作者感谢戴尔·莱特曼在使用瞬态透切检测来确定灵长类植物食物的雌激素活性方面进行了初步培训。感谢布拉德福德·韦斯特里奇和C·埃里克·约翰逊帮助建立实验室设备，并培训学生提取方法。最后，感谢印第安纳大学为这项研究提供资金。

1. 植物材料的准备
<ol><li>冷冻使用嗜酸剂新鲜收集的干燥植物物品。<ol>
<li>为了保护样品免受光线照射，在干燥过程中用铝箔盖住腔室。</li>
		<li>为了确保样品完全干燥，嗜酸，直到室不再感到寒冷触摸和植物材料不再失去质量时称重。</li>
		<li>将干燥的植物存放在无菌低残留袋中，在没有光线的情况下，直到研磨。</li>
	</ol>
</li>
	<li>使用带 0.85 mm 网格屏幕的研磨机精细研磨样品。<ol>
<li>在没有光线的情况下将地面样品存放在袋子里，直到提取。</li>
	</ol>
</li>
</ol>2. 提取植物二次代谢物
<ol><li>要提取二级植物代谢物，请使用 1 克干样品与 10 mL 的 HPLC 级甲醇的比例。<ol>
<li>在分析平衡上称量样品，并将其添加到大小合适的埃伦迈尔烧瓶（125-250 mL）中。然后添加适量的甲醇。记录提取的样本的质量。</li>
		<li>用铝箔盖住植物甲醇溶液，然后在室温 （RT） 下以 100 rpm 的速度在轨道摇床上旋转 3 天，使潜在的雌激素化合物溶解到甲醇中。</li>
		<li>使用滤纸（125毫米）将超细长剂倒入滴滤系统。</li>
		<li>使用旋转蒸发器，将植物提取物干燥，直到样品变厚，但可倒入 300 mL 的圆底烧瓶中。将样品倒入 50 mL 圆底烧瓶中，用少量甲醇冲洗大烧瓶。继续在小烧瓶中干燥样品，直到甲醇完全蒸发。</li>
		<li>使用分析平衡对样品残留物进行称重。记录残留质量。</li>
		<li>将植物提取物溶解在二甲基硫化物 （DMSO） 中，浓度为 0.1 克提取物到 2 mL DMSO。漩涡，直到同质化。</li>
		<li>将植物提取物-DMSO溶液在4°C下存放在琥珀色玻璃瓶中，直到检测。</li>
	</ol>
</li>
</ol>警告:植物可以产生未知的生物活性化学物质，DMSO是一种可以通过细胞膜运输它们的工具。在处理这些样品时，使用适当的个人防护设备并小心谨慎。
3. 人体雌激素受体β转位检测12
注:检测协议的第 1 天需要无菌技术和层流罩。
<ol><li>为标准曲线准备 17+ 雌二醇的稀释。<ol>
<li>将细胞恢复介质和复合筛选介质 （CSM） 从冰柜存储中转移，并在 37 °C 水浴中解冻。</li>
		<li>标记微中心管中间体 1 和 2 （INT1，INT2） 和 1-8。</li>
		<li>用 995 μL 的 CSM 填充 INT1，用 615 μL 的 CSM 填充 INT2，用 900 μL 的 CSM 填充 1 管，用 600 μL 的 CSM 填充 2-8。将管 8 放在一边。</li>
		<li>将 100 μM 17+Estradiol 库存的 5 μL 转入 INT1。丢弃提示。涡。</li>
		<li>每次传输前，冲洗移液器 3 次，然后从 INT1 传输 10 μL 到 INT2。丢弃提示。</li>
		<li>冲洗移液器3次，然后从INT2传输100μL到管1。丢弃提示。将 300μL 从 1 管转移到 2 管中。重复管 3 到 7。将 300μL 从 7 管中丢弃到废物容器中。管8是零，不接收雌二醇。镀膜标准的最终浓度为:400、133.3、44.44、14.815、4.938、1.646、0.5487和0 pM雌二醇。</li>
	</ol>
</li>
	<li>准备样品化合物。<ol>
<li>漩涡样本。</li>
		<li>以 DMSO 中每个植物样本的 4 μL 为例，并添加到 496 μL 的 CSM 中，以产生 0.8% 的 DMSO 解决方案。</li>
	</ol>
</li>
	<li>迅速解冻记者细胞。<ol>
<li>从 37 °C 水浴中取出细胞恢复介质管。使用 70% 乙醇对外部表面进行消毒。</li>
		<li>通过将预加热的 CRM 的 10 mL 传输到冷冻细胞管中，从 -80 °C 存储中检索记者细胞并解冻。</li>
		<li>关闭记者细胞管，转移到 37°C 的水浴室 5-10 分钟。</li>
		<li>从水浴中取出记者细胞悬浮管。轻轻倒置细胞管数次，以分解细胞聚集物并产生同质悬浮物。用 70% 乙醇清洁管子表面。</li>
	</ol>
</li>
	<li>检测电镀<ol>
<li>使用多通道移液器将 100 μL 的记者细胞悬浮液分配到每个井中。</li>
		<li>将 100 μL 的样品分三次放入适当的检测井中。</li>
		<li>将板转移到 37 °C 中，加湿 5% CO2 孵化器 22-24 小时。</li>
	</ol>
</li>
	<li>在黑暗的冰箱中通宵解冻检测基板和检测缓冲区，为第 2 天做准备。</li>
	<li>在板式孵化结束之前，从冰箱中取出检测基板和检测缓冲区，放置在低光区域，直到平衡到 RT。一旦在RT，轻轻倒置每个管几次，以彻底混合解决方案。<ol>
<li>在孵化完成之前，将检测缓冲区的全部内容倒入检测基板管中，以创建 Luciferase 检测试剂。轻轻混合，以免产生泡沫。</li>
		<li>孵化完成后，倒置板将内容倒置到适当的废容器中。轻轻敲击干净的吸水纸巾上的板，从井中取出最后一滴水滴。</li>
		<li>在每个油井中添加 100μL 的卢西费酶检测试剂。让检测板在RT上休息15分钟。不要摇盘子。</li>
	</ol>
</li>
	<li>使用 96 井板读数氧化铝计量化发光。</li>
</ol>

