重组B7-H1IgV融合蛋白的表达、纯化及活性鉴定

目的以B7-H1为靶点,研制肿瘤免疫治疗蛋白疫苗。方法将人B7-H1胞外片段IgV区基因插入pQE-30原核表达载体,转化大肠杆菌,经IPTG诱导表达。Ni2+-NTA亲和层析纯化蛋白,Westernblot鉴定。用纯化的rhB7-H1IgV蛋白免疫昆明小鼠,经ELISA、流式细胞术、免疫组化技术和CDC试验测定融合蛋白及其抗血清的生物学活性。结果所构建的pQE-30-TT-B7-H1IgV表达载体,能稳定表达rhB7-H1IgV蛋白,经纯化后免疫昆明小鼠,可获得高滴度抗B7-H1抗血清。经流式细胞术和免疫组化检测显示,其抗血清可与HT-29/B7-H1+及SP2/0肿瘤细胞结合,且在CDC试验中,可依赖补体杀伤HT-29/B7-H1+及SP2/0肿瘤细胞。结论rhB7-H1IgV融合蛋白不仅可引发小鼠体液免疫应答,而且其抗体还能与表达B7-H1的肿瘤细胞相结合,并介导补体依赖的体外杀伤作用。     

FABP7基因真核表达载体的构建及其对人乳腺癌细胞MCF-7增殖的影响

目的构建脑脂肪酸结合蛋白(BLBP/B-FABP,FABP7)基因的真核表达载体,并检测其对人乳腺癌细胞MCF-7增殖的影响。方法采用逆转录方法从星型细胞瘤组织中扩增FABP7基因,双酶切后插入线性化的pcDNA3.1载体真核启动子下游,构建重组真核表达载体,转染人乳腺癌细胞MCF-7后,采用半定量RT-PCR检测FABP7基因mRNA的表达,MTT法检测细胞的增殖活性、流式细胞术检测细胞的周期变化情况,并对细胞进行计数。结果FABP7基因重组真核表达质粒经双酶切及测序鉴定证明构建正确,转染MCF-7细胞后48、72和96h,pcDNA3.1-FABP7组的细胞数和细胞的A490值均比pcDNA3.1空质粒组明显降低,G1期细胞百分含量均明显升高。结论已成功构建了FABP7基因的真核表达载体,该载体可抑制人乳腺癌细胞MCF-7的增殖。     

Hsa-Let-7g miRNA Targets Caspase-3 and Inhibits the Apoptosis Induced by ox-LDL in Endothelial Cells

It has been well confirmed ox-LDL plays key roles in the development of atherosclerosis via binding to LOX-1 and inducing apoptosis in vascular endothelial cells. Recent studies have shown ox-LDL can suppress microRNA has-let-7g, which in turn inhibits the ox-LDL induced apoptosis. However, details need to be uncovered. To determine the anti-atherosclerosis effect of microRNA has-let-7g, and to evaluate the possibility of CASP3 as an anti-atherosclerotic drug target by has-let-7g, the present study determined the role of hsa-let-7g miRNA in ox-LDL induced apoptosis in the vascular endothelial cells. We found that miRNA has-let-7g was suppressed during the ox-LDL-induced apoptosis in EAhy926 endothelial cells. In addition, overexpression of has-let-7g negatively regulated apoptosis in the endothelial cells by targeting caspase-3 expression. Therefore, miRNA let-7g may play important role in endothelial apoptosis and atherosclerosis.     

