人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因的修正及修正基因在大肠杆菌中的表达

本文采用寡核苷酸介导的定位诱变技术修正了人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因中出现的合成错误,在该基因编码区的201位插入了原来缺失的G残基,从而使之恢复正确读框。经对修正基因进行DNA全序列测定,表明定位诱变的结果符合设计要求。在此基础上,利用原核高效表达载体pBV220在P_RP_L串联启动子的控制下在大肠杆菌中对该基因进行了表达,并测定了重组人嗜中性白细胞活化蛋白-1/白细胞介素-8的嗜中性白细胞趋化活性。本工作为开展人嗜中性白细胞活化蛋白-1/白细胞介素-8基因工程及蛋白质工程的研究奠定了基础。     

Cu(I)-thionein release from copper-loaded yeast cells

Summary The release of intact CU(I)₈-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.     

逆转录病毒载体介导的RNA干扰稳定抑制肌肉生长抑制素GDF-8的表达

为将肌肉生长抑制素的干扰序列表达盒hU6-siGDF-8有效导入肌成纤维细胞C2C12中, 以pAV-hU6 + 27载体为基础构建逆转录病毒载体pXSN-hU6-siGDF-8, 并使之与pVSV-G质粒共转染GP-293细胞, 用包装出的病毒粒子感染宿主细胞C2C12, G418筛选稳定整合逆转录病毒的抗性细胞库。2周后, Western Blotting和Real-Time PCR分析结果显示, 细胞内源性的GDF-8基因的表达得到了有效的抑制; MTT法和细胞流式仪分析表明, G418抗性细胞得到了更有效的增殖, 并且G0/G1期细胞数量减少了13.7%, S期细胞数量增加了14.9%。因此, 逆转录病毒载体的RNA干扰系统可以稳定抑制 GDF-8基因表达, 它将成为治疗肌肉萎缩疾病的一个强有力的工具。     

晚期糖基化终产物通过其受体促进内皮细胞分泌IL-8的研究

探讨晚期糖基化终产物(AGE)修饰蛋白对内皮细胞生成白介素8(IL-8)的作用,及晚期糖基化终产物受体(RAGE)在此病理过程中的作用.内皮细胞来自培养的人脐静脉内皮细胞(HUVEC).将内皮细胞与不同浓度的AGE修饰人血清白蛋白(AGE-HSA)在体外共同培养,或以可溶性晚期糖基化终产物受体(sRAGE)对AGE-HSA进行预处理后再与HUVEC共同培养.用蛋白质液相芯片法检测HUVEC培养上清中IL-8水平,并提取细胞RNA,进行RT-PCR反应,检测细胞中IL-8 mRNA的表达水平.结果表明,AGE-HSA以时间和剂量依赖的方式刺激HUVEC生成IL-8,未经修饰的HSA无此作用.AGE-HSA用sRAGE预处理后,刺激HUVEC生成IL-8的作用被抑制,并且此抑制作用呈剂量依赖的方式.AGE-HSA刺激HUVEC使IL-8 mRNA表达增高,未经修饰的HSA无此作用.sRAGE能够阻断AGE-HSA诱导HUVEC表达IL-8mRNA的作用.整个变化趋势与蛋白质水平一致.研究首次证实,AGE-HSA与细胞表面受体RAGE相互作用可刺激内皮细胞分泌IL-8,并上调IL-8 mRNA的表达.这为研究加速型血管病变的发病机制提供了新视角,也为治疗由AGE增多和潴留所引起的病理损害提供了新靶点.     

Synthetic studies on glycosphingolipids from Protostomia phyla: syntheses and biological activities of amphoteric glycolipids containing a phosphocholine residue from the earthworm Pheretima hilgendorfi

Two types of amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi, PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (1) and PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (2), and their derivatives (4, 5) were synthesized. These were examined for their ability to enhance production of interleukin-8 (IL-8), a potent inflammatory cytokine involved in neutrophil chemotaxis, in a TNFalpha-stimulated granulocytic HL-60 cells. Compounds 1 and 2 were found to be potent enhancers of IL-8 production.     

Scavenger receptors for oxidized and glycated proteins

Summary. Our present knowledge on chemically modified proteins and their receptor systems is originated from a proposal by Goldstein and Brown in 1979 for the receptor for acetylated LDL which is involved in foam cell formation, one of critical steps in atherogenesis. Subsequent extensive studies using oxidized LDL (OxLDL) as a representative ligand disclosed at least 11 different scavenger receptors which are collectively categorized as scavenger receptor family. Advanced glycation endproducts (AGE) and their receptor systems have been studied independently until recent findings that AGE-proteins are also recognized as active ligands by scavenger receptors including class A scavenger receptor (SR-A), class B scavenger receptors such as CD36 and SR-BI, type D scavenger receptor (LOX-1) and FEEL-1/FEEL-2. Three messages can be summarized from these experiments; (i) endocytic uptake of OxLDL and AGE-proteins by macrophages or macrophage-derived cells is mainly mediated by SR-A and CD36, which is an important step for foam cell formation in the early stage of atherosclerosis, (ii) selective uptake of cholesteryl esters of high density lipoprotein (HDL) mediated by SR-BI is inhibited by AGE-proteins, suggesting a potential pathological role of AGE in a HDL-mediated reverse cholesterol transport system, (iii) a novel scavenger receptor is involved in hepatic clearance of plasma OxLDL and AGE-proteins.     

Antioxidative Activity of 5,6,7,8-Tetrahydrobiopterin and its Inhibitory Effect on Paraquat-Induced Cell Toxicity in Cultured Rat Hepatocytes

The in vitro antioxidative activity of 5,6,7,8-tetrahydrobiopterin (BPH4) was measured and the ability of BPH4 to prevent paraquat-induced cell damage was examined in cultured hepatocytes. The scavenging activity of BPH4 against superoxide anion radicals was assayed in two systems, i.e., xanthine/xanthine oxidase (X/XOD) and rat macrophage/phorbol myristate acetate (MξPMA) radical-generating systems. BPH4 showed an extremely strong superoxide anion radical-scavenging activity in both assay systems. Biopterin (BP) itself did not show any activity in the X/XOD system, but was effective in the MξPMA system. The antioxidative activities of BPH4 against both superoxide anion and hydroxyl radicals were confirmed by spin trapping-ESR spectrometry. BPH4 also protected rat brain homogenate against auto-oxidation. We further examined the effect of BPH4 on paraquat-induced cell toxicity in cultured rat hepatocytes. The paraquat-induced elevation of the release of lactate dehydrogenase (LDH), a marker enzyme for cytotoxicity from cultured hepatocytes was suppressed by BPH4 in a dose-dependent manner. The elevation of lipid peroxides simultaneously induced by paraquat was also inhibited by BPH4 in the same manner. These results suggest that BPH4 might be useful in the treatment of various diseases whose pathogenesis is active oxygen-related.