FK506结合蛋白12.6调节小鼠嗜铬细胞分泌

FK506结合蛋白12.6(FKBP12.6)能够结合并调控钙离子释放通道兰尼碱受体2型(RyR2)的开放,可能是儿茶酚胺分泌的重要调控器.利用FKBP12.6敲除小鼠模型,我们研究了FKBP12.6在肾上腺嗜铬细胞胞吐中的作用.结果表明,FKBP12.6在小鼠肾上腺嗜铬细胞中表达,而敲除FKBP12.6小鼠的嗜铬细胞中有正常的去极化引起的钙电流和胞吐作用.然而,FKBP12.6敲除会导致嗜铬细胞中出现增强的咖啡因引起的细胞整体钙瞬变和咖啡因引起的胞吐作用.结果提示,FKBP12.6调控肾上腺嗜铬细胞儿茶酚胺的分泌,这种调控作用是通过调节钙离子的释放而实现的.FKBP12.6是嗜铬细胞分泌的重要蛋白.     

鼠伤寒沙门菌入侵宿主细胞机制研究进展*

鼠伤寒沙门菌(Salmonella typhimurium)是一种人畜共患的肠道病原菌,可引起肠道炎症。该病原菌主要通过其致病岛(SPIs)编码的III型分泌系统(T3SS)分泌效应因子,包括促炎因子和抗炎因子。其在入侵肠上皮细胞时会释放促炎因子引发炎症反应,同时,为防止促炎因子过度破坏宿主细胞影响菌体的生存和繁殖,鼠伤寒沙门菌会产生一系列抗炎因子来调节细胞内信号通路,与宿主共同繁殖并最终全身扩散造成严重感染。旨在对鼠伤寒沙门菌利用T3SS效应因子入侵并调节宿主细胞信号通路机制进行概述。     

嗜水气单胞菌AH-1 quorum sensing 负调控Ⅲ型分泌系统的表达

通过构建嗜水气单胞菌AH-1 Quorum Sensing(QS)2个关键调节基因ahyI,ahyR的突变菌株,来系统分析嗜水气单胞菌AH-1Ⅲ型分泌系统基因,揭示它们由QS系统调控.在ahyI突变菌中,TTSS分泌效应因子(effector)aexT量显著提高.通过构建LacZ-TTSS基因启动子融合表达,进一步表明QS系统负调控编码TTSS组分的基因.     

昆虫肠道微生物稳态的调控机制

稳定的肠道微生物内环境是肠道微生物与肠道免疫反应相互作用的结果。在不断的进食过程中,昆虫肠道微生物种类和数量不断发生变化,肠道微生物与肠道上皮细胞之间形成了复杂的、动态的平衡机制。昆虫肠道上皮细胞可以感知有益和有害条件并利用免疫调控通路来实现微生物种群稳态的动态调节,例如双重氧化酶-活性氧(dual oxidase-reactive oxygen species, Duox-ROS)系统和免疫缺陷(immunodeficiency, Imd)信号通路可以感知肠道微生物数量变化并参与到肠道微生物稳态调节过程。除此之外,肠道微生物群也会通过群体感应(quorum sensing, QS)释放相应的效应因子来调节菌群行为,间接性起到稳态调节的作用。因此,本文综述了昆虫肠道中物理防御、免疫信号通路以及肠道微生物通过QS在昆虫肠道微生物稳态维持中的作用,加深对肠道组织与肠道微生物互作关系的认识。未来将继续对更多种类昆虫体内微生物的稳态调控机制及调控机制间的作用关系进行研究,并基于调控机制设计开发改变肠道微生物稳态的新型农药,为实现有效害虫防治提供新的靶标和思路。     

磷脂酶C基因家族研究进展

磷脂酶C(Phospholipase C,PLC)基因是磷脂酶基因家族中的一个成员,它能够水解磷脂酰肌醇4,5-二磷酸生成两个重要的信使分子肌醇三磷酸和二酰甘油。在动物中磷脂酶C基因可以通过调节胞内钙离子的释放以及激活蛋白激酶C来起作用,而植物中磷脂酶C可以参与植物对生物及非生物胁迫的调节,但其作用的具体方式仍不清楚。综述了磷脂酶C基因的研究进展,主要包括基因的分类、结构以及其在不同逆境信号转导中的作用方式。     

