Name: detection of phospholipase c activity in the brain homogenate from the honeybee
Uploaded: 2018-09-14T13:30:00
Description: Watch this Scientific Journal Video about Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee at JoVE.com

图 4B - 4D从 Suenami等修改。24以生物开放的允许。作者感谢出版商的许可。这项工作得到了人类前沿科学计划 (RGY0077/2016) 的支持, 索塔 Suenami 和宫崎骏。

1. 捕食蜜蜂的捕获
<ol><li>从当地经销商那里购买蜜蜂殖民地。</li>
	<li>使用昆虫网, 捕捉只觅食蚂蚁蜜蜂返回蜂巢与花粉袋在他们的后腿。将蜜蜂转移到标准的50毫升塑料锥形管上, 并盖上管 (图 1)。把管子放在冰上麻醉蜜蜂。 注: 为养蜂人佩戴指定的夹克以避免蜂蜇。哺乳蜜蜂也可以根据实验收集。为了捉到护士蜜蜂, 观察蜜蜂在蜡梳上的行为, 抓住护士蜂, 用镊子将他们?...

<ol>
	<li>Winston, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biology of the Honey Bee. , (1991).</li><li>Szyszka, P., Galkin, A., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+and+non-associative+plasticity+in+Kenyon+cells+of+the+honeybee+mushroom+body.">Associative and non-associative plasticity in Kenyon cells of the honeybee mushroom body.</a> Frontiers in Systems Neuroscience. 2, 3 (2008).</li><li>M&#252;&#223;ig, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+disruption+of+the+NMDA+receptor+subunit+NR1+in+the+honeybee+brain+selectively+impairs+memory+formation.">Acute disruption of the NMDA receptor subunit NR1 in the honeybee brain selectively impairs memory formation.</a> The Journal of Neuroscience. 30 (23), 7817-7825 (2010).</li><li>Devaud, J. -. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+early+olfactory+processing+by+an+octopaminergic+reinforcement+pathway+in+the+honeybee.">Modulation of early olfactory processing by an octopaminergic reinforcement pathway in the honeybee.</a> The Journal of Neuroscience. 23 (12), 5370-5380 (2003).</li><li>Matsumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+nucleotide-gated+channels,+calmodulin,+adenylyl+cyclase,+and+calcium/calmodulin-dependent+protein+kinase+II+are+required+for+late,+but+not+early,+long-term+memory+formation+in+the+honeybee.">Cyclic nucleotide-gated channels, calmodulin, adenylyl cyclase, and calcium/calmodulin-dependent protein kinase II are required for late, but not early, long-term memory formation in the honeybee.</a> Learning &amp; Memory. 21 (5), 272-286 (2014).</li><li>Scholl, C., K&#252;bert, N., Muenz, T. S., R&#246;ssler, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+knockdown+affects+both+early+and+late+phases+of+olfactory+long-term+memory+in+the+honeybee.">CaMKII knockdown affects both early and late phases of olfactory long-term memory in the honeybee.</a> Journal of Experimental Biology. 218, 3788-3796 (2015).</li><li>Miyata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficient+long-term+synaptic+depression+in+the+rostral+cerebellum+correlated+with+impaired+motor+learning+in+phospholipase+C+&#946;4+mutant+mice.">Deficient long-term synaptic depression in the rostral cerebellum correlated with impaired motor learning in phospholipase C &#946;4 mutant mice.</a> European Journal of Neuroscience. 13 (10), 1945-1954 (2001).</li><li>Koh, H. -. Y., Kim, D., Lee, J., Lee, S., Shin, H. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+social+behavior+and+sensorimotor+gating+in+mice+lacking+phospholipase+C&#946;1.">Deficits in social behavior and sensorimotor gating in mice lacking phospholipase C&#946;1.</a> Genes, Brain and Behavior. 7 (1), 120-128 (2008).</li><li>Quan, W. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+behaviors+and+prepulse+inhibition+in+phospholipase+C&#949;-/-+mice.">Characteristics of behaviors and prepulse inhibition in phospholipase C&#949;-/- mice.</a> Neurology,Psychiatry and Brain Research. 18 (4), 169-174 (2012).</li><li>Rioult-Pedotti, M. -. S., Pekanovic, A., Atiemo, C. O., Marshall, J., Luft, A. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phospholipase+C&#949;+in+physiological+phosphoinositide+signaling+networks.">Role of phospholipase C&#949; in physiological phosphoinositide signaling networks.</a> Cellular Signalling. 24 (6), 1333-1343 (2012).</li><li>Dusaban, S. S., Brown, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLC&#949;+mediated+sustained+signaling+pathways.">PLC&#949; mediated sustained signaling pathways.</a> Advances in Biological Regulation. 57, 17-23 (2015).</li><li>Elgersma, Y., Sweatt, J. D., Giese, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+genetic+approaches+to+investigating+calcium/calmodulin-dependent+protein+kinase+II+function+in+plasticity+and+cognition.">Mouse genetic approaches to investigating calcium/calmodulin-dependent protein kinase II function in plasticity and cognition.</a> The Journal of Neuroscience. 24 (39), 8410-8415 (2004).</li><li>Giese, K. P., Mizuno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+protein+kinases+in+learning+and+memory.">The roles of protein kinases in learning and memory.</a> Learning &amp; Memory. 20 (10), 540-552 (2013).</li><li>Suenami, S., Iino, S., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+inhibition+of+phospholipase+C+in+the+brain+attenuates+early+memory+formation+in+the+honeybee+(Apis+mellifera+L.).">Pharmacologic inhibition of phospholipase C in the brain attenuates early memory formation in the honeybee (Apis mellifera L.).</a> Biology Open. 7 (1), (2018).</li><li>Zhu, L., McKay, R. R., Shortridge, R. 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蛋白质活性的生化检验对于理解大脑中的分子信号是非常重要的, 因为酶的活性受各种分子 (如基质和抑制剂) 的影响, 因此可以随动物行为 (例如, 学习和记忆)5。在蜜蜂研究中, 据报道, 在不同的脑区, 基于循环安培依赖性蛋白激酶 A、循环 GMP 依赖性蛋白激酶、PKC、磷酸化 CaMKII 和腺苷酸酶的酶有差异表达。免疫组化5,10,<s...

