Name: the identification of sea lamprey pheromones using bioassay-guided fractionation
Uploaded: 2018-07-17T15:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation at JoVE.com

我们感谢美国地质调查哈蒙德湾生物站的使用他们的研究设施和工作人员的美国鱼类和野生动物服务和渔业和海洋加拿大提供海洋鳗。这项研究得到了大湖渔业委员会给予未明李和科李的赠款的支持。

这里描述的所有方法都已获得密歇根州立大学机构动物保育和使用委员会 (AUF 03/14-054-00 和 02/17-031-00) 的批准。
1. 海洋鳗条件水的收集和提取
<ol><li>安置性成熟男性海鳗 (15-30 个动物) 在供应与 250 L 的被加气的湖休伦的水被维护在 16-18 °c 的坦克。</li>
	<li>每晚从琼到7月收集男性条件的水。 注: 海洋鳗产卵后自然死亡。如果一条鱼在它的生命中接近这一点, 用新鲜成熟的雄性代替它。</li>
	<li>固相萃取萃取条件水。<ol>
<li>将树脂浸泡在甲醇中4小时, 然后将其装入柱内。将树脂装入柱内, 然后泵入10升水通过柱, 消除有机溶剂。</li>
		<li>将有条件的水从水箱中通过泵浦系统传递到立柱顶部。水通过2公斤的中等极性聚合物离子交换树脂 (阳离子交换树脂 XAD-7 HP 树脂) 的床, 包含在一系列四 2.5 L 容量玻璃专栏。保持负载速度介于400和 600 mL/6 洗脱的代谢产物中, 甲醇的10升, 紧接着是丙酮的5升。 注意: 此步骤使用甲醇和丙酮。两者都是易燃和有毒的。 注: 池内残留物可存储在-80 °c, 直到进一步处理。</li>
	</ol>
</li>
	<li>在1周的浓缩后重新激活柱子, 用清水冲洗 (大约10升)。</li>
	<li>除去有机溶剂 (甲醇和水的混合物), 并在减压 (300 毫巴) 40 摄氏度的情况下, 以旋转蒸发的方法收获萃取液5小时。用冷冻干燥机将48小时的冻干干燥浓缩到干燥的-20 摄氏度。</li>
</ol>2. 用色谱法分离分数池
<ol><li>在初始移动阶段浸泡硅胶 (230-400 目), 30 分钟. 将凝胶与溶剂 (悬浮液) 转移到玻璃柱上。打开该列的阀门, 以允许移动阶段通过。允许硅胶沉淀, 形成硅胶床。</li>
	<li>将萃取物与硅胶 (70-230 目) 彻底混合, 将其装入硅胶床上的液相色谱柱上。洗脱他们与一个梯度从 95% CHCl3 (氯仿)/甲醇 (甲醇) 到100% 甲醇, 2.5 L 在总容量。收集淋洗在单独的瓶子 (每10毫升)。 注意: 这个步骤中使用的氯仿是一种有毒的试剂。</li>
	<li>用薄层色谱 (TLC) 分析方法将 eluents 的聚集性引导成20个组分。<ol>
<li>对预涂硅胶板进行薄层色谱实验, 将试样应用于起始线上, 将板材的起始线浸入显影溶剂中 (在密封玻璃中选择极性以 CHCl3与甲醇的比值为 100-0%)坦克。</li>
		<li>选择发展溶剂的比例, 使所有的薄层斑点与保留因子 (Rf) 范围从 0.3-0.8 不等。</li>
		<li>开发后的溶剂到达终点线后, 取出罐中的盘子, 等待10分钟的溶剂蒸发。</li>
		<li>在紫外线照射下, 先将斑点想象成 254 nm, 然后用一个色谱喷雾剂喷洒5% 茴香醛 (20 ul) 酸性甲醇溶液, 然后将它们加热到85摄氏度, 3 分钟。 注意: 薄层色板上的彩色斑点表示分数中的主要成分。</li>
		<li>根据专色和主成分的 Rf相似性, 将淋洗与20分数结合起来。</li>
	</ol>
</li>
	<li>在减少压力下 (300 毫巴根据溶剂的性质) 将分数集中到残留物上, 在40摄氏度左右30分钟。</li>
