The effects of P2X7 receptor antagonists on the formation and function of human osteoclasts in vitro

The P2X7 receptor (P2X7R) has been implicated in the process of multinucleation and cell fusion. We have previously demonstrated that blockade of P2X7Rs on osteoclast precursors using a blocking antibody inhibited multinucleated osteoclast formation in vitro, but that P2X7R KO mice maintain the ability to form multinucleated osteoclasts. This apparent contradiction of the role the P2X7R plays in multinucleation has prompted us to examine the effect of the most commonly used and recently available P2X7R antagonists on osteoclast formation and function. When added to recombinant RANKL and M-CSF human blood monocytes cultures, all but one compound, decreased the formation and function of multinucleated TRAP-positive osteoclasts in a concentration-dependent manner. These data provide further evidence for the role of the P2X7R in the formation of functional human multinucleated osteoclasts and highlight the importance of selection of antagonists for use in long-term experiments.     

人DFF45蛋白多克隆抗体制备及其初步应用

目的：表达、纯化带GST标签的DNA裂解因子45（DNA fragmentation factor 45 ,DFF45）融合蛋白并制备多克隆抗体。方法：构建pGEX-5X-1/DFF45原核表达质粒,转化大肠杆菌BL21,用IPTG诱导融合蛋白表达,经纯化后免疫日本大耳白兔得到多克隆抗体并用CNBr-activated Sepharose? 4B进行纯化。用间接ELISA法检测抗体效价,Western blot鉴定抗体特异性,同时用免疫荧光染色鉴定抗体特异性并观察DFF45的细胞定位。进一步检测DFF45在人类几种细胞系的表达差异情况。结果：成功构建原核表达质粒,表达、纯化DFF45蛋白并免疫动物后得到多克隆抗体。间接ELISA法显示抗体效价达1:20 000,Western blot确定抗体具有高度特异性。应用该抗体检测发现DFF45在人类几种细胞系中表达存在差异并观察到其细胞定位。结论：DFF45多克隆抗体的成功制备及其在人类细胞系的差异表达,为进一步研究DFF45基因与肿瘤及其相关疾病的关系奠定了基础。     

A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.     

腰椎多节段退变性滑脱的手术治疗

目的:研讨腰椎多节段退变性滑脱手术减压和植骨融合方式与疗效的关系。方法:20例腰椎多节段退变性滑脱患者行后路椎弓根螺钉复位内固定后,采用椎管减压、神经根松解,行椎间植骨和后外侧植骨融合术,采用日本整形外科学会(JOA)下腰痛评分标准评价疗效。结果:获得连续随访者18例,随访时间9月～3年,平均24个月,优良率90%。结论:可靠的内固定、复位,神经根松解,椎间植骨融合治疗腰椎多节段退变性滑脱效果满意,充分的神经根松解是手术治疗的关键。     

重组胰高血糖素样肽-1与人血清白蛋白融合蛋白的纯化及活性研究

利用构建的重组菌株Pichia pastoris GS115/GLP-1/HSA,在10L发酵罐中表达了胰高血糖素样肽-1与人血清白蛋白融合蛋白（GLP-1/HSA）,表达量为63.6mg/L。发酵液经中空纤维柱浓缩、疏水层析、阴离子交换层析和凝胶过滤分离纯化,获得了较高纯度的GLP-1/HSA,经HPLC分析纯度达95.8%。进一步的体内活性分析结果表明,GLP-1/HSA不仅具有天然GLP-1的生物活性,而且在给药后4 h仍能发挥显著性降血糖作用。以上结果表明,利用Pichia pastoris分泌型表达系统和建立的分离纯化方法,能获得大量较高纯度的GLP-1/HSA,为进一步研究和开发能够用于糖尿病临床治疗的长效GLP-1类似物奠定了基础。     

拟南芥未知功能基因At4g22890编码蛋白的叶绿体定位

蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208bp的DNA片段,与绿色荧光蛋白(GFP)基因构建重组表达载体pMON530-cTP-GFP,经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察,GFP荧光仅在叶绿体中观察到,表明所克隆的DNA序列编码的多肽能够将At4g22890编码蛋白质引导进入叶绿体,由此推测该蛋白质为叶绿体蛋白质。     

Determination of the minimal fusion peptide of bovine leukemia virus gp30

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.     

Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves

The production of recombinant proteins in plants is an active area of research and many different high-value proteins have now been produced in plants. Tobacco leaves have many advantages for recombinant protein production particularly since they allow field production without seeds, flowers or pollen and therefore provide for contained production. Despite these biosafety advantages recombinant protein accumulation in leaves still needs to be improved. Elastin-like polypeptides are repeats of the amino acids “VPGXG” that undergo a temperature dependant phase transition and have utility in the purification of recombinant proteins but can also enhance the accumulation of recombinant proteins they are fused to. We have used a 11.3 kDa elastin-like polypeptide as a fusion partner for three different target proteins, human interleukin-10, murine interleukin-4 and the native major ampullate spidroin protein 2 gene from the spider Nephila clavipes. In both transient analyses and stable transformants the concentrations of the fusion proteins were at least an order of magnitude higher for all of the fusion proteins when compared to the target protein alone. Therefore, fusions with a small ELP tag can be used to significantly enhance the accumulation of a range of different recombinant proteins in plant leaves. An erratum to this article can be found at     

A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor

The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins.     

Expression, purification and characterization of human urodilatin in E. coli

Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.