RNA干扰ABCE1基因后可增加肺癌95-D/NCI-H446细胞的E-钙黏附蛋白表达并减低细胞侵袭力

研究ABCE1对肺癌(95-D和 NCI-H446)细胞的作用．使用RNA干扰技术,抑制ABCE1基因的表达,通过Western blot 分析及FACS检测,观察ABCE1基因对E-钙黏附蛋白在95-D/NCI-H446细胞表达的影响;运用transwell 侵袭实验,观察M95-D/ NCI-H446细胞侵袭力的变化．RNA干扰ABCE1基因后,实验组与对照组相比,在48 h后可显著抑制肺癌(95-D和 NCI-H446)细胞ABCE1蛋白的表达,同时,伴随E-钙黏附蛋白的高表达,以及细胞侵袭力的降低． ABCE1基因与E-钙黏附蛋白相关,抑制ABCE1基因可增加肺癌95-D/NCI-H446细胞的E-钙黏附蛋白的表达,减低细胞的侵袭力．     

靶向糖基转移酶shRNA表达质粒对LoVo细胞侵袭和增殖能力的抑制作用

通过构建针对Ｎ-乙酰氨基葡萄糖转移酶Ⅴ(GnT-Ⅴ)的小片段发夹状RNA(shRNA)干扰表达质粒,研究了shRNA表达质粒沉默GnT-Ⅴ基因后对LoVo细胞增殖、黏附以及迁移、侵袭能力的影响．设计了靶向GnT-Ⅴ基因的小干扰RNA(siRNA)靶序列,构建shRNA表达载体并转染人结肠癌LoVo细胞,通过G418筛选建立稳定低表达GnT-Ⅴ基因的细胞株．分别采用半定量逆转录聚合酶链反应(RT-PCR)和蛋白质印迹(Western blot)检测shRNA对GnT-Ⅴ基因mRNA及蛋白质表达的影响．并通过CCK-8增殖实验、异质黏附实验、划痕愈合实验、趋化运动实验、细胞侵袭实验评价pGPU6/GFP/Neo GnT-Ⅴ shRNA对人结肠癌LoVo细胞增殖、黏附以及迁移、侵袭能力的影响．实验成功地构建了GnT-Ⅴ shRNA表达质粒,并且该质粒明显下调GnT-Ⅴ的表达,LoVo GnT-Ⅴ/1564和LoVo GnT-Ⅴ/2224的mRNA水平的抑制率分别为82%和71.5%,蛋白质水平的抑制率分别为68%和56%．选择干扰效率较高的LoVo GnT-Ⅴ/1564进行进一步实验．CCK-8增殖实验显示,与阴性对照组相比,LoVo GnT-Ⅴ/1564的增殖受到明显抑制(P < 0.001),尤以72 h为著;下调GnT-Ⅴ表达可增强LoVo细胞的黏附能力( t = -3.357,P < 0.01),而显著抑制LoVo细胞的趋化运动能力( t = 44.051,P < 0.001);划痕实验结果也显示抑制GnT-Ⅴ表达延长LoVo细胞的愈合时间;用Matrigel胶介导的细胞侵袭实验结果显示,LoVo GnT-Ⅴ/1564和LoVo GnT-Ⅴ/NC的穿膜细胞数分别为(5.10 ± 1.25)个和(39.55 ± 2.16)个,GnT-Ⅴ/1564组较阴性对照组明显减少( t = 61.626,P < 0.001)．结果表明,靶向GnT-Ⅴ的shRNA真核表达质粒可以显著降低GnT-Ⅴ的表达,从而抑制LoVo细胞的增殖、迁移和侵袭能力,因此,该GnT-Ⅴ的siRNA序列可能成为治疗结直肠癌的有效靶点．     