</ol>3. 电 olfactogram (EOG) 记录, 以识别气味的分数/化合物
<ol><li>拉硼玻璃毛细血管 (外径: 1.5 毫米; 内径: 0.86 毫米; 长度: 100 毫米), 与加热器水平设置为65的微拉出器。</li>
	<li>评分和削减开口在毛细管与钻石尖玻璃切割机, 并填补它与熔融0.4% 琼脂0.9% 盐水。毛细管尖端的开口应该直径约为10µm。</li>
	<li>填充从步骤 3.1-3.2 和固态电极持有人预先捏造与银/AgCl 丸 (见材料表) 与3米氯化钾使用微的拉毛毛细管电极。 注: 去除毛细管或电极支架内的气泡。</li>
	<li>将被拉电极插入电极夹中。</li>
	<li>在100毫升 10-5米 l-精氨酸在木炭过滤水从 10-2米精氨酸库存溶液在去离子水 (储存在4°c) 在一个容积烧瓶。将 10-5米精氨酸的20毫升转移到玻璃瓶中。</li>
	<li>对于浓度-反应曲线, 准备10毫升10倍稀释的分数池从步骤2.3 的玻璃瓶。在实验前每天准备新鲜稀释, 并在一天内使用。将 l-精氨酸和分数池稀释的工作溶液的玻璃瓶放在循环水浴中, 使温度平衡到8摄氏度。</li>
	<li>麻醉鳗与 3-苯甲酸酸乙酯 (MS222; 100 毫克/升), 并固定它与肌肉注射 gallamine triethiodide (3 毫克/公斤体重, 0.9% 生理盐水) 与无菌注射器。 注: 充分的麻醉深度是通过观察没有鳃运动, 不能保持直立姿势, 并没有吸入到坦克两侧使用其口服盘来确定的。在手术前和术中尽量减少任何微生物污染, 在使用前至少10分钟, 在70% 乙醇 (v: v) 中用无菌手套浸泡解剖工具。</li>
	<li>将麻醉鳗定向到 V 形支架上, 用湿纸巾包裹, 防止脱水。 注意: 不要阻碍鳃开口。</li>
	<li>将 MS222 50 毫克/升的循环加气水管插入颊腔内, 调整流速, 确保水通过鳃孔流出, 持续灌溉鳃。</li>
	<li>使用无菌手术刀和钳 [在立体显微镜下的1.25X 放大 (见材料表)], 以消除嗅囊表面的皮肤5毫米2切片, 以暴露嗅觉上皮。</li>
	<li>用过滤过的水冲洗气味输送管, 并将其连接到安装在机器人上的阀门上。将气味输送毛细管插入嗅上皮腔内, 使用机器人将过滤后的水输送到嗅觉上皮, 以防止不气味时干燥。</li>
	<li>在并联上安装录音和参考电极。将参考电极降低到鼻孔附近的外部皮肤。使用立体显微镜 (1.25X), 降低记录电极, 勉强触及嗅上皮的表面。</li>
	<li>将气味输送管的吸收从背景滤水转移到 10-5米精氨酸溶液中。</li>
	<li>打开计算机、放大器 (设置为 DC 模式)、滤镜和数字化器。使用阀门驱动程序软件, 通过检查 T1 盒, 设置 T 为 4, 并检查 T2 盒, 程序来管理 4 s 单脉冲气味。</li>
	<li>在数据采集软件 (见材料表) 中, 将采集模式设置为高速示波器, 单击播放图标, 然后单击启动阀驱动程序触发气味脉冲。 注: 数据采集软件自动记录二十年代的差分 EOG 响应振幅 (3 秒之前, 4 秒在气味脉冲和十三年代之后)。使用机器人来操纵记录电极、参考电极或气味输送管的位置, 以提高信噪比, 最大程度地响应 l-精氨酸标准, 对空白控制的响应最小 (过滤水)。在移动电极时, 通过将交流-直流放大器开关转换为接地, 使放大器接地。</li>
	<li>开始记录空白控制, l-精氨酸, 然后气味准备在步骤3.6 从低到高浓度和2分钟冲洗的过滤水在应用程序之间。</li>
	<li>在记录对所有要测试的气味的响应后, 接地放大器, 并仔细地收回电极和气味送毛管。继批准的机构动物护理和使用委员会 (IACUC) 方法后, 在 EOG 实验的终止, 弄死麻醉鳗的过量 MS222 (1 克/升)。 注: 检查成功的安乐死, 注意到没有鳃运动和心跳至少5分钟, 其次是 pithing 大脑。</li>
