秋水仙碱对LPS诱导巨噬细胞TNF—α基因表达的抑制作用

细胞微管聚合抑制剂秋水仙碱（Ｃｏｌｃｈｉｃｉｎｅ，Ｃｏｌ．，１０μｍｏｌ／Ｌ）可抑制ＬＰＳ激活的小鼠巨噬细胞株ＰＵ５－１．８产生ＴＮＦ－α及减少其基因表达，而Ｃｏｌ．衍生物β－Ｌｕｍｉｃｏｌｃｈｉｃｉｎｅ对微管无作用，对ＴＮＦ－α ｍＲＮＡ积累亦无影响，其它微管聚合抑制剂长春新碱、长春花碱等则有类似Ｃｏｌ．的抑制作用。用ｒｕｎ ｏｎ核转录试验和ｍＲＮＡ半寿期检测证实Ｃｏｌ．ＴＮＦ－α ｍＲＮＡ积累     

含人PDGF—B链基因不同上游序列报告基因的构建和表达

本研究构建了含人ＰＤＧＦＢ链基因不同上游序列的荧光素酶报告基因ｐＳＩＳ－１７５８／＋４３Ｌｕｃ和ｐＳＩＳ－４０２／＋４３Ｌｕｃ，经与ｐＳＶβＧａｌａｃｔｏｓｉｄａｓｅＣｏｎｔｒｏｌＶｅｃｔｏｒ共转染血管内皮细胞后，测定了基础水平和ＴＮＦα作用下细胞裂解液中的荧光素酶和β半乳糖苷酶活性。结果表明：在ＴＮＦα作用下，ＰＤＧＦＢ链基因上游序列上调内皮细胞荧光素酶的表达，其主要调控区可能位于－１７５８／－４０３ｂｐ之间。     

胶质瘤细胞c—fos和c—myc基因表达与血小板源生长因子B链的？…

目的 探讨胶质瘤细胞ｃ－ｆｏｓ和ｃ－ｍｙｃ基因表达和血小板源生长因子Ｂ链的纯合二聚体自分泌环活性的改变及其相互关系。方法 用原位杂交和免疫组化方法观察了６７例人胶质瘤组织。结果 ６７例中,ｃ－ｆｏｓ ｍＲＮＡ,ｃ－ｆｏｓ蛋白,ｃ－ｍｙｃ ｍＲＮＡ及ｃ－ｍｙｃ蛋白阳性率分别为;１００％,１００％,８５．１％,８３．６％。     

血小板源生长因子B链基因上游序列HMGI结合区的初步鉴定

目的：观察转录结构因子高迁移率蛋白I(HMGI)与血小板源生长因子B链(PDGF-B)基因上游序列的结合并初步鉴定HMGI的结合序列。 方法： 应用凝胶迁移率改变法（EMSA）观察重组人HMGI蛋白与PDGF-B基因上游-1 758/+43 bp片段的结合，并逐步确定与其结合的AT富含序列。 结果： 重组人HMGI蛋白能较特异地与PDGF-B链基因上游-1 758/+43 bp片段结合，分离到的两个HMGI结合片段，-1 393/-1 181 bp和-190/+43 bp，均含有AT富含序列TTTATAAA（-1 333/-1 326 bp，-1 314/-1 307 bp和-30/23 bp）。HMGI能与含TTTATAAA的合成寡核苷酸结合，并能促进转录因子NF-κB与拼接于旁侧的PDGF-B基因的切应力反应元件结合。 结论： HMGI在体外能与PDGF-B链基因上游的TTTATAAA序列结合，可能参与PDGF-B基因的转录调控。     

白细胞介素1,肿瘤坏死因子α和脂多糖对人脐静脉内皮细胞表达 …

目的 观察细胞因子白细胞介素１（ＩＬ－１）β、肿瘤坏死因子（ＴＮＦ）α和脂多糖（ＬＰＳ）是否诱导人脐静脉内皮细胞表达单核细胞趋化蛋白１（ＭＣＰ－１）ｍＲＮＡ及蛋白。方法 选取生长汇合的人脐胸脉内皮细胞,在其培养基中分别加入终浓度为２ｎｇ／ｍｌ的ＩＬ－１β、２０ｎｇ／ｍｌ的ＴＮＦβ和１００ｎｇ／ｍｌ的ＬＰＳ,３７℃共育４ｈ后,按照一步法提取其总ＲＮＡ,用γ－＾２２Ｐ标记的寡核苷酸ｄｏｔ ｂｌｏｔ分析     

