三氧化二砷下调MG-63骨肉瘤细胞IEX-1基因表达的转录调控机制研究

三氧化二砷(arsenic trioxide, As₂O₃)是中国传统中药砒霜的主要有效成分,最早应用于血液系统肿瘤的治疗,随后研究表明其对实体瘤也具有抑制细胞增殖并诱导凋亡的作用．早期研究发现,1.0 μmol/L的As₂O₃可以体外诱导骨肉瘤细胞系MG-63细胞凋亡,进一步的cDNA芯片分析、RT-PCR、RNA印迹证实细胞凋亡与As2O3干预后IEX-1基因表达下调有关．IEX-1为早期诱导应答基因,调节细胞生长和凋亡．通过荧光素酶分析,EMSA、蛋白质印迹等实验,发现As₂O₃干预骨肉瘤细胞系MG-63后能诱导p53蛋白表达上调,增加的p53蛋白通过与IEX-1的启动子结合,转录抑制IEX-1的转录,导致IEX-1基因表达下调．进一步证实了IEX-1与骨肉瘤的重要关系,同时也阐明As₂O₃诱导IEX-1基因表达下调的转录调控机制．     

竹红菌乙素作猝灭剂研究Fo构象方法的建立

线粒体F₁F_o复合体F_o部分a亚基的色氨酸荧光可被竹红菌乙素(hypocrellin B, HB)猝灭.不同温度下测定Stern-Volmer图的结果显示,猝灭常数(K_sv)随温度的增加而加大,时间衰变荧光的结果显示,荧光寿命随HB浓度的增加而减小,加入不同浓度的HB, F₁F_o复合体的吸收峰没有位移.这些实验结果支持动态猝灭机理.HB还具有有效猝灭浓度低,不影响酶的活力;在脂相和水相的分布比率可高达16 560∶1;实验操作简便等优点.因此HB可作为理想的疏水相荧光猝灭剂,研究与膜结合的F₁F_o复合体中镶嵌于膜脂内F_o的构象变化.     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

无机磷影响叠氮钠对ATP合成酶合成活性的抑制

利用ADP和放射性磷直接合成ATP的方法,研究了无机磷(P_i)和叠氮钠对猪心线粒体ATP合成酶（F₁F_O-ATPase）ATP合成活性的影响.结果发现无机磷除作为合成ATP的底物参与F₁F_O-ATPase的合成反应外,还对F₁F_O-ATPase的合成活性呈现抑制作用,在1 mmol/L ADP存在时,随着P_i浓度由0.01～10 mmol/L增加,抑制合成作用越来越强.与叠氮钠在低浓度时(小于1 mmol/L)只抑制ATP水解,不影响ATP合成的观点不同.实验结果显示0.1 mmol/L叠氮钠表观激活F₁F_O-ATPase的ATP合成活性,且激活程度与反应体系中所加P_i的浓度呈负相关.当固定P_i浓度（0.1 mmol/L）后,随着叠氮钠浓度的增加表观激活程度也在变化,叠氮钠与磷浓度相等时表观激活程度最大,直至叠氮钠浓度接近0.5 mmol/L时,开始呈现表观抑制现象,叠氮钠浓度高于1 mmol/L之后,就出现解偶联现象.     

Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用

为探讨Smac/DIABLO在过氧化氢(H₂O₂）所致C₂C₁₂肌原细胞凋亡中的作用,采用Hoechst 33258染色,观察H₂O₂ (0.5 mmol/L)处理C₂C₁₂肌原细胞不同时间后,细胞核形态学改变并计算凋亡核百分率,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带,利用细胞成分分离后蛋白质印迹分析H₂O₂是否导致Smac/DIABLO从线粒体释放,采用Caspase检测试剂盒及蛋白质印迹分析Caspase-3和Caspase-9的活化,转染Smac/DIABLO基因,观察Smac/DIABLO过表达对H₂O₂所致的C₂C₁₂肌原细胞凋亡的影响.结果表明:H₂O₂处理1 h后,Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放入胞浆,2 h更明显;H₂O₂处理4 h后,Caspase-3和Caspase-9活化,12 h达高峰;H₂O₂处理24 h后,C₂C₁₂肌原细胞显示特征性的凋亡形态改变,凋亡核百分率明显升高,DNA电泳出现明显“梯状”条带.与单纯过氧化氢损伤组相比,Smac/DIABLO高表达的C₂C₁₂肌原细胞经过氧化氢损伤组的Caspase-3和Caspase-9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显.结果表明,H₂O₂可导致Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放,促进Caspase-9和Caspase-3的活化而促进细胞凋亡的发生.     

