马铃薯块茎发育机理及其基因表达

马铃薯(Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成。对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日照、较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化。然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后。已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关。综述了这一领域现有的研究进展。     

植物CDPK在响应逆境胁迫中的作用及机制

钙依赖性蛋白激酶(calcium-dependent protein kinase,CDPK/CPK)是一类Ca²⁺敏感的Ser/Thr蛋白激酶,在植物生长发育和逆境胁迫响应中发挥重要作用。CDPK能够迅速感知细胞内瞬时Ca²⁺信号的变化,识别并磷酸化特异性底物,从而将Ca²⁺信号向下游传递并级联放大,广泛参与干旱、盐碱和伤害应激等逆境胁迫,调控植物生长发育以及相关基因表达、离子通道和气孔运动等。CDPK的自磷酸化会影响其酶活性以及底物的选择性。CDPK具有与多种底物结合并磷酸化的能力,除了参与呼吸暴发氧化酶同源物(respiratory burst oxidase homolog,RBOH)、丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)、植物激素等信号通路,CDPK还可以与14-3-3蛋白结合,调控植物应对逆境胁迫和促进生长发育。本研究综述了植物CDPK的发现、结构、分类及其在逆境胁迫响应中的作用等方面的研究成果,并对其未来研究方向进行展望,为农作物抗逆性遗传改良提供了基因资源和理论依据。     

CaCl2对铁棍山药块茎采后几种与膜脂过氧化相关的生理指标的影响

铁棍山药决茎采后经0．1％-1.0％（WIV）的CaCl2溶液处理后,其含水量增加,相对电导率减小,丙二醛（MDA）含量降低,超氧化物歧化酶（SOD）活性升高,超氧阴离子（O2^-）产生速率下降;而浓度为2．0％的CaCl2溶液则促进铁棍山药块茎贮藏后期SOD活性下降,对含水量增加和相对电导率、MDA含量以及O^2-产生速率下降的效果不稳定。     

山药异淀粉酶基因克隆及其在淀粉代谢中的作用

为了明确异淀粉酶基因(ISA 3)在山药淀粉代谢中的作用,该研究以‘毕克齐’和‘大和长芋’山药为试验材料,测定了块茎中淀粉及组分含量和异淀粉酶活性等;采用RT-PCR技术克隆了ISA 3,并进行生物学分析及山药块茎不同膨大期和不同组织间ISA 3基因的表达等。结果表明:(1)山药‘大和长芋’的直链淀粉、支链淀粉和总淀粉含量均显著高于‘毕克齐’,且两品种的淀粉含量随生长发育的变化均呈先升高后降低的趋势,并均于种植后120 d时达到最高,但‘毕克齐’的异淀粉酶(ISA)活性在整个膨大期均高于‘大和长芋’。(2)成功克隆获得山药ISA 3开放阅读框长1584 bp,编码527个氨基酸;ISA3为亲水性蛋白。(3)不同品种块茎在膨大时期的ISA 3基因表达趋势不同,‘毕克齐’中呈先显著上调随后下调,而在‘大和长芋’中表达总体下调,且在山药的叶、茎和块茎中均有表达,存在明显的组织特异性。(4)ISA活性与山药淀粉及支链淀粉含量呈显著和极显著正相关关系,但ISA活性与ISA 3的表达量呈负相关关系。研究表明,异淀粉酶参与了山药块茎中淀粉的合成,且主要对支链淀粉的合成起关键作用,ISA 3基因的表达可能对异淀粉酶活性和淀粉的合成起负调控作用。     

