作者感谢罗伯特Holdcraft技术援助。这项研究是由美国农业部CSREES特别津贴（2009-34155-19957）和孵化基金（NJ08192）部分。

1。本地诱导挥发物:草食性损伤<ol><li>二级分行的蓝莓植物袋装与涤纶套。 </li><li>六舞毒蛾的毛虫（2日-3 路龄）放在内袋，并允许挥发收集前2天的植物饲料。对照植株没有收到任何的毛毛虫。 </li><li>挥发性排放收集3天（图1）如下所述（协议＃7）。聚酯袖子留在植物上，以防止昆虫逃跑。 </li></ol> 2。本地诱导挥发物:机械损伤<ol><li>机械损伤对植物造成冲孔模仿叶面积舞毒蛾删除。 </li><li>百分之五植物叶片受伤位于在1和2天的月底的基础和叶的上部有两个7毫米的孔。对照处理没有收到机械性损伤。 </li><li&gt;挥发性排放，收集，如在下面的第3天描述。 </li></ol> 3。本地诱导挥发物:茉莉酸甲酯<ol><li>蓝莓植物用10毫升1或1.5毫米的茉莉酸甲酯在0.1％Tween - 20的解决方案解决方案。 </li><li>茉莉酸甲酯应用使用2盎司的喷雾瓶。对照植株喷洒10毫升0.1％Tween - 20的解决方案。 </li><li>植物被视为在16:00小时，并在温室中保存15小时内一个直径为17厘米和35厘米高的有机玻璃室前挥发集合。 </li><li>挥发性排放，收集，如下所述。 </li></ol> 4。系统性诱导挥发物:内部信号<ol><li>较低的一个蓝莓植物的分支，是袋装一个涤纶套和内袋放置六（2日-3路龄）吉普赛蛾的损坏。 </li><li>损坏的分支，挥发收集室以外的，而brancheS是上述损坏的分支放在箱内（ 图2）。 </li><li>毛毛虫饲料上2天的底部分支。 </li><li>在第3天开始，收集从完好的部分损坏的植物挥发物。 </li><li>连续7天收集挥发。控制植物都以类似的方式处理，但没有收到草食动物损害。 </li></ol> 5。血管连接<ol><li>本研究确定的血管连接在蓝莓植物的不同部门之间的程度。 </li><li>从植物中较低的分支部分切断支持花露水挑一个罗丹明B（Sigma - Aldrich公司），染料（0.25％W / V）Orians等，含6 mL的溶液。 </li><li>通过植物染料的运动是每天监测7天。 </li><li>经过7天，红染色的金额是视觉评估来自不同岗位的植物。 </li></ol> 6。曝光HIPVs:外部信号<ol><li>工厂内一个分支是从相邻的分支HIPVs或收到没有接触到HIPVs。 </li><li>为了揭露分行HIPVs，一个较低的分支袋装聚酯袖舞毒蛾的毛虫放在内袋（ 图3）和六（2日-3路龄） 。 </li><li>该工厂是一个类似上述的有机玻璃室内笼。昆虫饲料植物为2天，从诱导分支排放的挥发性物质接触到相邻的分支。 </li><li> （3天）曝光后，暴露分行放在挥发收集室，留下的昆虫受伤的分支外（ 如图2所示） 。 </li><li> HIPV暴露分行挥发性物质的收集，如下所述，从较低的分支消耗的叶面积量测使用接穗图像Softwa重。 </li></ol> 7。暴露到HIPVs:吸<ol><li>要确定植物是否"底漆"HIPV曝光后，植物被视为在协议＃5。 </li><li>一半的植物暴露诱导相邻的分支HIPVs;而另一半是裸露完好的分支机构。 </li><li>在第3天，四早2 届龄舞毒蛾幼虫放在每个工厂和挥发物收集如下所述（ 图4）。 </li><li>挥发后集合，最初叶损坏舞毒蛾的毛虫，以及那些在第3天损坏，切除，测量叶面积消耗的金额。 </li></ol> 8。挥发物的收集<ol><li> HIPV排放量进行采样，在使用推拉式系统的温室。