培养的心内膜内皮细胞形态和旁分泌特点的初步研究

心内膜内皮(EEC)是心脏功能重要的调节者。本实验观察培养的新生小牛右心室EEC形态特点及其血管活性物质的分泌功能。培养的EEC较同样条件下培养的肺动脉内皮细胞(PAEC)大而伸展。免疫组化发现EEC的VIII因子相关抗原(VIII_(Ag))、心钠素(ANP)染色较弱,而EEC的内皮素(ET-1)阳性反应染色较PAEC强。采用放免测定发现EEC可向培养液中释放分泌ET-1,其释放量是PAEC的2.6倍,其血管紧张素Ⅱ(ATⅡ)分泌量与PAEC类似,但ANP和内源性洋地黄因子(EDLF)的释放量显著低于PAEC(P<0.01)。上述观察揭示,右心室EEC能通过旁分泌血管活性成份特别是能分泌大量的ET-1,从而调节心肌功能,同时对肺循环也可能起重要的调节作用。     

5-羟色胺与Toll样受体及肠道菌群之间关系的研究

5-羟色胺(5-HT)是参与调节胃肠道运动、内脏敏感性、分泌等功能的重要神经递质和信号分子。肠道菌群对5-HT的产生有重要影响,已发现一些产芽孢细菌(spore-forming bacteria,SP)类肠道微生物能刺激肠嗜铬细胞(enterochromaffin cell,EC)产生5-HT。微生物通过Toll样受体(Toll-like receptors,TLR)激活神经分泌机制来调节胃肠运动,某些TLR通过作用于5-HT受体调节小鼠回肠的自主收缩和5-HT诱导的收缩反应。通过调节肠道菌群可以对5-HT综合征及其相关生理和病理状况产生重要影响。因此,对5-HT与TLR及肠道菌群相互之间的关系进行进一步的研究有重要意义。     

抗生素诱导革兰阴性菌外膜囊泡产生及发挥生理作用的机制

外膜囊泡是革兰阴性菌分泌的一种球形纳米颗粒,由外膜及其所含成分组成,是细菌在外界压力条件下分泌的具有生理活性的特殊结构。外界压力如抗生素、缺氧等可触发细菌释放外膜囊泡,甚至在正常生长周期中,一些革兰阴性菌也会释放囊泡。外膜囊泡与细菌的多种生理过程相关,如应激反应、毒素传递、致病、细胞间通讯、免疫调节、基因水平转移及维持微生物群稳态等。在使用抗生素治疗过程中,尤其是当人体微生物群处于低剂量抗生素环境时,细菌会大量分泌外膜囊泡。在肠道中,外膜囊泡释放后会通过多种机制刺激肠道而引发多种炎症。本文综述了外膜囊泡的产生、结构及生理作用,提出抗生素治疗不但会破坏人体正常菌群而导致菌群失调,还会诱导细菌产生大量外膜囊泡而引发慢性炎症。噬菌体治疗不破坏正常菌群,特异性杀灭细菌时也不引起外膜囊泡的产生,因此开发使用噬菌体靶向治疗细菌感染将大大减少不良反应。     

拆分获得(S)-酮基布洛芬脂肪酶基因在枯草芽孢杆菌中的克隆与表达

【目的】筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因,构建其表达分泌型工程菌,并进一步提高该脂肪酶的立体选择性。【方法】以自筛选出的一株具有不对称拆分消旋酮基布洛芬氯乙酯能力的菌株NK13为材料,通过构建其基因组文库,筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因。通过构建该脂肪酶基因的分泌型诱导表达载体pHY300-plk-sacR-gene,将其转入枯草芽孢杆菌WB600,获得基因重组菌WB600(pHY300-plk-sacR-gene)。用SDS-PAGE检测其表达和转化情况,采用非变性聚丙烯酰胺凝胶电泳的方法纯化脂肪酶;并利用TLC和HPLC检测该酶的立体选择专一性。【结果】得到了具有专一性拆分获得(S)-酮基布洛芬能力、长度为633bp的脂肪酶基因(GenBank登录号为:EU381317)。该脂肪酶在枯草芽孢杆菌WB600中得到了分泌表达。TLC和HPLC检测结果显示,纯化的脂肪酶对底物转化40h时转化率为30%,生成(S)-酮基布洛芬的e.e.%值最高,达60.02%,与未加Tween-80的枯草芽孢杆菌转化子体系相同。而在含Tween-80的环境下,枯草芽孢杆菌表达重组菌对底物转化36h时转化率约为45%,生成(S)-酮基布洛芬的e.e.%值最高,达93.64%,是野生菌NK13的16倍。【结论】从NK13号菌株中筛选得到的新的脂肪酶具有很高的不对称拆分获得(S)-酮基布洛芬的能力,实现了NK13菌中633bp脂肪酶基因在枯草芽孢杆菌中的分泌表达,研究证明Tween-80能提高该脂肪酶的拆分专一性。     