	<li>使用分析软件分析和绘制数据。确定检测气味22的阈值, 以确定引起反应大于空白水控制的气味分数。在行为分析 (步骤 4) 中, 可以得出不同于空白水控制的与浓度相关的嗅觉反应的分数池。</li>
</ol>4. 两种选择迷宫行为生物鉴定行为上活性组分/化合物
<ol><li>在迷宫的释放笼中适应性成熟的女性海鳗 (见补充图 1), 以5分钟的速度维持迷宫中河水的流速 注: 迷宫是由涂漆的海洋级木材建造的, 测量长度为6.5 米, 宽度为1.2 米, 分压器长度为2.7 米, 在上游端将迷宫分隔成两个通道。水暂时改道进入迷宫。水深应保持在0.19 米, 速度应保持在0.07 米/秒0.01。</li>
	<li>释放海洋鳗, 记录鳗在实验和控制通道中每含河水10分钟的累计时间。 注: 如果海鳗在10分钟内未能进入实验和控制通道, 则结束试验, 因为这是不活动或强侧偏倚的征兆。</li>
	<li>应用试验刺激 (即10-12 M 溶解在50% 甲醇/去离子水中的一种假定的信息素) 到随机分配的实验通道和车辆 (50% 甲醇/去离子水) 到控制通道使用蠕动泵的恒定速率为200毫升/分5分钟。 注: 测试刺激的检测阈值浓度与电 olfactogram 记录的确定应作为初始浓度的行为测试。</li>
	<li>将测试刺激和车辆加10分钟, 记录鳗在实验和控制通道中所花费的累计时间。</li>
	<li>下一次试验开始前, 用清水冲洗迷宫10分钟。如果有足够的测试刺激, 重复步骤 4.1-4.4, 至少有7鳗。</li>
	<li>计算每个试验的偏好22的索引, 并使用魏氏签名秩测试来评估其意义。 注意: 索引会产生一个数字, 可以是正数或负数。偏好指数的正值表示吸引力, 而偏好指数的负值表明排斥。如果首选项的索引与零显著不同, 则该分数被认为是活动的。 <img alt="Equation" src="/files/ftp_upload/58059/58059eq1v2.jpg"> 这里 Bc = 测试鳗在气味应用程序之前的控制通道中所花费的时间, 气味应用之前在实验频道所花费的时间, c = 在气味应用程序后的控制通道中所花费的时间, 以及 e = 气味应用程序后在实验通道中花费的时间。</li>
</ol>5. 活性馏分中纯化合物的色谱分离
<ol><li>重复步骤 2.1-2.4 与诱导嗅觉反应的分数池 (步骤 3) 和引出行为反应 (步骤 4)。</li>
	<li>用葡 LH-20 柱进一步将活性馏分提纯成具有大小排斥色谱的化合物。<ol>
<li>在初始移动相 [CHCl3-甲醇 (1:1) 或甲醇 100%] 中准备0.5 毫升的样品, 加载到相应的列 a CHCl3-甲醇 (1:1) 列, 然后是甲醇 (100%) 列, 并洗脱它们产生化合物。</li>
	</ol>
</li>
</ol>6. 纯质谱 (MS) 和核磁共振 (NMR) 的结构澄清
<ol><li>在最初的流动阶段 (通常, 甲醇到水, 1:1, v: v) 溶解和稀释高效液相色谱 (HPLC) 的纯化化合物, 形成 10 ul 溶液约1微克/毫升。<ol>
<li>将样品溶液转移到高效液相色谱瓶中, 并将其 autosampler。将样品 (10 ul) 注入电喷雾电离质谱仪 (ESIMS) 中, 用色谱质量光谱仪23记录高分辨电喷雾质谱 (HRESIMS) 谱。</li>
	</ol>
</li>
	<li>在质谱仪软件中根据数据库预测分子公式 (见材料表)24。<ol>
<li>通过选择色谱峰, 打开注射样品的色谱, 获得其质谱。</li>
		<li>将所测量的质量输入到刀具模块下元素组成中的离子 (m-/z) 值 (4 个小数)。将公差参数设置为 PPM < 5。</li>
		<li>调整符号参数, 以适应被测分子的元素组成。该软件对基于单质量分析的分子公式进行了预测。</li>
