK-IE-dbp and pSK-IE-BmRPSA) were constructed. The recombinant plasmids were then transfected into cultured B. mori N (BmN) cells, which had been infected with a recombinant bacmid containing the gene encoding luciferase (luc). The results showed that overexpression of either dbp or BmRPSA upregulated the polh promoter-driven transcription of luc in BmN cells. In addition, dbp or BmRPSA RNA interference (RNAi) resulted in the downregulation of luciferase reporter expression in BmN cells, demonstrating that DBP and BmRPSA are important for luc transcription. EMSA results further confirmed that DBP could directly bind to the conserved single-stranded polh promoter region in intro. However, EMSA assay also showed that BmRPSA did not bind to this region, precluding a direct DNA association. Conclusions: Both DBP and BmRPSA are important for polh transcription. DBP can regulate polh promoter activity by direct binding to the conserved single-stranded polh promoter region, BmRPSA may regulate polh promoter activity by indirect binding to this region. © 2012 Yu et al.; licensee BioMed Central Ltd.

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