Name: synthesis and evaluation of a ruthenium-based mitochondrial calcium uptake inhibitor
Uploaded: 2017-10-26T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor at JoVE.com

这项研究得到了康奈尔大学的支持。这项工作利用康奈尔材料研究中心的共享设施, 这是支持通过 NSF MRSEC 计划 (赠款 DMR-1120296)。S.R.N. 承认, 支持由 NSF 研究生研究奖学金 (赠款 DGE-1650441) 和 Dr. 戴夫 Holowka 协助钙实验。在这一材料中发表的任何意见、发现、结论或建议都是作者的观点, 不一定反映国家科学基金会的观点。

注意: 在这种合成中使用浓酸和碱。在执行反应时使用所有适当的安全措施, 包括使用工程控制 (油烟罩) 和个人防护用品 (PPE), 包括安全眼镜、手套、实验室大衣、全长长裤和闭脚趾鞋. 
 1. 准备 [(哦 2 ) (NH 3 ) 4 Ru (& #181;-O) 汝 (nh 3 ) 4 (OH 2 )] cl 5 <ol> <li> 合成 [ru (nh 3 ) 5 cl] cl 2 12 <ol> <li> 溶解1.00 克 RuCl 3 和 #183; n h 2 O (按重量 40% Ru, 4.1 摩尔) 在 5 mL h 2 o 将深褐色溶液冷却到0和 #176; C 在冰浴中。以滴的方式添加11毫升 (0.23 摩尔) 的80% 水合肼溶液。最初的反应将与演化气体一起剧烈, 导致棕色溶液。使所得到的溶液在室温下搅拌16小时;最终的解决方案将是深红色的. 
		注意: 肼具有剧毒和致癌性。此外, 这种试剂的无水形式是爆炸性的。与往常一样, 使用适当的 PPE 和通风罩时处理。不要将这些解决方案集中于干燥. </li>
		<li> 对此解决方案, 添加约5-10 毫升的浓缩 HCl, 以调整 pH 值为2。在这一点上, 解决方案将是黄褐色的颜色. </li>
		<li> 将此解决方案加热到105和 #176; 搅拌1-2 小时黄色固体会沉淀出溶液。当没有更多的沉淀明显的形式, 消除热量. </li>
		<li> 允许反应混合物冷却到室温, 然后放置在0和 #176; C 冰浴10分钟. 通过真空过滤, 用5毫升乙醇和乙醚洗涤, 收集黄色固体. </li>
		<li> 完全溶解在15-25 毫升热水中的原油产品。将浓缩的 HCl 溶液放在冰浴中冷却10毫升。将黄色溶液过滤到冷却的 HCl 溶液中, 以诱发一个淡黄色纯固体的沉淀。过滤这沉淀物和洗涤以5毫升每 0.5 M HCl, 乙醇并且乙醚. </li>
		<li> 用红外光谱表征化合物。通过在3226厘米 -1 、1604 cm -1 、1297 cm -1 和 801 cm -1 的拉伸频率标识来验证纯度。2069 cm -1 的常见次要杂质被分配给 [Ru (NH 3 ) 5 N 2 ] Cl 3 。
	</ol> </li> <li> 合成 [(哦 2 ) (NH 3 ) 4 Ru (和 #181;-O) 汝 (nh 3 ) 4 (OH 2 )] Cl 5 <ol> 溶解100毫克 (0.34 摩尔) [ru (nh <li> 3 ) 5 cl]Cl 2 在50毫升的1米 NH 4 OH 在200毫升重壁圆形底压力容器。用塞子将烧瓶松散地盖上, 将反应混合物加热75和 #176; C 为 6 h. 从热中取出, 室温下搅拌4天, 产生深绿色溶液. 
		谨慎!加热密封容器会导致压力积聚。确保使用适当的压力安全玻璃器皿。对于这个反应, 密封容器的目的是尽量减少气体 NH 的损失 3 。因此, 将塞子松散地放置, 以释放多余的压力. </li>
	</ol> </li> <li> 通过阳离子交换色谱 <ol> 在25毫升烧杯中进行纯化, 悬浮 5 g 阳离子交换树脂 (例如, 阳离子交换树脂 50WX2 200-400 目 (H <li> + 窗体) 在10毫升 0.1 M HCl 中. 
		<li> 将此浆料装入一个10毫升的柱子 (10 毫米直径, 15 厘米高) 贴附50毫升溶剂油藏。用大约20-30 毫升的0.1 米 HCl 清洗树脂, 直到洗无色. </li>
		<li> 返回到步骤1.2.1 中隔离的绿色反应解决方案。在此解决方案中, 添加浓缩 HCl 以调整 pH 值至 2, 此时溶液颜色会变为褐色. </li>
		<li> 将此酸化溶液加载到1.3.2 中的阳离子交换树脂柱上, 轻轻移在树脂上。让洗完全耗尽, 并继续加载解决方案。重复此过程, 直到添加了整个解决方案。树脂的顶端将是深褐色/黑色。树脂的体积会略有减少. </li>
		<li> 使用玻璃珠覆盖树脂的顶部。在添加新的解决方案时, 这些将防止树脂受到干扰. </li>
		<li> 洗为20毫升的1米 HCl 的列. </li>
		<li> 洗增加 HCl 浓度1.5 米 (和 #8776; 50 毫升) 的列。一个黄色的解决方案将开始从列中脱落。将 HCl 浓度提高到2米, 然后继续洗脱, 直到洗无色或呈淡绿色黄色。这个过程将需要150-200 毫升的总容量. </li>
		<li> 将 HCl 浓度提高到2.5 米 (20-50 毫升)。在试管中收集洗作为分数。增加到 3 M HCl。该产品将洗从该专栏作为一个绿色棕色的解决方案。红棕色的分数也可能会从柱子上脱落。由于这些分数是氧化钌红色杂质, 不池与绿色棕色的分数. </li>
