甲氧基黄酮类化合物对囊性纤维化跨膜电导调节因子的激活作用

囊性纤维化跨膜电导调节因子(CFTR)是一种c AMP依赖的Cl-通道蛋白,其在上皮液体分泌过程中具有重要作用。本研究组在前期工作中观察到两种甲氧基黄酮类化合物3’,4’,5,5’,6,7-六甲氧基黄酮(HMF)和5-羟基-6,7,3’,4’-四甲氧基黄酮(HTF)能够有效地激活CFTR Cl-通道,但是作用机制尚不清楚。本研究旨在利用细胞荧光淬灭模型和短路电流技术系统研究HMF和HTF对CFTR Cl-通道的激活作用。荧光淬灭实验结果显示两种化合物均能以剂量依赖的方式激活CFTR Cl-通道,该激活作用具有快速、可逆的特点,可被CFTR特异性抑制剂CFTRinh-172完全抑制;引人注目的是,HMF(EC50=2μmol/L)是迄今发现的亲和力最高的黄酮类CFTR Cl-通道激活剂。HMF和HTF对CFTR Cl-通道的激活作用具毛喉素(forskolin,FSK)依赖特性,与FSK和3-异丁基-1-甲基黄嘌呤(3-Isobutyl-1-methylx,IBMX)的作用存在相加效应,但是与三羟基异黄酮(genistein,GEN)的作用之间不存在协同效应。离体组织研究结果显示,HMF和HTF能够显著促进大鼠结肠粘膜Cl-电流及小鼠气管粘膜下腺液体分泌。以上结果提示,HMF和HTF能够通过提高c AMP水平和直接与CFTR蛋白作用两条途径发挥CFTR Cl-通道激活作用。本研究为深入揭示黄酮类CFTR Cl-通道激活剂结构与功能之间的关系奠定了基础。     

内含肽介导的氯离子通道蛋白CFTR的反式剪接

研究利用内含肽(intein)的蛋白质反式剪接功能在大肠杆菌中对囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane regulator, CFTR)的反式剪接作用.CFTR基因突变导致一种常染色体隐性遗传疾病囊性纤维化(cystic fibrosis, CF).将CFTR的cDNA于剪接反应所需的保守性氨基酸残基Ser-660前断裂为N端和C端,分别与split mini Ssp DnaB 内含肽的106个氨基酸残基的N端和48个氨基酸残基的C端编码序列融合,构建到原核表达载体pBV220 诱导表达后SDS-PAGE可见预期大小剪接形成的CFTR蛋白条带,Western印迹用CFTR特异性抗体进一步证明为剪接所产生的CFTR蛋白,表明内含肽可有效催化CFTR的反式剪接.     

氯离子通道CFTR在肝脏慢性炎症中的作用

目的分析囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)敲除小鼠肝组织中炎症相关因子的表达变化,为进一步探讨CFTR在调节肠肝微生态平衡中的作用奠定理论基础。方法利用CFTR基因敲除小鼠肝组织,采用Western blot检测炎性细胞因子JNK和AKT活性的变化。结果 CFTR敲除小鼠肝组织中炎性细胞因子JNK和AKT的活性表达均有显著提高。结论 CFTR具有抑制炎症发生发展的作用。     

CFTR基因在小鼠肠道组织中的差异表达

目的研究肠道组织CFTR基因表达与分泌性腹泻发生的关系。方法选取KM小鼠24只,雌雄各半,随机分为3组（每组8只）：对照组经小鼠腹腔注射0.2 mL生理盐水,实验组小鼠经腹腔注射LPS[6 mg/（kg·bw）]分别作用1 h、8 h,于注射后通过小鼠精神状态、肠道组织形态学判定分泌性腹泻模型的建立,利用荧光定量PCR法检测各段肠道组织CFTR基因的表达。结果 LPS成功诱导小鼠发生了分泌性腹泻;CFTR基因在小鼠十二指肠、空肠、回肠和结肠组织中均有不同的表达丰度,以结肠最高,但各段肠道间差异不显著;与对照组相比,LPS上调了十二指肠、空肠和回肠CFTR基因的转录,下调了结肠CFTR基因的转录。结论提示肠道组织CFTR基因转录水平的上调与LPS诱导分泌性腹泻的发生密切相关,且在各肠段发挥的作用不同,其中空肠在氯离子（Cl-）分泌中发挥主要作用,结肠的作用最弱。     

