Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system

The human adenovirus type 5 E1A, a tumor- suppressor gene[1], codes for two major related proteins of 243 amino acids (12S) and 289 amino acids (13S) by al-ternative splicing in two exons[2]. Studies have been shown that E1A can regulate expression of many genes and cell cycle[3]. Both in vitro and in vivo experiments indicated that E1A could induce tumor cells differentia-tion, convert tumor cells into an epithelial phenotype, in-hibit tumor cell growth and metastasis and strongly en-ha…     

Inhibitory effect of the adenovirus type 5 E1A protein expressed in the yeast system on the human tumor cell growth

Adenovirus 5 type E1A as a tumor suppressor gene can inhibit tumor growth and enhance the sensitivity of chemotherapy and radiotherapy. E1A have the ability to integrate into the host genome, resulting in long-time expression that induces Rb gene inactivation and animal cells immortalization. This prompted us to select the E1A protein for treatment of cancer in order to overcome the limitations of E1A gene therapy. Thus, we firstly constructed E1A eucaryotic expression vector (pPIC9/E1A), transformated the pichia pastoris yeast cells (GS115) and screened the high-expressing recombinant strains. The positive yeast strains were cultured in the shake flask, and induced for 3 d. The crude E1A protein was purified using two steps of column chromatography on HiTrap Q and HiTrap SP. The purified E1A protein was identified by SDS-PAGE and Western blot. E1A protein was mostly located at cellular nuclear when Chariot delivered E1A protein into cells. The analysis in vitro indicated that the E1A protein arrested LN686 cell cycle at G2/M phase, and significantly inhibited the growth of LN686 tumor cells. The current studies firstly provided an experimental basis to further develop E1A protein for tumor treatment.     

白高斯噪声对肿瘤增长系统的影响

目的研究受白高斯噪声驱动的肿瘤细胞增长系统的定态性质。方法从理论和数值两方面对系统的定态进行分析。结果该系统的定态概率密度随乘性噪声强度D的增长而减小,而加性噪声强度α对定态概率密度几乎没有影响。结论乘性噪声强度D的增长能够引起肿瘤数的减少。     

An integrative model and analysis of cell cycle in fission yeast

Cell cycle is a programmed process, during which a cell proliferates to two daughter cells. The eukaryotic or-ganisms share the same characters, such as four cycle phases G1, S, G2 and M, the evolutionally conserved cell cycle proteins and its dependent kinases, and the check-points mechanism[1,2]. Due to the different functions and the complicated interactions of these proteins involved in cell cycle, it is very difficult to understand the regulatory mechanism of cell cycle in a whole sense …     

人源CPP32基因表达对酵母细胞生长的影响

为研究人源ＣＰＰ３２基因的表达对酵母细胞生长的影响,了解其所编码的蛋白质在分子进行过程中的功能特性,将不原ＣＰＰ３２基因克隆到表达载体ｐＧＢＴ９中,得到重组质粒并命名为ｐＧＢＴ９／ＣＰＰ,再将其和对照质粒ｐ（ＧＢＴ９）分别转化到ＣＧ１９４５和ＨＦ７ｃ酵母细胞中,一定时间测定培养物的ＯＤ６００值并做出生长曲线。结果表明：诱导真核细胞程序性死亡的人源ＣＰＰ３２基因对不同种的酵母细胞的作用不同;转化到宿     

小鼠睾丸特异表达新基因(mtLR1)在大肠杆菌中的表达

根据已经克隆的mtLR1基因序列,采用RT-PCR的方法获得mtLR1基因。将所得的PCR产物插入原核表达载体pMAL-P-2x中,得重组质粒(pMAL-P-2x/mtLR1)并转化大肠杆菌(E．coli)DH5α．经IPTG诱导表达,SDS-PAGE电泳分析显示,重组蛋白得到了正确表达,表达的融合蛋白占菌体蛋白总量的52％,分子质量约为73 kDa。mtLR1蛋白的高效表达,为研究其生物学功能和制备单克隆抗体奠定了基础。     

