Expression of a fusion protein of human ciliary neurotrophic factor and soluble CNTF-Receptor and identification of its activity

Ciliary neurotrophic factor (CNTF) has pleiotropic actions on many neuronal populations as well as on glia. Signal transduction by CNTF requires that it bind first to CNTF-R, permitting the recruitment of gp130 and LIF-R, forming a tripartite receptor complex. Cells that only express gp130 and LIF-R, but not CNTF-R are refractory to stimulation by CNTF. On many target cells CNTF only acts in the presence of its specific agonistic soluble receptors. We engineered a soluble fusion protein by linking the COOH-terminus of sCNTF-R to the NH₂-terminus of CNTF. Recombinant CNTF/sCNTF-R fusion protein (Hyper-CNTF) was successfully expressed in COS-7 cells. The apparent molecular mass of the Hyper-CNTF protein was estimated from western blots to be 75 kDa. Proliferation assays of transfected BAF/3 cells in response to CNTF and Hyper-CNTF were used to verify the activity of the cytokines. The proliferative results confirmed that CNTF required homodimerization of the gp130, CNTF-R and LIF-R receptor subunit whereas Hyper-CNTF required heterodimerization of the gp130 and LIF-R receptor subunit. We concluded that the fusion protein Hyper-CNTF had superagonistic activity on target cells expressing gp130 and LIF-R, but lacking membrane-bound CNTF-R. Project supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholar, Zhejiang Province, China and SFB415, TP B5, Germany.     

亲和色谱法从融合蛋白GST-IL-6中纯化重组人白细胞介素-6

研究用亲和融合谷胱甘肽 S 转移酶 (GST)的方法纯化重组人白细胞介素 6(IL 6)的发酵和纯化工艺 ,使用含有质粒pHZl818的E .coliJMl0 9在 2XYT培养基中进行发酵表达 ,IL 6表达为与谷胱甘肽 S 转移酶 (GST)融合的融合蛋白GST IL 6.融合蛋白存在可溶的活性蛋白和不可溶的包含体两种形式 ,此包含体无活性且无法复性 ,无法用亲和层析回收 ,通过实验优化摇床发酵的诱导温度和转速 ,以增加可溶融合蛋白的表达 .菌体超声破碎液后 ,上清液用作亲和柱层析 ,可将融合蛋白提纯至 80 00 ,每升发酵液可得 10mg融合蛋白 ,用凝血酶裂解处理 6h ,亲和标志物GST被特异性切除 ,裂解得到的IL 6用离子交换柱层析可纯化至 95 00 ,MTT法测得纯化的IL 6生物学活性为 1.0 2× 10 8IU/mg .     

盐胁迫对白菜幼苗逆境指标及蛋白激酶活性的影响

研究NaCl对白菜幼苗叶片内SOD，POD，CAT活性，Vc、脯氨酸、可溶性糖、蛋白质含量和蛋白激酶(Protoin Kinaso，PK)活性的影响以及激酶活性与部分金属离子的关系．结果表明，0．5%的NaCl能使白菜体内SOD，POD，CAT活性及Vc的含量降低．与此同时体内脯氨酸、可溶糖的含量增加，反应体系的Ca^2 、Mn^ 2、Mg^2 对蛋白激酶活性的发挥有显影响．     

Semi-analytical Monte Carlo simulation of laser-induced fluorescence propagation in an optically participating spray

A semi-analytical Monte Carlo (SMC) simulation was developed to simulate the propagation of laser-induced fluo- rescence (LIF) in an optically participating spray, which simultaneously exhibits spectrally dependent emission, anisotropic scattering, absorption, and re-emission. The SMC simulation is described and then applied to an experimental configuration of a cloud of polydisperse droplets composed of water and sulforhodamine B dye. In the SMC simulation, the collected LIF flux on the remote receptor element is calculated as the global contribution from the emissive source, single, twice, … and nth collision events in any sequence. The effects on the fluorescence photons propagation of spray parameters like the dye concentration, droplets concentration, and droplets size are examined. Three spectral bands representing different optical properties are chosen to analyze the interference of absorption, scattering and re-emission on the detected LIF flux. The obtained spectral LIF flux distribution on the receptor demonstrates a “red shift” phenomenon.     