TNF—α诱导中性粒细胞呼吸爆发的信号传导

中性粒细胞是重要的炎性细胞,而ＴＮＦ－α则是一关键的促炎症细胞因子,可激活中性粒细胞,促使其粘附、吞噬、脱颗粒、呼吸爆发等。本文主要综述参与ＴＮＦ－α诱导中性粒细胞呼吸爆发的信号传递分子,包括ＰＫＡ、ｐ３８ＭＡＰＫ、ＰＴＫ等。     

5'上游序列对TNFα诱导内皮细胞血小板源生长因子-B链基因转录的调控作用

目的和方法:为探讨人血小板源生长因子(PDGF)-B链基因5'上游序列在TNFα诱导内皮细胞该基因转录中的调控作用,本研究将构建的一系列含人PDGF-B链基因不同上游序列荧光素酶报告基因质粒,与内参照质粒pSV-β-Gal共转染培养的人脐静脉内皮细胞(HUVECs),分别检测了不同浓度和不同持续时间TNFα作用下转染内皮细胞荧光素酶比活性,观察了5'上游序列的定向删切对TNFα诱导内皮细胞PDGF-B链基因转录的影响.结果和结论:不同浓度TNFα处理转染(重组报告基因质粒pSIS-1758/+43Luc)内皮细胞18h,荧光素酶的表达均有增加,在TNFα为200U/mL、400 U/mL和800 U/mL时,荧光素酶活性呈浓度依赖性增加(分别为P＜0.05,P＜0.05和P＜0.01);200U/mL TNFα分别处理9 h、18 h、36 h和72 h后,内皮细胞荧光素酶均有增加,自18 h开始其荧光素酶的表达量呈时间依赖性增加(分别为P＜0.05,P＜0.05和P＜0.01);在PDGF-B链基因上游序列-1758/+43 bp,-402/+43 bp或-189/+43 bp存在下,TNFα可使转染内皮细胞荧光素酶表达量明显增加,而在-84/+43 bp和-52/+43 bp存在时TNFα组与对照组荧光素酶表达量无明显差异,提示PDGF-B链基因上游序列-189/-85 bp范围内存在TNFα诱导反应的顺式调控元件.     

boxA及上游序列缺失突变对λ噬菌体抗转录终止功能的影响

目的 研究λ噬菌体表达调控机理，阐明PL操纵了boxA及邻近序列突变对抗转录终止作用的影响。方法 利用体内同源重组技术，使大肠杆菌染色体DNA上携带了CI857，PL，nutL[boxA*(*代表不同的boxA及临近序列缺失突变）,boxB],N-lacZ,ttt,galK单拷贝基因，构建了含boxA及邻近序列突变（boxA*)的一系列新菌株，模拟了λ噬菌体感染宿主菌后的生理状态，利用抗转录终止报道基因galK分析研究了boxA及临近序列突变对PL操纵子抗转录终止作用的影响。结果 证明在缺失了boxA及部分上游序列的情况下，大肠杆菌RNA聚合酶不能通读转录终止子。结论 λ噬菌体左向操纵子具有与右向操纵子不同的转录调控现象。     

Nucleotide sequence of the upstream regulatory region of BoLA–DRB†

The sequence of the proximal upstream regulatory region (URR) of the bovine DRB genes was amplified using oligonucleotide primers designed from the consensus among DRB sequences from different species. The obtained DNA sequence was 234 bp long and composed of highly conserved sequence motifs, showing the same organization as the HLA‐DRB, H2‐IAb, H2‐IEb and ELA‐DRB genes.     

An upstream regulatory sequence stimulates expression of the perfringolysin O gene of Clostridium perfringens.

The structural gene for perfringolysin O (pfoA), a thiol-activated hemolysin of Clostridium perfringens, was cloned into Escherichia coli JM109 on a 4.6-kilobase (kb) EcoRI-NdeI fragment which contained the 1.7-kb pfoA gene and an upstream 2.9-kb region. An E. coli strain transformed by this plasmid produced 20-fold more perfringolysin O than a strain containing only the 1.7-kb pfoA gene. The stimulatory effect of the upstream region on in vivo expression of the pfoA gene was further analyzed by using a set of deletion mutants. Stimulation was still observed with a 3.9-kb fragment, but stimulation was not observed with fragments that were 3.6 kb or less long, indicating that the upstream region between 3.9 and 1.7 kb was involved in activation of pfoA gene expression. Nucleotide sequencing showed that this region contained one open reading frame (pfoR) coding for 343 amino acids. The deduced amino acid sequence of pfoR possesses several motifs that are characteristic of DNA-binding proteins. When a region coding for a helix-turn-helix, one of the most important motifs of DNA-binding proteins, was deleted within pfoR, stimulation was completely abolished. These results indicate that pfoR positively controls expression of the pfoA gene.     