PLoS Gene：研究表明囊性纤维化是两种类型疾病

一项由美国匹兹堡大学医学院多中心团队完成的新研究提示：囊性纤维化（CF）其实是两种不同的疾病。一种影响多个器官包括肺，一种不影响肺。这项研究发表在PLOS Genetics杂志上，揭示了与囊性纤维化相关基因变异中的9个可导致胰腺炎。鼻窦炎和男性不育症。但却不对肺部造成伤害。 CF患者继承分别来自父母各方的CFTR基因的一个严重突变拷贝，CFTR生成那些构成通道来转运氯化物分子进出细胞的蛋白质，研究员David Whitcomb医学博士表示：没有功能性CFTR通道会导致出现问题，例如CF有关的慢性肺阻塞。     

基于CFTR的胞浆内第二信使cAMP检测方法的建立*

目的: 建立一种基于CFTR可敏感检测胞浆内第二信使cAMP的检测方法。方法: 构建CFTR和YFP-H148Q/I152L真核表达载体,应用脂质体转染法构建共表达CFTR和YFP-H148Q/I152L的FRT细胞,倒置荧光显微镜观察其表达情况,流式细胞仪检测细胞纯度;荧光淬灭动力学实验验证细胞模型的有效性;荧光淬灭动力学实验验证细胞模型可筛选CFTR调节剂;放射免疫法检测加入CFTR激活剂后细胞内的cAMP浓度。结果: 倒置荧光显微镜下观察到CFTR表达在细胞膜上,YFP-H148Q/I152L表达于胞浆中;成功构建共表达CFTR和YFP-H148Q/I152L的FRT细胞模型;荧光变化斜率值与CFTR调节剂浓度成剂量依赖关系,该模型可筛选CFTR调节剂;荧光变化斜率值可反映胞浆内cAMP浓度,该模型可敏感检测胞浆内cAMP浓度。结论: 此细胞模型可以高效敏感检测胞浆内第二信使cAMP浓度,为cAMP信号相关靶点的研究提供一种简便快捷的方法。     

TMD2前断裂CFTR翻译后的连接及氯离子通道功能

CFTR基因突变导致一种常染色体隐性遗传疾病——囊性纤维化(CF)。利用split Ssp DnaB intein的蛋白质反式剪接技术的真核细胞双载体转CFTR基因,旨在研究翻译后水平CFTR的连接,以及由其建立的氯离子通道功能。于CFTR膜内第2个跨膜结构域(TMD2)前的Glu838密码子后将其cDNA断裂为N端和C端两部分,与具有蛋白质反式剪接作用的split Ssp DnaB intein编码序列融合,分别插入到载体pEGFP-N1和pEYFP-N1,构建一对真核表达载体pEGFP-NInt和pEYFP-IntC。用脂质体将这对载体共转染至幼年仓鼠肾细胞(BHK),瞬时表达实验用Western blotting观察CFTR蛋白质的连接,并用膜片钳技术记录Cl-通道电流。结果显示,基因共转染细胞呈现完整的CFTR蛋白条带,膜片钳记录到全细胞Cl-电流和单个Cl-通道开放活性。结果表明split Ssp DnaB intein的蛋白质反式剪接技术可用于双载体共转移CFTR基因,为CF基因治疗应用双腺相关病毒载体(AAV)转运CFTR基因,克服AAV的容量限制提供了依据。     

囊性纤维化跨膜转运调节体氯离子通道——跨上皮离子转运的多功能引擎

囊性纤维化跨膜转运调节体（cystic fibrosis transmembrane conductance regulator,CFTR）是ATP结合转运体超家族（ATP-binding cassette transporter superfamily）的一名特殊成员,因为它是一个具有相当复杂调控机制的氯离子通道。CFTR由五个结构域（domain）组成：两个跨膜结构域（membrane-spanning domains,MSDs）,两个核苷酸结合域（nucleotide-binding domains．NBDs）和一个特殊的调控域（regulatory domain,RD）。MSDs构成一个低电导（6-12pS）的阴离子选择性孔道（pore）,其形状如同不对称的沙漏,胞外小胞内大,狭窄部分为离子筛。两个NBDs组成头尾相对的二聚体,在二聚体之间的接触面上有两个能和ATP结合的位点（位点1和位点2）。CFTR的门控机制是：ATP分子与位点1和2相互作用促使NBD二聚体的结合与解离,从而引起MSDs的构象发生变化进而使通道孔打开和关闭。RD具有多样化的结构,它含有多个磷酸化共有位点（consensus　phosphorylation　sites）。RD的磷酸化促进NBDs与ATP的结合,从而使CFTR得以激活。CFTR通过支架蛋白与其它膜受体以及蛋白激酶、磷酸酶形成大分子信号复合体。在复杂的细胞信号系统参与下,CFTR的功能活动在时间和空间上得到精确的调控。此外,CFTR的活动与细胞代谢有紧密联系：CFTR与代谢酶形成大分子复合体,当细胞能量需求增加时,CFTR活动会受到抑制而使细胞能量得以保存。CFTR广泛分布于机体上皮组织,它通过促进水盐转运而控制上皮细胞分泌物的量与组成。值得注意的是,在呼吸道,CFTR还对机体的防御机制起重要作用。CFTR功能失常严重影响跨上皮离子转运,进而引起或加重某些疾病。     