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.     

NIDDM合并肺部感染患者的免疫功能变化研究

[目的]通过对免疫球蛋白(IgA、IgG、IgM),补体(C3、C4),T淋巴细胞亚群(CD3、CD4、CD8),可溶性白细胞介素2受体(SIL-2R)的检测,研究这些免疫指标与NIDDM(非胰岛素依赖性糖尿病)合并肺部感染患者的关系.[方法]采用双抗夹心法测SIL-2R,速率散射比浊法测免疫球蛋白和补体,单克隆法测淋巴细胞亚群等免疫指标.[结果]①糖尿病及其合并感染组SIL-2R显著高于正常对照组(P<0.01).CD4/CD8显著低于正常对照组(P<0.05);②合并感染组较正常对照组IgG、IgA、C3、C4显著升高(P<0.05、P<0.01、P<0.05、P<0.05),IgM无显著变化(P<0.05).[结论]NIDDM合并肺部感染可加重糖尿病及机体免疫功能低下,NIDDM合并肺部感染T淋巴细胞亚群(CD3、CD4、CD8),补体(C3、C4)免疫球蛋白(IgA、IgG)可溶性白细胞介素2受体(SIL-2R)的检测,可作为糖尿病及其合并感染者的病情变化的客观指标.     

基于HAProxy的负载均衡选课系统应用研究

本文旨在采用负载均衡技术及虚拟机技术,解决目前高校选课系统存在的服务器瓶颈压力。该系统前端采用开源的负载均衡软件HAProxy,后端应用Windows Server 2008 R2中的Hyper-V虚拟机技术,部署了校园选课负载均衡系统。通过合理配置HAProxy的策略,使访问流量得以均衡分配,达到了预期目的。     

三叶木通化学成分和药理作用研究进展

对近年来三叶木通的化学成分和药理作用研究进展进行了综述.结果表明,该植物含有多种化学成分.其中藤茎含有多种齐墩果酸及常春藤皂苷类三萜皂苷;种子脂肪酸含量较高,不饱和脂肪酸含量达70.1%;果实中含有蛋白质、淀粉、可溶性糖、有机酸、氨基酸、矿质元素等化学物质.三叶木通果实不同部位的乙醇提取物对酪氨酸酶活性均有一定的抑制作用.此外,该植物还具有抗炎、利尿、抗菌作用.     

熊果酸和齐墩果酸 D-苯甘氨酸缀合物的合成、表征及抗癌活性研究

熊果酸和齐墩果酸为同分异构体,同属五环三萜类化合物,具有多种生物活性,如抗肿瘤、抗HIV、抗炎、降糖、保肝、抗疟等生物活性。本文以具有一定生物活性的熊果酸和齐墩果酸为先导化合物,通过对它们C．28位甲酯化及C．3位导入D-苯甘氨酸,共设计合成了4种未见报道的熊果酸、齐墩果酸衍生物,利用IR和1HNMR波谱技术对其结构进行了表征。然后对目标产物进行了人神经胶质瘤U87AGEFR细胞和人肺癌细胞PC9／G体外抗癌活性测试。结果表明U-3对两种癌细胞的抑制作用明显优于熊果酸和齐墩果酸。     

油菜素内酯对硝酸钙胁迫下黄瓜幼苗抗氧化系统及可溶性蛋白表达的影响

目的:研究硝酸钙胁迫下24-表油菜素内酯(EBR)对黄瓜幼苗抗氧化性能及可溶性蛋白表达的影响。方法:通过单因素随机区组设计,从其抗氧化性能及可溶性蛋白表达的变化来探讨EBR对硝酸钙胁迫下黄瓜幼苗的影响。结果:与对照相比,硝酸钙胁迫导致黄瓜幼苗的丙二醛含量及细胞膜透性显著提高了115.1%和105.6%,膜脂过氧化加剧,黄瓜根系中可溶性蛋白含量降低且至少有35种蛋白发生明显变化,而叶片的可溶性蛋白含量升高且至少有29种蛋白发生明显变化;而硝酸钙胁迫下喷施EBR后黄瓜幼苗的SOD、POD、CAT分别比单纯的硝酸钙胁迫增加了20.2%、20.3%、34.4%,并提高了其可溶性蛋白含量及其表达的变化幅度。结论:24-表油菜素内酯可通过调节硝酸钙胁迫下黄瓜幼苗抗氧化性、可溶性蛋白含量及其表达,进而降低其膜脂过氧化程度,以缓解硝酸钙胁迫对植株的伤害。     