Egr-1对APP基因表达的调控作用

该研究构建了含有APP启动子的荧光素酶报告质粒以及缺失突变的报告质粒,将该质粒与Egr-1真核表达载体p CDNA3-Egr-1共转染到HEK293与U87MG细胞中进行荧光素酶活性测定,以确定Egr-1对APP基因表达的调控作用。通过染色质免疫共沉淀实验(chromatin immunoprecipitation assay,Ch IP)和凝胶电泳迁移实验(electrophoretic mobility shift assay,EMSA)确定Egr-1蛋白在APP启动子上的结合部位,同时利用组蛋白去乙酰化酶抑制剂TSA(trichostatin A)和butyrate诱导内源性Egr-1的表达以及Egr-1抑制剂suramin来作用于细胞,通过蛋白免疫印迹分析(Western blot)检测Egr-1对APP蛋白表达的作用。结果显示,Egr-1蛋白在APP基因启动子上5′UTR区域有特异性的结合部位,去除该结合部位则Egr-1失去对APP基因启动子的调控作用,Ch IP和EMSA的结果也证实了该结合位点;通过HDAC抑制剂和suramin干扰内源性Egr-1的表达则显著影响了Egr-1对APP基因表达的调控作用。     

钙调蛋白激酶Ⅱ在神经病理痛中的作用及其痛觉调控通路概况

钙调蛋白激酶Ⅱ(Ca^2+/calmodulin-dependent protein kinase,CaMKⅡ)是一种多功能的丝氨酸/苏氨酸蛋白激酶,在神经元中大量存在,广泛参与疼痛调制。神经病理痛是一种由疾病或躯体感觉系统的损伤引起的慢性难治性疼痛。钙调蛋白激酶Ⅱ在中枢、外周神经病理痛、代谢型神经病理痛和药物引起神经病理痛等各种类型的神经病理痛的发生发展中发挥着重要的作用。本文拟将从钙调激酶Ⅱ介导的各型神经病理痛及其上下游的调控两个方面进行综述,以期为今后钙调蛋白激酶Ⅱ在神经病理痛领域的研究提供一定参考。     

不同产地浙贝母生物碱含量及其合成相关基因表达研究

为了探究浙贝母(Fritillaria thunbergii)药效成分积累量与生物碱合成相关基因表达水平之间的关系,该研究采用UPLC-MS、qPCR技术分别测定不同产地11个样品中总生物碱(贝母素甲和贝母素乙之和)含量和3个参与生物碱合成途径相关基因(HMGR、FPS和DXR)的表达量,同时运用生物统计学方法分析成熟期鳞茎生物碱含量与各基因表达量之间的相关性。结果表明:不同产地浙贝母成熟期鳞茎总生物碱含量存在显著差异(P<0.05),为0.2105%～0.4612%; HMGR和FPS基因在盛花期组织表达、盛花期至成熟期鳞茎表达变化趋势同生物碱含量变化趋势基本一致; DXR基因在成熟期鳞茎中表达量最高,盛花期组织表达、盛花期至成熟期鳞茎表达变化趋势同生物碱含量变化趋势大体不一致; HMGR、FPS基因表达量分别与贝母素甲、贝母素乙和总生物碱含量呈显著或极显著正相关性(P<0.05或P<0.01),以FPS基因表达量与生物碱含量相关系数为最高,相关系数分别为0.672,0.631,0.664,DXR基因与生物碱含量呈低度相关性。由此可以推断,生物碱的积累受MVA途径中...     

Twist对小鼠乳腺癌细胞基因表达谱的调控研究

摘要 Twist是一个bHLH（basic Helix-loop-Helix）类型的转录因子,近年来研究发现,Twist在乳腺癌中的表达显著升高,并能促进乳腺癌的转移。为了探索Twist促进乳腺癌转移的分子机制,本文采用RNA干扰技术在小鼠乳腺癌细胞株4T1中沉默Twist的表达,通过全基因组基因芯片技术检测了Twist沉默前后4T1细胞基因表达谱的差异性。体内实验结果证明Twist表达被沉默后4T1细胞的肺转移能力明显被抑制。芯片结果表明：表达差异显著的基因有167条,其中与肿瘤相关的基因有26条,包括15条上调基因和11条下调基因。这些基因中可能存在能被Twist调控并与肿瘤转移相关的基因,为以后研究Twist影响乳腺癌转移的分子机制提供了帮助。     