	</ol>
</li>
	<li>根据协议25 , 选择氘溶剂 (600 ul, CH3OHd4或亚砜-d6) 来溶解样品。<ol>
<li>在选定的氘溶剂中完全溶解样品, 形成溶液, 浓度约从0.1 到10毫克/毫升不等。将样品溶液转换成核磁共振管以记录 1D (1小时、 13C) 和2D 核磁共振 [1h-1H 相关光谱学 (舒适性)、异单量子相干性 (HSQC)、异多键相关性(HMBC)]光谱在900兆赫核磁共振光谱仪26。</li>
	</ol>
</li>
	<li>将核磁共振管与样品放在微调涡轮内。使用深度测量仪确保样品高度在测量窗口的中间。打开核磁共振软件 (见材料表), 点击升降机按钮, 改变磁铁中的样品。 注: 握住磁铁顶部的一只手, 感觉到来自磁铁顶部的气体。同时, 吹口哨的声音应该是发声的。</li>
	<li>轻轻地将样品放在磁铁顶部的气垫上, 再按下升降机按钮, 将样品降落到核磁共振磁体中。 注: 侦听 "单击" 噪音以指示示例处于正确的位置。</li>
	<li>创建新数据集并加载核磁共振仪器推荐的默认参数 (见材料表)。<ol>
<li>键入 "锁定" 以调用自动锁定过程, 并在提示页上选择溶剂。</li>
		<li>要进行微调, 请在 "操作" 面板中, 调整解锁模型中的按钮, 调整操作面板上的光标, 然后再次锁定磁铁。</li>
		<li>在 "提示Atma " 页上, 将 "操作" 面板中的参数调整为优化过程, 这可能需要几分钟时间。请稍候, 直到调整完成才能继续。</li>
		<li>在提示中键入 "topshim", 启动自动匀例程。等到匀完成后再继续。</li>
		<li>键入 "rga" 设置自动接收增益调整, 然后键入 "d1" 设置脉冲之间的延迟, 然后键入 "zg" 开始采集, 并等到采集完成。</li>
		<li>键入 "ef" 或 "efp" 来处理数据, 然后键入 "apk" 自动分阶段。处理核磁共振处理软件上的数据。</li>
	</ol>
</li>
	<li>通过对核磁共振数据分析的解释, 阐明化学结构。</li>
	<li>计算13C 核磁共振谱中的碳信号。从高分辨率质谱 (HR) 的预测结果中选择匹配的分子公式。</li>
	<li>将质子信号集成在1h-核磁共振谱中, 并根据 HSQC 光谱中的信号分配碳和质子的连通性。</li>
	<li>根据1小时-1小时舒适和 HMBC 谱的相关关系, 分配碳骨架的连通性。</li>
	<li>根据结构原理, 初步分配化学结构27。在化学结构数据库中搜索初步结构。将暂定结构与参照中的类似物进行比较。</li>
	<li>用核 Overhauser 效应光谱学 (NOESY) 谱分配相对构型。 注: 由于在光谱和光谱方法上显示的对映体的身份属性相同, 某些化合物的绝对构型, 特别是 2 '-酒精和 2 '-NH2, 必须用衍生反应。在衍生反应之后, 在光谱上表示的差异促进了大多数化合物的绝对构型的明确赋值28。</li>
</ol>7. EOG 和生物鉴定确认纯化合物有异味和行为上活性
<ol><li>重复步骤 3.1-3.5。</li>
	<li>对于浓度-反应曲线, 准备10毫升10倍稀释的纯化合物从 10-6米-10-13 m 的 10-3米信息素库存解决方案, 50% 甲醇/水存储在-20 °c 基于分子量确定的步骤6.2。将玻璃瓶与 l-精氨酸和纯化合物的工作溶液放在循环水浴中, 使温度平衡到8摄氏度。</li>
	<li>重复步骤 3.7-3.18。</li>
	<li>重复步骤 4.1-4.2。</li>
	<li>将纯化合物在 EOG 检测阈值集中到随机分配的实验通道和车辆 (50% 甲醇/去离子水) 到控制通道, 使用蠕动泵, 恒定率为200毫升/分钟5分钟。</li>
	<li>重复步骤 4.4-4.6。</li>
</ol>