NaHCO3胁迫对紫根水葫芦形态学指标和光合参数的影响

为研究NaHCO₃胁迫对紫根水葫芦的形态学指标及光合参数的影响,该文以紫根水葫芦为材料,采用不同浓度碱性盐NaHCO₃溶液处理成株紫根水葫芦,测定在NaHCO₃胁迫下紫根水葫芦的植株株高、根长、根冠比、生物量、含水量和光合参数[净光合速率(P_n)、胞间CO₂浓度(C_i)、蒸腾速率(T_r)、气孔导度(G_s)]。结果表明:紫根水葫芦在20 mmol·L^-1 NaHCO₃浓度下的水溶液pH值最为平缓; 在低浓度NaHCO₃溶液中(≤40 mmol·L^-1),相比CK,紫根水葫芦的形态学指标呈现增长或无显著影响情况,而在高浓度NaHCO₃溶液中(≥60 mmol·L^-1),随着NaHCO₃浓度的升高,紫根水葫芦形态学指标显著降低,且与NaHCO₃浓度呈负相关; NaHCO₃胁迫对紫根水葫芦的光合参数影响显著,随着NaHCO₃浓度的增加及试验处理时间的延长,紫根水葫芦的P_n呈持续下降的趋势,C_i、T_r和G_s整体呈上升趋势,其光合作用受到的主要是非气孔限制。综合分析显示,紫根水葫芦具有一定的耐NaHCO₃能力,能正常生存在不超过40 mmol·L^-1 NaHCO₃浓度的水体中,且能改善低NaHCO₃浓度下的水体pH值。     

人可溶性CD14基因克隆表达及功能研究

利用反转录PCR技术,从U₉₃₇细胞总RNA中,扩增编码人可溶性CD₁₄的基因序列,构建了重组表达质粒pEF1/HisC/sCD₁₄^348aa;用脂质体转染法,实现了在真核细胞中的高效表达;用免疫亲和层析纯化表达产物,纯度达90%以上;LPS刺激U₉₃₇细胞产生CD₁₄的变化,证明了表达产物具有结合LPS的功能。     

猪瘟病毒E2基因主要抗原区的克隆及原核表达

利用反转录PCR（RT-PCR）和套式PCR（nest Polymerase Chain Reaction ,nPCR）技术扩增出当前猪瘟流行毒（广西玉林株,GXYL）与中国猪瘟兔化弱毒（C-株）兔脾组织毒E₂基因的主要抗原区,将其克隆到表达载体pP_ROEX-HTb中,获得重组质粒pP_ROEX-GXYL和pP_ROEX-C,经PCR、酶切和序列分析鉴定表明,插入的位置、大小和读码框均正确。 SDS-PAGE检测表明,经重组质粒pP_ROEX-GXYL和pP_ROEX-C转化、诱导的受体菌能表达E₂基因主要抗原区蛋白,Western-blot检测表明,诱导表达的抗原蛋白能与猪瘟阳性血清发生特异性反应。      

血红蛋白A2现象发生机制的研究——“红细胞HbA2”为HbA2与HbA的结合产物

为了弄清血红蛋白A₂现象的发生机制,我们对“红细胞HbA₂”的化学组成进行了分析。“红细胞HbA₂”的双向电泳结果表明,它含有两种血红蛋白成分:一种相当于HbA,另一种很可能是溶血液HbA₂。其单向二次电泳结果也证明,它是由溶血液HbA₂和HbA所组成。结果初步说明,盘红细胞中HbA₂可能与HbA结合存在。两者可能有相互作用,也许这是产生血红蛋白A₂现象的原因。     

