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Development and Identification of a Live Attenuated Influenza B Virus Vaccine Candidate

Penghui YANG ; Wenqi AN ; Xinfu SHI ; Yueqiang DUAN ; Deyan LUO ; Pengfei ZHANG ; Chong TANG ; Li XING ; Yujing ZHANG ; Xiufan LIU ; Xiliang WANG

Progress in Biochemistry and Biophysics.2009;36(3):358-363. doi:10.3724/SP.J.1206.2008.00542

A cold-adapted (ca), temperature sensitive (ts), live attenuated influenza B virus strain B/Ann Arbor/1/66 was chosen for influenza virus rescue research, in which six internal gene segments, PB1, PB2, PA, NP, M, NS, were fully synthesized and nine amino acid substitutions were artificially alter by human intervention. The resultant B/Ann Arbor/1/66 plasmids were named as pAB121-PB1, pAB122-PB2, pAB123-PA, pAB124-HA, pAB125-NP, pAB126-NA, pAB127-M and pAB128-NS, respectively. A recombinant influenza A virus was previously generated entirely from cloned cDNA. An infectious recombinant influenza B virus was generated here, and designated as rMDV-B, by plasmid-based reverse genetics. The rMDV-B virus contained HA and NA genes from an epidemic influenza B vires strain B/Malaysia/2506/2004 in the background of internal genes derived from influenza B virus strain B/Ann Arbor/1/66. HA titer of rMDV-B in MDCK cells and embryonated chicken eggs ranged from 1 : 64 to 1 : 512. The results may allow an effective live influenza B vaccine to be produced from a single master strain, providing a model for the design of future live human influenza vaccines.

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Screening and Identification of The Proteins Interacting With NLS-RARα Protein

Chong WANG ; Liang ZHONG ; Dongsheng WANG ; Beizhong LIU ; Fei LIAO ; Po HAO ; Chang LIU ; Danting JIN ; Chunguang WANG

Progress in Biochemistry and Biophysics.2009;36(4):500-505. doi:10.3724/SP.J.1206.2008.00424

Acute promyelocytic leukemia (APL) is characterized by the generation of the prototypic promyelocytic leukemia-retinoicacid receptor alpha (PML-RARα), an oncogenic fusion protein due to chromosomal translocation. In a human myeloid cell line,PML-RARα is cleaved by neutrophil elastase (NE) to produce the mutational PML [nuclear localization signal (NLS) deletion andRARα (NLS-RARα, containing NLS of PML), both of which may play an important role in APL pathogenesis. The yeast two-hybridtechnique was used to screen the intracellular proteins interacting with NLS-RARα, which may be involved in NLS-RARα signaling. The NLS-RARα coding sequence was amplified by polymerase chain reaction method and was cloned into the bait plasmid pGBKT7vector, which, after the confirmation by sequencing, was transformed into yeast AH109 and the subsequent expression of bait plasmidwas proved by Western-blot. The transformed yeast AH109 was mated with yeast Y187 (containing leukemia cDNA library plasmidspACT2) in medium. Diploid yeast was plated on synthetic dropout nutrient medium containing X-α-gal for screening. After beingreintroduced into yeast AH109 and sequenced to verify the expression of ORF, eight positive colonies were obtained, among whichonecontaining JTV-1 was cloned. The interaction between NLS-RARα and JTV-1 was further supported by indirect immunofluorescence,GST pull-down and co-immunoprecipitation, respectively. These findings brought some new clues for the further exploration ofNLS-RARα signaling to APL.

