Biochemical properties of C78SC96S rhFGF-2: A double point-mutated rhFGF-2 increases obviously its activity

Fibroblast growth factor-2 (FGF-2) is a multifunctional polypeptide that affects many cellular functions and phenomena. The wild-type recombinant human fibroblast growth factor rhFGF-2(W) and the mutant C78SC96S rhFGF-2(M) were expressed in Escherichia coli and their products were purified. The results by the means of fluorescence spectroscopy and CD spectrums, suggested that due to its decreased hydrophobicity rhFGF-2 is not deposited as an inclusion body. The mitogenic activity of the expressed rhFGF-2(M) on 3T3 fibroblasts was shown to be 10-fold more than the expressed rhFGF-2(W) of which the biological activity was a little less than that of the standard rhbFGF(W), indicating that the increased biological activity was due to the change of its secondary structure, dimerization and affinity binding to FGF receptor (FGFR).

棕色固氮菌突变种(UW45、DJ54和DJ35)的缺失FeMoco钼铁蛋白的纯化、特性鉴定及结晶研究

一．棕色固氮菌突变种UW45、缺失nifH（DJ54）和缺失nifE(DJ35)突变种的钼铁蛋白的纯化、特性鉴定及结晶研究 棕色固氮菌突变种UW45的菌体破碎后，所得粗提物经两次DEAE 52柱层析后得到部分纯的nifB- MoFe蛋白和Fe蛋白。再经Sephacryl S-300和 DEAE柱的进一步纯化，便使nifB- MoFe蛋白基本达到SDS凝胶电泳纯。SDS-PAGE结果表明，nifB- MoFe蛋白具有与野生型棕色固氮菌（OP）MoFe蛋白相同的亚基种类和组成。此粗提物可为用NMF抽提的 OP MoFe蛋白的FeMoco激活，所得Fe蛋白具有与OP Fe蛋白相似的互补活性，可使OP MoFe的比活性达到2192 nmol C2H2/min/mg蛋白。FeMoco可使无互补活性的 nifB- MoFe蛋白与nifB- Fe蛋白组成具有可观放氢活性的固氮酶，使FeMoco显出的比活性接近文献报道的还原乙炔的最高值。对nifB- MoFe蛋白的结晶及晶体生长进行了的研究，初步探讨了结晶溶液各组分的种类和浓度、结晶方法和实验操作等与能否出现晶体及晶体的数目、大小、质量、形状和出晶时间等的相互关系。在结晶实验时，一次就得到了国内外尚未报道的该蛋白的短斜四棱柱的棕色晶体。目前所得的最大的晶体的二维边长都为0.1mm。初步结果表明，这种晶体可能就是nifB- MoFe蛋白的晶体。 从棕色固氮菌突变种DJ54中得到了ΔnifH MoFe蛋白;并参与了棕色固氮菌突变种DJ35的ΔnifE MoFe蛋白的分离纯化，所用方法与nifB- MoFe蛋白的分离纯化相似。对这两种突变种蛋白的特性和结晶进行了初步研究。在结晶实验时，也是一次就得到了国内外尚未报道的ΔnifH MoFe蛋白和ΔnifEMoFe蛋白的晶体。 二．新型固氮酶MnFe蛋白和CrFe蛋白的特性与结晶研究 在已有的工作基础上，分离纯化了几批MnFe蛋白和CrFe蛋白，并用部分纯的nifB- Fe蛋白进行活性互补，分别测定了MnFe蛋白和CrFe蛋白的底物还原活性。不断优化MnFe蛋白和CrFe蛋白晶体生长条件，获得了晶质良好的MnFe蛋白和CrFe蛋白的较大晶体。 在2001年的“神舟2号”飞船搭载实验中，MnFe蛋白的出晶率达到100%，所获得的晶体也比地面对照略厚些。继续进行MnFe蛋白和CrFe蛋白的空间计划的地面匹配实验，以满足对蛋白质样品的要求，以保证宇宙飞船“神舟3号”的蛋白质搭载实验获得更好的结果。

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG(20)k-S7C, PEG(20)k-K173C, and PEG(20)k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life. (C) 2004 Elsevier Inc. All rights reserved.

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

Construction of cytoplasmic molecular markers distinguishing Danio rerio from Gobiocypris rarus at high identity domains based on MP-PCR strategy and Sybr Green I detection

To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio female x G. rarus male) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity.

