黄鳝两种抗病毒基因的克隆与组织表达分析

[目的]干扰素调节因子(IRFs)-5,IRF-7在抗病毒免疫反应中发挥重要作用,研究黄鳝(Monopterus albus)干扰素调节因子的结构及表达有助于阐明黄鳝抗病毒的机理。[方法]通过PCR法扩增黄鳝IRF-5和IRF-7两个基因c DNA序列,分析其氨基酸序列,并利用荧光定量PCR技术研究黄鳝不同组织这两个基因的表达。[结果]已扩增到黄鳝IRF5 c DNA序列887bp,编码295aa;IRF7 c DNA序列1 181bp,编码378aa。荧光定量显示:IRF-5在脑中表达量很高,IRF5/β-actin高达120.3×10-2,而在主要免疫器官头肾、尾肾、肠和脾脏中这个比值仅分别为:0.17×10-2、0.11×10-2、0.69×10-2和0.60×10-2。IRF-7在皮肤中表达量很高,IRF7/β-actin比值达68.3×10-2。[结论]IRF-5可能在黄鳝神经系统中发挥重要作用,而IRF-7在黄鳝粘膜免疫中发挥重要作用。     

血管损伤的新靶点：干扰素调节因子

内膜增生是血管损伤后动脉重塑过程中普遍存在的现象。血管平滑肌细胞（vascular smooth muscle cells,VSMCs）的增殖、迁移、表型转换是血管损伤性疾病高血压、动脉粥样硬化、血管成形术后再狭窄等的共同病理生理学过程。干扰素调节因子（interferon regulatory factors,IRFs）是一类能对干扰素基因表达起到免疫调节作用的转录因子。近来研究发现,其在血管损伤病理过程具有调节作用,其中IRF1与细胞生长、分化和损伤密切相关,IRF3与IRF7可以抑制新生内膜的形成,而IRF8和IRF9则促进VSMCs增殖、迁移及血管内膜增生。本文重点介绍了IRFs的结构特征、信号途径及在血管重塑过程中作为新型调控因子的功能。     

干扰素调节因子家族和免疫调控

干扰素(IFN)是一类能够抵抗病毒感染、抑制细胞周期和行使免疫调节功能的细胞因子。干扰素调节因子家族(IRF)成员因在病毒感染时能够结合到IFN启动子上诱导、调节IFN的表达而得名。最近的研究表明:IRF是一类多功能的转录因子,在干扰素转录调控,病原体免疫反应,细胞因子信号转导,细胞增殖调控;以及造血干细胞的发育、淋巴细胞的分化,自稳平衡,先天性免疫和适应性免疫调控等方面都有着重要的作用。IRF的功能及其分子机制的揭示将对抗病毒,免疫疾病和恶性肿瘤的治疗提供新的靶向策略。     

干扰素调节因子的抑制肿瘤作用

干扰素（interferon,IFN）具有调节机体免疫功能、抗病毒、抗肿瘤等多种作用,是机体防御系统的重要组成部分。目前,基因工程生产的干扰素已广泛应用于临床治疗多种肿瘤。干扰素调节因子（IRF）家族在IFN的诱导表达中起着重要的调节作用。已发现了9个IRF成员,其中IRF-1和IRF-2的表达水平与宫颈癌、乳腺癌等有关;IRF-3与食管癌、宫颈癌等密切相关;IRF-5可以抑制B细胞淋巴瘤;IRF-7与乳腺癌相关;IRF-8和IRF-4参与慢性髓细胞性白血病的发病机制。     

干扰素调控因子3：细胞抗病毒反应的核心转录因子

固有免疫系统是宿主抵御病毒入侵的第一道防线．Ⅰ型干扰素是关键的抗病毒细胞因子,它在细胞建立抗病毒状态的过程中发挥了核心的作用．Ⅰ型干扰素的诱导表达是固有免疫的重要调节与效应机制．已有的研究表明：多种转录因子(NF-kappa B、ATF-2/c-Jun、IRF3、IRF7)通过在Ⅰ型干扰素的转录调控区形成稳定的转录增强复合物(enhanceosome),迅速并大量地诱导Ⅰ型干扰素表达．体内与体外的生物学分析已确立,干扰素调控因子3 (IRF3)是介导细胞表达Ⅰ型干扰素最关键的转录因子,其转录活力与生物学功能直接影响细胞的抗病毒的能力．近年来,IRF3相关的细胞信号转导与调控机制等研究取得重大进展．围绕IRF3的结构、功能以及分子调控机制等方面,概述相关的研究进展,并做前沿展望．     