Analysis of Phosphoinositide-Binding Properties and Subcellular Localization of GFP-Fusion Proteins

Specific protein‐phosphoinositide (PI) interactions are known to play a key role in the targeting of proteins to specific cellular membranes. Investigation of these interactions would be greatly facilitated if GFP‐fusion proteins expressed in mammalian cells and used for their subcellular localization could also be employed for in vitro lipid binding. In this study, we found that lysates of cells overexpressing GFP‐fusion proteins could be used for in vitro protein‐PI binding assays. We applied this approach to examine the PI‐binding properties of Aplysia Sec7 protein (ApSec7) and its isoform ApSec7(VPKIS), in which a VPKIS sequence is inserted into the PH domain of ApSec7. EGFP‐ApSec7 but not EGFP‐ApSec7(VPKIS) did specifically bind to PI(3,4,5)P₃ in an in vitro lipid‐coated bead assay. Overexpression of EGFP‐ApSec7 but not EGFP‐ApSec7(VPKIS) did induce neurite outgrowth in Aplysia sensory neurons. Structure modeling analysis revealed that the inserted VPKIS caused misfolding around the PI(3,4,5)P₃‐binding pocket of ApSec7 and disturbed the binding of PI(3,4,5)P₃ to the pleckstrin homology (PH) domain. Our data indicate that plasma membrane localization of EGFP‐ApSec7 via the interaction between its PH domain and PI(3,4,5)P₃ might play a key role in neurite outgrowth in Aplysia.     

生长抑素(SS)基因在大肠杆菌pT7ZZ表达系统中的克隆与表达

目的 构建和筛选既能稳定、高效表达SS融合蛋白，又能使这种融合蛋白保持良好的SS抗原性和高水溶性等特点的重组质粒。方法 用 BamH I/Xho I (B/X）和 BamH I/EcoR I（B/E）双酶切，将含有SS基因的片段由pThioHis A中切出，然后分别克隆到 pT7ZZa中的相应酶切位点，得到pSSB/X（短尾）和pSS-B/E(长尾)两个重组质粒，用常规表达技术在大肠杆菌中对其进行表达。结果 pSS-B/X和pSS-B/E两个重组质粒在宿主菌BL21（DE3）中均获得表达，pSS-B/X获得高效表达后融合蛋白占菌体总蛋白的30．6％。两个表达菌经超声裂解后电泳发现，SS－B/X－ZZ和SS－B/E－ZZ这两种融合蛋白基本为可溶性蛋白，沉淀中含量极低。二者均具有良好的抗原性，结论pSS-B/X重组质粒很有希望成为SS基因疫苗的候选质粒。     

Cover Picture: A Fusion of Disciplines: Chemical Approaches to Exploit Fusion Proteins for Functional Genomics (ChemBioChem 9/2003)

The cover picture shows a living cell inside which a protein is labeled with fluorescein. The labeling is based on the unusual mechanism of the DNA repair protein O⁶‐alkylguanine‐DNA alkyltransferase (AGT, structure on the right), which irreversibly transfers the alkyl group from O⁶‐alkylguanine to its reactive cysteine residue. By using O⁶‐benzylguanine derivatives such as the fluorescein derivative shown, AGT fusion proteins can be covalently labeled in vivo. The method is one example of a growing number of approaches that aim at equipping proteins with functionalities that can not be genetically encoded, which opens up new ways to study proteins in vivo. Further information can be found in the article by N. Johnsson and K. Johnsson on p. 803 ff.     

Urea-Modified Soy Globulin Proteins (7S and 11S): Effect of Wettability and Secondary Structure on Adhesion

This investigation characterized wettability and adhesive properties of the major soy protein components conglycinin (7S) and glycinin (11S) after urea modification. Modified 7S and 11S soy proteins were evaluated for gluing strength with pine, walnut, and cherry plywood and for wettability using a bubble shape analyzer. The results showed that different adhesives had varying degrees of wettability on the wood specimens. The 7S soy protein modified with urea had better wettability on cherry and walnut. The 11S soy protein modified with 1M urea had better wettability on pine. The 1M urea modification gave 11S soy protein the greatest bonding strength in all the wood specimens. The 3M urea modification gave 7S soy protein stronger adhesion on cherry and walnut than did 11S protein; but with pine, 11S soy protein had greater adhesion strength than 7S soy protein. Measurement of protein secondary structures indicated that the β-sheet played an important role in the adhesion strength of 3M urea-modified soy protein in cherry and walnut, while random coil was the major factor reducing adhesion strength of 7S soy protein modified with 1M urea.     