桑黄菌质多糖体外抑瘤及抗环磷酰胺致突变的作用

采用MTT法研究桑黄固态发酵菌质多糖（PISPS）对肝癌细胞（HepG-2）、乳腺癌细胞（MCF-7）生长的抑制作用，测定出多糖对细胞生长的最高抑制率为59．18％、55．39%，半数抑制浓度为196.9、162．5μg／mL，且随药物浓度的增加抑制率有增大的趋势，与正常组比较作用较为显著（P〈0．05）；选用小鼠骨髓嗜多染红细胞微核试验和小鼠精子畸形试验，对多糖的抗环磷酰胺（CP）致突变作用进行评价，结果表明桑黄菌质多糖使环磷酰胺诱发的细胞微核率和精子畸形率明显降低（P〈0．01）．     

Expression, purification of a synthetic fuse-protein-TSF from PF4 and TSP1 fragments and its effect on angiogenesis and tumor growth

Sequences encoding PF4 (58–70) and TSP1 (429–459) were linked to yield a single gene TSF which encodes the fuse-protein of TSF. The gene was cloned into a pGEX-2T expression vector to generate a protein GST-TSF, which was strongly expressed inE. coli. The purified GST-TSF was degraded with thrombin to generate the protein TSF. With the methods of MTT and wound repair assay, the effects of TSF on the proliferation and migration of EC were detected, respectively. The results showed that TSF significantly suppressed BAEC proliferation and migration in a dose-dependent manner. The fuse protein GST-TSF, and the peptides PF4 (58–70) and TSP1 (429–459) also inhibited BAEC proliferation and migration, respectively, but their inhibition rates were not as high as TSF. Using the CAM assay, it was shown that TSF, GST-TSF, PF4 (58–70) and TSP1 (429–459) inhibited angiogenesis in chick CAM potentially, the effect of TSF was the highest.In vivo, the growth of Lewis lung carcinoma was potently inhibited by TSF treatment, and the inhibition rate was 68.75% at a dose of 1.00 μmol/kg · d. These findings suggest that the design on TSF gene was successful, and TSF with its anti-angiogenic and anti-tumor activity, should be a useful source of the inhibitor.     

关联色噪声对具有免疫监视下肿瘤细胞生长系统稳定性和瞬态性质的影响

基于具有催化作用的Michaelis-Menten反应模型,得到一种更全面的具有免疫监视下肿瘤细胞生长系统的一维朗之万方程.考虑肿瘤细胞增殖过程中生长环境等因素的变化而导致生长率和死亡率的波动,通过线性化近似和最快下降等方法计算系统的稳态概率分布函数和平均首次通过时间,并以此作为观察肿瘤细胞生长过程中的稳定性和瞬态性质的窗口,分析噪声对肿瘤细胞生长过程的影响.结果表明：1)当改变噪声强度D和Q时,肿瘤细胞数概率分布函数Pst(x)的结构会发生改变,系统产生多极值和单极值之间的结构转换,导致系统产生类相变;2)平均首次通过时间T＋(xu→x＋)是乘性噪声强度D的单调增函数,T－(xu→x－)却是D的单调减函数;3)T＋(xu→x＋)随加性噪声强度Q变化的图像出现一个类似“共振峰”的极大值,而T－(xu→x－)是关于Q的减函数;4)T＋(xu→x＋)随噪声间关联时间τ变化时,表现出单调递减和“共振峰”两种状态的转换,而T－(xu→x－)的图像随τ的变化呈现出单峰值样态;5)T＋(xu→x＋)随噪声间关联强度λ的变化出现类似于“共振峰”的极大值,T－(xu→x－)是λ的单调减函数.上述结果有助于人们掌握肿瘤细胞的生长动力学特性,不仅能够为临床上肿瘤细胞生长研究提供一定理论基础,而且还能为临床医学上肿瘤检测和治疗提供理论指导,对控制和治疗肿瘤疾病有一定的指导意义.     