欧洲蜜蜂 (api 蜜蜂L) 是一种群居昆虫, 雌性蜜蜂显示种姓依赖的生殖和年龄依赖性的分工。例如, 在被称为 "工人" 的蜜蜂的无菌种姓中, 年轻的个体喂养巢, 而年长的人则在蜂巢1外觅食花蜜和花粉。学习和记忆能力在蜜蜂的生命中至关重要, 因为觅食必须反复往返于食物来源和巢穴之间, 然后通过舞蹈将好的食物来源的位置传达给他们的 nestmates。通信1。以前的研究表明, MB, 一个更高的大脑中心, 昆虫, 参与的学习和记忆能力的蜜蜂2,3,4。不同的表达基因和蛋白质已经在蜜蜂的各个脑区被发现5,6,7,8,9,10 ,11, 表明它们与每个脑区的独特功能有关。虽然药物的药理抑制或活化蛋白的兴趣是一个良好的方法来揭示蛋白质的功能在蜜蜂行为12,13,14, 它是未知的是否所有药物在蜜蜂大脑的不同区域有功能效应。这种药物的功能验证将加强行为药理学研究的结论。
在这里, 我们专注于 PLC, 其中一项涉及老鼠认知的酶15,16,17,18。PLC 通过将磷酸肌醇 45-bisphosphate (PIP2) 降解为肌醇14、5-trisphosphate (IP3) 和甘油 (达格·哈马舍尔德)19、20、21, 从而触发钙信号。ip3在内质网 (er) 上打开 ip3受体, 导致钙离子从 ER 中释放出来。释放钙激活钙/钙调素依赖性蛋白激酶 II (CaMKII) 与钙调素和蛋白激酶 C (PKC) 在存在的达格·哈马舍尔德。两种蛋白激酶都参与学习和记忆22,23, 符合 PLC 参与这个过程。PLCs 分为子类型, 包括 PLCβ、PLCγ和 PLCε, 其结构为20。每个 PLC 子类型在不同的上下文20中激活, 编码这些亚型的基因在不同的组织中表现出差异。我们以前证明, 蜜蜂 MBs 表达基因编码 PLCβ和 PLCε亚型在较高的水平比其余的大脑区域24, 和两个 pan-PLC 抑制剂 (edelfosine 和新霉素硫酸 [新霉素]) 降低 plc 活动不同的大脑区域, 事实上, 影响蜜蜂的学习和记忆能力24。
传统上, PLC 的酶活性已被测量使用核素 PIP225, 这需要适当的培训, 设备和设施。目前, 已建立了 plc 的合成荧光基板26, 便于对标准实验室的 plc 活动进行评估。在这里, 我们提出了一个详细的协议, 以检测在不同的大脑区域的蜜蜂使用荧光基质, 并随后测试 edelfosine 和新霉素对 plc 在这些组织的抑制效应。因为该协议只需要基本的操作, 它可能适用于对不同的社会任务分配给蜜蜂的其他组织或脑区的 PLC 活动的研究。