	</ol> </li> <li> 的特性和验证 [(OH 2 ) (NH 3 ) 4 汝 (和 #181;-O) 汝 (NH 3 ) 4 (OH 2 )] "Cl 5 <ol> <li> 测试所有分数步1.3。8紫外可见光谱学。为了完成这项任务, 添加100和 #181; 将给定分数的 L 升为2毫升的3米 NH3, 并通过紫外可见光谱进行分析。含有纯产品的分数将有一个大的吸光度带在 360 nm 和较低的强烈吸光度在 600 nm。480或 533 nm 的吸光度分别指示氧化钌红和钌红色杂质. </li>
		<li> 包含纯产品的池馏分, 并通过旋转蒸发将溶液蒸发干燥。该产品将作为一个绿色棕色固体隔离。产量通常在5-15 毫克 (10-20% 收益率) 的顺序。单晶体, 适用于 X 射线衍射, 可以获得的水蒸气扩散的乙醇水溶液的化合物. </li>
		<li> 为了验证纯度, 在 pH 值为7.4 的磷酸盐缓冲盐水 (PBS) 溶液中通过紫外-可见光谱分析化合物。纯度可以通过采取 360 nm 和 600 nm 峰的强度比值来评估。这个比率是31为一个纯净的化合物。对于不纯化合物, 比例将会更小. </li>
		<li> 用红外光谱法对 solid-state 样品进行分析。诊断带是在 3234 cm -1 , 3151 cm -1 , 1618 cm -1 , 1313 cm -1 , 和 815 cm -1 。常见的杂质带见于 1762 cm -1 和 1400 cm -1 , NH 4 Cl. 钌红的特征可以通过带在 1404 cm -1 、1300 cm -1 、1037 cm -1 和 800 cm -1 中标识. </li>
	</ol> </li> <li> 荧光光谱法评价线粒体钙的摄取抑制 警告!以下过程使用哺乳动物细胞。工作应在适当的层流罩, 是认证的生物安全等级 2 (BSL2) 研究. 
	#8203; 注意: [(哦 2 ) (NH 3 ) 4 Ru (#181;-O) 汝 (NH 3 ) 4 (OH 2 )] Cl 5 将被称为 [Ru] 在本节 <ol> <li> 中使缓冲含葡萄糖的盐水溶液 (BGSS) 作为化验介质。BGSS 是一个解决方案, 包括110毫米氯化钾, 1 mm 2 PO 4 , 1 毫米氯化镁 2 , 20 毫米 4-(2-羟基乙基)-1-piperazineethanesulfonic 酸 (HEPES), 5 毫米琥珀酸钠, 30 和 #181; M 乙二醇双 (和 #946; 乙氨基醚)- n, n, n 和 #39;, n 和 #39; -乙酸酸 (EGTA)。除了 EGTA, 将 pH 值调整到7.4。添加 EGTA 并调整 pH 值至7.4。50毫升的测定培养基中添加0.5 毫升的1毫克/毫升葡萄糖. </li>
		<li> 500 cm 中的培养 HeLa 细胞 2 Dulbecco 和 #39 的改良鹰培养基 (DMEM) 和10% 胎牛血清 (FBS) 在湿式恒温箱中, 5% CO 2 37 和 #176; c. 通过播种增强 100 mm Petri 皿中生长的 hela 细胞他们在 500 cm 2 培养皿。大盘中的总介质量为115毫升。每个大盘子将产生大约1800万细胞, 足够的两个荧光光谱实验。
		<ol> <li> 将单元格增长到 90-95% 70-100。取出培养基, 用15毫升 pH 7.4 PBS 冲洗细胞。在 PBS 中加入15毫升的1毫米乙酸酸 (EDTA), 孵育10分钟以分离细胞。将电池转移到14毫升圆底猎鹰管 </li> </ol> </li> <li> 使用台盼蓝和例的计数单元, 并用倒置显微镜计算每1.8 毫升容量达到750万细胞所需的细胞总数和培养基数量 中等。将细胞离心10分钟, 在5310和 #215; 醒酒上清液, 加入 BGSS 的计算量。重细胞轻轻。
		<ol> <li> 用于此检测, 准备40毫米 digitonin 在二甲基亚砜 (亚砜), 1 毫米钙 Green-5N 在 h 2 o 和10毫米 CaCl 2 在 h 2 o 中的库存解决方案. [汝] 库存解决方案, 在纯净水中, 可以从1-3 毫米不等。
			#8203; 注意: Green-5N 钙是光敏的。存储在黑暗中, 尽量减少曝光. </li>
		</ol> </li> <li> 设置 fluorimeter 以在 506 nm 处激发, 并在 532 nm 上读取试管控制在37和 #176 上的发射器; c. 用搅拌棒或轮子制作丙烯酸试管, 1.8 mL 细胞悬浮1.5.2 以上, 1.8 和 #181; L digitonin 解决方案, 3。6#181;l 钙 Green-5N (溶液), 9 和 #181; l [汝] (1 毫米的股票溶液, 5 和 #181; M 最终浓度)。在 fluorimeter 孵育细胞15分钟。
		<ol> <li> 将数据读取为激发/发射比而不是原始吸收。这种做法最大限度地减少了与光源强度波动相关的误差. </li>
			<li> 在没有 [Ru] 的情况下执行第一个样本分析, 以测量在单元格的响应中添加 CaCl 2 的效果. </li>
			<li> 使用1.5.4 中描述的设置开始分析 fluorimeter。等待约2分钟建立一个稳定的排放基线, 然后添加1.8 和 #181; L CaCl 2 (10 和 #181; M 最后的浓度)。在加入 CaCl 2 后, 发射强度会立即增加, 然后随着钙离子进入线粒体而在几分钟内衰变。等到衰变结束 (#8776; 5 分钟)。添加额外的钙 boluses, 以确定不处理的细胞线粒体钙吸收反应 [Ru]. </li>
		</ol> </li> <li> 在包含5和 #181 的另一个试管中; M [Ru], 重复上述1.5.4.3 中所述的实验。在抑制剂的存在下, 排放强度会增加, 但不会衰减。这种观察意味着阻断线粒体钙的摄取. </li>
	</ol>
	</li>
</ol>

