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Analysis of a Novel Transcription Mode of Fhx/P25 Gene in Bombyx mori

Chun LIU ; Ping ZHAO ; Tingcai CHENG ; Xingfu ZHA ; Qingyou XIA ; Zhonghuai XIANG

Progress in Biochemistry and Biophysics.2005;32(8):740-746.

Fhx/P25 in silkworm, Bombyx mori, one of the main components of silk fibroin, is presumed in previous reports to be expressed exclusively in the posterior silk gland (PSG) of the animal with strict territorial and developmental specificities. On the basis of a large-scale analysis ofthe silkworm EST data, it was found that Fhx/P25 gene is transcribed not only in the posterior silk gland, but in the ovary and in other tissues of the larvae at day 3 of the fifth-instar as well and that this gene has distinct transcription start sites (TSSs) in the posterior silk gland and the ovary. The TSS in the ovary is located about 115 bp upstream sequence of that in the posterior silk gland. Subsequent RT-PCR, FQ-PCR and sequencing have verified the validity of this presumption. In addition, alternative splicing is predicted in pre-mRNA of Fhx/P25 gene and confirmed by RT-PCR. In conclusion, Fhx/P25 gene is not a gene with strictly tissue-specific transcription.Complicated regulation mechanisms may exist for its transcription and expression and it may have other functions to perform.

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Efficient Protection of H5N1 Influenza Virus DNA Vaccine Delivering by Electroporation in Mammalian and Avian System

Qiangzhe ZHANG ; Ximing QIN ; Haili DONG ; Rong LIANG ; Hongxuan HE ; Xi LI ; Beiyu JIANG ; Xiangjun LIU ; Mingxinng DUAN

Progress in Biochemistry and Biophysics.2005;32(8):726-733.

To investigate the protection effect of DNA vaccine in mammalian and avian systems, the DNA vaccine was inoculated in both BALB/c mice and SPF chickens immunized with DNA vaccines encoding hemagglutinin (HA) from A/Goose/GuangDong/1/96 (H5N1) virus. The mice and chickens were immunized twice, 3 weeks apart, by electroporation into muscles or intramuscular injection. Two weeks after the second immunization, the mice and chickens were challenged with a lethal dose of homologous virus. The mice and chickens immunized by electroporation obtained completely protection against the virus, and could effectively inhibited viruses to replicating in mouse lung and chicken cloaca. At the same time, these protections were companied by high levels specific antibody to H5N1 AIV, while the blank plasmid controls experience 100 percent mortality following challenge. Furthermore, in the experiment of mice by eletroporation,stronger obviously CTL activity were observed after challenge. Thus, the cellular immune responses of the mice immunized by electroporation were exhibited. These results strongly demonstrate that HA DNA vaccines provide effective protection against influenza virus infection in mammalian and avian, and suggest that electroporation is one of the efficient gene delivery systems for the transfer of influenza DNA vaccine in both humoral immunity and cellular immunity.

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Abundance and Distribution of Microsatellites in The Entire Mosquito Genome

Quanyou YU ; Bin LI ; Guanrong LI ; Shoumin FANG ; Hong YAN ; Xiaoling TONG ; Jifeng QIAN ; Qingyou XIA ; Cheng LU

Progress in Biochemistry and Biophysics.2005;32(5):435-441.

Microsatellite is a genetic marker, explored recently. In order to improve related studies on genetics of Anopheles gambiae, simple sequence repeats of the entire mosquito genome with 1～6 bp nucleotide motifs were analyzed.Abundance and distribution of microsatellites across the A.gambiae genome were analyzed and compared between various (exons, introns and intergenic) regions of all the chromosomes. About 2.14% of the mosquito genome was occupied by SSRs. Chromosome X had the maximum density of SSRs. Abundance orA repeats was similar to C repeats. AC was a little more than two times as abundant much as AG. However, AT and CG repeats were rare. For tri- and tetramer repeats, AGC,AAAC and AAAT predominated while ACG, ACT, AGG, CCG, ATGC, CCCG, ACTG, AACT, ACGT, AGAT, CCGG,ACCT and AGCT were rare. For some pentamer repeats, one was completely absent on a certain chromosome, even on several chromosomes. SSRs in exons of all chromosomes were less abundant than in introns and intergenic regions except for mono- and dimer repeats in exons of chromosome 2L. Abundance and distribution of SSRs on the two arms of each chromosome showed much in common.

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The Thermodynamic Evidence of Two Native Conformations Coexisting in Solution For Apoazuirn: a DSC and CD Study

Hongjie ZHANG

Progress in Biochemistry and Biophysics.2005;32(3):239-244.