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作者没有什么可透露的。

鱼类生活在一个充满化合物的化学世界中, 但尚未确定。生物鉴定-引导分馏已证明是必不可少的识别和表征活性分子, 调解许多化学相互作用, 如观察鲑鲑鱼31, 亚洲大象32, 海鳗33, 34,35。生物测定引导分馏是一种有效的方法, 可以准确地从起始提取物到纯化活性化合物中精确地跟踪和定位活性?...

信息素是个人发布的特定化学信号, 帮助他们找到食物来源, 检测捕食者, 并调解 conspecifics1的社会互动。昆虫信息素的传播研究已有2;然而, 水生脊椎动物信息素的化学鉴定和生物学功能还没有得到广泛的研究。对所释放的信息素的特性和功能的了解可以应用于促进被威胁物种3、4或控制害虫物种5、6的恢复。这些技术的应用需要对生物活性素成分进行分离和表征。
信息素识别是天然产物化学的一个分支。由于信息素分子本身的性质, 信息素研究的进展受到部分限制。信息素往往不稳定, 很少释放, 只有少数抽样技术存在, 以检测微量的挥发性7,8或水溶性化合物9。识别信息素的方法包括 1) 目标筛选已知化合物, 2) 新陈代谢和 3) 生物测定引导分馏。对已知化合物进行有针对性的筛选, 可以检验被推测为费洛蒙功能的生理过程的商业性代谢副产品。这种方法是有限的, 因为研究人员只能测试已知和可用的化合物。然而, 它导致了在金鱼中的性激素的成功识别功能作为费洛蒙10,11,12。新陈代谢是第二种信息素识别方法, 它区分了生物系统中潜在的小分子代谢产物13。对两组代谢剖面 (即活性与非活性提取物) 进行比较, 可以识别出代谢产物纯化的潜在新陈代谢剖面, 阐明结构, 以及生物活性证实14。复杂配方的添加剂或协同效应更可能被发现与新陈代谢, 因为代谢物被考虑在一起, 而不是作为一系列的分数13。然而, 新陈代谢的实施依赖于合成参照的可用性, 因为由此产生的数据不利于澄清新结构。
生物鉴定-引导分馏是一个综合的, 迭代的方法, 跨越两个领域: 化学和生物学。该方法利用生理和行为生物鉴定的结果来指导活性信息素化合物的分离和鉴定。粗萃取物由化学性质 (即分子量、极性等) 分馏, 并用电 olfactogram (EOG) 记录和/或在生物测定中进行测试。通过重复这些步骤的分馏和 EOGs 和/或生物鉴定, 筛选出活性成分。用光谱和光谱方法阐明了纯活性化合物的结构, 提供了分子量和结构信息, 生成了合成的模板。生物测定引导分馏可以产生不同的代谢物和潜在的新的信息素与独特的化学骨骼, 是不可能预测的生物合成途径。
在这里, 我们描述了用于分离和表征雄性海鳗性信息素化合物活性的生物测定制导分馏协议。海鳗 (Petromyzon marinus) 是研究信息素传播的理想脊椎动物模型, 因为这些鱼类大量依赖化学线索的嗅觉检测, 以调解其溯河产卵生命史, 包括三个不同阶段:幼虫, 幼仔和成年。海洋鳗幼虫钻入淡水溪流的沉积物中, 经历剧烈的蜕变, 转化为迁徙到湖泊或海洋的幼鱼, 它们寄生大的寄主鱼类。从宿主鱼分离后, 成虫迁移回产卵流, 由流驻留幼虫15、16、17、18、19 所释放的迁移信息素引导..成熟雄性上升到产卵场, 释放多组分性信息素吸引配偶, 间歇产卵约一周, 然后死15,20。海洋鳗信息素的识别是很重要的, 因为对劳伦琴大湖21的入侵性海洋鳗的控制, 是一种调控费洛蒙传播系统的方法。

