| A Method to Quantify Drosophila Behavioral Activities Induced by GABAA Agonist Muscimol
|
|
Author:
Date:
2021-04-20
[Abstract] Muscimol is a psychoactive isoxazole derived from the mushroom Amanita muscaria. As a potent GABAA receptor agonist, muscimol suppresses the activity of the central nervous system, reduces anxiety and induces sleep. We investigated the effects of muscimol on Drosophila behavior. Drosophila behavioral assays are powerful tools that are used to assess neural functions by focusing on specific changes in selected behavior, with the hypothesis that this behavioral change is due to alteration of the underlying neural function of interest. In this study, we developed a comparatively simple and cost-effective method for feeding adult flies muscimol, a pharmacologically active compound, and for quantifying the phenotypes of “resting” and “grooming+walking”. This protocol ...
[摘要] [摘要] Muscimol是一种精神活性的异恶唑，衍生自蘑菇鹅膏菌（Amanita muscaria）。作为有效的GABA A受体激动剂，muscimol抑制中枢神经系统的活动，减轻焦虑并诱导睡眠。我们调查了麝香酚对果蝇行为的影响。果蝇行为分析小号是强大的工具，小号是用来评估通过着眼于选择行为的具体变化，与假设，这种行为的改变是由于利益的根本神经功能改变神经功能。在这项研究中， 我们开发了一种相对简单且经济高效的方法来喂养成年蝇麝香酚（一种药理活性化合物），并定量“休息”和“修饰+行走”的表型。该协议可以为研究人员提供一种便捷的方法来表征苍蝇中小分子诱导的行为输出。
[背景]越来越多的证据小分子可用于神经元病症的治疗（Rissman和莫布里，2011） 。因此，知识的小分子诱导行为的神经生物学人的变化和阐发IR底层机制将促进我们的理解的神经回路的功能机制。但是，大多数以前的行为研究都以小鼠或大鼠为模型，完成此类研究需要大量资源。果蝇已被用作研究许多遗传疾病的易处理的遗传模型（Mackay和Anholt，2006），近年来，它们被用作行为研究的模型变得越来越普遍（Zhukovskaya等，2013； Chen等。，2014； Hampel等人，2015； Kaur等人，2015； Pitmon等人，2016； Hidalgo等人，2017）。 ...
|
|
|
| Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry
|
|
Author:
Date:
2016-11-20
[Abstract] Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal ...
[摘要] 专业吞噬细胞通过吞噬作用内化自身和非自身颗粒以启动先天免疫应答。内化后，形成的吞噬体通过与内体和溶酶体的融合和裂变事件成熟，以获得更酸性，氧化和水解的环境用于其货物的降解。有趣的是，吞噬体成熟动力学在细胞类型和细胞活化状态之间不同。该协议允许使用流式细胞术定量不同类型的吞噬细胞中单个细胞器水平上的吞噬体成熟动力学。在这里，卵白蛋白（OVA）耦合的颗粒用作吞噬作用模型系统在树突状细胞（DC），其通过吞噬内化。在不同的时间点之后，吞噬体成熟参数，例如OVA的吞噬体降解和溶酶体蛋白（例如LAMP-1）的获得，可以通过流式细胞器细胞计数以高度定量的方式同时测量。这些读出可以与其他吞噬体功能相关，例如抗原降解，在DC中的加工和负载。
[背景] ...
|
|
|
| Purification of a Protein Exhibiting Isoleucine 2-epimerase Activity from Lactobacillus otakiensis JCM 15040
|
|
Author:
Date:
2015-10-20
[Abstract] Prominent accumulation of D-leucine, D-allo-isoleucine and D-valine was observed in the culture medium of the heterofermentative bacterial species, Lactobacillus otakiensis (L. otakiensis) JCM 15040. The racemase enzyme that resulted in this accumulation, isoleucine 2-epimerase, was purified from the bacterial cells. This is the first reported observation of such production of D-branched chain amino acids in lactic acid bacteria, and the first example of a racemase with isoleucine 2-epimerase activity in any organisms. In the described protocol, we introduce methods for purification of this protein from L. otakiensis JCM 15040. Because no specific ligand that has high affinity for this enzyme has been identified, the purification was performed using ...
[摘要] 在异质发酵细菌物种，例如otakiensis的乳酸杆菌（Lactobacillus otakiensis）的培养基中观察到D-亮氨酸，D-异亮氨酸 - 异亮氨酸和D-缬氨酸的显着积累。 otakiensis）JCM 15040.从细菌细胞中纯化导致这种积累的消旋酶，异亮氨酸2-差向异构酶。这是首次报道的在乳酸菌中这种D-支链氨基酸的产生的观察结果，以及在任何生物体中具有异亮氨酸2-差向异构酶活性的消旋酶的第一个实例。在所述的方案中，我们介绍从L中纯化该蛋白质的方法。因为没有鉴定到对该酶具有高亲和力的特异性配体，所以使用硫酸铵级分，四种类型的柱层析和制备型Native-PAGE，不使用亲和柱层析进行纯化。我们希望协议将提供有用的信息用于纯化不能容易地使用亲和柱层析纯化的酶。
|
|
|