盆栽植物的地上部分（干，枝，叶），包括在草食动物损伤治疗的毛毛虫，被放在一个4大号VOLA瓦收集室。 2件断头台支持植物的基地。净化后的空气进入每个室顶部在2升/分钟的速度。 </li><li>挥发物收集在充满奥特奇超级Q值吸附剂拉分庭在1 L / min的的速率空气30毫克的陷阱。 </li><li>挥发性收集装置由四个腔室，允许同时从四个不同的植物（图1）的挥发性物质的集合。 </li><li>集合进行白天09:00至17:00小时</li><li>收集后，所有从植物叶子收获，烘箱干燥，在60 ° C，并称重，以及商会是用自来水和70％的乙醇冲洗。 </li></ol> 9。挥发物的分析<ol><li>从超级Q值陷阱收集的挥发物被洗脱二氯甲烷（150μL）和400 N -辛烷值吴是作为内部标准添加。 </li><li>在惠普6890系列化合物的分离和量化气相色谱仪（GC）（图5），配备火焰离子化检测器（FID）和安捷伦的HP - 1柱（10 × 0.53毫米× 2.65微米），并使用他作为载气（恒流量= 5毫升/分钟，速度= 39厘米/秒）。温度程序开始在40 °彗星保持1分钟，同比增长在14 ° C /分钟至180 ° C（2分钟），然后在40 ° C /分钟至200 ° C，并保持在200 ° C为2分钟。 </li><li>作为载气耦合到一个Finnigan公司MAT 8230质谱仪（MS）在Varian 3400气相色谱法，配有一个Supelco公司的MDN - 5S柱（30米× 0.32毫米× 0.25微米），并与他进行了初步鉴定化合物。 MS的电子电离（EI）和总离子流图（TIC）模式运作，在250 ° C（源温度）。该计划开始于35℃（1分钟），增加在4 ° C /分钟至170 ° C，然后在15 ° C /分钟至280 ° C。 </li><li>与NIST谱库和B的光谱数据进行比较，初步确定化合物Ÿ气相色谱保留指数，并通过比较与市售的化合物的保留时间。 </li></ol> 10。代表性的成果: 第二十二条挥发物确定了从蓝莓叶（ 图6）。图7显示了一个完好的蓝莓叶代表色谱仪和叶饲养舞毒蛾损坏。机械损伤和舞毒蛾的毛虫喂养增加蓝莓叶挥发性物质排放的本地控制（ 图8） 。卡特彼勒喂养相比，茉莉酸甲酯处理诱导11舞毒蛾（ 图9）诱导的17个化合物。有，但是，没有完好的叶舞毒蛾损坏投饵损害（即缺乏内部信号）（ 图10）后七天植物的系统性诱导挥发物的证据。此外，一个星期后，非常缓慢移动红色染料中观察到的蓝莓植物的分支（ 图11） 。有高血管单支叶片之间的连接。然而，在拍摄垂直排列的两个分支，和上一拍两侧的两个分支之间的低连接之间的连接，中间低。 有没有分行的暴露不暴露HIPVs（ 图12）与那些HIPVs排放的挥发性物质的金额之间的差异。然而，HIPVs作为蓝莓中的外部防御信号。舞毒蛾喂毛毛虫在树叶上以前暴露消耗少叶71％的材料比那些未曝光的控制叶片（ 图13）美联储HIPVs 。此外，每排放量叶面积HIPV暴露的分支消耗的挥发物的数额相比，未曝光的分支（图14）高4倍，这表明，叶HIPV - EX<html>构成分支机构，更加适应草食动物（即他们是催芽）。 <img src="/files/ftp_upload/3440/3440fig1.jpg" alt="图1"/> 图1。推挽系统是用来收集来自蓝莓植物的挥发物。植物放在玻璃商会和清新的空气是通过对它们的。含有吸附材料的过滤器是连接到每个室的一侧，从工厂排放的陷阱挥发物。真空是用来拉腔内部空气通过过滤器。 <img src="/files/ftp_upload/3440/3440fig2.jpg" alt="图2"/> 图2，研究的系统性蓝莓植物挥发反应，降低蓝莓分行损坏，舞毒蛾毛虫（右室）或左（左完好无损室） 。经过2天（3天），从完好的上部分支损坏和完好的植物挥发物收集。 <html> ontent"&gt; <img src="/files/ftp_upload/3440/3440fig3.jpg" alt="图3"/> 图3，为了测试是否完好的树枝叶回应HIPVs从损坏的分支，我袋装蓝莓植物的一个分支。然后，我一半的植物袋放在舞毒蛾的毛虫。袋我用允许的挥发物从内到外袋的运动。植物放在有机玻璃商会揭露完好的分支机构HIPVs。然后收集挥发物暴露的分支。 <img src="/files/ftp_upload/3440/3440fig4.jpg" alt="图4"/> 图4为了测试是否完好的树枝叶"底漆"暴露后，以HIPVs，实验重复在图3中描述，但吉普赛蛾的HIPV暴露和未暴露分行放在每个挥发收集室。 G"ALT ="图5"/&gt; 图5。挥发后的集合，样品注射到气相色谱仪（GC）的确定和量化，从蓝莓植物排放的挥发性。 <img src="/files/ftp_upload/3440/3440fig6.jpg" alt="图6"/> 图6，至少有22个化合物排放蓝莓叶。 <img src="/files/ftp_upload/3440/3440fig7.jpg" alt="图7"/> 从完好的蓝莓典型色谱图7。叶片和叶吉普赛蛾的损坏。挥发物排放量非常低，从完好的蓝莓叶。然而，当叶子是吉普赛蛾的损坏，挥发物排放量急剧增加。 <img src="/files/ftp_upload/3440/3440fig8.jpg" alt="图8"/> 图8图表显示完好的树叶，米22挥发物排放量。echanically损坏叶片，叶舞毒蛾的毛虫损坏。人为损坏模仿毛虫删除的叶面积增加了从蓝莓叶挥发排放，但叶舞毒蛾喂养的挥发性反应不同的反应。 <img src="/files/ftp_upload/3440/3440fig9.jpg" alt="图9"/> 图9:测试是否JA途径调节蓝莓叶的挥发排放。茉莉酸甲酯不同的植物进行喷洒。我发现，外生的应用茉莉酸甲酯浓度的增加增加了从蓝莓叶挥发物的排放。 <img src="/files/ftp_upload/3440/3440fig10.jpg" alt="图10"/> 图10:图表显示挥发物的排放控制蓝莓分行和舞毒蛾损坏植物（全身反应）完好无损分行的总金额。挥发物被收集到连续7天的TAL。我发现后，初始伤害，降低分支机构的植物不挥发物，甚至7天的全身感应。 <img src="/files/ftp_upload/3440/3440fig11.jpg" alt="图11"/> 图11，我用罗丹明B（红色染料），以确定蓝莓植物内分支机构之间的血管连接程度。我发现，约。叶含有染料的分支机构的80％，20％，5％和0％，直接以上含有染料，整个含染料的分支的分支，和在不同的拍摄在蓝莓植物分行遍布的分支的分支，分别是完全与染料染色。 <img src="/files/ftp_upload/3440/3440fig12.jpg" alt="图12"/> 图12。图表显示暴露HIPVs和未分支的分支排放的挥发性物质的量。我发现，接触到HIPVs没有影响邻居的挥发性排放枯燥完好的蓝莓分行。 <img src="/files/ftp_upload/3440/3440fig13.jpg" alt="图13"/> 图13:图表显示暴露HIPVs和未分公司蓝莓分行的吉普赛蛾的喂养量。分行HIPV暴露的毛毛虫消耗少，枝叶量，与那些未曝光的分支机构。 <img src="/files/ftp_upload/3440/3440fig14.jpg" alt="图14"/> 图14，当我计算每消耗领域的排放率，我发现HIPV，暴露分行增加了挥发物的排放率比未曝光的分支机构，这表明，暴露于通货膨胀蓝莓树枝叶HIPVs增加的挥发性反应。

