鸡髓样分化因子88的原核表达及单克隆抗体制备

目的:克隆、表达、纯化鸡髓样分化因子88(MyD88),制备其单克隆抗体。方法:从脾脏cDNA中扩增857bp的MyD88基因片段,插入pMAL-c5X表达载体,转化大肠杆菌BL21(DE3)获得表达菌株,IPTG诱导表达,用SDS-PAGE分析MBP(麦芽糖结合蛋白)-MyD88重组融合蛋白的表达,切胶纯化目的蛋白;免疫BALB/c小鼠,制备针对MyD88的单克隆抗体,Western印迹检测抗体特异性,制备腹水并进行抗体亚型鉴定和效价测定。结果:构建了鸡MyD88原核表达载体pMAL-MyD88,并在大肠杆菌中获得高表达,目的蛋白以可溶性和包涵体两种形式存在;建立了3株抗鸡MyD88单克隆抗体细胞株,制备了腹水,亚型分别为IgG1、IgG1和IgG2a,轻链均为κ,腹水抗体的效价均为1∶2×105。结论:在原核表达系统中表达、纯化了重组鸡MyD88,制备了针对鸡MyD88的单克隆抗体,为后续的MyD88定量和功能研究奠定了基础。     

彩色多普勒超声对晚孕期羊水过少胎儿肾动脉血流的研究

目的:应用彩色多普勒血流显像(CDFI)观察晚孕期羊水过少胎儿肾动脉(RA)血流阻力参数的变化,为临床预测羊水过少时胎儿宫内缺氧及评价胎盘功能提供相关信息。方法:应用CDFI检测100例晚孕期孕妇(孕36～40周)RA的搏动指数(PI)、阻力指数(RI)及收缩期峰值流速与舒张末期流速比值(S/D)。其中羊水过少A组(5cm<羊水指数AFI≤8cm)40例,羊水过少B组(AFI≤5 cm)20例,正常对照C组(8cm相似文献   

IL-1beta epitope mapping using site-directed mutagenesis and hydrogen-deuterium exchange mass spectrometry analysis

Hu007, a humanized IgG1 monoclonal antibody, binds and neutralizes human, cynomolgus, and rabbit IL-1beta but only weakly binds to mouse and rat IL-1beta. Biacore experiments demonstrated that Hu007 and the type-I IL-1 receptor competed for binding to IL-1beta. Increasing salt concentrations decrease the association rate with only moderate effects on the dissociation rate, suggesting that long-range electrostatics are critical for formation of the initial complex. To understand the ligand-binding specificity of Hu007, we have mapped the critical residues involved in the recognition of IL-1beta. Selected residues in cynomolgus IL-1beta were mutated to the corresponding residues in mouse IL-1beta, and the effects of the changes on binding were evaluated by surface plasmon resonance measurements using Biacore. Specifically, substitution of F150S decreased binding affinity by 100-fold, suggesting the importance of hydrophobic interactions in stabilizing the antibody/antigen complex. Substitution of three amino acids near the N- and C-terminal regions of cIL-1beta with those found in mouse IL-1beta (V3I/S5Q/F150S) decreased the binding affinity of Hu007 to IL-1beta by about 1000-fold. Conversely, mutating the corresponding residues in mouse IL-1beta to the human sequence resulted in an increase in binding affinity of about 1000-fold. Hydrogen-deuterium exchange/mass spectrometry analysis confirmed that these regions of IL-1beta were protected from exchange because of antibody binding. The results from this study demonstrate that Hu007 binds to a region located in the open end of the beta-barrel structure of IL-1beta and blocks binding of IL-1beta to its receptor.     

Bioaccumulation and Biovolatilisation of Pentavalent Arsenic by Penicillin janthinellum, Fusarium oxysporum and Trichoderma asperellum Under Laboratory Conditions

Some fungi are able to control and remediate arsenic (As)-contaminated soil, sediment, or water. Here, we investigate potential accumulation and volatilisation of As by three fungi strains. Results indicated that the highest level of As was accumulated by Penicillin janthinellum with 39.54 μg after 10 days in the culture system amended with 2,500 μg As(V), which represents 50 mg/l As. Fusarium oxysporum showed the highest amount of volatilised As with 304.06 μg after 15 days. The As content in the treated system (filter paper + As + fungi) was significantly higher than that in the control (filter paper + As; filter paper + fungi; filter paper). Trichoderma asperellum and F. oxysporum showed superior abilities for the absorption of extracellular As and accumulation of intracellular As, which accounted for 82.2 and 63.4% of the total accumulated As, respectively. However, P. janthinellum presented an equal distribution of intracellular and extracellular As. Scanning electron microscope (SEM) analysis suggested that little impact on mycelium growth of the three fungal strains was seen after exposure to 50 mg/l As(V) for 5 days, while the growth of fungi in the control was inhibited. The present results demonstrate that P. janthinellum, F. oxysporum, and T. asperellum would be expected to tackle As-contaminated environments.     

The effect of receptor protein tyrosine phosphatase kappa on the change of cell adhesion and proliferation induced by N‐acetylglucosaminyltransferase V

N‐acetylglucosaminyltransferase V (GnT‐V) has been reported to be positively associated with tumor progression, but its mechanism still remains unknown. In the present study, we found that GnT‐V overexpression not only changed the glycosylation of receptor protein tyrosine phosphatase kappa (RPTPκ) but also decreased its protein level. Moreover, GnT‐V overexpression decreased cell calcium‐independent adhesion and increased the tyrosine phosphorylation level of β‐catenin, in which RPTPκ played an important role. Since RPTPκ has an RXKR motif, which is a favored cleavage site for furin, we used furin inhibitor to further explore the effect of RPTPκ on the change of cell adhesion and β‐catenin signaling induced by GnT‐V. Our results showed that preventing RPTPκ cleavage rescued the above effects of GnT‐V, suggesting that furin cleavage could be one of the factors for RPTPκ to regulate cell adhesion and β‐catenin signaling in GnT‐V overexpression cell lines. In addition, the increased tyrosine phosphorylation level of β‐catenin was associated with the increased nuclear level of β‐catenin and downstream signaling molecules such as c‐myc and cyclin D1 that were associated with cell proliferation. Our results suggest that GnT‐V could decrease human hepatoma SMMC‐7721 cell adhesion and promote cell proliferation partially through RPTPκ. J. Cell. Biochem. 109: 113–123, 2010. © 2009 Wiley‐Liss, Inc.     