RecA-mediated, targeted mutagenesis in zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into I-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in I or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

~(12)C~(6+)离子束诱导HeLa细胞DNA损伤效应

本文研究了HeLa细胞经过12C6+离子束辐照之后的DNA损伤效应,及辐照后p53激活的分子机制。运用中性单细胞电泳技术,检测了HeLa细胞经过4Gy 12C6+离子束辐照间隔0、3、6和12h之后DNA的损伤情况,及0.5、1、2和4Gy 12C6+离子束辐照后即时的DNA损伤情况。同时运用细胞生长实时监测仪监测了HeLa细胞在经过0、0.5和1Gy 12C6+离子束辐照之后的生长变化,并运用AO/EB双染检测了辐照细胞24h后的凋亡情况。另外,利用8mmol/L的咖啡因[抑制ATM(ataxia-telangiectasia,mutated)和ATR(ATM and Rad3-related kinase)]和20μmol/L的wortmannin[抑制ATM和DNA-PK(DNA-dependent protein kinase)]处理HeLa细胞后再进行1Gy 12C6+离子束辐照,通过westernblot检测p53的表达。结果显示,12C6+离子束辐照可造成HeLa细胞的DNA损伤,损伤随剂量升高而升高但随测定间隔时间降低,诱导HeLa细胞发生凋亡;而且辐照后p53表达升高。结果证明12C6+离子束辐照可造成HeLa细胞的DNA损伤并诱导损伤修复及凋亡等效应,损伤效应相关因子p53被激活,并且激活依赖于ATM。

重离子束辐照诱变啤酒酵母线粒体DNA研究

摘要 II 5. 在不同注量离子束辐照后筛选出来的呼吸缺陷型酵母菌株中扩增获得位 于第 12 染色体上的 SOF1 基因，而在同样的扩增体系中没有得到野生型 菌株的该基因。 6. 选取离子束辐照后筛选出来的呼吸缺陷型酵母菌株再次进行辐照，发现 其在低剂量范围（＜0.93Gy）辐照下非常敏感，而在高剂量范围（＞ 0.93Gy）又表现出一定程度的辐射抗性。 结论： 1. 离子束辐照酵母细胞，直接或间接作用于酵母线粒体DNA，导致线粒体 DNA损伤，形成呼吸缺陷的酵母菌株。 2. I 类内含子和 II 类内含子对于离子束辐照的敏感性不同： II 类内含子比较 稳定，II 类内含子可能利用自身编码的反转录酶通过目的DNA引导的反 转录机制对受到辐照损伤的II 类内含子进行修复。 3. 离子束辐照后 SOF1 基因可能发生了突变，影响酵母细胞的生长。 4. 呼吸缺陷型酵母菌株因其线粒体 DNA发生变化及线粒体功能的改变， 使 呼吸缺陷型酵母菌株在不同剂量区的离子束辐照下表现不同辐射敏感 性。目的： 研究啤酒酵母的线粒体 DNA 在重离子辐照作用下的突变效应及其突变机 理。 材料与方法： 利用兰州重离子研究装置(HIRFL)加速的氖、碳离子辐照酵母细胞，用 TTC 显色培养基筛选呼吸缺陷型酵母菌株，并用 mtDNA 限制性酶切手段分析其突变 规律。采用 PCR扩增并对目的产物测序的方法对辐照后线粒体DNA上的 I 类内 含子和 II类内含子进行研究。 结果： 1. TTC 显色实验表明：离子束辐照导致酵母线粒体上的电子传递链发生改 变，产生的还原氢减少，造成呼吸缺陷。 2. 利用限制性酶切实验对线粒体 DNA进行研究，结果表明：离子束辐照诱 变筛选出来的呼吸缺陷型酵母菌株其线粒体DNA变化明显： 主要表现为 酶切条带缺失严重。即使在同一注量下筛选出来的呼吸缺陷型酵母菌株， 其酶切图谱也不相同。 3. 通过 PCR 手段对辐照后酵母线粒体 DNA 碱基序列进一步进行分析，发 现经不同注量离子束辐照后筛选出来的呼吸缺陷型酵母菌株，其I 类内含 子（ai4 and ai5）经设计不同引物进行扩增，没有获得目的条带，说明此 序列发生了突变，可能对离子束辐照比较敏感。 4. 经不同注量离子束辐照后筛选出来的呼吸缺陷型酵母菌株，其 II 类内含 子（ai2）的碱基序列与野生型相比没有变化，表现出在离子束辐照作用 下比较稳定的特性。

Purification and structure identification of hyaluronic acid

Polysaccharide produced by mutated strain of Streptococcus zooepidemicus was purified by the procedures including Savage method, quaternary ammonium compound precipitation, DEAE-cellulose(DE52) chromatography and Sephadex G-75 gel filtration. The structure of the purified polysaccharide has been characterized by means of chemical composition analysis, C-13 NMR spectrum, infrared spectrum and circular dichroism (CD). All the results showed that the purified polysaccharide was hyaluronic acid (HA). The single helix conformation of the purified HA was determined by Congo red experiment. The molecular weight of the HA was about 1.16x10(6)D, which was measured by viscosity method.

Human catalytic antibodies with glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens. Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library. The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25. In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E. coli. After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/mu mol and 28.8U/mu mol, respectively. With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied. (C) 2001 Elsevier Science BY. All rights reserved.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e. g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e. g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo-induced antigen technology (IVIAT). Methods and Results: The convalescent-phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo-induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion-mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo-expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.