人细胞周期蛋白D1在大肠杆菌BL21中的表达及纯化

将人细胞周期蛋白D1全长cDNA克隆入原核表达载体pET-28c(+)中, 经酶切和测序鉴定正确的重组质粒转化E.coli BL21(DE3)后获得表达菌株. 该菌株经IPTG诱导高效表达出带有组氨酸标签的以包涵体形式存在的融合蛋白, 表达量占菌体总蛋白的23%. 包涵体经洗涤和溶解， 在变性条件下利用Ni²⁺螯合柱纯化、 尿素梯度复性后， 得到纯度达98%以上的纯化蛋白. SDS-PAGE显示纯化蛋白的分子量约为43 ０００， Western-b lot分析表明， 在相应分子量处有一特异性条带， 说明成功表达和纯化重组人细胞周期蛋白D1.     

Effect of GbKTN1 from Gossypium barbadense on cell elongation of fission yeast（Schizosaccharomyces pombe）

The GbKTN1 gene was isolated from 10 DPA fiber cells of Gossypium barbadense using 5′RACE/3′RACE.Full-length cDNA of this gene is 2006 bp, including a 113 bp of 5′untranslated region, a 1563 bp of an open reading frame(ORF), and a 327 bp of 3′untranslated region (excluding the stop codon TAA). The ORF of GbKTN1 encodes a 521-amino acid protein with a predicted size of 55 kD. Near C-terminal of the deduced protein there is a putative ATP binding site between amino acid residues from 233 to 414. Southern blot analysis indicated that the GbKTN1 was a single copy gene in G barbadense. Combining semi-quantitative RT-PCR with Southern blot hybridization revealed that GbKTN1 expressed in all the organs detected such as roots, stems, leaves and fibers. However, the mRNA of GbKTN1 was the most abundant in fiber cells, while it was the lowest in leaves. The GbKTN1 cDNA was transformed into S. pombe to verify its function on cell elongation. Results showed that most yeast cells over expressing GbKTN1 gene were elongated dramatically with an average length increase of 2.18 times than that of the non-induced cells. Even the morphology of some yeast cells appeared irregularly. To the best of our knowledge this is the first evidence that KTN1 is correlated with cell elongation in vivo.     

流加不同物质对大肠杆菌DB15(pAET-8)表达人表皮生长因子的影响

考察了在大肠杆菌DB15(pAET-8)表达人表皮生长因子(hEGF)过程中流加不同的物质——磷酸盐、蛋白胨和酵母抽提物等对人表皮生长因子表达的影响.在表达阶段单独流加磷酸盐虽然增加了表达初期的比生长速率,延长了菌体生长期,提高了菌体浓度,但hEGF产量却比对照组减少了57%.在表达阶段流加蛋白胨或酵母抽提物也未能提高hEGF的表达水平.表明氨基酸或核苷酸合成并不是hEGF表达的限速步骤,提高大肠杆菌DB15(pAET-8)合成三磷酸腺苷(ATP)的能力或许是提高hEGF表达的关键.     

截短型HCV NS5B RNA聚合酶在大肠杆菌中表达纯化及鉴定

为构建HCV截短型ns5bΔ21基因的原核表达质粒;在大肠杆菌中表达NS5B蛋白并纯化,制备其抗体。运用软件设计引物,以BB7复制子为模板PCR扩增ns5bΔ21基因;克隆入原核表达载体pRSETA。将重组表达质粒pRSETA-ns5bΔ21转化BL21大肠杆菌,并诱导表达、可溶性分析及纯化;用纯化的NS5B蛋白免疫小鼠制备多克隆抗体。结果酶切鉴定和序列测定表明成功的构建了重组表达质粒pRSETA-ns5bΔ21;其经IPTG诱导后行SDS-PAGE可见新生表达条带,Western-blot证实了其特异性和抗原性良好;利用镍离子亲和层析和电洗脱方法获得了纯化NS5B蛋白;用纯化蛋白免疫小鼠制备出了多价抗血清。说明表达和纯化的截短型HCV NS5B RNA聚合酶可用于下一步筛选单链抗体。     

Regulation of activin A in cell proliferation and hormone secretion by human normal trophoblast cells

Regulation of activin A in cell proliferation as well as hCG and progesterone secretion was investigated using primary cultured cytotrophoblast cells and normal placenta origin cytotrophoblast cell line-NPC cells in serum-free system. It was shown that activin A promoted hCG and progesterone secretion in primary cultured cytotrophoblast cells as well as progesterone secretion in NPC cells, while it had no effect on cell proliferation and hCG secretion in NPC cells. Important evidence is provided for the autocrine regulatory mechanism of activin A on hormone secretion in placental trophoblast cells at early pregnancy.     

