Unique Expression of HRF20 (CD59) in Human Nervous Tissue

Damage to autologous tissue by complement is limited by several widely distributed membrane-associated glycoproteins which restrict the action of the complement in homologous species. These include decay accelerating factor (DAF), membrane cofactor protein (MCP) and 20 kDa homologous restriction factor (HRF20,CD59). Using immunohistochemical techniques, we examined the localization of these proteins in the centra] nervous system (CNS) and peripheral nervous system (PNS) using non-neurological human nervous tissue since some complement components have been demonstrated to be synthesized in the CNS. There was no evidence of parenchymal staining by anti-DAF or anti-MCP antibodies in either type of tissue except for the staining of the endothelium in capillaries. On the other hand, anti-HRF20 antibody clearly stained myelinated axons in the CNS as well as Schwann cells in the PNS. In addition, we detected positive staining by anti-DAF antibody in the PNS of a Paroxysmal nocturnal hemoglobinuria (PNH) patient who is genetically deficient in HRF20.     

Characterisation of the complement-regulatory proteins decay-accelerating factor (DAF,CD55) and membrane cofactor protein (MCP,CD46) on a human colonic adenocarcinoma cell line

 To avoid destruction by complement, normal and malignant cells express membrane glycoproteins that restrict complement activity. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and protectin (CD59), which are all expressed on colonic adenocarcinoma cells in situ. In this study we have characterised the C3/C5 convertase regulators DAF and MCP on the human colonic adenocarcinoma cell line HT29. DAF was found to be a glycosyl-phosphatidylinositol-anchored 70-kDa glycoprotein. Blocking experiments with F(ab′)₂ fragments of the anti-DAF monoclonal antibody BRIC 216 showed that DAF modulates the degree of C3 deposition and mediates resistance to complement-mediated killing of the cells. The expression and function of DAF were enhanced by tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β). Cells incubated with interferon γ (IFNγ) did not alter their DAF expression. Two MCP forms were expressed, with molecular masses of approximately 58 kDa and 68 kDa, the lower form predominating. MCP expression was up-regulated by IL-1β, but not by TNFα or IFNγ. Expression of DAF and MCP promotes resistance of colonic adenocarcinoma cells to complement-mediated damage, and represents a possible mechanism of tumour escape. Received: 18 July 1995 / Accepted: 4 January 1996     

Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b

The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 Å limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.     

Immunophysical exploration of C3d-CR2(CCP1-2) interaction using molecular dynamics and electrostatics

The formation of the complex between the d-fragment of the complement component C3 (C3d) and the modular complement receptor-2 (CR2) is important for cross-linking foreign antigens with surface-bound antibodies and C3d on the surface of B cells. The first two modules of CR2, complement control protein modules (CCPs), participate in non-bonded interactions with C3d. We have used computational methods to analyze the dynamic and electrostatic properties of the C3d-CR2(CCP1-2) complex. The interaction between C3d and CR2 is known to depend on pH and ionic strength. Also, the intermodular mobility of the CR2 modules has been questioned before. We performed a 10 ns molecular dynamics simulation to generate a relaxed structure from crystal packing effects for the C3d-CR2(CCP1-2) complex and to study the energetics of the C3d-CR2(CCP1-2) association. The MD simulation suggests a tendency for intermodular twisting in CR2(CCP1-2). We propose a two-step model for recognition and binding of C3d with CR2(CCP1-2), driven by long and short/medium-range electrostatic interactions. We have calculated the matrix of specific short/medium-range pairwise electrostatic free energies of interaction involved in binding and in intermodular communications. Electrostatic interactions may mediate allosteric effects important for C3d-CR2(CCP1-2) association. We present calculations for the pH and ionic strength-dependence of C3d-CR2(CCP1-2) ionization free energies, which are in overall agreement with experimental binding data. We show how comparison of the calculated and experimental data allows for the decomposition of the contributions of electrostatic from other effects in association. We critically compare predicted stabilities for several mutants of the C3d-CR2(CCP1-2) complex with the available experimental data for binding ability. Finally, we propose that CR2(CCP1-2) is capable of assuming a large array of intermodular topologies, ranging from closed V-shaped to open linear states, with similar recognition properties for C3d, but we cannot exclude an additional contact site with C3d.     

Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor Alpha in Patients with Human Immunodeficiency Virus (HIV) Type 1 Infection

Variations in cytokine production in patients with human immunodeficiency virus (HIV) infection could be involved in the physiopathology and in the progression of the disease. Therefore we studied the level of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor α (TNFα) produced in patients with HIV infection at stage II (asymptomatic seropositives) and stage IV (AIDS) of the CDC classification, by using an enzyme amplified sensitivity immunoassay. We measured the level of GM-CSF and TNFα in supernatant of phytohemagglutinin-activated peripheral blood mononuclear cells from patients and healthy individuals. In one out of 10 stage II patients and 4 out of 14 stage IV patients, we obtained higher levels of GM-CSF than the mean + 2 S.D. of controls, but in 3 stage IV patients with very low CD4+ T lymphocyte counts (< 50/mm^–3) compared to other patients, the GM-CSF values were very low. High levels of TNFα were detected in 3 out of 10 stage II and 6 out of 11 stage IV patients. The high values of TNFα were associated with high values of GM-CSF in stage II and in most of AIDS patients except those with very low CD4⁺ T cell counts, who produced low levels of GM-CSF. Plasma levels of cytokines were evaluated in 10 stage II, 22 stage IV patients and 20 controls. Increased levels of GM-CSF (more than 9 pg/ml) were observed in the plasma from 8 out of 10 stage II patients and 17 out of 22 stage IV patients. The tendency that increased levels of GM-CSF were associated with increased levels of TNFα was observed in plasma from stage IV patients. We report a disarray of GM-CSF production in patients with HIV infection that could be involved in clinical manifestations and progression of the disease.     

人钙周期蛋白结合蛋白（hCacyBP）基因的克隆与表达

筛选cDNA文库得到了人的钙周期蛋白结合蛋白基因 ,将此基因的全编码区克隆到原核表达载体pET2 8上 ,诱导目的蛋白质表达以后将重组蛋白质用亲和层析的方法进行纯化 ,得到了纯度很好的重组的目的蛋白质 ,以此作为抗原免疫动物 ,得到抗钙周期蛋白结合蛋白的特异多克隆抗体。Western印迹的结果表明 ,该基因在小鼠多种组织中广泛表达 ;免疫组化的结果表明 ,BT32 5细胞诱导分化后钙周期蛋白结合蛋白分布有变化 ,由分布于胞质中转向分布于胞核和核周胞质     

人呼吸道合胞病毒感染的SPC-A1细胞相关基因 zzz171con的电子克隆与分析

采用mRNA差异显示RT-PCR方法克隆到人呼吸道合胞病毒感染的SPC-A1细胞中相关的50个表达序列标签,其中一个片段g17-1在HRSV感染的细胞中高表达。利用生物信息学的方法对g17-1进行分析鉴定,推测是一个新基因。利用电子克隆,得到一全长2101bp的cDNA,对其ORF、启动子、电子表达谱、染色体定位以及所编码的蛋白进行分析,结果表明,该cDNA含有1个ORF,与人类假定蛋白XP_097121同源性达90%,定位于人17号染色体,在肿瘤细胞、腺癌细胞、扁桃体初级B细胞中表达,其编码的蛋白预测定位于细胞核内,推测其与细胞抗病毒感染有一定的关系。     

人呼吸道合胞病毒感染的SPC-A1细胞相关基因 zzz171con的电子克隆与分析

采用mRNA差异显示RT-PCR方法克隆到人呼吸道合胞病毒感染的SPC-A1细胞中相关的50个表达序列标签,其中一个片段g17-1在HRSV感染的细胞中高表达。利用生物信息学的方法对g17-1进行分析鉴定,推测是一个新基因。利用电子克隆,得到一全长2101bp的cDNA,对其ORF、启动子、电子表达谱、染色体定位以及所编码的蛋白进行分析,结果表明,该cDNA含有1个ORF,与人类假定蛋白XP_097121同源性达90%,定位于人17号染色体,在肿瘤细胞、腺癌细胞、扁桃体初级B细胞中表达,其编码的蛋白预测定位于细胞核内,推测其与细胞抗病毒感染有一定的关系。     

Molecular Cloning of emip,a Member of the Major Intrinsic Protein (MIP) Gene Family,Preferentially Expressed in Epidermal Cells of Barley Leaves

Cloning and characterization of a barley gene named emip is reported that encodes a member of the major intrinsic protein family. A λ-unizap cDNA library synthesized from poly A⁺?mRNA of leaf epidermis was screened differentially with epidermis-versus mesophyll-derived probes. One of the clones epi 3-2 was sequenced and further analyzed. The open reading frame of the full length clone codes for a polypeptide of 288 amino acids with a molecular mass of 30,634 Da exhibiting a high degree of homology with members of the major intrinsic protein family. Hydropathy analysis predicts six potential membrane-spanning helices. mRNA levels were high in the growing zone of barley leaves and declined towards the tip of the fully expanded leaf blade. Expression was high in epidermal strips, lower in roots and very low in the leaf mesophyll. In order of decreasing response, wilting, salt shock and heat shock resulted in stimulated expression. mRNA levels remained low during slow salting up experiments. The expressional pattern suggests a role of EMIP in turgor regulation, particularly under stress.     