干扰素调节因子新剪接异构体IRF7-e的结构及功能

干扰素调节因子7是诱导玉型干扰素表达的最主要的转录因子。寻找IRF7新的剪接异构体,研究其结构及功能,为探索IRF7参与调控玉型干扰素机制的多样性提供基础。通过PCR和Sanger测序获得了IRF7一种新的剪接形式IRF7-e,并通过RACE获取了IRF7-e 基因全长。IRF7-e全长为1994 bp,含5'-UTR 410 bp,3'-UTR 120 bp,开放阅读框1464 bp,编码487个氨基酸的蛋白,预测其等电点为6.659,蛋白分子量为52.8 kD。双荧光素酶报告分析表明过表达IRF7-e能够提高玉型干扰素IFNα和IFNβ启动子的活性,其中对IFNα启动子活性提高了12.18倍,对IFNβ的启动子活性提高了2.99倍。表明IRF7-e可能参与玉型干扰素的调控。     

缬沙坦改善小鼠动脉粥样硬化的作用机制研究

本研究通过给予动脉粥样硬化Apo E-/-小鼠不同剂量的缬沙坦(1 mg/kg,10 mg/kg),连续灌胃2周,观察其治疗效果,并分析正常饲养小鼠、动脉粥样硬化模型小鼠以及不同缬沙坦剂量组治疗小鼠干扰素系统蛋白和炎症反应蛋白水平的变化。研究表明,小鼠动脉粥样硬化管腔面积明显降低,斑块面积、血管内膜厚度及脂质中心面积明显升高,血清中干扰素(IFNα,IFNβ,IFNγ)、干扰素调节蛋白(IRF1,IRF2,IRF4,IRF8)、炎症反应因子(IL2,IL4,IL6,IL10,IL15)、质金属蛋白酶(MMP1,MMP9,TIMP1和TIMP3)等的表达水平均显著升高(p0.01),且与正常饲养小鼠、动脉粥样硬化模型小鼠相比有显著的统计学差异(p0.01),缬沙坦治疗后缬沙坦10 mg/kg组明显优于缬沙坦1 mg/kg组(p0.01)。因此,缬沙坦可能通过调控干扰素系统蛋白及炎症反应蛋白水平来改善小鼠动脉粥样硬化。此结果为探讨缬沙坦在改善动脉粥样硬化小鼠中的作用机制进一步深入研究提供理论依据,为临床治疗和新药开发提供参考方向。     

IRF6在颅面部发育中的作用

干扰素调节因子6(interferon regulatory factor 6, IRF6)是一种在胚胎发育过程中广泛表达的转录激活因子。研究发现, IRF6能够调控口腔上皮细胞的增殖与分化,影响唇腭的正常融合、牙釉质的形成以及牙齿形态,同时还参与颌骨成骨细胞的分化。IRF6基因的缺失或者突变会导致腭裂、牙齿缺失、颅面畸形、骨骼缺陷等颅面部异常。该文综述了IRF6在颅面部发育中的研究进展,旨在了解其在颅面部发育以及颅面畸形中的作用,以期为今后相关研究提供参考。     