幽门螺杆菌黏附素保守区蛋白的免疫原性、安全性和黏附作用的体外评价

探讨幽门螺杆菌（Hp）保守区（AB）蛋白的体外安全性、免疫原性和黏附作用,以确定AB在Hp疫苗研制中的应用价值.ELISA法测定Hp感染者血清中抗AB抗体,四唑盐比色法（MTT）测定T细胞对AB的增殖反应,流式细胞术检测AB致T细胞表达FasL的作用,二苯胺（DPA）法测定AB致T细胞凋亡率,光镜计数法研究AB抗体对Hp与胃癌细胞黏附的影响.ELISA法共检测了55份血清,以快速尿素酶实验(RUT)作为平行对照,两法的评价判断一致性程度的指标卡帕系数为0.76. 同时,低剂量AB即可刺激Hp⁺T细胞的增殖.体外安全性实验表明,AB无明显调节T细胞表达FasL的作用以及无明显致Hp^－T细胞凋亡的作用. AB抗血清能部分阻断Hp与胃癌细胞系的黏附,在光镜下表现为经抗AB兔血清预处理后,每细胞周围黏附的细菌数较免疫前兔血清预处理组显著减少（P＜0.05）.研究表明AB是安全的、具有免疫原性的Hp菌体成分,既可以刺激体液免疫,又能够提高细胞免疫,并且其抗体还可防止Hp与胃上皮细胞的黏附.     

Argonaute亚族蛋白对人类肿瘤细胞周期的影响

Argonaute(Ago)家族蛋白与小的非编码RNAs(siRNAs, miRNAs, piRNAs)生物发生和细胞功能密切相关．它可以结合小RNAs，调控蛋白质的合成或影响mRNA的稳定性，即RNA干扰(RNAi)，并且参与Piwi相关RNAs(piRNAs)的生成．人类Argonaute蛋白家族包括8个成员，对其中的4个(Ago1～Ago4)mRNAs进行了实时定量PCR(qRT-PCR)检测，其在许多细胞中共表达，与细胞生长紧密相关．针对人乳腺癌MCF7和子宫颈癌HeLa细胞系，通过下调Ago亚族蛋白表达量，研究其在细胞周期中的调控作用．MTT实验证明，Ago蛋白表达缺失导致细胞增殖活性显著下降(P < 0.01)，生长受阻．进一步研究揭示，细胞周期在G0/G1期发生停滞，其中Ago1(P < 0.01)和Ago4(P < 0.05)的作用尤为明显，但并不引发凋亡．虽然具体调控机制尚不清晰，但在人类肿瘤细胞中Argonaute蛋白可直接或间接地参与细胞周期进程的调控．     

口蹄疫病毒样颗粒诱导表达型载体的构建及其BHK-21细胞池的筛获

瞬时表达是目前利用哺乳动物细胞表达口蹄疫病毒(foot-and-mouth disease virus, FMDV)衣壳蛋白的主流方法。为实现染色体稳定表达FMDV衣壳蛋白并高效组装出病毒样颗粒(virus like particles, VLPs)，本研究构建了piggyBac (PB)转座-组成型表达、PB转座-四环素(tetracycline, Tet)诱导型表达两套质粒。利用荧光蛋白标记技术，验证了质粒的功能。通过抗生素筛选得到了组成型表达P12A3C (WT/L127P)基因的BHK-21细胞池(C-WT、C-L127P)和诱导型表达P12A3C (WT/L127P)基因的BHK-21细胞池(I-WT、I-L127P)。荧光观察和PCR检测证明了绿色荧光蛋白、3C蛋白酶、反向四环素转录激活因子等基因的稳定整合。Western blotting、酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)实验证明了细胞池I-L127P具有更强的衣壳蛋白和VLPs生产能力。本研究首次实现了哺乳动物细胞染色体诱导表达FMDV衣壳蛋白，有助于推动哺乳动物生产FMDV VLPs疫苗的技术工艺，也为构建其他蛋白的哺乳动物细胞诱导型表达系统提供了参考。     