Protective effect of recombinant Lactobacillus plantarum against H2O2-induced oxidative stress in HUVEC cells

This study probed the protective effect of recombinant Lactobacillus plantarum against hydrogen peroxide(H_2O_2)-induced oxidative stress in human umbilical vein endothelial cells(HUVECs). We constructed a new functional L. plantarum(NC8-p SIP409-alr-angiotensin-converting enzyme inhibitory peptide(ACEIP)) with a double-gene-labeled non-resistant screen as an expression vector. A 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-tetrazolium bromide(MTT) colorimetric assay was carried out to determine the cell viability of HUVEC cells following pretreatment with NC8-p SIP409-alr-ACEIP. Flow cytometry(FCM) was used to determine the apoptosis rate of HUVEC cells. Cysteinyl aspartate specific proteinase(caspase)-3/8/9 activity was also assayed and western blotting was used to determine protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), inducible nitric oxide synthase(i NOS), nicotinamide adenine dinucleotide phosphate oxidase 2(gp91 phox), angiotensin II(Ang II), and angiotensin-converting enzyme 2(ACE2), as well as corresponding indicators of oxidative stress, such as reactive oxygen species(ROS), mitochondrial membrane potential(MMP), malondialdehyde(MDA),and superoxide dismutase(SOD). NC8-p SIP409-alr-ACEIP attenuated H_2O_2-induced cell death, as determined by the MTT assay. NC8-p SIP409-alr-ACEIP reduced apoptosis of HUVEC cells by FCM. In addition, compared to the positive control, the oxidative stress index of the H_2O_2-induced HUVEC(Hy-HUVEC), which was pretreated by NC8-p SIP409-alr-ACEIP, i NOS,gp91 phox, MDA, and ROS, was decreased obviously; SOD expression level was increased; caspase-3 or-9 was decreased, but caspase-8 did not change; Bcl-2/Bax ratio was increased; permeability changes of mitochondria were inhibited; and loss of transmembrane potential was prevented. Expression of the hypertension-related protein(Ang II protein) in HUVEC cells protected by NC8-p SIP409-alr-ACEIP decreased and expression of ACE2 protein increased. These plantarum results suggested that NC8-p SIP409-alr-ACEIP protects against H_2O_2-induced injury in HUVEC cells. The mechanism for this effect is related to enhancement of antioxidant capacity and apoptosis.     

CD33分子信号传递及其功能的研究进展

CD33分子为一种跨膜受体,主要存在于血液、骨髓和淋巴细胞中.它属于免疫球蛋白超家族成员,并且是能够结合唾液酸的免疫球蛋白样的凝集素家族成员.在这一膜受体内部存在免疫受体酪氨酸抑制基序(ITIM).由于ITIM能够特异性的与酪氨酸磷酸酶shp-1和shp-2结合并向胞内传递抑制性信号,从而达到抑制细胞活性的目的,所以CD33分子主要在临床上作为一种靶位点用于急性骨髓系白血病(AML)的治疗.     

素质教育契合点：人文教育与科学教育的融合

人文教育与科学教育的融合是素质教育的核心内容,也是高等教育改革和发展的必然趋势。科学教育与人文教育融合的主要路径为：确立有利于人文教育与科学教育相融合的人才培养目标,构建有利于人文教育与科学教育相融合的学科专业结构和课程体系,建立有利于人文教育与科学教育相融合的教师队伍,营造有利于人文教育与科学教育相融合的校园文化场。     

Lymphotactin enhances the in-vitro immune efficacy of dendritoma formed by dendritic cells and mouse hepatocellular carcinoma cells