杂交石斑鱼干扰素调节因子3(IRF3)基因的克隆及表达分析

干扰素调节因子家族(IRFs)具有抗病毒、免疫调节功能,干扰素调节因子3(Interferon regulatory factor 3,IRF-3)是IRFs家族中的一员,也是免疫相关因子。为了解IRF3基因在杂交石斑鱼(Epinephelus. fuscoguttatus,♀)×清水石斑鱼(E.polyphekadion,♂)应对外源病毒刺激的免疫反应,利用cDNA末端快速扩充(Rapid-amplification of cDNA ends,RACE)技术克隆了杂交石斑鱼IRF3基因,该基因cDNA全长为2 529 bp,包含5'非编码区(5'-UTR)325 bp,3'非编码区(3'-UTR)916 bp,开放阅读框(Open reading frame,ORF)1 377 bp,可编码458个氨基酸。氨基酸序列包含N-末端DNA结合区(N-terminal DNA binding region,DBD)(1-108 aa)、1个C-末端干扰素相关区(C-terminal interferon related region,IAD)(255-435 aa)及色氨酸富含区(Tryptophan rich region,SRD)(440-450 aa)3个结构域。系统进化分析结果显示,杂交石斑鱼IRF3与花鲈(Lateolabrax maculatus)IRF3聚为一支,亲缘关系较近。利用实时荧光定量PCR检测IRF3基因在杂交石斑鱼肝、胃、鳃、肠和脾脏等9种组织表达情况,以及在外周血淋巴细胞(Peripheral blood lymphocytes,PBL)的时序性表达情况,结果显示,IRF3基因在9种组织中均有表达,肝、胃和肠中的表达量明显高于其他组织,头肾表达量最低。PBL在PolyI:C刺激1 h后IRF3基因表达量逐渐升高,4 h时表达量达到最大值(约为对照组的9.3倍),8 h后逐渐下降。     

α干扰素和利巴韦林抗丙型肝炎病毒作用的比较研究

α干扰素(IFN-α)联合利巴韦林是目前临床治疗丙型肝炎病毒(HCV)感染的标准方案.本研究以体外培养的感染性病毒HCVcc为研究对象,比较IFN-α和利巴韦林对HCV复制的影响,以及对干扰素调节因子9(IRF9)和干扰素刺激基因15(ISG15)等抗病毒基因的调节能力.结果显示,100 u/ml IFN-α显著降低HCV RNA水平,利巴韦林剂量依赖性抑制HCV复制,且IFN-α联合利巴韦林对HCV复制及HCV NS3和E2蛋白表达具有协同抑制作用.5～80 u/ml IFN-α剂量依赖性诱生IRF9和ISG15;1和5μg/ml利巴韦林不促进IRF9和ISG15水平升高;而利巴韦林联合IFN-α对两者亦无促进作用.     

5'-Fluoro-5'-deoxyaristeromycin

5'-Fluoro-5'-deoxyaristeromycin (2) has been prepared via a Mitsunobu coupling of (1S,2S,3R,4S)-2,3-(cyclopentylidenedioxy)-4-fluoromethylcyclopentan-1-ol with N6-bis-boc protected adenine. This procedure is adaptable to preparing a number of 5'-fluoro-5'-deoxycarbocyclic nucleoside analogs with diversity in the heterocyclic base. Antiviral analysis found promising activity for 2 toward measles but no other viruses. No cytotoxicity was observed for 2.     

Enzyme-catalyzed redox reactions with the flavin analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphte, and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'-adenosine ester.

The ability of 5-deazaisoalloxazines to substitute for the isoalloxazine (flavin) coenzyme has been examined with several flavoenzymes. Without exception, the deazaflavin is recognized at the active site and undergoes a redox change in the presence of the specific enzyme substrate. Thus, deazariboflavin is reduced catalytically by NADH in the presence of the Beneckea harveyi NAD(P)H:(flavin) oxidoreductase, the reaction proceeding to an equilibrium with an equilibrium constant near unity. This implies an E0 of -0.310 V for the deazariboflavindihydrodeazariboflavin couple, much lower than that for isoalloxazines. With this enzyme, both riboflavin and deazariboflavin show the same stereospecificity with respect to the pyridine nucleotide, and despite a large difference in Vmax for the two, both have the same rate-determining step (hydrogen transfer). Direct transfer of the hydrogen is seen between the nicotinamide and deazariboflavin in both reaction directions. DeazaFMN reconstituted yeast NADPH: (acceptor) oxidoreductase (Old Yellow Enzyme), and deazaFAD reconstituted D-amino acid:O2 oxidoreductase and Aspergillus niger D-glucose O2 oxidoreductase are all reduced by substrate at approximately 10(-5) the rate of holoenzyme; none are reoxidized by oxygen or any of the tested artificial electron acceptors, though deazaFADH-bound to D-amino acid:O2 oxidoreductase is rapidly oxidized by the imino acid product. Direct hydrogen transfer from substrate to deazaflavin has been demonstrated for both deazaFAD-reconstituted oxidases. These data implicate deazaflavins as a unique probe of flavin catalysis, in that any mechanism for the flavin catalysis must account for the deazaflavin reactivity as well.     