智力低下相关蛋白(FXR1P)与CMAS相互作用的生物学效应研究

脆性X综合征(FXS)是一种遗传性智力低下疾病,其发病率仅次于21三体综合征．脆性X智力低下蛋白(FMRP)是FXS的关键性致病因子,该蛋白由脆性X智力低下基因1(FMR1)编码所得．FMR1在神经肌肉和睾丸组织中广泛表达．脆性X相关蛋白1(FXR1P)则是由FMR1的同源基因脆性X相关基因1(FXR1)编码所得,并且与蛋白质和RNAs之间存在着相互作用．许多疾病都涉及到FXR1表达的改变．为了了解FXR1P与CMAS(胞嘧啶单核苷酸-N-乙酰神经氨酸合成酶)相互作用所产生的的生物学效应,我们构建了FXR1的过表达载体,并观察其在PC12细胞(大鼠鼠肾上腺嗜铬细胞瘤细胞)和VSMC(血管平滑肌细胞)中的表达以及继而对于细胞形态和CMAS活性相关的许多细胞指标的效应．我们证实,FXR1基因的过表达可以提高PC12细胞中CMAS的活性,并对于该类细胞的生长提供一定程度的保护作用．PC12细胞是一种较为常见的用于研究神经系统疾病的细胞系．结论：我们推测FXR1P是一个组织特异调节因子,可以改变PC12细胞而非VSMC细胞中神经节苷酯(GM1)的浓度．     

67kD层粘连蛋白受体的克隆、表达及其对肝癌细胞侵袭能力的影响

探讨细胞膜表面 6 7kD层粘连蛋白受体 (6 7kDlamininreceptor ,6 7LR)在肝癌细胞侵袭转移中的作用 ,从肝癌细胞中提取RNA ,通过RT PCR扩增 6 7LR的前体——— 37kD层粘连蛋白受体前体(37kDlamininreceptorprecursor,37LRP)基因并定向克隆到真核表达载体pcDNA3.1 myc His(- )A ,采用脂质体将重组质粒转染到HepG2肝癌细胞中 ,通过G4 1 8筛选和RT PCR、流式细胞术鉴定 ,获得了细胞膜表面 6 7LR高表达 (阳性率为 6 9.2 % )和低表达 (阳性率为 1 1 .7% )的细胞克隆 ,采用体外细胞侵袭实验测定不同细胞的侵袭能力 ,发现膜表面 6 7LR高表达的细胞侵袭能力明显高于低表达及不表达细胞 ,说明 6 7LR在肝癌细胞侵袭转移过程中可能具有重要意义     

人67kD层粘连蛋白受体在毕赤酵母中的表达及活性分析

为实现人67kD层粘连蛋白受体(Human 67kD Laminin Receptor,67LR)蛋白的分泌表达,采用DNA重组技术将67LR cDNA片段插入分泌型酵母表达载体pPIC9K中,构建了相应的重组表达质粒pPIC9K-67LR并在GS115毕赤酵母菌株中表达,每升培养基经亲和层析可纯化目的蛋白12.56mg。纯化的目标蛋白能够与肺癌A549细胞竞争性结合其配体分子LN-1,具有相应的生物学活性,从而为深入研究人67LR的结构与功能奠定了基础。     

共轭亚油酸单体诱导乳腺癌细胞SKBr3凋亡的PPARγ信号通路研究

cis9,trans11-CLA和trans10,cis12-CLA是共轭亚油酸(CLA)二种抑瘤活性最强的主要单体．在以前报道二者诱导乳腺癌细胞凋亡的工作基础上,进一步探讨共轭亚油酸单体诱导乳腺癌细胞SKBr3凋亡的途径及机制．采用RT-PCR和　　Western blot等方法,证实了CLA在SKBr3细胞中可显著提高PPARγ 的转录及蛋白质表达水平,并发现CLA对PPARγ与凋亡相关蛋白Bax、Bcl-2和caspase3的表达影响呈同步相关性,并表现出时间和剂量依赖性．通过PPARγ 抑制剂GW9662实验表明它们之间存在协同关系．首次提出了PPARγ -Bcl-2-Caspase3信号通路的SKBr3细胞凋亡途径,为CLA有望作为PPARγ新型调节剂诱导肿瘤细胞凋亡在临床应用提供实验证据．     