伪狂犬病病毒gE基因主要抗原表位区的原核表达及其在疫苗接种和自然感染鉴别诊断中的应用

根据伪狂犬病病毒（PRV）Min-A株gE基因序列,利用PCR方法扩增了PRV-gE基因不含信号肽、胞内区和跨膜区的主要抗原表位区,并克隆到原核表达载体pGEX-6p-1中,获得的重组质粒命名为pGEX-tgE。经SDSPAGE电泳分析证实克隆的部分gE基因获得了表达,融合表达产物大小约为63kD,并在终浓度为0.6mmol/L的IPTG诱导下,3.5h其表达量达到高峰。通过改变诱导条件,有效抑制了包涵体形成,提高了重组蛋白的溶解性。Western blot分析证实表达的重组gE蛋白具有抗原反应活性。将表达产物利用亲和层析法纯化后作为ELISA抗原,通过对其特异性、敏感性及工作条件的优化试验,和对48份PRV阴性血清样品的检测结果的统计学分析,建立了猪伪狂犬病tgE-ELISA鉴别诊断方法。通过对400份送检血清样品的检测结果分析,表明其与PRV全病毒ELISA试验的符合率高达95%以上,与基于抗gE蛋白单抗竞争性ELISA的符合率达94%。此方法可用于gE基因缺失PRV疫苗免疫动物和PRV自然感染动物的鉴别诊断。     

Lateral diffusion of plasma membrane receptors labelled with either platelet-derived growth factor (PDGF) or wheat germ agglutinin (WGA) in human polymorphonuclear leukocytes and fibroblasts

The aims of the present investigation were (a) to compare the lateral mobility of membrane receptors of human fibroblasts and polymorphonuclear leukocytes (PMNL) labelled with either platelet-derived growth factor (PDGF), or the lectin wheat germ agglutinin (WGA), and (b) to study effects of serum or PDGF on the mobility of these receptor molecules in human fibroblasts. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard (10%) or under serum-free conditions yielding normal and starved cells, respectively. The receptor mobility was studied in response to exposure to PDGF, or serum, in short time or prolonged incubations. Human polymorphonuclear leukocytes (PMNL) were adhered to microscope slides by clotting drops of blood. They were stained with rhodaminated PDGF or fluoresceinated WGA. The diffusion of labelled receptors was assessed with fluorescence recovery after photobleaching (FRAP). It was found that (a) fibroblasts grown at normal serum concentration had a lower diffusion coefficient (D=3×10^–10 cm² s^–1) for the PDGF-receptor and a slightly lower mobile fraction (R=60%) than starved cells (D=5×10^–10 cm²s^–1 and R=73%), (b) addition of serum to starved cells increased both D and R for the PDGF receptor to 12×10^–10 cm² s^–1 and 96%, respectively, (c) a similar pattern was obtained for WGA-labelled glycoconjugates indicating general membrane effects of serum-induced cell stimulation, and (d) in PMNL the PDGF receptor displayed motility characteristics (D=3–4×10^–10 cm² s^–1 and R=59%) similar to those in fibroblasts, possibly suggesting equivalent anchorage mechanisms in the membrane.     

甘蓝型油菜钙依赖蛋白激酶6(BnaCPK6)的定位与互作蛋白分析

为了探究甘蓝型油菜中钙依赖蛋白激酶(calcium dependent protein kinase, CPK)在植物对逆境响应中的作用和机制,同时为油菜品质的升级改良发掘新的基因资源,开展了对于BnaCPK6研究的分子生物学试验。首先,通过在本氏烟草中瞬时表达BnaCPK6与GFP融合蛋白来检测它的亚细胞定位情况;其次,利用双分子荧光互补(Bimolecular fluorescence complementation)试验来检测BnaCPK6与ABA信号通路中转录因子BanABF1/3/4、BnaABI5、BnaAREB3的相互作用情况。结果显示BnaCPK6具有典型的钙依赖蛋白激酶特征,N端具有潜在的棕榈酰化和豆蔻酰化位点,并且与AtCPK6在进化上有着很高的同源性。亚细胞定位的结果发现BnaCPK6主要分布于细胞膜和细胞核中。同时,双分子荧光互补试验还发现BnaCPK6与调控ABA信号转导的关键转录因子BnaABF3/4、BnaABI5以及BnaAREB3之间存在相互作用。本研究为进一步研究BnaCPK6在ABA信号通路中的作用提供了依据。     