鼻咽癌细胞中ezrin基因启动子上游序列转录调控特性的研究

目的:研究鼻咽癌细胞中ezrin基因启动子上游序列的转录调控特性。方法:构建一系列携带ezrin基因-1541/-706序列的报告基因表达载体,ezrin基因-1541/-706序列以正向和反向分别连接至不含启动子的报告基因上游、ezrin启动子上游、SV40启动子上游、ezrin启动子或SV40启动子控制的报告基因下游,将质粒转染CNE2细胞,检测荧光素酶活性。结果:CNE2细胞中,当-1541/-706序列正向位于报告基因上游时表现出启动子活性,其转录激活作用约为ezrin启动子的50%;反向连接时无启动子活性。而且,当-1541/-706序列正向位于ezrin启动子或SV40启动子上游时,显著提高荧光素酶表达;当反向位于启动子下游、正向或反向位于启动子控制的报告基因下游时,转录增强作用消失。结论:CNE2细胞中ezrin基因启动子上游序列具有转录激活和转录增强作用,这种作用具有DNA序列位置和方向依赖性。     

The effect of upstream platelet-fibrinogen interactions on downstream adhesion and activation

Circulating activated platelets roll and make transient contacts before ultimately adhering to a substrate. However, despite the dynamic nature of platelet adhesion, most in vitro adhesion and activation studies have focused on establishing local cause and effect relationships. Here, we determined the effect of exposing platelets to immobilized upstream human fibrinogen on downstream adhesion and activation. Microcontact printing was used to prepare substrates that contained well defined fibrinogen priming regions. Washed platelets were perfused over the substrates and adhesion and activation in a downstream capture region were compared with samples that did not contain a fibrinogen priming region. It was found that samples containing an upstream priming region resulted in higher adhesion, platelet spreading areas and aggregation than samples that lacked the priming region. Also, when the priming region was selectively blocked with a polyclonal anti-fibrinogen antibody, the platelet response was attenuated. To characterize this phenomenon further, flow cytometry was used to assess bulk platelet activation following fibrinogen priming. The expression of two activation markers, PAC-1 and P-selectin were quantified. Expression of both activation markers was found to be higher after perfusion over fibrinogen versus albumin-coated substrates.     

New polymorphisms for the BoLA-DRB3 upstream regulatory region

Two new alleles, named BoLA-DRB3-P*06 and BoLA-DRB3-P*07, have been identified for the upstream regulatory region of the BoLA-DRB3 gene. The 228-bp nucleotide sequences of the promoter comprising the W, X, Y, CAAT and TATA regulatory boxes were analysed. The BoLA-DRB3-P*06 exhibits one insertion between the W and X boxes, and one transition between the X and Y boxes. On the other hand, the BoLA-DRB3-P*07 showed one insertion in the X box.     

The zinc finger protein Gfi1 acts upstream of TNF to attenuate endotoxin-mediated inflammatory responses in the lung

Gfi1 is a 55-kD nuclear zinc finger protein that is differentially expressed in lymphoid and myeloid cells. Gfi1(-/-) mice show a very strong systemic response to the endotoxin LPS and die rapidly within 36 h with symptoms of septic shock. Here we report that the pathohysiological processes for this exaggerated inflammatory response take place in the lung. After LPS treatment, lungs of Gfi1(-/-) mice showed a rapid accumulation of mononuclear cells and a significant overproduction of inflammatory cytokines such as TNF, IL-1beta and IL-6. Increased cytokine production was also observed in blood-free perfused lungs from Gfi1(-/-) mice exposed to either LPS or overventilation. Alveolar macrophages but not airway epithelial cells from Gfi1(-/-) mice were found to be responsible for the enhanced cytokine production. Strikingly, when the TNF gene was deleted, Gfi1(-/-) animals were completely rescued from LPS hypersensitivity and had significantly lower IL-1beta and IL-6 levels. We conclude that the unrestrained endotoxin response of Gfi1(-/-) mice occurs mainly in the lung and that Gfi1 represents a novel factor limiting the inflammatory immune response of this organ, and propose that Gfi1 exerts its regulatory function in alveolar macrophages downstream of the LPS receptor (TLR4) and upstream of TNF.