山药蛋白多糖体外抗氧化作用的研究

目的:测定山药蛋白多糖体外抗氧化作用.方法:山药经破碎匀浆,水浸提,离心收集上清液减压浓缩,用Sevag法除去游离蛋白质,收集沉淀复溶,透析48h,冷冻干燥得山药蛋白多糖.采用辣根过氧化酶法、核黄素光照法、Fenton反应2-脱氧-D-核糖法等生物化学方法分别测定了山药蛋白多糖对H2O2、O2-、·OH的清除能力,用硫代巴比妥酸法、分光光度法分别测定山药蛋白多糖对小鼠肝组织脂质过氧化反应和小鼠红细胞溶血的抑制作用.结果:山药蛋白多糖对活性氧自由基如H2O2、O2·-、·OH具有良好的清除作用,可减少红细胞溶血和抑制小鼠肝匀浆脂质过氧化反应,在一定范围内和剂量成正比.结论:山药蛋白多糖具有明显的体外抗氧化作用,其体外抗氧化能力与蛋白多糖浓度呈正相关性.     

豌豆质膜内源CDPK对植物转脂蛋白CaMBP-10的磷酸化及CaMBP-10对激酶自磷酸化的影响

植物转脂蛋白（LTPs）是多基因编码的蛋白家族,广泛分布于高等植物.虽然LTPs的确切功能至今仍不完全清楚,但它参与植物生物、非生物胁迫反应以及它的抗性功能已成为近年来的研究热点.关于LTPs功能的调节机制目前几乎一无所知.最近,从白菜中分离的钙调素结合蛋白-10（CaMBP-10）被鉴定为植物转脂蛋白家族成员,并且,体外实验证明钙调素（CaM）调节其脂质结合活性.为了深入了解转脂蛋白功能的调节机制,本文研究了CaMBP-10的磷酸化作用,发现CaMBP-10可被豌豆质膜内源性蛋白激酶磷酸化,钙离子（Ca2＋）能刺激磷酸化,钙螯合剂EGTA以及CaM拮抗剂W-7和TFP均能显著抑制磷酸化.免疫印迹分析最终确定该激酶为CDPK家族成员.构建突变体进一步研究了CaMBP-10的磷酸化位点,发现其位于蛋白的C-末端区域,并与已确定的CaM结合位点重合.同时,分析结果表明CaM能抑制CaMBP-10的磷酸化.反之,CaMBP-10的磷酸化又能阻断其与CaM的结合,显示出两种调节方式相互竞争的特点.为深入研究磷酸化作用对CaMBP-10脂质结合活性的影响,构建突变体（Ser83Asp,Ser85Asp）以模拟磷酸化状态.实验结果显示,磷酸化作用能显著增强CaMBP-10的脂质结合活性,而且突变体的脂质结合活性不受CaM的影响.采用胶内磷酸化测定法（in-gelkinaseassay）研究了激酶的自磷酸化特点以及CaMBP-10对激酶自磷酸化的影响,发现CaMBP-10能激活激酶的自磷酸化,激酶的自磷酸化又能促进其对底物的磷酸化作用.这样,激酶的自磷酸化与底物的磷酸化形成一种＂正反馈环＂的调节模式.综合研究结果,本文首次证明了LTP受CaM结合和CDPK磷酸化的双重调节.而且,CaM结合位点与磷酸化位点的重合预示可能存在特殊的调节机制,以协同应答胞内的Ca2＋信号.     

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: Role of calcium and protein kinase A and C

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E₂ (PGE₂) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE₂ provoked a 3.9 ± 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca⁺⁺ channel blockers, verapamil and nifedipine, and the Ca⁺⁺/calmodulin inhibitor, W-7. The Ca⁺⁺ ionophore, ionomycin, mimicked the effects of PGE₂ as did the phorbol ester PMA, which activates Ca⁺⁺-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE₂ did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE₂ secretagogue, IL-1β, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 ± 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE₂ does not mediate the effect of its secretagogue and that IL-1β signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE₂ receptor signalling pathways. Taken together, our results suggest that PGE₂ modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism. © 1996 Wiley-Liss, Inc.     