<table border="0" cellpadding="0" cellspacing="0" style="width:479px;" width="478">
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:113px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:101px;">ThermoFisher Scientific</td>
			<td style="width:139px;">23227</td>
			<td style="width:125px;">The reagent kit for measurement of protein concentration</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:113px;">Pierce Bovine Serum Albumin Standard Ampules 2mg/mL</td>
			<td style="width:101px;">ThermoFisher Scientific</td>
			<td>23209</td>
			<td style="width:125px;">The standard samples used in BCA assay</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Paraffin wax</td>
			<td style="width:101px;">GC</td>
			<td>13B1X00155000141</td>
			<td style="width:125px;">Dental wax used as dissection stage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Insect pin</td>
			<td style="width:101px;">Shiga</td>
			<td>No. 0</td>
			<td style="width:125px;">Stainless, solid head</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">PLCglow</td>
			<td style="width:101px;">KXT Bio</td>
			<td>KCH-0001</td>
			<td style="width:125px;">A fluorogenic substrate of PLC</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:113px;">384-well microplate</td>
			<td style="width:101px;">Corning</td>
			<td>4511</td>
			<td style="width:125px;">Low-volume, round-bottom plate in black color</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:113px;">Gemini EM microplate reader</td>
			<td style="width:101px;">Molecular Devices</td>
			<td></td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:113px;">Edelfosine</td>
			<td style="width:101px;">Santa Cruz Biotechnology</td>
			<td>sc-201021</td>
			<td style="width:125px;">pan-PLC inhibitor</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:113px;">Neomycin sulfate</td>
			<td style="width:101px;">Santa Cruz Biotechnology</td>
			<td>sc-3573</td>
			<td style="width:125px;">pan-PLC inhibitor</td>
		</tr>
	</tbody>
</table>