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线粒体钙吸收抑制剂 [(OH2) (nh3)4汝 (µ O) ru (nh3)4(OH2)] cl5可以从 [Ru (NH3)5cl] cl2中合成众所周知的钌 (III)起始材料, 如本程序所述。合成的 [Ru (NH3)5cl] cl2很容易实现, 但难度很小。在水合肼中搅拌 RuCl3 16 小时后, 溶液的 pH 值应调整为2与 HCl 的数值。pH 值下降是实现所需产品的关键。如果?...

线粒体钙是对正常细胞功能, 包括能量生产和细胞凋亡至关重要的一些过程的关键调节器。1,2,3线粒体钙 uniporter (MCU), 一种离子转运蛋白, 驻留在线粒体膜上, 调节钙离子流入线粒体。4,5,6单片机的化学抑制剂是继续努力研究这种转运蛋白和线粒体钙的功能和细胞作用的宝贵工具。化合物 [(小贩2) (nh3)4ru (µ) 汝 (nh3)4(o2CH)3 +, Ru360, 是唯一已知的单片机的选择性抑制剂之一, 其报告的 Kd值为24µM.7 ,8,9,10此复合物是在钌红色 (RuRed) 的商业配方中发现的常见杂质, triruthenium µ-羰基桥接 hexacation 的公式 [(NH3)5ru (µ) 汝 (nh3)4(µ) ru (nh3)5)6 +, 这也被用作钙吸收抑制剂。虽然 Ru360 是商用的, 但成本很高。此外, Ru360 的合成和分离受到困难的纯化程序和模糊的表征方法的挑战。
我们最近报告的替代程序访问一个 Ru360 模拟, [(OH2) (nh3)4汝 (µ) ru (新罕布什尔州3)4(oh2)] Cl5。11此复合抑制具有高亲和力的 MCU, 类似于 Ru360。在本协议中, 我们将描述这一化合物的最有效的合成, 它从 [Ru (NH3)5cl] cl2开始。使用强酸性阳离子交换树脂提纯产品, 并对此程序的常见缺陷进行了详细介绍。我们还提出了表征和评估化合物纯度的方法, 并描述了一个简单的方法来测试它的有效性, 阻断线粒体钙摄取。