7-氨基头孢烷酸中蛋白质含量的检测

7-氨基头孢烷酸(7-ACA)为生产半合成头孢类抗生素的重要母核,其质量情况直接决定了各类合成头孢产品生产质量水平。为监控产品质量,采用考马斯亮蓝(Coomassie Brilliant Blue)法对7-氨基头孢烷酸(7-ACA)中蛋白质浓度进行检测,并对检测方法进行验证:检测波长为595 nm,蛋白质含量为0.000～100.000μg呈良好线性,相关系数r=0.999 3,平均回收率为99.3%(n=3)。采用考马斯亮蓝法对7-氨基头孢烷酸(7-ACA)中蛋白质浓度进行检测,灵敏度高,准确、可靠,可作为7-氨基头孢烷酸的质量控制方法。     

Oxidative Stress-Mediated Apoptosis Induced by Ethanolic Mango Seed Extract in Cultured Estrogen Receptor Positive Breast Cancer MCF-7 Cells

Breast cancer has become a global health issue requiring huge expenditures for care and treatment of patients. There is a need to discover newer cost-effective alternatives for current therapeutic regimes. Mango kernel is a waste product with potential as a source of anti-cancer phytochemicals, especially since it is non-toxic towards normal breast cell lines at concentrations for which it induces cell death in breast cancer cells. In this study, the anti-cancer effect of mango kernel extract was determined on estrogen receptor-positive human breast carcinoma (MCF-7) cells. The MCF-7 cells were cultured and treated with 5, 10 and 50 μg/mL of mango kernel extract for 12 and 24 h. In response to treatment, there were time- and dose-dependent increases in oxidative stress markers and pro-apoptotic factors; Bcl-2-like protein 4 (BAX), p53, cytochrome c and caspases (7, 8 and 9) in the MCF-7 cells treated with the extract. At the same time, there were decreases in pro-survival markers (Bcl-2 and glutathione) as the result of the treatments. The changes induced in the MCF-7 cells by mango kernel extract treatment suggest that the extract can induce cancer cell apoptosis, likely via the activation of oxidative stress. These findings need to be evaluated further to determine whether mango kernel extract can be developed as an anti-breast cancer agent.     

氟吗啉对人乳腺癌细胞株MCF-7细胞增殖的影响

研究氟吗啉对MCF-7细胞增殖的影响.实验中设定溶剂对照组、雌激素组(10-7～10-12 mol/L)和氟吗啉各剂量组(10-5～10-9 mol/L),染毒7 d,采用噻唑蓝(MTT)法对人乳腺癌MCF-7细胞增殖情况进行分析,酶标仪检测吸光度(490 nm).氟吗啉各剂量组的增殖率在87.1%～129%之间,与溶剂对照组相比均无显著性差异.氟吗啉可能不具有拟雌激素样活性.     

重组人B7-H4IgV融合蛋白的表达、纯化及活性

目的表达、纯化重组人B7-H4IgV融合蛋白,并检测其活性。方法将人B7-H4胞外片段IgV区基因插入pQE-30原核表达载体,转化大肠杆菌,IPTG诱导表达。表达产物经Ni2+-NTA柱亲和层析纯化,并进行Westernblot鉴定。用纯化的rhB7-H4IgV蛋白免疫昆明小鼠,经ELISA、流式细胞术测定小鼠抗血清的活性;免疫SP2/0荷瘤小鼠,观察对肿瘤细胞生长的抑制作用。结果表达的rhB7-H4IgV蛋白约占菌体总蛋白的25%,纯化后蛋白纯度为93%,浓度约为0.5mg/ml,与抗His单抗可产生特异性反应条带。免疫昆明小鼠产生的抗血清效价可达1∶10000,可与SP2/0肿瘤细胞结合。rhB7-H4IgV蛋白对SP2/0移植瘤生长具有一定的抑制作用。结论已成功表达并纯化了重组人B7-H4IgV融合蛋白,纯化蛋白可诱发小鼠体内免疫应答,产生的抗体能与表达B7-H4的肿瘤细胞SP2/0结合,rhB7-H4IgV蛋白在一定程度上能抑制SP2/0移植瘤的生长。     