炭疽芽胞杆菌BslA(260－652)蛋白的表达纯化与黏附功能鉴定

【目的】克隆表达炭疽芽胞杆菌BlsA的功能区片段并对其生物学功能进行鉴定。【方法】以炭疽芽胞杆菌A16R基因组DNA为模板PCR扩增bslA(260-652)基因片段,克隆至pET-28a(+)载体。将成功构建的重组质粒转化入大肠杆菌Rosetta(DE3)中,诱导表达后收集菌体经超声破碎后,对可溶表达部分用镍柱进行亲和层析纯化。以纯化后的蛋白为抗原,免疫BALB/c小鼠制备该蛋白的多抗,用ELISA和Western blot检测抗血清;使用间接免疫荧光实验和细菌黏附实验研究目标蛋白及其抗体的生物学功能。【结果】BslA(260-652)获得了可溶性表达,纯化后纯度约为87.4%。以纯化蛋白为抗原,免疫BALB/c小鼠制备的抗血清ELISA效价可达1∶20000。将BslA(260-652)蛋白与Hela细胞共孵育后,能够直接和Hela的细胞膜结合。细菌黏附实验表明BslA(260-652)蛋白及其相应的多抗血清都能够显著地抑制炭疽芽胞杆菌A16R对Hela细胞的黏附。【结论】大肠杆菌表达得到的炭疽芽胞杆菌BslA(260-652)蛋白具有与天然蛋白相似的生物活性,为深入研究BslA蛋白在炭疽芽胞杆菌致病过程中的作用奠定实验基础。     

Structural Basis for Ubiquitin Recognition by Ubiquitin-Binding Zinc Finger of FAAP20

Several ubiquitin-binding zinc fingers (UBZs) have been reported to preferentially bind K63-linked ubiquitin chains. In particular, the UBZ domain of FAAP20 (FAAP20-UBZ), a member of the Fanconi anemia core complex, seems to recognize K63-linked ubiquitin chains, in order to recruit the complex to DNA interstrand crosslinks and mediate DNA repair. By contrast, it is reported that the attachment of a single ubiquitin to Rev1, a translesion DNA polymerase, increases binding of Rev1 to FAAP20. To clarify the specificity of FAAP20-UBZ, we determined the crystal structure of FAAP20-UBZ in complex with K63-linked diubiquitin at 1.9 Å resolution. In this structure, FAAP20-UBZ interacts only with one of the two ubiquitin moieties. Consistently, binding assays using surface plasmon resonance spectrometry showed that FAAP20-UBZ binds ubiquitin and M1-, K48- and K63-linked diubiquitin chains with similar affinities. Residues in the vicinity of Ala168 within the α-helix and the C-terminal Trp180 interact with the canonical Ile44-centered hydrophobic patch of ubiquitin. Asp164 within the α-helix and the C-terminal loop mediate a hydrogen bond network, which reinforces ubiquitin-binding of FAAP20-UBZ. Mutations of the ubiquitin-interacting residues disrupted binding to ubiquitin in vitro and abolished the accumulation of FAAP20 to DNA damage sites in vivo. Finally, structural comparison among FAAP20-UBZ, WRNIP1-UBZ and RAD18-UBZ revealed distinct modes of ubiquitin binding. UBZ family proteins could be divided into at least three classes, according to their ubiquitin-binding modes.     

Variation in microsomal subfractions obtained from normal animal tissues and transformed cells following cell disruption by nitrogen cavitation

Summary Microsomal membranes were obtained from MPC-11 cells, L-cells, Krebs II ascites cells and various normal animal tissues following cell disruption by nitrogen cavitation. Membrane preparations were applied to discontinuous sucrose gradients designed to separate three fractions — heavy rough (HR), light rough (LR) and smooth (S) microsomes. In each of the transformed cell lines all three fractions were found whilst in the normal tissues tested the HR fraction was absent. Of the normal tissues liver and pancreas were rich in both LR and S microsomes, the presence of large amounts of LR indicating a rich protein synthesizing activity on membrane-bound polysomes. Kidney also contained appreciable LR but much less than both liver and pancreas. Both heart and lung contained virtually only S microsomal material — a reflection of low protein synthetic activity on membrane-bound polysomes. Attempts to promote the appearance of the HR fraction in liver, kidney and pancreas by incubation in tissue culture medium, or, in the case of pancreas, by cholecystokinin/pancreozymin/secretin, stimulation bothin vivo andin vitro were unsuccessful.     