<table><tbody><tr><td>Ruthenium Trichloride hydrate</td><td>Pressure Chemical</td><td>3750</td><td></td></tr><tr><td>Concentrated hydrochloric acid</td><td>J.T. Baker</td><td>9535</td><td></td></tr><tr><td>Concentrated ammonium hydroxide</td><td>Mallinckrodt Chemical Works</td><td>A669C-2 1</td><td></td></tr><tr><td>Dowex 50 WX2 200-400 Mesh</td><td>Alfa Aesar</td><td>13945</td><td></td></tr><tr><td>Calcium Green 5N</td><td>Invitrogen</td><td>C3737</td><td></td></tr><tr><td>Digitonin</td><td>Aldrich</td><td>260746</td><td></td></tr><tr><td>DMSO</td><td>Aldrich</td><td>471267</td><td></td></tr><tr><td>EGTA</td><td>Aldrich</td><td>E3889</td><td></td></tr><tr><td>KCl</td><td>USB</td><td>20598</td><td></td></tr><tr><td>KH2PO4</td><td>Aldrich</td><td>P3786</td><td></td></tr><tr><td>MgCl2</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>HEPES</td><td>Fluka</td><td>54466</td><td></td></tr><tr><td>Sodium Succinate</td><td>Alfa Aesar</td><td>33386</td><td></td></tr><tr><td>EDTA</td><td>J.T. Baker</td><td>8993-01</td><td></td></tr><tr><td>Glucose</td><td>Aldrich</td><td>G5000</td><td></td></tr><tr><td>200 Round bottom flask</td><td>ChemGlass</td><td>CG-1506-14</td><td></td></tr><tr><td>Glass stopper</td><td>ChemGlass</td><td>CG-3000-05</td><td></td></tr><tr><td>10 mm x 15 cm glass column with reservoirs</td><td>Custom - similar to Chemglass columns</td><td>Similar to CG-1203-20</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Round bottom Falcon tubes</td><td>Fisher Scientific</td><td>14-959-11B&amp;nbsp;</td><td></td></tr><tr><td>500 cm&lt;sup&gt;2&lt;/sup&gt; petri dishes</td><td>Corning</td><td>431110</td><td></td></tr><tr><td>Trypan blue</td><td>ThermoFisher Scientific</td><td>15250061</td><td></td></tr><tr><td>Hemacytometer</td><td>Aldrich</td><td>Z359629</td><td></td></tr><tr><td>Acrylic Cuvettes</td><td>VWR&amp;nbsp;</td><td>58017-875</td><td></td></tr><tr><td>UV-Vis spectrometer</td><td>Agilent Model Cary 8454&amp;nbsp;</td><td></td><td></td></tr><tr><td>Spectrofluorimeter</td><td>SLM Model 8100C</td><td></td><td></td></tr><tr><td>IR spectrometer</td><td>Bruker Hyprion FTIR with ATR attachment</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>ALC Model PM140R</td><td></td><td></td></tr><tr><td>Inverted light microscope</td><td>VWR&amp;nbsp;</td><td>89404-462</td><td></td></tr></tbody></table>

synthesis and evaluation of a ruthenium-based mitochondrial calcium uptake inhibitor

我们详细介绍了线粒体钙吸收抑制剂的合成和纯化, [(oh2) (nh3)4汝 (µ) ru (新罕布什尔州3)4(OH2)5 +。这种化合物的优化合成开始从 [Ru (NH3)5cl] cl2在 1 M NH4OH 在一个封闭的容器, 产生一个绿色的解决方案。纯化是用阳离子交换色谱法完成的。这种化合物的特点和验证是纯紫外-可见光和红外光谱。荧光光谱法对化 HeLa 细胞线粒体钙摄取抑制特性进行了评价。

该方法描述了线粒体钙吸收抑制剂的合成 [(OH2) (nh3)4汝 (µ O) ru (nh3)4(OH2)] cl5从 [Ru (NH3)5cl] cl2开始, 一众所周知的钌 (III) 起动材料。[Ru (NH3)5Cl]Cl2的特征是红外光谱, 具有 3200 cm-1、1608 cm-1、1298 cm-1和 798 cm-1 (图 1) 的...