囊性纤维化跨膜电导调节体:ATP结合和水解门控Cl-通道

囊性纤维化跨膜电导调节体（cystic fibrosis transmembrane conductance regulator,CFTR）是一种Cl^-通道,属于ATP结合（ATP-binding cassette,ABC）转运体超家族。CFTR功能缺陷是高加索人种中普遍存在的致死性常染色体隐性遗传疾病囊性纤维化（cystic fibrosis,CF）发生的主要原因。这种疾病患者各组织上皮细胞内Cl^-转运失调。目前,与CF相关的不同突变超过1400种。CFTR调节（regulatory,R）域负责调控,核苷酸结合域（nucleotide-binding domains,NBDs）NBD1和NBD2负责ATP结合和水解门控。近期研究发现CFFR的NBDs与其它ABC蛋白一样可以二聚化。二聚化过程中,NBD1和NBD2首-尾相连,一个NBD上的WalkerA和B模块与另一个NBD提供的标签序列（signature sequence）形成ATP结合袋（ATP-binding pockets,ABPs）ABP1和ABP2。ABPs中与ATP结合相关的氨基酸突变实验揭示,ABP1和ABP2在CFTR的ATP依赖门控中发挥不同作用。ABP2由NBD2上的WalkA和B模块与NBD1提供的标签序列形成,它与ATP结合催化通道开放,而ABP1单独与ATP结合不能促进通道开放,只能稳定通道构象。有一些CFrR突变相关疾病的特征就是门控失调,进一步深入研究CFTR的NBD1和NBD2如何通过相互作用而达到通道门控,将为药理学研究提供更多所需的机制信息,有利于为CF治疗的药物设计铺平道路。     

CFTR型氯离子通道研究进展

囊性纤维化跨膜传导调节因子（CFTR）是一种重要的氯离子通道,突变易引起囊性纤维化病变,故得名。一系列研究表明,CFTR由5个结构域组成：两个跨膜结构域形成氯离子通道;两个核苷酸结合结构域调节通道的开闭;一个调节结构域主要影响氯通道的活动。这些结构域通过协同作用共同控制了氯离子的跨膜流动,而一些突变可以影响细胞功能而导致囊性纤维化的发生。本文通过介绍CFTR基本结构、调节机制、与囊性纤维化病变的关系及针对CFTR的治疗而对CFTR型氯离子通道有一个的全面的理解。     

On the origin of the Hirudinea and the demise of the Oligochaeta

The phylogenetic relationships of the Clitellata were investigated with a data set of published and new complete 18S rRNA gene sequences of 51 species representing 41 families. Sequences were aligned on the basis of a secondary structure model and analysed with maximum parsimony and maximum likelihood. In contrast to the latter method, parsimony did not recover the monophyly of Clitellata. However, a close scrutiny of the data suggested a spurious attraction between some polychaetes and clitellates. As a rule, molecular trees are closely aligned with morphology-based phylogenies. Acanthobdellida and Euhirudinea were reconciled in their traditional Hirudinea clade and were included in the Oligochaeta with the Branchiobdellida via the Lumbriculidae as a possible link between the two assemblages. While the 18S gene yielded a meaningful historical signal for determining relationships within clitellates, the exact position of Hirudinea and Branchiobdellida within oligochaetes remained unresolved. The lack of phylogenetic signal is interpreted as evidence for a rapid radiation of these taxa. The placement of Clitellata within the Polychaeta remained unresolved. The biological reality of polytomies within annelids is suggested and supports the hypothesis of an extremely ancient radiation of polychaetes and emergence of clitellates.     