<ol>
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作者没有什么可透露的。

ER+记者检测开发为单独筛选药物制剂也适用于筛选植物性植物雌激素通过 ER+生物活性的食物。协议中的重要考虑因素包括小心处理植物样品:需要迅速干燥新鲜植物材料以防止成型或其他生物退化，并且需要远离光线以防止化合物18的光解。制造商提供的检测协议12 是明确的，需要很少的修改进行筛选。制造商建议的标准曲线已在此协议中进行了修改，以增加曲...

植物是许多动物必需的食物来源，提供对生存、繁殖、生长、发育和行为至关重要的卡路里和营养物质。植物产生成千上万的化学物质，许多是适应自身生长、植物维持和繁殖的。其他化合物，被视为植物二次代谢物（PSM），其功能不太清晰，虽然有些是有毒的，并可能用作防御食草动物和寄生虫（如生物碱，单宁）2，3.其中一些化学物质有能力影响动物的长期生理过程，如内分泌功能，虽然为什么这些内分泌活性植物化学物质与脊椎动物内分泌系统相互作用仍不清楚2，4。
植物雌激素，研究最充分的内分泌活性植物化学物质，是多酚类PSM，在结构和功能上模仿雌激素，直接与脊椎动物内分泌系统5的假体-垂体淋巴轴相互作用。在人类饮食中摄入植物雌激素与预防某些癌症、心脏病和更年期症状有关，尽管其他影响包括生育问题。事实上，这些化合物的生理影响是在20世纪40年代发现的，当时绵羊的不育归因于它们对富含植物雌激素的三叶草（三叶草）的放牧。摄入后，植物雌激素可以进入细胞并模仿雌激素的作用。虽然植物雌激素对绵羊的生育能力有负面影响，但植物雌激素与生理学之间的关系并不简单。与绵羊一样，南方白犀牛对源自大量大豆和紫花花的饲料中的雌激素化合物表现出敏感性。在怀孕期间喂养这种饮食的女性的女儿不太可能繁殖7。然而，其他研究表明，植物雌激素也可能有积极的影响，包括卵巢卵泡的成熟在老老鼠8，预防某些癌症，抗氧化活性和抗增殖作用9。
植物雌激素的影响范围并不奇怪，因为雌激素影响广泛的生物功能，包括生长，发育和调节生殖和中枢神经系统10。虽然有许多作用机制，植物雌激素往往有能力修改，增强或破坏雌激素信号，通过他们的能力，作为配体的核内雌激素受体阿尔法和β（ER+和ER+）。许多植物雌激素具有类似于雌激素的酚环结构，允许它们结合雌激素受体。那些具有痛苦的雌激素活性功能，如雌激素，形成一个激活的ER-ligand复合物，可以变暗和结合雌激素反应元素（ERE），并触发基因转录11。因此，雌激素和植物雌激素通过它们作为转录因子的行为来调节细胞活动和系统功能。
在这里，我们介绍了一种新颖的利用基于细胞的检测方法来筛选植物提取物，以发现具有雌激素生物活性的化合物的存在。这种检测使用中国仓鼠卵巢CHO细胞设计，以高度表达ER+，已被转染与萤火热病毒（Photinus皮拉里斯）荧光酶基因链接到ERE促进者12。当雌激素化合物存在时，它们与急ER结合，变暗，并结合到ERE，导致荧光酶基因的转录。加入基板溶液后，卢西费酶催化导致光子发射的反应。因此，阳性样品产生的光和阴性样品不。
这种市售的检测无需实验室用报告基因和雌激素受体13、14来传染哺乳动物细胞，这些受体的功效不稳定且变化无常。该检测提供了一个稳定的转染平台，能够快速而简单地通过受体结合确定植物是否具有雌激素活性。
我们测试了这样一个假设，即大豆的雌激素活性高于所有其他食物，因为大豆已知浓度为雌激素异黄酮15， 使用来自当地杂货店的人类食物。

