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Liu Z.W.,Chinese Academy of Sciences | Yin H.X.,Chinese Academy of Sciences | Yi X.P.,Chinese Academy of Sciences | Zhang A.L.,Chinese Academy of Sciences | And 6 more authors.
Molecular Biology Reports | Year: 2012

a-amy gene amplified from barley genome was cloned into MCS of pGAP9K to generate pGAP9K-a-amy which was then transformed into Pichia pastoris GS115 by electroporation. Transformants with multi-copies and high expression for the foreign gene were selected on G418 containing plate and expression analysis. The fermentation was carried out in a 50 l bioreactor with 20 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 54 h at 30°C. Under the control of GAP promoter (pGAP), a-amy gene was constitutively expressed. At the end of the fermentation, the a-AMY expression reached 125 mg/l, while the biomass growth was 186 as measured by absorption of 600 nm. The secreted a-AMY was purified to 97.5% by SP-Sepharose FF ion-exchange chromatography and affinity purification. The recombinant a-AMY showed activity on hydrolysis of starch. © Springer Science+Business Media B.V. 2011.

Li B.,University of Texas Medical Branch | Li B.,Hainan Provincial Institute for Drug Control | Shi Y.,University of Texas Medical Branch | Shu J.,University of Texas Medical Branch | And 4 more authors.
Journal of Biological Chemistry | Year: 2013

Background: HIV-1 infection causes chronic neuroinflammation in the central nervous system (CNS). Results: The spinal cytokine up-regulation induced by HIV-1 gp120 protein depends on Wnt5a/CaMKII and/or Wnt5a/JNK pathways. Conclusion: gp120 stimulates cytokine expression in the spinal cord dorsal horn by activating Wnt5a signaling. Significance: The finding reveals Wnt signaling-mediated novel mechanisms by which HIV-1 may cause neuroinflammation. Copyright © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Yin H.X.,Chinese Academy of Sciences | Zhang Z.H.,Chinese Academy of Sciences | Shen J.C.,Chinese Academy of Sciences | Zhang A.L.,Chinese Academy of Sciences | And 8 more authors.
Molecular Biology Reports | Year: 2013

Hemangioma is a tumor that causes vascular endothelial cell hyperplasia, which commonly occur in newborns. Angiogenesis inhibitor targets the processes of angiogenesis, including the proliferation of vascular endothelial cells. A DNA sequence named Scl was designed, recombined into Pichia Pastoris, expressed by fermenting the engineered strain in a bioreactor, and purified the recombinant Scl by SP-sepharose fast flow. Scl can inhibit CAM angiogenesis. Only 1 μg of Scl significantly suppressed the growth of CAM blood vessel, similar to that of 25 μg of angiostatin. Scl showed a strong cytotoxicity on hemangioma cell (ATCC CRL No. 2587). After the drug acted for 24 h, the OD 570 measured value of the PBS control group averaged 1.873, whereas that of the Sc1 drug group was 0.692 (P < 0.01). Using the DeadEndTM Fluorometric TUNEL System, the detection results showed that 92 % of hemangioma cell apoptosis was observed in the Scl protein group, but only 1.3 % in the PBS control group (P < 0.01). After 2 weeks of treatment with the hemangioma model (cock's wattle) of the PBS group, 151 blood vessels with 100 views (40×) were obtained, whereas 250 in the PBS group (P < 0.01). During the two-week medication, the hemangioma model of the PBS group increased by 1.18 cm, whereas only 0.58 cm in the Scl drug group (P < 0.01). © 2012 Springer Science+Business Media Dordrecht.

Yin H.,Chinese Academy of Sciences | Liu Z.,Chinese Academy of Sciences | Zhang A.,Chinese Academy of Sciences | Zhang T.,Sun Yat Sen University | And 7 more authors.
Gene | Year: 2012

Canstatin-N DNA fragment amplified from human genome was inserted into the MCS of pGAP9K*, an intracellular expression vector of Pichia pastoris, to generate pGAP9K*-can-N which was then transformed into P. pastoris GS115 by electroporation. A transformant was chosen as an engineering strain from the plate containing G418 (700μg/ml). d-sorbitol was selected as the only carbon source. The fermentation was carried out in a 50L bioreactor at a 20L working volume. After 48h fermentation with continuous feeding of 25% (w/v) d-sorbitol and 0.8% PTM4, the cell grew to A 600=178 and intracellularly expressed Canstatin-N reached 780mg/L. Snail enzyme was combined with water to crack P. pastoris and to release intracellular proteins. The purified recombinant Canstatin-N inhibited CAM angiogenesis and induced significant apoptosis of the human umbilical vein endothelial cell (EVC340). © 2012 Elsevier B.V.

Wang X.-C.,CAS Shanghai Institute of Materia Medica | Wang X.-C.,Hainan Provincial Institute for Drug Control | Ouyang X.-W.,CAS Shanghai Institute of Materia Medica | Hu L.-H.,CAS Shanghai Institute of Materia Medica
Journal of Asian Natural Products Research | Year: 2010

Three new lupane-type triterpenes, 3α-O-trans-feruloylbetulinic acid (1), 3α-O-trans-coumaroylbetulinic acid (2) and 3β-O-cis- feruloylbetulin (3), together with 10 known triterpenes (4-13), were isolated from the aerial parts of the mangrove plant Ceriops tagal. The structures of the three new compounds were established by means of spectroscopic data analyses and chemical methods. © 2010 Taylor & Francis.

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