颗粒裂解肽抗菌结构域在大肠杆菌中表达水平的分析

为实现颗粒裂解肽抗菌结构域的高效表达并避免其对宿主菌的毒性,研究设计了共表达阴离子配体重组生产阳离子抗菌肽的方法。结果证明该方法可有效提高阳离子抗菌肽的表达产量。同时研究分析了颗粒裂解肽不同抗菌结构域在同一原核表达载体pACYCDuet-1中表达水平的实验数据。通过计算机程序对mRNA二级结构的模拟分析,计算并比较出其二级结构生成自由能之间的差异,提出了预测颗粒裂解肽抗菌结构域在E.coli中表达水平的几点依据,参考这些依据可以对不同抗菌肽基因进行改造以获得高效表达。     

Corticosteroid Treatment Ameliorates Acute Lung Injury Induced by 2009 Swine Origin Influenza A (H1N1) Virus in Mice

Background

The 2009 influenza pandemic affected people in almost all countries in the world, especially in younger age groups. During this time, the debate over whether to use corticosteroid treatment in severe influenza H1N1 infections patients resurfaced and was disputed by clinicians. There is an urgent need for a susceptible animal model of 2009 H1N1 infection that can be used to evaluate the pathogenesis and the therapeutic effect of corticosteroid treatment during infection.

Methodology/Principal Findings

We intranasally inoculated two groups of C57BL/6 and BALB/c mice (using 4- or 6-to 8-week-old mice) to compare the pathogenesis of several different H1N1 strains in mice of different ages. Based on the results, a very susceptible 4-week-old C57BL/6 mouse model of Beijing 501 strain of 2009 H1N1 virus infection was established, showing significantly elevated lung edema and cytokine levels compared to controls. Using our established animal model, the cytokine production profile and lung histology were assessed at different times post-infection, revealing increased lung lesions in a time-dependent manner. In additional,the mice were also treated with dexamethasone, which significantly improved survival rate and lung lesions in infected mice compared to those in control mice. Our data showed that corticosteroid treatment ameliorated acute lung injury induced by the 2009 A/H1N1 virus in mice and suggested that corticosteroids are valid drugs for treating 2009 A/H1N1 infection.

Conclusions/Significance

Using the established, very susceptible 2009 Pandemic Influenza A (H1N1) mouse model, our studies indicate that corticosteroids are a potential therapeutic remedy that may address the increasing concerns over future 2009 A/H1N1pandemics.     

Profilin1 facilitates staurosporine-triggered apoptosis by stabilizing the integrin β1-actin complex in breast cancer cells

Profilin1 (Pfn1) functions as a tumour suppressor against malignant phenotypes of cancer cells. A minimum level of Pfn1 is critical for the differentiation of human epithelial cells, and its lower expression correlates with the tumourigenesis of breast cancer cells and tissues. However, the molecular mechanisms underlying its anti-tumour action remain largely unknown. In this study, we found that stable expression of ectopic Pfn1 sensitized the breast cancer cell line MDA-MB-468 to apoptosis induced by staurosporine, a widely used natural apoptosis-inducing agent. Pfn1 overexpression could also up-regulate the expression of integrin α5β1, which has been shown to inhibit the transformed phenotype of cancer cells. Furthermore, the Pfn1-facilitated apoptosis induced by staurosporine was blocked in cells attached to a supplementary fibronectin substrate, which serves as a ligand of integrin α5β1. These results suggest that the insufficient fibronectin caused by the integrin α5β1 up-regulation might activate a signalling pathway leading to an increase of cellular apoptosis. Moreover, Pfn1 that primarily functions to promote local superstructure formation involving actin filaments and integrin β1 may contribute to its promotion on apoptosis. Our study indicated a previously uncharacterized role of Pfn1 in mediating staurosporine-inducing apoptosis in breast cancer cells via up-regulating integrin α5β1, and suggested a new target for breast cancer therapy.     

胃癌肝转移术中射频消融治疗的临床意义

目的：评估胃癌根治术同时性射频消融治疗在治疗胃癌肝转移的临床意义。方法：收集2008年1月至2010年12月19例胃癌肝转移患者,合计转移灶33个,转移灶小于4厘米,且合计不超过4个转移灶。射频消融治疗在术中超声引导下完成。Kap-lan—Meier法分析无病生存期,无肝转移生存期及总生存期。对临床病理学因子及肝转移相关因子采用Log-rank法Cox风险比例模型解析。结果：中位无病生存、无肝转移生存及总生存期为355,394及488天。有3例患者获得长期的无复发生存。单因素分析显示,较早的T分期、N分期,较少的转移灶数量,较小的转移灶直径与良好的预后相关。多因素分析提示,病灶数量与病灶直径为独立预后因子。结论：肝转移射频消融治疗安全性好,对单发的小于4cm转移灶,以及多发病灶合计直径小于4cm者射频消融治疗较为适用,可有显著生存获益。部分患者可迭临床治愈。对于胃癌肝转移的合适病例,射频消融治疗值得推广。