BMP7 Drives Human Adipogenic Stem Cells into Metabolically Active Beige Adipocytes

Adult humans have a substantial amount of inducible‐brown (or beige) fat, which is associated with increased energy expenditure and reduced weight gain via thermogenesis. Despite the identification of key regulators of beige adipogenesis, impacts of dietary factors on adaptive thermogenesis are largely unknown, partly due to a lack of validated human cell models. Bone morphogenetic protein 7 (BMP7) is known to promote brown adipogenesis in rodent and human progenitor cells. However, controversy still surrounds the cellular identity in BMP7‐mediated transition of white to brown adipocytes. The aim of this study was to confirm BMP7‐derived human adipocytes as a relevant in vitro model of human beige adipocyte by verifying the cellular lineage and metabolic activity. In this study, we hypothesized that pre‐exposure of the stromal vascular (SV) fraction of primary human adipogenic precursor cells (hASC) to BMP7 would convert metabolically active brown adipocytes. Our results showed that exposure of hASC to human BMP7 was associated with significant escalation of (1) UCP1 gene expression, a signature gene of brown adipocytes, (2) beige specific marker gene expression (i.e., CD137 and TMEM26), (3) glucose and fatty acid uptake, and (4) basal and cAMP‐stimulated oxygen consumption rate compared to white adipocyte control. Taken together, we demonstrated that BMP7 mediates conversion of hASC into metabolically active beige adipocytes. By confirming the cellular identity and metabolic activity, this BMP7‐induced human beige adipocytes from hASC should aid in the discovery and assessment of bioactive molecules to promote adaptive thermogenesis.     

Condensation of 7 H -Benzo[ de ]naphthacene-7-one (9,10-Dibenzanthrone)

We have synthesized undecacyclic aromatic hydrocarbons using condensation of dibenzoanthrones. For example, dibenzo[ a,o ]dinaphtho[3,2,1- cd ;1,2,3- lm ]perylene, diphenanthra[1,2,3- cd ; 3,2,1- lm ]perylene and diphenanthra[2,3,4- cd ;4,3,2- lm ]perylene were produced from 5,6-, 8,9- and 10,11-dibenzoanthrones, respectively. In the present study, we have extended our work and aimed to synthesize new undecacyclic aromatic hydrocarbons by the use of 9,10-dibenzoanthrone (7 H -benzo[ de ]naphthacene-7-one) as a starting material. 9,10-Dibenzoanthrone was prepared by the cyclization of 1,2'-dinaphthylketone in aluminum chloride anhydride and sodium chloride, and the structure was confirmed by NMR measurements. It dissolves into ethanol and crystallizes in pale yellow needles; its melting point is 198.2-199.3°. The condensation of 9,10-dibenzoanthrone should give undecacyclic aromatic hydrocarbons such as benzo[ vwx ]naphtha[12,1,2- cde ] hexaphene.     

3种茶提取物对MCF-7细胞增殖、凋亡的影响及其与蛋白激酶B的相关性

目的 探讨红茶、绿茶及普洱茶提取物对人乳腺癌细胞MCF-7增殖、凋亡的影响及其与蛋白激酶B(Protein ki-nase B,PKB)的相关性。方法用不同浓度的3种茶提取物处理MCF-7细胞不同时间,MTT法检测其对MCF-7细胞增殖的影响;Annexin V-FITC/PI双染流式细胞术检测其对细胞凋亡的影响;实时定量PCR检测细胞PKB基因mRNA的转录水平。结果 3种茶提取物对MCF-7细胞的增殖均具有明显的抑制作用,且呈时间、剂量依赖性;3种茶提取物处理的细胞早期凋亡率均高于未处理细胞,PKB基因mRNA的表达量明显降低。结论 3种茶提取物均含有可以抑制PKB转录活性的天然成分,并可诱导MCF-7细胞凋亡;其中绿茶诱导MCF-7细胞凋亡效果最明显。     

