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).


Sun C.,Zhejiang Sci-Tech University | Sun C.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Wang R.-J.,Zhejiang University | Su Y.,Zhejiang University | And 11 more authors.
International Journal of Systematic and Evolutionary Microbiology | Year: 2017

A Gram-stain-negative, aerobic, non-spore-forming, non-motile, oval to rod-shaped, prosthecate bacterium, designated strain WM6T, was isolated from a seawater sample collected from the South China Sea at a depth of 150 m and subjected to a polyphasic taxonomic investigation. Cells of strain WM6T were approximately 0.5–0.6μm in width and 0.8–1.2μm in length, and colonies were smooth, circular, convex and whitish yellow. Strain WM6T was found to grow at 10–45 °C (optimum, 30 °C), at pH 6.5–9.0 (optimum, pH 7.5–8.5) and with 1–6% (w/v) NaCl (optimum, 1–2%). Chemotaxonomic analysis showed the predominant respiratory quinone and the major fatty acid of strains WM6T were ubiquinone-10 and C18: 1ω7c, respectively. The polar lipids of strain WM6T were phosphatidylglycerol, glucuronopyranosyldiglyceride, monoglycosyldiglyceride, sulfoquinovosyl diacylglycerol, seven unknown glycolipids and two unknown lipids. The DNA G+C content of strain WM6T was determined to be 59.8 mol% by HPLC. 16S rRNA gene sequence similarities showed that strain WM6T was related most closely to the genus Maricaulis with a similarity range from 92.3 to 93.8%. Phylogenetic trees reconstructed with the neighbour-joining and maximum-likelihood methods using MEGA and maximum-likelihood methods using ARB showed that strain WM6T constituted a separated branch in the family Hyphomonadaceae. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain WM6T is clearly distinct from any validly published genus. On the basis of these features, strain WM6T represents a novel species of a new genus with the name Hyphobacterium vulgare gen. nov., sp. nov. The type strain of Hyphobacterium vulgare is WM6T (=MCCC 1K03222T=KCTC 52487T). © 2017 IUMS.


Lu Y.,Zhejiang Sci-Tech University | Lu Y.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Zhang T.-F.,Zhejiang Sci-Tech University | Zhang T.-F.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 15 more authors.
Scientific Reports | Year: 2016

LF11-322 (PFWRIRIRR-NH 2) (PFR peptide), a nine amino acid-residue peptide fragment derived from human lactoferricin, possesses potent cytotoxicity against bacteria. We report here the discovery and characterization of its antitumor activity in leukemia cells. PFR peptide inhibited the proliferation of MEL and HL-60 leukemia cells by inducing cell death in the absence of the classical features of apoptosis, including chromatin condensation, Annexin V staining, Caspase activation and increase of abundance of pro-apoptotic proteins. Instead, necrotic cell death as evidenced by increasing intracellular PI staining and LDH release, inducing membrane disruption and up-regulating intracellular calcium level, was observed following PFR peptide treatment. In addition to necrotic cell death, PFR peptide also induced G 0/G 1 cell cycle arrest. Moreover, PFR peptide exhibited favorable antitumor activity and tolerability in vivo. These findings thus provide a new clue of antimicrobial peptides as a potential novel therapy for leukemia.


Su X.,Zhejiang Sci-Tech University | Geng X.,Zhejiang Sci-Tech University | Fu M.,Zhejiang Sci-Tech University | Wu Y.,Zhejiang Sci-Tech University | And 4 more authors.
Protein Expression and Purification | Year: 2017

EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the Mollusca, Ampullaria crossean. In this study, the EG27I mature peptide gene fused to HFBII secretion signal of Trichoderma reesei was expressed under GAP promoter of Pichia pastoris in SMD1163 strain. Bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into the culture medium. When high cell density fermentation of the recombinant Pichia pastoris was performed by a fed-batch strategy for totally 132 h in a 7.5 L fermenter, the OD600 and the hydrolytic activity reached final values of 333 and 1.28 U/mL, respectively. The recombinant protein concentration was 47.7 mg/L of fermentation supernatant. EG27I was purified from the fermentation supernatant by ultrafiltration, cation exchange and hydrophobic interaction, consecutively. The specific activity of the recombinant EG27I was 26.8 U/mg. The optimal pH and temperature for enzyme were pH 5 and 50 °C, respectively. The half-life of enzyme activity at 100 °C was able to reach 40 min. N-terminal amino acid sequence analysis of purified recombinant protein confirmed that the amino-terminal sequence was consistent with natural structure. The high quantity and purity of the EG27I pave the way for future structural and functional studies. © 2017 Elsevier Inc.