Whether protein could adopt multiple conformations coexisting in solution is disputable. In a previous report,the conformation heterogeneity of apoazurin mutant M121L had been identified. The thermal unfolding of wild type apoazurin from Pseudomonas aeruginosa is re-investigated with differential scanning calorimetry (DSC) and circular dichroism (CD) methods. The results show that unfolding in the pH range from 4 to 9 is associated with two heat capacity maxima. The low temperature transitions are reversible at all pH conditions used, while the high temperature transitions are irreversible. The two unfolding transitions were analyzed by the two-interchangeable-conformation model with the fraction for the first transition (N1) from 64% at pH 4.0 to 55% at pH 9.0. Temperature induced unfolding monitored at 219 nm shows also two separate transitions. The ratio of the signal changes is consistent with the fractions obtained from the corresponding DSC measurements. These results provide further support for the hypothesis that at least two conformations of apoazurin coexists in solution.

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A LIF Mutation at The 29th Amino Acid Totally Abolished The Biological Functions

Hongxiu NING ; Yue CHEN ; Yuanjiang ZHANG ; Yu RONG ; Xiaojun WU ; Xiufang ZHANG ; Zhijie CHANG

Progress in Biochemistry and Biophysics.2005;32(4):318-324.

Leukemia inhibitory factor (LIF) plays important roles in varieties of biological processes. This factor is highly conserved in mammalian animals and only one heterozygous LIF mutation was reported to cause the infertility of women. A LIF mutation was generated and the evidences were provided that the mutation of mature LIF at the 29th amino acid totally abolished its functions, including stimulation of STAT activation assayed by Luciferase reporter gene expression and EMSA experiments. In addition, the mutated LIF failed to inhibit the proliferation of M1 cells. The data indicated that the mutation of LIF did not have a dominant negative effect but lost the biological functions, suggesting that the 29th amino acid is critical for maintaining the activities of LIF.

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Protein Preparation, Crystallization and Preliminary X-ray Crystallographic Analysis of Smu.260 From Streptococcus mutans--a Cariogenic Dental Pathogen

Xiaoyan ZHANG ; Wei MI ; Yanfeng ZHOU ; Xiangyu LIU ; Lanfen LI ; Yuhe LIANG ; Shicheng WEI ; Xiaodong SU

Progress in Biochemistry and Biophysics.2005;32(3):217-220.

Smu. 260 encodes a putative protein of 200 residues in Streptococcus mutans, a primary pathogen for human dental caries. Smu. 260 was cloned into expression vector pET28a and expressed in good amount trom the E. coli strain BL21 (DE3). Smu.260 protein was purified to homogeneity in a two-step procedure of Ni2+ chelating and size exclusion chromatography. The purified protein exists in two forms, a dimer form about 46 ku with yellow color and a tetramer form without apparent color. Crystals were obtained from the dimer protein by hanging-drop vapor-diffusion method. The crystals diffracted to about 2.3 A resolution and belong to orthorhombic space group P212121 with cell dimensions of a = 89.88A, b = 90.91 A, c = 105.17 A. The asymmetric unit is expected to contain two dimers with solvent content of 53%.

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Insight Into Folding,Binding and Stability of Insulin by NMR

Qingxin HUA

Progress in Biochemistry and Biophysics.2004;31(1):1-26.

Insulin is one of the most important hormonal regulators of metabolism. Since the diabetes patients increase dramatically, the chemical properties, biological and physiological effects of insulin had been extensively studied. In last decade the development of NMR technique allowed us to determine the solution structures of insulin and its variety mutants in various conditions, so that the knowledge of folding, binding and stability of insulin in solution have been largely increased. The solution structure of insulin monomers is essentially identical to those of insulin monomers within the dimer and hexamer as determined by X-ray diffraction. The studies of insulin mutants at the putative residues for receptor binding explored the possible conformational change and fitting between insulin and its receptor. The systematical studies of disulfide paring coupled insulin folding intermediates revealed that in spite of the conformational variety of the intermediates, one structural feature is always remained: a "native-like B chain super-secondary structure", which consists of B9-B19 helix with adjoining B23-B26 segment folded back against the central segment of B chain, an internal cystine A20-B19 disulfide bridge and a short α-helix at C-terminal of A chain linked. The "super-secondary structure" might be the "folding nucleus" in insulin folding mechanism. Cystine A20-B19 is the most important one among three disulfides to stabilize the nascent polypeptide in early stage of the folding. The NMR structure of C.elegans insulin-like peptide resembles that of human insulin and the peptide interacts with human insulin receptor. Other members of insulin super-family adopt the "insulin fold" mostly. The structural study of insulin-insulin receptor complex, that of C.elegans and other invertebrate insulin-like peptide, insulin fibril study and protein disulfide isomerase (PDI) assistant proinsulin folding study will be new topics in future to get insight into folding, binding, stability, evolution and fibrillation of insulin in detail.