Amino substituted derivatives of 5'-amino-5'-deoxy-5'-noraristeromycin

The potent antiviral potential of 5'-amino-5'-deoxy-5'-noraristeromycin (2) is limited by associated toxicity. To seek derivatives of 2 that circumvent this undesirable property, three amino substituted derivatives (acetyl, 3; formyl, 4; and methyl, 5) of 2 have been prepared in 4-7 steps from the same intermediate, (1S,4R)-4-(6-chloropurin-9-yl)cyclopent-2-en-1-ol (6). Key steps involved an improved Pd(0)-catalyzed allylic azidation and a novel Pd(0)-catalyzed allylic amidation. The three target compounds were evaluated against a large number of viruses and found to be inactive except for a very weak effect of 5 on human cytomegalovirus, varicella zoster virus, and Epstein-Barr virus. There was also no noteworthy cytotoxicity associated with the new derivatives. Thus, these results indicate variation of the cyclopentyl amine of 2 does not offer a means to improve upon its antiviral potential.     

Mechanism of inactivation of S-adenosyl-L-homocysteine hydrolase by 5'-deoxy-5'-S-allenylthioadenosine and 5'-deoxy-5'-S-propynylthioadenosine

5'-Deoxy-5'-S-allenylthioadenosine 1 and 5'-deoxy-5'-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II "mechanism-based" inhibitors.     

Independence of brachymesophalangia-5 from brachymesophalangia-5 with cone mid-5

Analysis of hand radiographs of juvenile siblings of juvenile propositi indicates that brachymesophalangia-5 alone (without cones) is separately inherited without apparent sex bias while brachymesophalangia-5 with the cone-epiphysis of mid-5 and the cone-epiphysis of mid-5 alone are both apparently inherited as a complex and with a marked excess of females over males.     

Stat5及JAK-Stat5通路

信号转导与转录因子（Stats）家族是一个由7个成员组成的转录调控家族,其中Stat5由于在多种细胞因子刺激时均可起到重要作用而引起了广泛的关注。Stat5具有两种不同类型,即Stat5a和Stat5b,它们均可被多种细胞因子所激活,启动JAK-Stat5信号通路,从而调节相应基因的表达。在两种Stat5的激活中,特殊位点的丝氨酸和酪氨酸的磷酸化对Stat5的激活起重要的作用。对信号通路严格的控制对于生物体来说具有重要的意义,在JAK-Stat5信号通路中,以SOCS3为代表的SOCS家族对JAK-Stat5的负反馈调节具有重要作用。     

Enzymatic synthesis of 5-azacytidine 5'-triphosphate from 5-azacytidine.

5-Azacytidine 5′-monophosphate (5-aza-CMP) was synthesized enzymatically from 5-azacytidine (5-aza-C) in a reaction catalyzed by uridine-cytidine kinase. In a second step, 5-azacytidine 5′-triphosphate (5-aza-CTP) was synthesized enzymatically from 5-aza-CMP using CMP kinase and nucleoside diphosphokinase. Due to the chemical instability of the triazide ring of 5-azacytosine at neutral and alkaline pH, the enzymatic synthesis and purification of the nucleotides by ion exchange chromatography were performed at acid pH. The enzymatically synthesized 5-aza-CTP had an ultraviolet absorbance spectrum at pH 5.5 similar to the spectrum of 5-aza-C. In the DNA-dependent RNA polymerase reaction, 5-aza-CTP inhibited the incorporation of [³H]CTP, but [³H]UTP, into RNA.     

Enzymatic conversion of deoxycytidine 5'-monophosphate to 5-methyldeoxycytidine 5'-triphosphate

A simple procedure for the synthesis of chiral acetic acids has been developed. The key step is an enzymatic exchange reaction which introduces ³H from ³H-labeled water into ethane 1,2-diol. The method involves no resolution of racemic intermediates and the products are of high specific radioactivity and optical purity.