detection of phospholipase c activity in the brain homogenate from the honeybee

蜜蜂是一种评估复杂行为和更高的大脑功能的模型有机体, 如学习、记忆和分工。蘑菇体 (MB) 是一个更高的大脑中心, 被建议成为复杂蜜蜂行为的神经基质。尽管以前的研究发现了在 MBs 和其他脑区表达差异的基因和蛋白质, 但每个区域的蛋白质的活动尚未完全理解。为了揭示这些蛋白质在大脑中的作用, 药理学分析是一种可行的方法, 但首先必须确认药理操作确实改变了这些脑区的蛋白质活动。
我们以前发现在 MBs 中, 编码磷脂酶 C (PLC) 的基因的表达比其他脑区更高, 药理评估了 PLC 参与蜜蜂行为的情况。在这项研究中, 我们生化测试了两个药理剂, 并证实他们降低了在 MBs 和其他大脑区域的 PLC 活动。在此, 我们详细介绍了如何检测蜜蜂脑匀浆中的 PLC 活性。在该检测系统中, 由不同脑区产生的组织匀浆与合成荧光基板反应, 并对由 PLC 活动引起的荧光进行量化, 并对脑区进行比较。我们还描述了我们对某些药物对 PLC 活动的抑制作用的评估, 使用相同的系统。虽然该系统可能受到其他内源荧光化合物和/或测定成分和组织的吸收, 使用该系统的 PLC 活动的测量比使用传统的检测更安全和容易, 这需要核素基板。通过简单的程序和操作, 我们可以检查在不同社会任务中参与的蜜蜂大脑和其他组织中的 PLC 活动。

脑组织匀浆中的蛋白质浓度: 我们准备用只觅食蚂蚁蜜蜂组织匀浆。在原组织匀浆中计算出的蛋白质浓度如图 3所示。原始匀浆中的近似蛋白质浓度如下: 1.5 毫克/毫升在 MBs 和2.3 毫克/毫升在其他脑区。我们用了两只蜜蜂, 并分析了六多。
脑组织匀浆中 PLC 活动的检测: 在实验?...

Watch this Scientific Journal Video about Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee at JoVE.com

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

蜜蜂匀浆中磷脂酶 C 活性的检测

为了检验药理学药物对蜜蜂脑不同区域磷脂酶 C (plc) 的抑制作用, 我们提出了一种生化检测方法来测量这些地区的 plc 活性。该方法可用于比较组织中的 PLC 活动, 以及不同行为的蜜蜂之间的对比。

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

这种方法可以帮助回答神经科学领域的关键问题，例如PLC在不同大脑区域的活动是否和如何参与蜜蜂的大脑功能。这项技术的主要优点是，研究人员可以很容易地用标准实验室设施进行实验，因为该协议只需要基本的操作。首先，在冰上放置一个50毫升的管子，里面装着蜜蜂30分钟，对蜜蜂进行麻醉。接下来，准备解剖阶段，将一块牙蜡折叠成两半，放在塑料盘中。在双目显微镜下，用钳子将蜜蜂的头与身体分开。然后，将头部固定在牙蜡上，并在每个天线底部插入昆虫针，使头部内部处于向上位置。接下来，将足够的盐水溶液倒入头部以盖住它。使用钳子卸下天线。在此之后，使用手术刀在天线底座附近进行水平切割，并在复合眼的边界进行纵向切割。在头顶上做一个水平切口，取出角质层，打开大脑的窗户。接下来，使用钳子从骨囊和气管中去除大脑内表面的视网膜眼。然后，将钳子直接插入眼皮下，以去除复合眼皮和视网膜眼之间的结缔组织。之后，去除复合眼的角质层和视网膜眼。接下来，使用钳子小心地从大脑内部表面取出剩余的气管。然后，在大脑之间和组织周围切割结缔组织，从头部胶囊解剖大脑。使用钳子，从大脑后表面剥下气管。然后使用手术刀切断蘑菇体或MB和垂直叶旁边的其他大脑区域之间的连接。将解剖的MB和任何剩余的脑组织放入1.5ml管中，用干冰快速冷冻。首先，在冷冻脑组织中加入10ml均质缓冲液。使用塑料 peselle 手动使 1.5ml 管中的组织均质化。在此之后，将均质组织溶液转移到地段的下一个组织，并重复均质化过程。然后，添加 30

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

从真核细胞蛋白质复合体串联亲和纯化

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

个性化肽阵列在器官移植 HLA 抗体检测中的研究

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

用氢-氘交换质谱法定义 Hsp33's 氧化还原调节陪护活动和 Hsp33 映射构象变化

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

格拉菲克斯用于检测 糖精 的瞬态相互作用器

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...