蓖麻毒素和凝集素LD50值的简易测定法

本文介绍了一种蓖麻毒素和凝集素LD₅₀测定的新方法。与经典LD₅₀测定法比较,该法简易、经济、快速,结果可靠,特别适用于蓖麻毒素和凝集素这类作用慢、毒性强的毒蛋白的急性毒性试验。     

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C(H)3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C(H)3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C(H)3 dimer were similar, but differences were observed. The reduced C(H)3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C(H)3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C(H)3 domain. Hence, these results may have implications for the stability of the intact antibody.     

Pharmacokinetics of engineered human monomeric and dimeric CH2 domains

Therapeutic monoclonal antibodies have several advantages over small molecule drugs and small proteins and peptides, including a long serum half-life. The long serum half-life of IgG is due, in part, to its molecular weight (150kDa) and its ability to bind FcRn. Both the CH2 and CH3 domains of Fc are involved in FcRn binding. Antibody fragments and antibody-like scaffolds have improved penetration into tissues due to their small size, yet suffer from a short serum half-life of less than one hour. The human CH2 domain (CH2D) of IgG1 retains a portion of the FcRn binding site, is amenable to modification for target binding, and may represent the smallest antibody-like scaffold retaining a relatively long serum half-life. Here we describe the generation of a dimeric CH2D (dCH2D) and determination of its pharmacokinetics (PK), as well as the PK of wild-type monomeric CH2D (mCH2D) and a short stabilized CH2D variant (ssCH2D) in normal B6 mice, human FcRn transgenic mice and cynomolgus macaques. The elimination half-life of dCH2D was 9.9, 10.4 and 11.2 hours, and that of ssCH2D was 13.1, 9.9 and 11.4 hours, in B6 mice, hFcRn mice and cynomolgus macaques, respectively. These half-lives were slightly longer than that of mCH2D (6.9 and 8.8 hours) in B6 and hFcRn mice, respectively. These data demonstrate that engineered CH2D-based variants have relatively long serum half-lives, making them a unique scaffold suitable for development of targeted therapeutics.     

The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis

Lysins are peptidoglycan hydrolases that are produced by bacteriophage and act to lyse the bacterial host cell wall during progeny phage release. Here, we describe the structure and function of a novel bacteriophage-derived lysin, PlyB, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342. This molecule comprises an N-terminal catalytic domain (PlyB(cat)) and a C-terminal bacterial SH3-like domain, SH3b. It is shown that both domains are required for effective catalytic activity against ATCC 4342. Further, PlyB has specific activity comparable to the phage lysin PlyG, an amidase being developed as a therapeutic against anthrax. In contrast to PlyG, however, the 1.6 A X-ray crystal structure of PlyB(cat) reveals that the catalytic domain adopts the glycosyl hydrolase (GH)-25, rather than phage T7 lysozyme-like fold. PlyB therefore represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.     

Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.     

The structure of human apolipoprotein E2, E3 and E4 in solution 1. Tertiary and quaternary structure

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer-dimer-tetramer-octamer for apoE2, monomer-dimer-tetramer for apoE3 and monomer-dimer-tetramer-octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273-278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230-260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.     

Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain

Synaptotagmin I has two tandem Ca(2+)-binding C(2) domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C(2)B domain, one of them in the cation-free form at 1.50 A resolution, two in the Ca(2+)-bound form at 1.04 A (two bound Ca(2+) ions) and 1.65 A (three bound Ca(2+) ions) resolution and one in the Sr(2+)-bound form at 1.18 A (one bound Sr(2+) ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca(2+) ions (sites 1 and 2). At higher Ca(2+) concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca(2+) coordination site (site 3). Divalent cation binding sites 1 and 2 in the C(2)B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca(2+) sites in the crystal structures, while it is coordinated to Ca(2+) in site 2 in the NMR structure. In the presence of Sr(2+), which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.     

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.     

Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels

All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.