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Molecular Mechanisms of Cell-cell Recognition

Jiahuai WANG

Progress in Biochemistry and Biophysics.2004;31(5):385-392.

Cell-cell recognition is the key for multicellular organisms to survive. This recognition critically depends on protein-protein interactions from opposing cell surfaces. Recent structural investigations reveal unique features of these cell surface receptors and how they interact. These interactions are specific, but usually relatively weak, with more hydrophilic forces involved in binding. The receptors appear to have specialized ways to present their key interacting elements for ligand-binding from the cell surface. Cell-cell contacts are multivalent. A large group of cell surface molecules are engaged in interactions. Characteristic weak interactions make possible for each individual molecule pair within the group to constantly associate-dissociate-reassociate, such that the cell-cell recognition becomes a dynamic process. The immunological synapse is a good example for immune receptors to be orchestrated in performing immunological function in a collective fashion.

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Roles of Calmodulin-dependent Protein Kinase Ⅱ in Meiotic Maturation and Fertilization of Oocytes

Hengyu FAN ; Lijun HUO ; Qingyuan SUN

Progress in Biochemistry and Biophysics.2003;30(2):171-174.

Calmodulin-dependent protein kinase (CaMK), activated by auto-phosphorylation at the presence of calcium and calmodulin, is widely distributed in eukaryotes. CaMKs are important mediators of calcium signal in eukaryotes. Recent researches have suggested that CaMKⅡ is involved in the regulation of meiotic cell cycle of oocytes. It plays functional roles in meiotic maturation, polar body extrusion, fertilization and egg activation. As one of the down-stream signaling molecules of calcium, CaMKⅡ facilitates the inactivation of maturation promoting factor (MPF) and cytostatic factor (CSF) following fertilization, as well as the spindle microtubule organization and centrosome duplication. Although the functions of CaMKⅡ in oocyte meiosis are versatile and essential, the present results are primarily obtained from low vertebrates and mouse. In future studies, the function and regulation of this kinase in other mammals should be stressed.

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High-level Expression of Foreign Genes In vivo and In vitro by Improved DNA-Based Replicon Vector Derived From Semliki Forest Virus

Yunzhou YU ; Zhiwei SUN ; Zhigang LIU ; Weiyuan YU

Progress in Biochemistry and Biophysics.2006;0(01):-.

The design of DNA-based alphavirus vectors significantly improves the utility of these replicon vectors. The DNA-based replicon vectors can be used in expressing foreign genes and preparing RVP in virto efficiently, also in developing replicon vaccines and gene therapy vectors in vivo. The approach involved the conversion a RNA-based replicon vector into a layered DNA-based replicon vector by the RNA polymerase Ⅱ promoter and transcription termination/polyadenylation signal transcribed replicon RNA from DNA. When DNA-based alphavirus vector tranfected into cells, the first layer includes a eukaryotic RNA polymerase Ⅱ expression cassette that initiates transcription of RNA in nucleus. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the RNA vector corresponds to virus RNA replication cycle and results in high level expression of foreign gene. DNA and RNA-based bifunctional replicon expression vector pSCTA and helper vector pSHCTA were successfully constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with CMV promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3′-untranslated region (UTR). In order to obtain DNA-based highly efficient replicon vectors, they were further modified to construct additional three DNA-based SFV replicon expression vectors and corresponding helper vectors. To investigate the efficiency of foreign gene expression level by the four different DNA-based SFV expression vectors and recombinant virus particle (RVP) prepared by cotranfecting with corresponding helper vectors, improved DNA-based replicon vectors pSCAR and pSHCAR derived from SFV were developed. high level protein could be generated using the new vector system by transfecting DNA into BHK21 cells and High titer of RVP produced by cotranfecting with helper vector. Antigen genes were also expressed in cells by the replicon expression vector. Additionally, reporter gene expression was observed in mice muscle following injection with SFV DNA vector. Anti-?-Gal antibody response and cell-mediated immune response were induced after intramuscular inoculation of the ?-Gal-encoding SFV replicon DNA. The results suggested that highly efficient DNA-based replicon vectors pSCAR and pSHCAR were constructed by modifying the SFV vectors. The improved DNA-based replicon vectors enhance the utility of them, and can be developed as potentially replicon vaccines and gene therapy vectors.

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