Li Y.,Zhejiang Sci-Tech University | Li Y.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Wang W.,Zhejiang Sci-Tech University | Wang W.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 17 more authors.
Marine Drugs | Year: 2015

APSL (active peptide from shark liver) is a hepatic stimulator cytokine from the liver of Chiloscyllium. It can effectively protect islet cells and improve complications in mice with alloxan-induced diabetes. Here, we demonstrate that the APSL sequence is present in the N-terminus of novel TBC (Tre-2, Bub2 and Cdc16) domain family, member 15 (TBC1D15) from Chiloscyllium plagiosum. This shark TBC1D15 gene, which contains an ORF of 2088 bp, was identified from a cDNA library of regenerating shark liver. Bioinformatic analysis showed that the gene is highly homologous to TBC1D15 genes from other species. Moreover, the N-terminus of shark TBC1D15 contains a motif of unknown function (DUF3548), which encompasses the APSL fragment. Rab-GAP activity analysis showed that shark TBC1D15 is a new member of the TBC1D15 family. These results demonstrated that shark TBC1D15 possesses Rab-GAP activity using Rab7 as a substrate, which is a common property of the TBC1D15 family. The involvement of APSL at the N-terminus of TBC1D15 also demonstrates that this protein might be involved in insulin signaling and may be associated with the development of type 2 diabetes. The current findings pave the way for further functional and clinical studies of these proteins from marine sources. © 2015 by the authors; licensee MDPI.


Zha Z.,Zhejiang Sci-Tech University | Zha Z.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | Li C.,Zhejiang Sci-Tech University | Li C.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine | And 6 more authors.
Scientific Reports | Year: 2016

Outer membrane proteins (OMPs) are unique to Gram-negative bacteria. Several features, including surface exposure, conservation among strains and ability to induce immune responses, make OMPs attractive targets for using as vaccine antigens and immunotherapeutics. LptD is an essential OMP that mediates the final transport of lipopolysaccharide (LPS) to outer leaflet. The protein in Vibrio parahaemolyticus was identified to have immunogenicity in our previous report. In this study, broad distribution, high conservation and similar surface-epitopes of LptD were found among the major Vibrio species. LptD was further revealed to be associated with immune responses, and it has a strong ability to stimulate antibody response. More importantly, it conferred 100% immune protection against lethal challenge by V. parahaemolyticus in mice when the mice were vaccinated with LptD, and this finding was consistent with the observation of efficient clearance of bacteria in vaccination mice. Strikingly, targeting of bacteria by the LptD antibody caused significant decreases in both the growth and LPS level and an increase in susceptibility to hydrophobic antibiotics. These findings were consistent with those previously obtained in lptD-deletion bacteria. These data demonstrated LptD is a promising vaccine antigens and a potential target for antibody-based therapy to protect against Vibrio infections. © 2016 The Author(s).


Zhang C.,Zhejiang Sci-Tech University | Wu Y.-Q.,Zhejiang Sci-Tech University | Fang Q.,Zhejiang Sci-Tech University | Wang Y.-D.,Zhejiang Sci-Tech University | And 2 more authors.
Chinese Journal of Pharmaceutical Biotechnology | Year: 2016

Thymosins, one family of thymic hormones, play an important role in physiological functions such as immune regulation and development of organisms.Thymosins widely exist in vertebrates and invertebrates.Thymosins has been highly conserved in the progress of evolution.Nowadays, the function of vertebrates thymosins has been studied thoroughly.Vertebrates thymosins have already been developed for clinical applications.Emerging evidence revealed that vertebrates thymosins were widely used as pharmacological agents in clinical practice to treat some diseases including immunodeficiency related diseases, chronic hepatitis and certain types of cancers.However, there was less research on invertebrates thymosins especially on insects, such as Bombyx mori.Only twenty related proteins composed of thymosin conservative structural domains from lepidoptera can be found in NCBI website.These proteins are highly homologous to β-thymosin (Tβ) and are distributed in Drosophila melanogaster, Bombyx mori and other species.In this paper, research progress on the homology, evolution analysis, functions and characteristics of thymosin super-family proteins from different insects in recent 20 years have been reviewed. © 2016, Editorial Board of Pharmaceutical Biotechnology. All right reserved.

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