Distinct signalling pathways control Toxoplasma egress and host‐cell invasion

Calcium signalling coordinates motility, cell invasion, and egress by apicomplexan parasites, yet the key mediators that transduce these signals remain largely unknown. One underlying assumption is that invasion into and egress from the host cell depend on highly similar systems to initiate motility. Using a chemical‐genetic approach to specifically inhibit select calcium‐dependent kinases (CDPKs), we instead demonstrate that these pathways are controlled by different kinases: both TgCDPK1 and TgCDPK3 were required during ionophore‐induced egress, but only TgCDPK1 was required during invasion. Similarly, microneme secretion, which is necessary for motility during both invasion and egress, universally depended on TgCDPK1, but only exhibited TgCDPK3 dependence when triggered by certain stimuli. We also demonstrate that egress likely comes under a further level of control by cyclic GMP‐dependent protein kinase and that its activation can induce egress and partially compensate for the inhibition of TgCDPK3. These results demonstrate that separate signalling pathways are integrated to regulate motility in response to the different signals that promote invasion or egress during infection by Toxoplasma gondii.     

Organ-specific distribution of the calcium sensor CaMKII in Locusta migratoria

The Ca(2+)/calmodulin-dependent kinase CaMKII is a key signaling component in Ca(2+)-dependent physiological processes. The expression and function of CaMKII in insect brain is well documented but less investigated for other tissues of insects. The present study demonstrates that in the locust Locusta migratoria CaMKII is widely expressed in various tissues. Relatively high expression levels of CaMKII were found in the brain, upper part of the digestive tract (pharynx, esophagus), and the flight and leg muscles. The different expression patterns of CaMKII in various tissues, as well as different molecular masses of CaMKII between 48 and 60 kDa indicate a tissue-specific expression of CaMKII variants. The expression was monitored with a polyclonal anti-(rat)CaMKII antibody. About 60% of total CaMKII activity in flight muscle cells is associated to the myofibril-rich, particulate fraction suggesting an important role of CaMKII in sarcomeric function.     

拟南芥钙依赖蛋白激酶参与植物激素信号转导

在植物信号通路中,涉及到钙应答的蛋白激酶大多是钙依赖蛋白激酶。钙依赖蛋白激酶作为钙信号转导因子,参与了包括激素信号转导途径在内的很多传递过程。本工作在前人研究的基础上,对拟南芥AtCPK30基因的功能进行了深入的研究。RT-PCR分析结果表明:AtCPK30在植物根中的表达量很高,其在幼苗中的转录水平分别受ABA、IAA、2,4-D、GA_3和6-BA等激素的诱导调节。AtCPK30基因过表达的转基因株系幼苗的主根比野生型的长,同时发现转基因植株幼苗的根在缺钙的MS培养基上生长较野生型植株长,表明缺钙对转基因幼苗影响较小。用ABA、IAA、GA_3和BA处理时,转基因植株幼苗的根对激素更敏感。当野生型和转基因植株生长在含有生长素抑制剂NPA的MS培养基上时,NPA对转基因植株侧根的抑制比对野生型弱。GFP-CPK30融合蛋白的亚细胞定位研究结果表明:CPK30蛋白定位在细胞壁和细胞膜上。这些研究结果说明了AtCPK30作为钙信号转导因子,参与了多种激素调节植物根生长的过程。     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

苹果和葡萄果实蛋白激酶特性分析

以组蛋白Ⅲ S作苹果和葡萄果肉蛋白激酶制剂底物时 ,反应体系中加EGTA可抑制蛋白激酶活性 ,而加Ca2 可激活蛋白激酶的活性 ,表明苹果和葡萄果实中有依赖钙的蛋白激酶存在。而且 ,葡萄果实微粒体蛋白激酶呈热稳定性 ,苹果果实微粒体蛋白激酶对热敏感。以髓鞘碱性蛋白 (MBP)作底物 ,在苹果和葡萄果实微粒体中都检测出很高的蛋白激酶活性 ,并且不依赖于钙 ,说明苹果和葡萄果实中可能有分裂原激活的蛋白激酶 (MAP激酶 )的存在。苹果和葡萄果实MAP激酶的活性都表现出对二价阳离子Mg2 或Mn2 的依赖 ,并对高温处理表现出了激活效应