载脂蛋白M与脂蛋白的代谢及其调节

载脂蛋白M(apoM)是lipocalin家族成员,其表达具有组织特异性.血小板活化因子(PAF)能增加apoM以及apoM mRNA在Hepg2细胞中的表达,而PAF受体拮抗剂(1exipafant)能抑制apoM的表达.apoM血清水平的高低与肝细胞核因子(HNF-1a)浓度有关.胰岛素在体内也调节apoM的生成.瘦素(Levtin)特异性调节apoM的分泌.apoM大部分存在于高密度脂蛋白(HDL)中,影响HDL的代谢并具有抗动脉粥样硬化作用.通过对apoA-I缺陷小鼠研究发现:正常小鼠体内apoM与apoA-I代谢有关.在低密度脂蛋白(LDL)与HDL中apoM存在5个亚型.在汉族人群中apoM基因的核苷酸778C等位基因以及855C均能增加患冠状动脉粥样硬化的危险性.apoM似乎与2型糖尿病患者的动脉粥样硬化的因子相关.     

Calcium-dependent activator protein for secretion 2 interacts with the class II ARF small GTPases and regulates dense-core vesicle trafficking

The Ca(2+) -dependent activator protein for secretion (CAPS) family consists of two members (CAPS1 and CAPS2) and regulates the exocytosis of catecholamine-containing or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites such as nerve terminals. A large fraction of CAPS1, however, is localized in the cell soma, and we have recently shown the possible involvement of somal CAPS1 in DCV trafficking in the trans-Golgi network. CAPS1 and CAPS2 are differentially expressed in various regions of the mouse brain but exhibit similar expression patterns in other tissues, such as the spleen. Thus, in the present study we analyzed whether CAPS2 displays similar subcellular localization and functional roles in the cell soma as CAPS1. We found that somal CAPS2 is associated with the Golgi membrane, and mediates binding and recruitment of the GDP-bound form of ARF4 and ARF5 (members of the membrane-trafficking small GTPase family) to the Golgi membrane. CAPS2 knockdown and overexpression of CAPS2-binding-deficient ARF4/ARF5 both induced accumulation of the DCV resident protein chromogranin?A around the Golgi apparatus. CAPS2 knockout mice have dilated trans-Golgi structures when viewed by electron microscopy. These results for CAPS2 strongly support our idea that the CAPS family proteins exert dual roles in DCV trafficking, mediating trafficking at both the secretion site for exocytosis and at the Golgi complex for biogenesis.     

UNC-10在致密核心囊泡分泌过程中的作用

Rim是囊泡分泌活性区中的重要组成蛋白,它与细胞分泌和突触可塑性相关．在秀丽隐感线虫中只存在一种编码Rim的基因即unc-10．我们的研究发现,在线虫中Rim的基因突变unc-10(md1117)会导致致密核心囊泡的分泌缺陷．在活体中,unc-10突变虫系的神经多肽分泌显著下降．此外,在主要分泌致密核心囊泡的ALA神经元内,钙光解释放促发的快相分泌也比野生型减少．运用全内反射荧光显微成像技术,我们观察在unc-10缺失的情况下ALA 神经元中致密核心囊泡的锚定过程,结果显示在细胞膜附近停留的囊泡数目减少,表明囊泡锚定受到阻碍．上述试验结果表明,UNC-10能够影响致密核心囊泡的分泌过程,其机制可能是影响了囊泡的锚定过程．     

Regulation of fusion pore closure and compound exocytosis in neuroendocrine PC12 cells by SCAMP1

During exocytosis, neuroendocrine cells can achieve partial release of stored secretory products from dense core vesicles (DCVs) by coupling endocytosis directly at fusion sites and without full discharge. The physiological role of partial secretion is of substantial interest. Much is known about SNARE-mediated initiation of exocytosis and dynamin-mediated completion of endocytosis, but little is known about coupling events. We have used real-time microscopy to examine the role of secretory carrier membrane protein SCAMP1 in exo-endocytic coupling in PC12 cells. While reduced SCAMP1 expression is known to impede dilation of newly opened fusion pores during onset of DCV exocytosis, we now show that SCAMP1 deficiency also inhibits closure of fusion pores after they have opened. Inhibition causes accumulation of fusion figures at the plasma membrane. Closure is recovered by restoring expression and accelerated slightly by overexpression. Interestingly, inhibited pore closure resulting from loss of SCAMP1 appears to increase secondary fusion of DCVs to already-fused DCVs (compound exocytosis). Unexpectedly, reinternalization of expanded DCV membranes following compound exocytosis appears to proceed normally in SCAMP1-deficient cells. SCAMP1's apparent dual role in facilitating dilation and closure of fusion pores implicates its function in exo-endocytic coupling and in the regulation of partial secretion. Secondarily, SCAMP1 may serve to limit the extent of compound exocytosis.     