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Sec-10 Knockout Increases The Neuroactive-drug Responses Without Affecting Function of The Postsynaptic Ionotropic Receptors in Neuromuscular Junctions

Lei ZHANG ; Jiangli LI ; Shangbang GAO ; Zhengxing WU ; Rongying ZHANG ; Tao XU

Progress in Biochemistry and Biophysics.2009;36(4):410-416. doi:10.3724/SP.J.1206.2009.00100

Exocyst complex is known to function in the exocytosis network, however, the molecular mechanism is unclear yet. Using UV/trimethylpsoralen mutagenesis, the sec-10 (one component of the exocyst complex) knockout mutant of C. elegans was obtained for the first time. The drug sensitive assays revealed clearly that the sec-10 gene affected the neural signal transmission, however, the electrophysiological assay showed the function of the ionotropic receptors in the neuromuscular junctions (NMJs) were unaltered compared with the wild type (WT). Thus it was assumed that the sec-10 gene might not influence the known ionotropic receptors in the NMJs, but some other pathways instead.

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Enzymological Characteristics of Catalytic Antibody-catalyzed enantioselective Hydrolysis of Ibuprofen Ester in Water-in-oil microemulsion

Gensheng YANG ; Yingdan QI ; Zhimin OU ; Shanjing YAO

Progress in Biochemistry and Biophysics.2009;36(2):182-189. doi:10.3724/SP.J.1206.2008.00347

The asymmetric hydrolyzation of racemic ibuprofen ester is one of the most important methods for chiral separation of ibuprofen. A catalytic antibody that accelerates the rate of enantioselective hydrolysis of ibuprofen methyl ester was successfully elicited against an immunogen consisting of tetrahedral sulfate hapten attached to bovine serum albumin (BSA). The rate constant enhancement factor Kcat/Kuncat was about 1.6x104. The catalytic activity of the catalytic antibody in a reverse micelle reaction system based on sodium b/s (2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane was studied. Kinetic analysis of the catalytic antibody-catalyzed reaction was found to be possible in this system. Kinetic studies showed that hydrolysis in the microemulsion system follow Michaelis-Menten kinetics. The catalytic antibody can also accelerate catalysis of S-ibuprofen methyl ester in the microemulsion system. Temperature effects, the pH profile, Km,app and Kcat were determined. The dependence of the catalytic antibody hydrolytic activity on the Wo (molar ratio of water to surfactant) showed a bell-shaped curve, presenting a maximum at about wo = 21.

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Apoptosis Effects of ODC and AdoMetDC biantisense Virus on Esophageal Cancer Cell Ecal09

Hui TIAN ; Jie XU ; Xianxi LIU ; Bing ZHANG ; Wenjun LI ; Xu SONG

Progress in Biochemistry and Biophysics.2009;36(2):238-243. doi:10.3724/SP.J.1206.2008.00398

Polyamine biosynthesis is controlled primarily by omithine Decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). To study the inhibitory effects of Ad-ODC-AdoMetDCas on polyamine biosynthesis and esophageal cancer cell apoptosis, adenovirus-mediated gene tmnsduction efficiency was assessed with counting GFP-positive cells using MTT. The malignant phenotype of Eca109 cells was assessed by growth curve. Western blot and HPLC were used to detect ODC and AdoMetDC expression and polyamine content in Ecal09 cells. TUNEL was used to analyze cell apoptosis. The change of morphology of apoptotic cells was observed by electron microscope. It was demonstrated approximate 70% of Eca 109 cells were infected with Ad-ODC-AdoMetDCas when MOI reached 50. The expression of ODC was inhibited in the infected tumor cells. Ad-ODC-AdoMetDCas could inhibit Ecal09 cell growth and invasive ability. TUNEL proved that Ad-ODC-AdoMetDCas can lead to cell apoptosis. Characterized morphology was observed by electronmicroscope (ehromatincondensation,nuclear disintegration,formation of apoptoticbodies).It was suggested Ad-ODC-AdoMetDCas has significant inhibitory effects on esophageal cancer cell proliferation, leads to cell apoptosis and bears therapeutic potential for the treatment of esophageal cancer.