Watch this Scientific Journal Video about Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor at JoVE.com

Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

钌线粒体钙吸收抑制剂的合成与评价

提出了一种用于线粒体钙吸收的钌抑制剂的合成、纯化和表征的协议。本文介绍了一种评价其在化哺乳动物细胞中的有效性的方法。

We detail the synthesis and purification of a mitochondrial calcium uptake inhibitor, [(OH2)(NH3)4Ru(&#181;-O)Ru(NH3)4(OH2)]5+. The optimized synthesis of ...

synthesis-evaluation-ruthenium-based-mitochondrial-calcium-uptake

Research

JoVE Journal

Chemistry

7.8K 次观看.  Cornell University. 提出了一种用于线粒体钙吸收的钌抑制剂的合成、纯化和表征的协议。本文介绍了一种评价其在化哺乳动物细胞中的有效性的方法。

视频: Synthesis and Evaluation of a Ruthenium-based Mitochondrial Calcium Uptake Inhibitor

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

一系列钌的合成，表征和反应<em&gt;Ñ</em&gt; -triphos<sup&gt;博士</sup&gt;配合

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

科研

化学

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

生物化学

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy.
Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action.
Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control.
The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide.
This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties.

一个<em&gt;在体外</em&gt;酶测定来测量转录抑制作用镓（Ⅲ）和H<sub&gt; 3</sub&gt; -5,10,15-三（五氟苯基）corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

生物工程

Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model.
One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS).
ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.

The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry.

通过电感耦合等离子体质谱法对斑马鱼中铂基和钌基化合物暴露的剂量吸收，具有更广泛的应用

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the ...

<meta charset="utf8"></head><body>改变线粒体功能以用于治疗和生物杀虫剂应用

Unveiling Xenobiotic Transport and Effects in Isolated Mitochondria: Insights from Respirometric and Enzymatic Assays

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). However, their significance extends far beyond energy production. Dysregulation of mitochondrial bioenergetics can trigger intracellular cascades that impact health and cellular function. Given their physiological importance, there is growing interest in exploring the pharmacological potential of mitochondrial function for developing new therapies and bioproducts. Modulating mitochondrial bioenergetics could provide innovative approaches to address challenges such as neurodegenerative disorders, metabolic diseases, and cancer, as well as to develop bioproducts for controlling pests and vector-borne diseases.
This work presents a method for assessing the effects of xenobiotics on the electron transport chain (ETC) using various substrates, including glutamate and NADH for complex I, succinate for complex II, and cytochrome c (both oxidized and reduced) for complexes III and IV. This methodology allows the activation or inhibition of electron transport through mitochondrial complexes to be evaluated using a respirometer and spectrophotometer. The mitochondria can be sourced from isolated mitochondria, fragmented cells, or homogenized tissue from various species.
In the laboratory, mitochondrial function has been analyzed in Aedes aegypti and Wistar rats, but this method is also applicable to other species, such as Rhipicephalus microplus and Rhodnius prolixus. This approach provides the basis for theorizing about the existence of uncoupler proteins and species-specific oxidizable substrate preferences influenced by their unique energetic demands. Recent findings offer valuable insights into innovative bioinsecticide design strategies that target mitochondrial function, holding significant potential for effectively controlling vector-borne diseases and pest infestations.

<meta charset="utf8"></head><body>揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

This study introduces a methodology for studying xenobiotic effects on mitochondrial function via respirometric and enzymatic assays. It examines electron transport chain modulation in various species, offering insights for targeted biopesticide development against insect vector-borne diseases.

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

Mitochondria are often referred to as the cell's powerhouse due to their crucial role in energy production through the electron transport chain (ETC). ...

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

线粒体 Ca2 +保留容量检测和 Ca2 +触发的线粒体肿胀试验

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

神经科学

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

生物学

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

钌线粒体钙吸收抑制剂的合成与评价

摘要

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此视频中的章节

一系列钌的合成，表征和反应

线粒体钙在分离线粒体和培养细胞中的流入分析

一个

通过电感耦合等离子体质谱法对斑马鱼中铂基和钌基化合物暴露的剂量吸收，具有更广泛的应用

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

揭示离体线粒体中的异种生物运输和影响:来自呼吸测定和酶测定的见解

线粒体 Ca^{2 +}保留容量检测和 Ca^{2 +}触发的线粒体肿胀试验

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在