<ol>
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我没有披露

这里描述的推挽挥发收集仪器代表了植物挥发物的顶空集合的标准方法。该仪器是用来确定由吉普赛蛾的草食动物蓝莓叶挥发响应，也让我HIPVs在工厂内信号的作用提供新的证据。 本文结果表明，卡特彼勒喂养，外生的应用茉莉酸甲酯，机械伤人增加损害的部位虽然不同，但挥发性排放。这些结果表明，JA途径中起着关键作用，在诱导蓝莓HIPVs。 HIPV暴露?...

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>挥发性收集箱</td><td>分析研究系统公司</td><td> VCC - G6X12DT - 1P </td><td>佛罗里达州盖恩斯维尔， </td></tr><tr><td> 20加仑，空气压缩机，无油，2马力</td><td>向西</td><td> 3JR71 </td><td>格兰杰，公司出售</td></tr><tr><td>空气输送系统</td><td>分析研究系统公司</td><td> VCS - ADS - 4AFM4C </td><td>佛罗里达州盖恩斯维尔， </td></tr><tr><td>空气收集系统</td><td>分析研究系统公司</td><td> VCS - MVCS - 4CX1P </td><td>佛罗里达州盖恩斯维尔， </td></tr><tr><td>真空泵¼ HP 100 - 150V， </td><td>加斯特制造公司</td><td> <a href="http://www.grainger.com/Grainger/GAST-Vacuum-Pump-4F740">4F740</a> </td><td>格兰杰，公司出售</td></tr><tr><td>茉莉酸甲酯</td><td> Sigma - Aldrich公司&lt;/ TD&gt; <td> J2500 </td><td>密苏里州圣路易斯</td></tr><tr><td>吐温20 </td><td> Sigma - Aldrich公司</td><td> 93773 </td><td>密苏里州圣路易斯</td></tr><tr><td>罗丹明- B </td><td> Sigma - Aldrich公司</td><td></td><td>密苏里州圣路易斯</td></tr><tr><td>塑料喷雾瓶，2盎司</td><td> Setco公司</td><td></td><td>克林伯利，新泽西州</td></tr><tr><td>涤纶袖</td><td>罗金厄姆机会公司</td><td></td><td> Reidsville，NC </td></tr><tr><td>超级Q值挥发收集陷阱</td><td>分析研究系统公司</td><td> VCT-1/4X3-SPQ </td><td>佛罗里达州盖恩斯维尔， </td></tr><tr><td> Scion的图像软件</td><td>接穗公司</td><td></td><td>冯检基，MD </td></tr><tr><td>二氯甲烷</td><td> Sigma - Aldrich公司</td><td> 270997 </td><td>密苏里州圣路易斯</td></tr><tr><td>惠普6890气相色谱仪</td><td>惠普</td><td></td><td></td></tr><tr><td>瓦里安3400气相色谱仪</td><td>瓦里安</td><tD&gt; </td><td></td></tr><tr><td> 正辛烷</td><td> Sigma - Aldrich公司</td><td> 296988 </td><td>密苏里州圣路易斯</td></tr><tr><td>质谱仪MAT 8230 </td><td>菲尼根</td><td></td><td>加利福尼亚州圣何塞， </td></tr><tr><td> HP - 1 GC色谱柱</td><td>安捷伦科技公司</td><td></td><td>帕洛阿尔托， CA </td></tr><tr><td> MDN - 5S GC柱</td><td> Supelco公司，公司</td><td></td><td> Bellefonte，PA </td></tr></table>

herbivore-induced blueberry volatiles and intra-plant signaling

虫害诱导植物挥发物（HIPVs）通常发出的从植物，草食动物攻击后 1,2 。这些HIPVs都主要受防守的植物激素茉莉酸（JA）和其挥发衍生茉莉酸甲酯（茉莉酸甲酯）3,4,5 。在过去的三十年中，研究人员已经证明，HIPVs可以排斥或吸引食草动物，吸引食草动物的天敌，在某些情况下，他们可诱发或草食动物攻击前总理植物防御。在最近的一篇论文6，我报，饲喂舞毒蛾的毛虫，外源茉莉酸甲酯的应用，以及机械损伤，诱发蓝莓植物挥发物的排放量，尽管不同的。此外，蓝莓分行从同一工厂邻近分行排放的增加JA和抗食草动物（即直接植物的防御）水平HIPVs，吸挥发性排放（​​即间接植物防御）。类似的发现ings报道，近日已对杨树山艾树7，8，和利马豆9 .. 在这里，我描述了一个推拉式的方法，收集舞毒蛾（草食动物）喂养，外源茉莉酸甲酯的应用，和机械损伤引起的蓝莓挥发物。挥发的收集装置由一个4升的挥发性收集室，2件断头台，净化进气空气输送系统，真空系统连接到一个充满陷阱与超级Q值吸附剂收集挥发物5,6,10 。在超级- Q陷阱收集的挥发物用二氯甲烷洗脱，然后用气相色谱（GC）分离和量化。这个动荡的收集方法是使用ñ我的研究调查内蓝莓植物虫害损坏分行完好的分支机构接触到挥发物的挥发反应。这里将介绍这些方法。简言之，完好的蓝莓分行暴露HIPVs来回邻近的分行在同一个工厂的米。使用上述相同的方法，从暴露后，以HIPVs分行排放的挥发性物质的收集和分析。

Watch this Scientific Journal Video about Herbivore-induced Blueberry Volatiles and Intra-plant Signaling at JoVE.com

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

一个推拉式的方法收集的植物挥发是描述。该方法允许草食动物喂食，外源茉莉酸甲酯和机械损伤诱导挥发物的比较。这种技术也被用于调查从蓝莓植物内的虫害损坏分行接触到挥发物的挥发反应完好的分支机构。

蓝莓虫害诱导挥发物和植物内信令

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

herbivore-induced-blueberry-volatiles-and-intra-plant-signaling

Research

JoVE Journal

Biology

16.3K 次观看.  Rutgers University. 一个推拉式的方法收集的植物挥发是描述。该方法允许草食动物喂食，外源茉莉酸甲酯和机械损伤诱导挥发物的比较。这种技术也被用于调查从蓝莓植物内的虫害损坏分行接触到挥发物的挥发反应完好的分支机构。

视频: Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

科研

生物学

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

偏光易位荧光蛋白爪蟾外胚层响应Wnt信号

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

血管钙的可视化 2 +信号旁分泌源性活性氧引发

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

机械刺激引起的钙波传播的细胞单层:牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

隔离成年小鼠心肌细胞的细胞信号传导和文化及<em&gt;在体外</em&gt;心肌肥厚

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...