A theoretical study of the initial stages of the hydrogenation of Si(111) 7×7

We have used the periodic extended Hückel method to examine the initial stages of the hydrogenation of Si(111) 7×7. Energies, atomic charges and reduced overlap populations are considered, and we discuss the inequivalence of the adatoms. The adatom dangling bond (DB) level positions with respect to E_F are also discussed. On the basis of our results, we propose the following adsorption sequence: first, saturation of the five occupied adatom DBs, then hydrogenation of the remaining atoms by pairs, the hydrogenation of an adatom being coupled with that of a restatom or that of the corner atom. We also show that the atomic charges of the surface become more uniform when the 19 DBs are saturated, and that this modification occurs in discontinuous stages.     

Modified Polyethersulfone (PES) Ultrafiltration Membranes for Enhanced Filtration of Whey Proteins

Abstract

Application of membrane technology to whey protein separation is an interesting development that has seen growth in recent years. In particular, modification of existing membranes to impart charge properties on the membrane surface or in the pores has been shown to improve membrane selectivity, product purity, and throughput of protein solutions. This paper focuses on exploring the effects of membrane charge and solution pH on filtration of the major whey proteins α‐lactalbumin (14.1 kDa) and β‐lactoglobulin (18.4 kDa) using functionalized PES membranes. The membranes have an open pore structure containing charged sulfonated grafted polymer chains that allows for greater protein retention. The modified membranes were synthesized by polymerization of styrene in the membrane pores followed by sulfuric acid treatment of the resulting polystyrene grafts. The charged membrane gave a calculated selectivity of five times better than the raw membrane at pH 7.2 based on data from single protein transmission experiments. The enhanced selectivity of the tailor‐made membrane was due to increased retention of β‐lactoglobulin due to a reduction in molecular sieving combined with electrostatic repulsion between negatively charged β‐lactoglobulin and the negatively charged membrane.     

壬基酚聚氧乙烯（7）醚磷酸酯的合成研究及单,双酯含量的测定

本文合成了壬基酚聚氧乙烯（７）醚磷酸酯（ＮＥＰ－７），研究了合成条件对单、双酯含量的影响，并介绍了一种测量单、双酯含量的新方法。     

N-取代(4R,7S)-4-甲基-7-异丙基己内酰胺的合成研究

从薄荷酮出发经肟化反应,贝克曼重排反应,以及酰基化反应,合成了一系列标题化合物,其中N-乙酰基取代的产物的产率高达94.8％.通过1HNMR和13CNMR对各步产物进行了表征.     

Pro-Apoptotic Effect of Rice Bran Inositol Hexaphosphate (IP6) on HT-29 Colorectal Cancer Cells

Inositol hexaphosphate (IP₆), or phytic acid is a natural dietary ingredient and has been described as a “natural cancer fighter”, being an essential component of nutritional diets. The marked anti-cancer effect of IP₆ has resulted in our quest for an understanding of its mechanism of action. In particular, our data provided strong evidence for the induction of apoptotic cell death, which may be attributable to the up-regulation of Bax and down-regulation of Bcl-xl in favor of apoptosis. In addition, the up-regulation of caspase-3 and -8 expression and activation of both caspases may also contribute to the apoptotic cell death of human colorectal adenocarcinoma HT-29 cells when exposed to IP₆. Collectively, this present study has shown that rice bran IP₆ induces apoptosis, by regulating the pro- and anti-apoptotic markers; Bax and Bcl-xl and via the activation of caspase molecules (caspase-3 and -8).