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The demethylating Agent 5-Aza-2'-Deoxycytidine (5-AZA-CdR) Inhibits The Development of preimplantation Mouse Embryos

Jianning YU ; Shaohua LI ; Danqiu WANG ; Meng WANG ; Fei LIN ; Honglin LIU

Progress in Biochemistry and Biophysics.2009;36(2):228-237. doi:10.3724/SP.J.1206.2008.00394

DNA methylation is crucial for mammalian development, and DNA methylation is always in the dynamic status during preimplantation mouse embryos development. The effects of 5-AZA-CdR on the development of preimplantation mouse embryos were evaluated. Preimplantation mouse embryos created by in vitro fertilization were cultured continuously in 5-AZA-CdR (0.2, 1.0, or 5.0 μmol/L). Fertilized oocytes exposed to CZB containing 5-AZA-CdR at the pronuclear stage were unable to form morulae (0.2 and 1.0 μmol/L) or 4-cell embryos (5.0 μmol/L), while 2-cell stage embryos exposed to 5-AZA-CdR developed into uncompacted 8-cell (0.2 and 1.0 μmol/L) or 3/4-cell (5.0 μmol/L) stage embryos. The rate of morula formation was significantly lower in 4-cell embryos cultured in 5-AZA-CdR (1.0 or 5.0 μmol/L) than that in control embryos (P < 0.05). These data indicate that 5-AZA-CdR inhibits the development of mouse preimplantation embryos. Apoptosis, DNA methylation, and transcriptional activity were analyzed to determine the reason for these developmental defects. An aunexin V-PI assay revealed that high doses of 5-AZA-CdR led to apoptosis. Compared to the controls, DNA methylation was significantly reduced in uncompacted 8-cell embryos and morulae (p < 0.05) in a dose- dependent manner, whereas no significant change was detected in 2-or 4-cell embryos (P > 0.05). The observed changes in transcriptional activity, determined by measuring the incorporation of BrUTP, were similar to the observed alterations in DNA methylation. Therefore, the developmental defects induced by 5-AZA-CdR appear to bc mediated by alterations in DNA methylation and transcriptional activity in preimplantation mouse embryos.

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A Novel Neutralizing epitope of Human cytomegalovirus glycoprotein M Screened by Phage Display

Benxu WANG ; Yu LIU ; Zhan LIU ; Yaping GAO ; Fang WANG ; Heping PAN ; Guang YANG ; Hua XU ; Beifen SHEN ; Chuan LIU ; Ningsheng SHAO

Progress in Biochemistry and Biophysics.2009;36(2):220-227. doi:10.3724/SP.J.1206.2008.00391

Human cytomegalovirus glycoprotein complex Ⅱ (gC Ⅱ ) consists of two glycoproteins, gM and gN. Although gC Ⅱ specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report on the generation of virus-neutralizing antibodies by immunizing with one epitope of gM. The epitope, termed MAD, was screened from random phage peptide library by subtractive strategy. The peptide sequence of MAD was highly homologous with 32~38 amino acids of HCMV gM. Mice immunized with MAD coupled with keyhole limpet hemocyanin (KLH) could produce specific antibodies against MAD, and the antibodies obtained could bind not only native HCMV particles, but also the recombinant gM30～78 peptide. ELISA analysis results showed that MAD could specifically bind HCMV-positive human serum samples. Virus-neutralizing assay results demonstrated that the antibodies against MAD could inhibit HCMV strain AD169 entering the human embryonic lung cells. The results suggested that MAD could be used as a new potential protective antigen in the development of HCMV vaccine.

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Tissue and Induction Expression Profiles of Rice Phospholipid Hydroperoxide Glutathione Peroxidase at Protein Level

Tian LI ; Xiaodong YANG ; Jinyuan LIU

Progress in Biochemistry and Biophysics.2009;36(1):77-82. doi:10.3724/SP.J.1206.2008.00335