Objective: To investigate the in-vitro antitumor immune responses of dendritoma formed by mouse hepatocellular carcinoma (HCC) cells and lymphotactin (Lptn) gene modified dendritic cells (DCs). Method: DCs prepared from mouse bone marrow were genetically modified by lymphotactin adenovirus, and fused with H22 cells by polyethylene glycol (PEG). RT-PCR and ELISA were employed to identify lymphotactin expression at mRNA and protein level. Cell phenotypes and fusion efficiency was detected by FACS. The stimulatory effect of DC on T cells was detected by mixed lymphocyte reaction. The cytotoxicity activity against H22 cells was assayed by LDH method. Results: Lymphotactin could be efficiently expressed by DCLptn/H22 hybridoma. DCLptn/H22 cells could induce potent T cell proliferation effect and generate strong cytotoxic T lymphocyte (CTL) reaction against allogenic H22 cells. Conclusion: Lymphotactin genetic modification could enhance the in vitro immune activity of the dendritoma.     

小麦可溶性和不溶性PGIP分离纯化的初步研究

以小麦黄化苗为材料,采用不同离子强度的缓冲液抽提,通过硫酸铵分级沉淀、离子交换层析和分子筛层等步骤分离出两种不同分布的PGIP.经SDS-PAGE分析:不溶性PGIP出现两条谱带,分子量为41KD和39KD,而可溶性PGIP出现一条谱带,分子量为41KD.     

Anti-hepatocarcinoma activity of TT-1, an analog of melittin,combined with interferon-α via promoting the interaction of NKG2D and MICA

Hepatocarcinoma is one of the malignant cancers with significant morbidity and mortality. Immunotherapy has emerged in clinical treatment, owing to the limitation and severe side effects of chemotherapy. In the immune system, natural killer (NK) cells are important effectors required to eliminate malignant tumor cells without the limitation of major histocompatibility complex (MHC) molecule issues. Hence, treatment which could stimulate NK cells is of great interest. Here, we investigated the efficacy of the combined therapy of TT-1 (a mutant of melittin) and interferon-α (IFN-α) on NK cells and human liver cancer HepG-2/Huh7 cells in vitro and in vivo, as well as the mechanism involved. The combination therapy significantly inhibited the growth of HepG-2/Huh7 cells in vivo, but this effect was impaired after depleting NK cells. TT-1 not only up-regulated MHC class I-related chain molecules A (MICA) expression, but also prevented the secretion of soluble MICA (sMICA). Both the mRNA and protein of a disintegrin and metallopeptidase 10 (ADAM 10) in HepG-2/Huh7 cells were decreased after TT-1 treatment. The combined therapy of TT-1 and IFN-α could suppress the growth of HepG-2/Huh7 xenografted tumor effectively via promoting the interaction of NK group 2, member D (NKG2D) and MICA, indicating that TT-1+IFN-α would be a potential approach in treating liver cancer.     

农垦58A花粉败育过程花药吲哚乙酸氧化酶活性

光敏感核不育水稻,在长日诱导的不育花药中,可溶性蛋白质含量,比短日诱导的可育花药为低;在花粉母细胞发育期和单核早期,不育花药中吲哚乙酸氧化酶总活性和比活性,均较可育花药高得多;单核晚期以后,酶活性仍以不育花药较高。     

雌激素对阿尔茨海默病神经元保护作用研究进展

阿尔茨海默病是一种由多种因素引起的神经系统性退化疾病,其表现为β-淀粉样蛋白沉积和Tau蛋白高度磷酸化等。雌激素可以通过抑制天冬氨酸蛋白水解酶（cysteinyl aspartate specific proteinase,caspase）的活性、调节Bcl相关X蛋白（Bcl-associated X protein,Bax）和B淋巴细胞瘤2（B-cell lymphoma 2,Bcl-2）的表达及其比值、提高下丘脑神经元和神经胶质细胞的环磷酸腺苷（cyclic adenosine monophosphate,c AMP）含量、促进c AMP反应元件结合蛋白（c AMP response element-binding protein,CREB）快速磷酸化、影响海马细胞内N-甲基-D-天门冬氨酸（N-methl-D-aspartate,NMDA）受体的表达而抑制神经元的凋亡;雌激素还可通过影响脑源性神经营养因子和突触素,发挥对神经元的直接保护作用。