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			<td height="21" style="height:21px;width:215px;">1000 &#181;L pipette</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">200 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">37 &#8451; water bath</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">37 &#8451;, humidified 5% CO2 incubator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70% ethanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">analytical balance</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">cell culture-rated laminar flow hood</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">dimethyl sulfoxide</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">disposable media basin, sterile</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">drip filtration system</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Erlenmeyer flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>125 mL and 250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HPLC grade methanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Human ER&#946; Reporter Assay System, 1 x 96-well format assays</td>
			<td style="width:80px;">Indigo Biosciences</td>
			<td style="width:119px;">IB00411</td>
			<td>Assay kit - analyzes 24 samples plus standard curve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">lyophilizer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">multi-channel pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">orbital shaker</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">plate-reading luminometer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>ex. Bioteck Synergy HTX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">rotory evaporator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">round bottom flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>50 mL and 300 mL</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">sterile microcentrifuge tubes or sterile multi-channel media basins</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">sterile tips</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>200 &#181;L and 1000 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Whatman grade 1 paper</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">whirl-pak bags</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>sterile polyethylene bags</td>
		</tr>
	</tbody>
</table>

screening for phytoestrogens using a cell-based estrogen receptor  &#946; reporter assay

植物是许多动物的食物来源，它们可以产生成千上万的化学物质。其中一些化合物影响消耗它们的脊椎动物的生理过程，如内分泌功能。植物雌激素，研究最充分的内分泌活性植物化学物质，直接与脊椎动物内分泌系统的下丘脑-皮质淋巴轴相互作用。在这里，我们介绍了一种新颖的利用基于细胞的检测方法来筛选植物提取物，以发现具有雌激素生物活性的化合物的存在。这种分析使用哺乳动物细胞设计，以高度表达雌激素受体β（ER+），并且已经与露西费酶基因转染。接触具有雌激素活性的化合物会导致细胞产生光。这种检测是测试生物雌激素活性的可靠和简单方法。它比瞬态透变检测有几种改进，最明显的是易于使用、细胞的稳定性和检测的灵敏度。

在人类饮食中常见的22种水果和蔬菜提取物被筛选出雌激素化合物的存在。各种食物被检测，包括豆类，如大豆，雪豌豆和豌豆，因为豌豆家族是已知的植物雌激素16的来源，以及无花果，枣，玉米，胡萝卜，苹果，香蕉，草莓，番茄，甘蓝和卷心菜。内分泌破坏化合物存在于常见物质（例如塑料和杀虫剂）中，有些通过红外体17具有生物活性。如果可能，对有?...

Watch this Scientific Journal Video about Screening for Phytoestrogens using a Cell-based Estrogen Receptor  ÃŽÂ² Reporter Assay at JoVE.com

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

我们优化了一种市售雌激素受体β记者检测，用于筛查人类和非人类灵长类动物的食物，以进行雌激素活性活动。我们通过显示已知的雌激素人类食物大豆注册高，而其他食物显示没有活性来验证这一分析。

使用基于细胞的雌激素受体对植物雌激素进行筛查β记者分析

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

screening-for-phytoestrogens-using-cell-based-estrogen-receptor

Research

JoVE Journal

Biochemistry

4.7K 次观看.  Indiana University. 我们优化了一种市售雌激素受体β记者检测，用于筛查人类和非人类灵长类动物的食物，以进行雌激素活性活动。我们通过显示已知的雌激素人类食物大豆注册高，而其他食物显示没有活性来验证这一分析。

视频: Screening for Phytoestrogens using a Cell-based Estrogen Receptor  β Reporter Assay

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

用于筛查绝经后心血管功能障碍的外源雌激素治疗的活体雌激素缺乏小鼠模型

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

一种基于报告基因的细胞检测，用于监测剪接效率

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

用于重组G蛋白偶联受体高通量筛选的"双加法"钙荧光测定

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

<meta charset="utf8"></head><body>研究维生素 A 受体动力学和功能对类维生素 A 稳态的影响

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. 
These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. 
Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

<meta charset="utf8"></head><body>作者聚焦：解开维生素 A 运输机制 — 通过 RBPR2 和 RBP4 相互作用将肝脏受体与视力健康联系起来

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

研究维生素 A 膜受体和视蛋白与其配体在产生视黄基蛋白中的相互作用的方法

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...