HIV—1长末端重复序列对重组痘苗病毒表达Gag蛋白的影响

将系列缺失的ＨＩＶ１长末端重复序列（ＬＴＲ）和全长的ｇａｇＯＲＦ置于痘苗病毒载体中,经同源重组和血球吸附试验,成功地构建了６株重组痘苗病毒。免疫印迹和免疫酶试验检测均表明,６株重组病毒的Ｇａｇ蛋白表达量因ＬＴＲ不同而有明显差异,表明ＨＩＶ１的ＬＴＲ及其下游基因置于痘病毒启动子控制下,在痘苗病毒中表达时有下述特点：（１）不同的痘苗病毒启动子与全长ＬＴＲ相互作用,对ｇａｇ基因表达有显著不同的调控效果;（２）ＮＲ序列对Ｇａｇ蛋白表达没有明显影响;（３）ＥＮ序列不能被重组痘苗病毒表达系统识别;（４）ＴＡＲ序列可提高Ｇａｇ蛋白的表达量;（５）Ｕ５区及下游非翻译序列不影响Ｇａｇ蛋白的表达。     

喜树的分泌结构及其与喜树碱积累的关系

为了揭示喜树碱(camptothecin,CPT)在喜树(Camptotheca acuminata Decne.)中的积累部位及积累规律,运用组织化学技术和高效液相色谱技术对喜树茎、叶中喜树碱的积累部位和含量进行了相关性分析.结果发现两类分泌结构与喜树碱积累密切相关:一类是分布于幼茎和幼叶表面的单细胞腺毛;另一类是分布于喜树幼茎和幼叶中的由1～2层细胞包围而成的分泌道.由此推断,喜树中的分泌结构为喜树碱的主要积累部位.     

喜树的分泌结构及其与喜树碱积累的关系(英文)

为了揭示喜树碱(camptothecin,CPT)在喜树(Camptotheca acuminata Decne.)中的积累部位及积累规律,运用组织化学技术和高效液相色谱技术对喜树茎、叶中喜树碱的积累部位和含量进行了相关性分析。结果发现两类分泌结构与喜树碱积累密切相关:一类是分布于幼茎和幼叶表面的单细胞腺毛;另一类是分布于喜树幼茎和幼叶中的由1～2层细胞包围而成的分泌道。由此推断,喜树中的分泌结构为喜树碱的主要积累部位。     

Bax/Bak-dependent,Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.     

IBV青岛腺胃分离株（SD／97／02）S1蛋白基因的序列测定和分析

参考Genbank上发表的IBV S1纤突蛋白基因序列,设计了一对引物,对鸡传染性支气管炎病毒青岛腺胃分离株（SD／97／02）RNA进行RT－PCR扩增。将PCR产物克隆入pMD18-T载体中进行序列测定和分析。序列分析表明,该毒株的S1基因的G＋C％含量较少,为37.0%,存在HindⅢ,BamHⅠ,BglIⅠ,SacⅠ和SalⅠ位点,无EcoRⅠ位点,与其他毒株的同源性在87.02%－94.21%之间,在第154－429nt处为高度的变异区;将基因序列翻译成氨基酸后,假定的S1蛋白由540个氨基酸组成,等电点8.24,在蛋白质内部存在18个Cys,在S1与S2蛋白之间的剪切位点为HRRRR,这与大多数IBV毒株（RRF／SRR）不一样,有三个区域的氨基酸序列高度保守;169－181aa,230-250aa,485-506aa;与其他毒株进行抗原性比较后发现,在该毒株的320－326aa及390－401aa处的抗原表位消失,而在325－345aa、379－389aa处则出现了很强的抗原表位;第438－444aa处,其他IBV毒株（除ZJ971株外）原来存在的强抗原位点在本毒株中消失。在53－65位的氨基酸抗原性与其他毒株相比明显变弱。