Interaction of calcium-dependent activator protein for secretion 1 (CAPS1) with the class II ADP-ribosylation factor small GTPases is required for dense-core vesicle trafficking in the trans-Golgi network

Ca(2+)-dependent activator protein for secretion (CAPS) regulates exocytosis of catecholamine- or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites, such as nerve terminals. However, large amounts of CAPS protein are localized in the cell soma, and the role of somal CAPS protein remains unclear. The present study shows that somal CAPS1 plays an important role in DCV trafficking in the trans-Golgi network. The anti-CAPS1 antibody appeared to pull down membrane fractions, including many Golgi-associated proteins, such as ADP-ribosylation factor (ARF) small GTPases. Biochemical analyses of the protein-protein interaction showed that CAPS1 interacted specifically with the class II ARF4/ARF5, but not with other classes of ARFs, via the pleckstrin homology domain in a GDP-bound ARF form-specific manner. The pleckstrin homology domain of CAPS1 showed high affinity for the Golgi membrane, thereby recruiting ARF4/ARF5 to the Golgi complex. Knockdown of either CAPS1 or ARF4/ARF5 expression caused accumulation of chromogranin, a DCV marker protein, in the Golgi, thereby reducing its DCV secretion. In addition, the overexpression of CAPS1 binding-deficient ARF5 mutants induced aberrant chromogranin accumulation in the Golgi and consequently reduced its DCV secretion. These findings implicate a functional role for CAPS1 protein in the soma, a major subcellular localization site of CAPS1 in many cell types, in regulating DCV trafficking in the trans-Golgi network; this activity occurs via protein-protein interaction with ARF4/ARF5 in a GDP-dependent manner.     

ESYT-2 在致密核心囊泡分泌中起到的作用

目的:研究秀丽隐杆线虫(简称线虫)中一种扩展的Synaptotagmin同源物ESYT-2在致密核心囊泡分泌过程中起到的作用。方法:以线虫为研究对象,运用线虫腔胞吸收ANF-GFP的基本原理来确定ESYT-2与分泌相关,之后又进一步使用全内反射荧光显微镜技术(TIRFM)来研究ESYT-2对致密核心囊泡的具体调控。结果:①ESYT-2功能缺失影响线虫神经细胞致密核心囊泡分泌。②ESYT-2影响致密核心囊泡分泌的栓系过程。结论:ESYT-2调控了致密核心囊泡的分泌过程。     

RIC-7 promotes neuropeptide secretion

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV-mediated secretion.     

CAPS acts at a prefusion step in dense-core vesicle exocytosis as a PIP2 binding protein

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.     

ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIgamma restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIgamma with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.     

Intrapulmonary neuro-epithelial bodies in newborn rabbits

Summary Neonatal rabbit neuro-epithelial bodies (NEB) were investigated under various experimental conditions with light microscopy, microspectrography, morphometry and electron microscopy. (1) Hypoxia causes a decreased amine fluorescence intensity and an increased secretory exocytosis of dense core vesicles (DCV). Otherwise the NEB appear structurally normal. (2) Hypercapnia also produces a decreased fluorescence and an increased exocytosis; ultrastructurally, however, the dense core of DCV fragmentizes. (3) Hyperoxia does not appear to affect significantly either fluorescence or exocytosis. (4) The uptake of biogenic amines such as 5-HTP and L-DOPA was demonstrated by fluorometry and electron microscopy. (5) Reserpine, on the other hand, provokes an amine depletion with a decrease of the NEB fluorescence and an ultrastructural palor of the DCV. (6) Intratracheally administered nicotine is accompanied by a decreased fluorescence and a distinct exocytosis of fragmented DCV.The reaction of NEB to hypoxia and hypercapnia suggests that these corpuscles could be intrapulmonary chemoreceptors (in addition to the classically known central and peripheral chemoreceptors), inducing a reflex reaction through the liberation of DCV at the corpuscular sensible nerve endings and via the CNS. In addition, they may subserve a local intrapulmonary effect by modulating directly the hypoxic and hypercapnic pulmonary vasoconstriction and thus the V/Q ratio. Acknowledgment. This study was supported by a grant from The Council for Tobacco Research, U.S.A., and the Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium. We thank M.R. Van Hamme, R. Renwart and K. Armee for technical, G. Pison and St. Ons for photographical and N. Tyberghien and G. Verbeeck for secretarial assistanceMartin Deleersnyder (deceased May 11, 1976) was Candidate of the Nationaal Fonds voor Wetenschappelijk Onderzoek, Belgium     

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.