Effect of P15INK4b expression on the cell cycle and G1 phase related regulatory genes in human melanoma cells

Using the transfeetion teehnique. P15INK4b was introduced into P15INk4b gene deleted human melanoma A375 cells,and a cell model MLED6 overexpressing P15INK4b WAS CONSTRUCTED.Comparing with the control cells MLC2,MLEK6cells in G1phase increased by 11%,but those in Sphase decreased by 15%by FCM.By the method of thymidine(TdR)and N2O arresting,the proportions of synchronized Mphase cells of MLEK6 ana MLC23 were measured and found to be 89.1% and 76.8%respectively ,and the cells in G1phase were 74.3% for MLID6 AND 76. 4% forMLC2.The result of3 H-TdR incorporation indicated that the transition of G1/Sof MLEK6 cell was delayed 2h as compared with that of MLC2 cells,and incorporation rate also decreased.The observation on exprissions of some G1/ S-resates relatory rigusating genes showed that in MLIK6 cells the protein leves of P27KIPI increased with the decreasing expressions of cyclinD1,cyclinE and c-myc,especially cyclinD1 in late G1phade.The expression of cyclinE obviously decreased at G1/S transition ,and c-myc wad inhibited throughout all the process of G1 S phase.All the risults suggest that P15INK4b can delayG1/S transition of MLEK6 cells by inhibiting the cell cycle engine ,and by increasing the expression of Cdk ingibitor P27KIPI in different stages of G1 phase.     

RBM5蛋白的RNA识别区在肿瘤细胞周期调控中作用的研究

为分析RNA识别区(RNA recognition motif,RRM)在RBM5蛋白调控肿瘤细胞周期中的作用,本研究利用低表达RBM5的A549细胞系构建了A549/Vector、A549/RBM5-WT、A549/RBM5-△RRM三种稳定细胞株,通过流式细胞术检测,发现仅有A549/RBM5-WT能显著阻滞细胞于G1期,说明RRM区是RBM5抑制细胞周期进程必不可少的区域.利用Western Blot检测细胞中cyclin A与Phospho-Rb(ser 795)的蛋白水平差异,证明了RBM5蛋白的RRM能够参与其介导的pRb去磷酸化以及抑制cyclin A表达,从而抑制细胞周期进程.     

The effect of pmpR on the type III secretion system in Pseudomonas aeruginosa

The type III secretion system(T3SS) plays important roles in Pseudomonas aeruginosa pathogenicity.Previously,we reported that the uncharacterized protein PmpR could regulate pqsR,an important regulator in the quorum-sensing system,by directly binding to its promoter region.As the T3SS is controlled by the quorum-sensing system,here,we investigated the relationship between PmpR and the T3SS.Our data showed that expression of the T3SS genes exoS,exoY,exoT,and exsD was dramatically increased in a pmpR-deletion mutant compared with that in the wild-type P.aeruginosa strain PAO1.Data from DNA mobility assays indicated that PmpR affects the T3SS indirectly.It is unlikely that PmpR controls the T3SS via the Pseudomonas quinolone signal(PQS) because the PQS negatively regulates the T3SS,while pmpR negatively regulates the PQS.The effect of PmpR on the T3SS seems to be independent of the PQS;further investigation is required to uncover the underlying regulatory pathways.     

高校体育理论网络课程教学系统的开发与研制

着重介绍了高校体育理论网络课程教学系统的开发与研制,旨在探(?)高校体育理论课教学的新途径。