Identification of FAAP24, a Fanconi anemia core complex protein that interacts with FANCM

The Fanconi anemia (FA) core complex plays a crucial role in a DNA damage response network with BRCA1 and BRCA2. How this complex interacts with damaged DNA is unknown, as only the FA core protein FANCM (the homolog of an archaeal helicase/nuclease known as HEF) exhibits DNA binding activity. Here, we describe the identification of FAAP24, a protein that targets FANCM to structures that mimic intermediates formed during the replication/repair of damaged DNA. FAAP24 shares homology with the XPF family of flap/fork endonucleases, associates with the C-terminal region of FANCM, and is a component of the FA core complex. FAAP24 is required for normal levels of FANCD2 monoubiquitylation following DNA damage. Depletion of FAAP24 by siRNA results in cellular hypersensitivity to DNA crosslinking agents and chromosomal instability. Our data indicate that the FANCM/FAAP24 complex may play a key role in recruitment of the FA core complex to damaged DNA.     

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.     

The Expression of Membrane-Associated 67-kDa Laminin Receptor (67LR) Is Modulated in Vitro by Cell-Contact Inhibition

The interaction of cells with their substratum is an important determinant of cell behaviour, influencing attachment, proliferation, and motility. Such interactions are mediated by cell surface receptors which bind to attachment factors, like the glycoprotein laminin in basement membranes. We have previously shown that expression of the 67-kDa laminin receptor (67LR) is elevated in proliferating retinal microvasculature compared with mature, quiescent vessels. Here, we examined 67LR mRNA and protein expression in primary cultures of retinal microvascular endothelial cells (RMEC) and in the breast cancer cell-line T47D during stages of contact inhibition. In both cell types, the expression levels of 67LR mRNA and membrane-associated 67LR protein were significantly increased during the proliferative phases of monolayer formation. As the cells achieved contact inhibition, 67LR expression was reduced to comparatively low levels. Thus, the differential expression of 67LR between dividing and contact-inhibited cells may indicate a role for this receptor during proliferative processes.     

Structure analysis of FAAP24 reveals single-stranded DNA-binding activity and domain functions in DNA damage response

The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)₂ domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation.     

Ultrastructure and polysome content of the microsomal subfractions of mouse plasmacytoma cells

Summary The endoplasmic reticulum (ER) of MPC-11 cells released as vesicles upon cell disruption by nitrogen cavitation was separated from the bulk of mitochondria, lysosomes and plasma membranes by a low speed centrifugation. The ER membranes were fractionated on discontinuous sucrose gradients into heavy rough (HR), light rough (LR) and smooth (S) membranes. The morphology of subcellular fractions was studied by electron microscopy and the ER membranes were shown to be virtually free of contaminating organelles. The S fraction was easily distinguishable because of the lack of ribosomes but there were no apparent morphological differences between the HR and LR fractions. Of total activity in the microsomal subfractions, 70% of the UDPase and 67% of the 5′-nucleotidase activity was associated with the S fraction. Polysomes were present in the HR, LR and nuclear-associated ER fractions but not in the S fraction. The HR and LR fractions did not appear to be contaminated to any great extent with free polysomes. RNA/protein and RNA/phospholipid ratios of the HR fraction were higher than those of the LR fraction, indicating a greater density of ribosomes in the former fraction. These ratios were much lower in the S fraction reflecting the low ribosome content.     

A ubiquitin-binding protein, FAAP20, links RNF8-mediated ubiquitination to the Fanconi anemia DNA repair network

The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.     

FANCM and FAAP24 function in ATR-mediated checkpoint signaling independently of the Fanconi anemia core complex

The Fanconi anemia (FA) pathway is implicated in DNA repair and cancer predisposition. Central to this pathway is the FA core complex, which is targeted to chromatin by FANCM and FAAP24 following replication stress. Here we show that FANCM and FAAP24 interact with the checkpoint protein HCLK2 independently of the FA core complex. In addition to defects in FA pathway activation, downregulation of FANCM or FAAP24 also compromises ATR/Chk1-mediated checkpoint signaling, leading to defective Chk1, p53, and FANCE phosphorylation; 53BP1 focus formation; and Cdc25A degradation. As a result, FANCM and FAAP24 deficiency results in increased endogenous DNA damage and a failure to efficiently invoke cell-cycle checkpoint responses. Moreover, we find that the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, is required for its role in ATR/Chk1 signaling. Our data suggest that DNA damage recognition and remodeling activities of FANCM and FAAP24 cooperate with ATR/Chk1 to promote efficient activation of DNA damage checkpoints.