<table><tbody><tr><td>Premium standard wall borosilicate capillaries with filament&amp;nbsp;</td><td>Warner Instruments</td><td>G150F-4</td><td>recording and reference electrode (OD 1.5 mm, ID 0.86 mm)</td></tr><tr><td>Pipette puller instrument</td><td>Narishige</td><td>PC-10</td><td>pulls electrodes for EOGs</td></tr><tr><td>Diamond-tipped glass cutter</td><td>Generic</td><td></td><td>cut tip of electrodes for EOG</td></tr><tr><td>Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B150-4</td><td>odorant delivery tube for EOG</td></tr><tr><td>Recording electrode holder E Series straight body with Ag/AgCl pellet for glass capillary OD 1.5 mm</td><td>Warner Instruments</td><td>ESP-M15N</td><td>recording electrode holder</td></tr><tr><td>Reference electrode holder E Series with handle with&amp;nbsp; Ag/AgCl pellet&amp;nbsp; for glass capillary OD 1.5 mm</td><td>Warner Instruments</td><td>E45P-F15NH</td><td>reference electrode holder</td></tr><tr><td>1 mm pin</td><td>Warner Instruments</td><td>WC1-10</td><td>to bridge reference and recording electrode holders</td></tr><tr><td>2 mm pin</td><td>Warner Instruments</td><td>WC2-5</td><td>to bridge reference and recording electrode holders</td></tr><tr><td>Agar</td><td>Sigma</td><td>A1296</td><td>molten agar to fill electrodes</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma</td><td>P9333</td><td>3M KCl to fill electrodes and electrode holders</td></tr><tr><td>Micropipette microfil</td><td>World Precision Instruments</td><td>MF28G-5</td><td>to fill electrodes and electrode holders</td></tr><tr><td>L-Arginine</td><td>Sigma</td><td>A5006</td><td>positive control odorant for EOG</td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td></td></tr><tr><td>Water bath</td><td>Custom made</td><td>N/A</td><td>holds odorants for EOG</td></tr><tr><td>3-aminobenzoic acid ethyl ester (MS222)</td><td>Syndel USA</td><td>Tricaine1G</td><td>EOG anesthetic</td></tr><tr><td>Gallamine triethiodide</td><td>Sigma</td><td>G8134-5G</td><td>EOG paralytic</td></tr><tr><td>1 mL syringe</td><td>BD Biosciences</td><td>301025</td><td>to administer paralytic</td></tr><tr><td>Subcutaneous needle 26G 5/8</td><td>BD Biosciences</td><td>305115</td><td>to administer paralytic</td></tr><tr><td>Roller clamp</td><td>World Precision Instruments</td><td>14043-20</td><td>adjust flow rate of anesthic into lamprey&#039;s mouth</td></tr><tr><td>Sodium chloride (NaCl)</td><td>J.T. Baker</td><td>3624-05</td><td>for preparation of 0.9% saline</td></tr><tr><td>V-shaped plastic stand as specimen stage</td><td>Custom made</td><td>N/A</td><td>holds lamprey during EOG</td></tr><tr><td>Plastic trough</td><td>Custom made</td><td>N/A</td><td>holds V-shaped plastic stand during EOG</td></tr><tr><td>Scalpel Blades - #11</td><td>Fine Science Tools</td><td>10011-00</td><td>for EOG dissection</td></tr><tr><td>Scalpel Handle - #3</td><td>Fine Science Tools</td><td>10003-12</td><td>for EOG dissection</td></tr><tr><td>Straight ultra fine forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>for EOG dissection, Dumont #5SF Forceps</td></tr><tr><td>Curved ultra fine forceps</td><td>Fine Science Tools</td><td>11370-42</td><td>for EOG dissection, Moria MC40B</td></tr><tr><td>Straight pring Scissors</td><td>Fine Science Tools</td><td>15003-08</td><td>for EOG dissection</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Discovery V8</td><td>for EOG dissection</td></tr><tr><td>Illuminator light</td><td>Zeiss</td><td>CL 1500 ECO</td><td>for EOG dissection</td></tr><tr><td>Plastic tubing</td><td>Generic</td><td></td><td>to connect re-circulating EOG setup and water baths</td></tr><tr><td>Odorant delivery tubing</td><td>Custom made</td><td>N/A</td><td></td></tr><tr><td>In line filter and gasket set</td><td>Lee Company</td><td>TCFA1201035A</td><td></td></tr><tr><td>Micromanipulators</td><td>Narishige</td><td>MM-3</td><td>to position electrodes and odorant delivery capillary tube</td></tr><tr><td>Magnetic holding devices</td><td>Kanetec</td><td>MB-K</td><td></td></tr><tr><td>Valve driver</td><td>Arduino</td><td>custom made</td><td>to control the opening of the valve for odor stimulation</td></tr><tr><td>Electromagnetic valve</td><td>Lee Company</td><td>LFAA1201618H</td><td>valve for odor stimulation</td></tr><tr><td>NeuroLog AC/DC amplifier</td><td>Digitimer Ltd.