On the ontogeny of the haptor and the evolution of the Monogenea

Data on the ontogeny of the posterior haptor of monogeneans were obtained from more than 150 publications and summarised. These data were plotted into diagrams showing evolutionary capacity levels based on the theory of a progressive evolution of marginal hooks, anchors and other attachment components of the posterior haptor in the Monogenea (Malmberg, 1986). 5 + 5 unhinged marginal hooks are assumed to be the most primitive monogenean haptoral condition. Thus the diagrams were founded on a 5 + 5 unhinged marginal hook evolutionary capacity level, and the evolutionary capacity levels of anchors and other haptoral attachement components were arranged according to haptoral ontogenetical sequences. In the final plotting diagram data on hosts, type of spermatozoa, oncomiracidial ciliation, sensilla pattern and protonephridial systems were also included. In this way a number of correlations were revealed. Thus, for example, the number of 5 + 5 marginal hooks correlates with the most primitive monogenean type of spermatozoon and with few sensillae, many ciliated cells and a simple protonephridial system in the oncomiracidium. On the basis of the reviewed data it is concluded that the ancient monogeneans with 5 + 5 unhinged marginal hooks were divided into two main lines, one retaining unhinged marginal hooks and the other evolving hinged marginal hooks. Both main lines have recent representatives at different marginal hook evolutionary capacity levels, i.e. monogeneans retaining a haptor with only marginal hooks. For the main line with hinged marginal hooks the name Articulon-choinea n. subclass is proposed. Members with 8 + 8 hinged marginal hooks only are here called Proanchorea n. superord. Monogeneans with unhinged marginal hooks only are here called Ananchorea n. superord. and three new families are erected for its recent members: Anonchohapteridae n. fam., Acolpentronidae n. fam. and Anacanthoridae n. fam. (with 7 + 7, 8 + 8 and 9 + 9 unhinged marginal hooks, respectively). Except for the families of Articulonchoinea (e.g. Acanthocotylidae, Gyrodactylidae, Tetraonchoididae) Bychowsky's (1957) division of the Monogenea into the Oligonchoinea and Polyonchoinea fits the proposed scheme, i.e. monogeneans with unhinged marginal hooks form one old group, the Oligonchoinea, which have 5 + 5 unhinged marginal hooks, and the other group form the Polyonchoinea, which (with the exception of the Hexabothriidae) has a greater number (7 + 7, 8 + 8 or 9 + 9) of unhinged marginal hooks. It is proposed that both these names, Oligonchoinea (sensu mihi) and Polyonchoinea (sensu mihi), will be retained on one side and Articulonchoinea placed on the other side, which reflects the early monogenean evolution. Except for the members of Ananchorea [Polyonchoinea], all members of the Oligonchoinea and Polyonchoinea have anchors, which imply that they are further evolved, i.e. have passed the 5 + 5 marginal hook evolutionary capacity level (Malmberg, 1986). There are two main types of anchors in the Monogenea: haptoral anchors, with anlages appearing in the haptor, and peduncular anchors, with anlages in the peduncle. There are two types of haptoral anchors: peripheral haptoral anchors, ontogenetically the oldest, and central haptoral anchors. Peduncular anchors, in turn, are ontogenetically younger than peripheral haptoral anchors. There may be two pairs of peduncular anchors: medial peduncular anchors, ontogentically the oldest, and lateral peduncular anchors. Only peduncular (not haptoral) anchors have anchor bars. Monogeneans with haptoral anchors are here called Mediohaptanchorea n. superord. and Laterohaptanchorea n. superord. or haptanchoreans. All oligonchoineans and the oldest polyonchoineans are haptanchoreans. Certain members of Calceostomatidae [Polyonchoinea] are the only monogeneans with both (peripheral) haptoral and peduncular anchors (one pair). These monogeneans are here called Mixanchorea n. superord. Polyonchoineans with peduncular anchors and unhinged marginal hooks are here called the Pedunculanchorea n. superord. The most primitive pedunculanchoreans have only one pair of peduncular anchors with an anchor bar, while the most advanced have both medial and lateral peduncular anchors; each pair having an anchor bar. Certain families of the Articulonchoinea, the Anchorea n. superord., also have peduncular anchors (parallel evolution): only one family, the Sundanonchidae n. fam., has both medial and lateral peduncular anchors, each anchor pair with an anchor bar. Evolutionary lines from different monogenean evolutionary capacity levels are discussed and a new system of classification for the Monogenea is proposed.In agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. EditorIn agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. Editor