Proteins arc the major molecules performing life activities, and their spatial and temporal expression profiles in organisms are very important for understanding their accurate functions. Phospholipid hydroperoxide glutathione pcroxidase (PHGPx) is a unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes and plays a vital role in defense against mernhrane peroxidation damage. The protein expression profiles of rice PHGPx (OsPHGPx) were investigated in different rice tissues and under various stress treatments by using Western blot analysis. The results showed that in mature rice plants, OsPHGPx was mainly distributed in leaves, especially flag leaves, and in rice seedlings, OsPHGPx was detected in shoots and leaves. Moreover, OsPHGPx expression in rice seedlings could be markedly induced by H2O2 and NaCI, but weakly influenced by several plant hormones. Time- and dose-dependent effects were observed in both H2O2 and NaCI treatments, and the strongest induction was observed when rice seedlings were treated with 0.5 mmol/L H2O2 for 12 h or 500 mmol/L NaCI for 24 h. Additionally, dimethylthiourea, a H2O2 trap, inhibited H2O2-enhanced expression of OsPHGPx, but did not impair the enhanced effect of NaCI, implying that NaCl-induced OsPHGPx expression was not mediated by H2O2. These results will contribute greatly to further study the exact physiological function of OsPHGPx in rice.

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UDP-Ga1NAc: polypeptide α-N-acetygalactosaminyltransferase 2 Localized on Both cis and trans Side of Golgi Stacks in SGC7901 Cells

Yinghui ZHOU ; Saiyu HANG ; Hao QIU ; Wei JIA ; Lan XU ; Zhi JIANG ; Shiliang WU

Progress in Biochemistry and Biophysics.2009;36(1):49-57. doi:10.3724/SP.J.1206.2008.00299

Uridine diphosphate (UDP)-GalNAc : polypeptide N-acetylgalactosaminyltransfemse (ppGalNAcT) catalyzes the initial step in mucin type O-glycosylation in the Golgi apparatus. Here generation and characterization of a polyclonal antibody to human ppGalNAcT2 were described. The subcellular location of ppGalNAeT2 in SGC7901 cell line was investigated using Western blot analysis of fractionated cell extracts and confocal microscopy with this antibody and two Golgi markers: Golgi SNARE (soluble N-ethylmalemide-sensifive factor attachment protein receptor) of 28 ku (GS28) and trans-Golgi network (TGN) 38, markers for the c/s- and trans-Golgi apparatus, respectively. Morphometric analyses indicated that ～60% of the ppGalNAcT2 signal colocalized with the GS28, while～36% of the c/s-Golgi marker colocalized with the ppGalNAeT2. Approximately 34% of the ppGalNAcT2 signal colocalized with the TGN38, whereas 38% of the trans-Golgi marker colocalized with the ppGalNAcT2. The results provide unequivocal evidence for the location ofppGalNAcT2 within the Golgi apparatus, and further highlight the importance of this organelle in the initiation of O-linked glycosylation.

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Fucosylated Glycans Associated With Development and Metastasis of Hepatocellular Carcinoma Cells

Lihui WU ; Ping HU ; Wei WU ; Xingzhong WU

Progress in Biochemistry and Biophysics.2009;36(1):33-41. doi:10.3724/SP.J.1206.2008.00291

Dynamic fucosylation of glycoprotein especially 80 ku which bound to UEA and LCA during the course of rat hepatocarcinogenesis was investigated. In patient hepatocellular carcinoma, more UEA- and LCA-bound proteins were also observed in patients with high metastatic potential than those with low metastatic potential. Fueosylated glycans constitute important adhesion molecules such as Lewis antigens. A differential expression pattern of Lewis antigens was further conf'Lrmed on various metastasis potential hepatocellular carcinoma cells (HCC). High metastatic hepatocellular carcinoma cell line (HMCC97H) expressed much more Lewis x and b than low metastasis HMCC97L cells. Moreover, surface Lewis x, or b expression level declined significantly after the cells were treated by retinoie acid. Not only in experimental metastasis foei, but in HCC as well, both α1,3/1,2 and α1,6 fucosyltransferase activities were quite high. After retinoie acid treatment, α1,3/1,2 fueosyltransferase activities were significantly inhibited, Lewis x on epidermal growth factor receptor reduced, and the EGFR was less phosphorylated. These results suggested that fucosylated glycans such as Lewis x played an important role in HCC development and metastasis.

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