</td><td>NL106</td><td>to increase the amplitude of the elictrical signal</td></tr><tr><td>NeuroLog DC pre-amplifier with headstage</td><td>Digitimer Ltd.</td><td>NL102G</td><td>to increase the amplitude of the elictrical signal</td></tr><tr><td>Low-pass 60 Hz filter</td><td>Digitimer Ltd.</td><td>NL125</td><td></td></tr><tr><td>Digitizer</td><td>Molecular Devices LLC</td><td>Axon Digidata 1440A</td><td></td></tr><tr><td>Dell computer (OptiPlex 745) running Axoscope data acquistion software</td><td>Molecular Devices LLC</td><td>AxoScope version 10.4</td><td></td></tr><tr><td>Faraday cage</td><td>Custom made</td><td>N/A</td><td>Electromagnetic noise shielding</td></tr><tr><td>Two-choice maze</td><td>Custom made</td><td>N/A</td><td>waterproofed marine grade plywood covered with plastic liner</td></tr><tr><td>Trash pump</td><td>Honda</td><td>WT30XK4A</td><td>fills maze with water from nearby river</td></tr><tr><td>Peristaltic pump with tubing</td><td>Cole Parmer</td><td>Masterflex 07557-00</td><td>to adminster odorants in maze</td></tr><tr><td>Inverter Generator</td><td>Honda</td><td>EU1000i</td><td>powers perstaltic pump</td></tr><tr><td>Release cage</td><td>Custom made</td><td>N/A</td><td>used to acclimate lamprey in the maze</td></tr><tr><td>Mesh</td><td>Generic</td><td></td><td>used to contain the dimensions of the maze and minimize water turbulance with mesh rollers</td></tr><tr><td>Buckets (5 gallon)</td><td>Generic</td><td></td><td>to mix odorants</td></tr><tr><td>Flow meter</td><td>Marsh-McBirney</td><td>Flo-Mate 2000</td><td>to measure discharge</td></tr><tr><td>XAD 7 HP resin</td><td>Dow chemical</td><td>37380-43-1</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>Water bath</td><td>Yamato</td><td>BM 200</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>Freeze dryer</td><td>Labconco</td><td>CentriVap Concentrator</td><td>for extraction of conditioned water</td></tr><tr><td>chloroform</td><td>Sigma</td><td>CX1050</td><td>for isolation of fraction pools</td></tr><tr><td>Silica gel 70-230 mesh</td><td>Sigma</td><td>112926-00-8</td><td>for isolation of fraction pools</td></tr><tr><td>Silica gel 230-400 mesh</td><td>Sigma</td><td>112926-00-8</td><td>for isolation of fraction pools</td></tr><tr><td>Pre-coated silica gel TLC plates</td><td>Sigma</td><td>99571</td><td>for isolation of fraction pools</td></tr><tr><td>anisaldehyde</td><td>Sigma</td><td>A88107</td><td>for isolation of fraction pools</td></tr><tr><td>Sephadex LH-20</td><td>GE Healthcare</td><td>17-0090-01</td><td>for isolation of fraction pools</td></tr><tr><td>Amberlite XAD 7 HP resin</td><td>Sigma</td><td>XAD7HP</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>4, 2.5L capacity glass columns</td><td>Ace Glass Inc.</td><td>5820</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>Acetone</td><td>Sigma</td><td>650501</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>TQ-S TOF LC Mass spectrometer (or equivalent)</td><td>Waters Co.</td><td>N/A</td><td>for structure elucidation</td></tr><tr><td>Binary HPLC pump</td><td>Waters Co.</td><td>1525</td><td>for isolation of fraction pools/compounds</td></tr><tr><td>Agilent NMR spectrometer, 900MHz (or equivalent)</td><td>Agilent</td><td>N/A</td><td>for structure elucidation</td></tr><tr><td>Rotovap drying system</td><td>Buchi</td><td>RII</td><td>for extraction of conditioned water&amp;nbsp;</td></tr><tr><td>UV lamp (254 nm)</td><td>Spectronics Co.</td><td>ENF-240C</td><td>for thin layer chromatography&amp;nbsp;</td></tr></tbody></table>

the identification of sea lamprey pheromones using bioassay-guided fractionation

生物测定引导分馏是利用生理和行为生物鉴定的结果来指导活性信息素化合物的分离和鉴定的一种迭代方法。这种方法的成功表征了化学信号的功能作为信息素在广泛的动物物种。海洋鳗依赖嗅觉来检测介导行为或生理反应的费洛蒙。我们利用这一知识的鱼类生物学的假设的功能, 假设的信息素, 并指导的分离和鉴定活性的费洛蒙成分。色谱用于从条件水中提取、浓缩和分离化合物。电 olfactogram (EOG) 记录, 以确定哪些分数引出嗅觉反应。两个选择迷宫行为化验, 然后用来确定是否有任何气味的分数也行为上活跃和诱发偏好。光谱和光谱方法提供了分子量和结构信息, 以协助结构的澄清。通过 EOG 和行为学测定, 证实了纯化合物的生物活性。在迷宫中观察到的行为响应最终应在字段设置中进行验证, 以确认它们在自然流设置中的作用。这些生物鉴定发挥双重作用到 1) 指导分馏过程和 2) 确认并进一步确定分离组分的活性。在这里, 我们报告了一个海洋鳗信息素鉴定的代表性结果, 证明了生物测定指导分馏方法的效用。海洋鳗信息素的识别是特别重要的, 因为它是控制劳伦琴大湖侵入性海鳗的选择之一。该方法可以很容易地适应于广泛的分类群中的化学通信, 并揭示了水化学生态学的特点。

图 1中概述了生物测定导向分馏的协议中描述的步骤。该协议涉及的步骤, 以分离和特征的结构, 嗅觉能力和行为活动的5假定海鳗信息素 (图 2)。利用质谱和核磁共振数据 (图 3和图 4), 从条件与成熟的雄性海鳗22、29的水中阐明了 petromyzene a-B...

Watch this Scientific Journal Video about The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation at JoVE.com

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

在这里, 我们提出了一个协议, 以分离和描述的结构, 嗅觉效力, 和行为反应的推测信息素化合物的海洋鳗。

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

the-identification-sea-lamprey-pheromones-using-bioassay-guided

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.9K 次观看.  Michigan State University. 在这里, 我们提出了一个协议, 以分离和描述的结构, 嗅觉效力, 和行为反应的推测信息素化合物的海洋鳗。

视频: The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

科研

环境科学

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

爬行动物皮肤脂类的化学分离、定量和分离

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and response to external stressors. Yet, the biochemical mechanisms and full complement of neuropeptides and their functional roles remain poorly understood. Characterization of these endogenous peptides is hindered by the immense diversity within this class of signaling molecules. Additionally, neuropeptides are bioactive at concentrations 100x - 1000x lower than that of neurotransmitters and are prone to enzymatic degradation after synaptic release. Mass spectrometry (MS) is a highly sensitive analytical tool that can identify, quantify, and localize analytes without comprehensive a priori knowledge. It is well-suited for globally profiling neuropeptides and aiding in the discovery of novel peptides. Due to the low abundance and high chemical diversity of this class of peptides, several sample preparation methods, MS acquisition parameters, and data analysis strategies have been adapted from proteomics techniques to allow optimal neuropeptide characterization. Here, methods are described for isolating neuropeptides from complex biological tissues for sequence characterization, quantitation, and localization using liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. A protocol for preparing a neuropeptide database from the blue crab, Callinectes sapidus, an organism without comprehensive genomic information, is included. These workflows can be adapted to study other classes of endogenous peptides in different species using a variety of instruments.

Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.

卡利内克特氏菌中神经肽的多方面质谱研究

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and ...

化学

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

生物化学

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

通过直接生物测定法鉴定海葵粗提取物中的溶血和磷脂酶活性

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

<meta charset="utf8"></head><body>秀丽隐杆线虫中挥发性信息素反应的提取和定量

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

<meta charset="utf8"></head><body>作者聚焦：检查挥发性信息素对雄性 秀丽隐杆线 虫行为的影响

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

秀丽隐杆线虫中的挥发性信息素提取和趋化试验

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

行为学

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 ...

生物学

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

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Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

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