Neuroprotocols最新文献

英文中文

Adrenergic Receptor Autoradiography and in Situ Hybridization 肾上腺素能受体放射自显影和原位杂交

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/NCMN.1994.1009

J. Saffitz, S. Beau

Abstract The distribution of α- and β-adrenergic receptors in slices of anatomically complex tissues can be delineated autoradiographically with great precision and resolution. This brief review highlights methodological aspects of adrenergic receptor autoradiography and in situ hybridization at the light microscopic level of resolution. It focuses on technical differences between autoradiographic analysis in tissue sections and conventional radioligand binding assays in membranes prepared from tissue homogenates. it emphasizes strategies for characterizing quantitatively the distribution of specific receptor subtypes and classes using autoradiography and ways of detecting naturally occurring low-abundance adrenergic receptor mRNAs using in situ hybridization.

α-和β-肾上腺素能受体在解剖结构复杂的组织切片中的分布可以用放射自显影技术精确地描绘。这篇简短的综述强调了肾上腺素能受体放射自显影和原位杂交在光显微镜分辨率水平的方法学方面。它侧重于组织切片的放射自显影分析与组织匀浆制备的膜中的常规放射配体结合测定之间的技术差异。它强调了使用放射自显影技术定量表征特定受体亚型和类别分布的策略，以及使用原位杂交技术检测自然发生的低丰度肾上腺素能受体mrna的方法。

引用次数: 2

Development of Novel Irreversible Ligands 新型不可逆配体的发展

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/NCMN.1994.1008

S. Baker, Malgorzata D. Deyrup

Abstract Alkylating affinity labels are reactive compounds that bind receptors in a covalent manner. This property makes them useful tools with which to study the structure and function of these important biological recognition molecules. To familiarize investigators with the use of these compounds, an overview of their general properties in terms of design, pharmacology, and chemistry is presented. In addition, some experimental approaches to characterizing their irreversible effects are described. Finally, several uses of alkylating affinity labels for the determination of a receptor reserve, receptor turnover, and studies on receptor structure are briefly outlined. important limitations with the use of these compounds are discussed, and examples focusing mainly on catecholamine receptors are given.

烷基化亲和标签是以共价方式结合受体的活性化合物。这种特性使它们成为研究这些重要生物识别分子的结构和功能的有用工具。为了使研究人员熟悉这些化合物的使用，本文概述了它们在设计、药理学和化学方面的一般特性。此外，还描述了表征其不可逆效应的一些实验方法。最后，简要概述了烷基化亲和标记在确定受体储备、受体周转和受体结构研究方面的几种用途。讨论了使用这些化合物的重要限制，并给出了主要集中在儿茶酚胺受体上的例子。

引用次数: 9

Development of Novel Irreversible Ligands 新型不可逆配体的研究进展

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/ncmn.1994.1008

Baker Stephen P., Deyrup Malgorzata D.

Alkylating affinity labels are reactive compounds that bind receptors in a covalent manner. This property makes them useful tools with which to study the structure and function of these important biological recognition molecules. To familiarize investigators with the use of these compounds, an overview of their general properties in terms of design, pharmacology, and chemistry is presented. In addition, some experimental approaches to characterizing their irreversible effects are described. Finally, several uses of alkylating affinity labels for the determination of a receptor reserve, receptor turnover, and studies on receptor structure are briefly outlined. important limitations with the use of these compounds are discussed, and examples focusing mainly on catecholamine receptors are given.

烷基化亲和标记物是以共价方式结合受体的反应性化合物。这种特性使它们成为研究这些重要生物识别分子结构和功能的有用工具。为了使研究人员熟悉这些化合物的用途，概述了它们在设计、药理学和化学方面的一般性质。此外，还介绍了一些表征其不可逆效应的实验方法。最后，简要概述了烷基化亲和标记在测定受体储备、受体周转和受体结构研究中的几种用途。讨论了这些化合物使用的重要限制，并给出了主要关注儿茶酚胺受体的例子。

引用次数: 9

Adrenergic Receptors: Data and Programs on the Internet 肾上腺素能受体:互联网上的数据和程序

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/NCMN.1994.1006

R. J. Hughes

Abstract The adrenergic receptors belong to a gene superfamily whose members share a great deal of sequence homology. To date, over 60 members of this superfamily have been cloned and sequenced. Ready access to this wealth of sequence information, together with the software tools to analyze it, can facilitate experimental design and interpretation as well as expedite the realization of experimental goals. Many investigators have one or more computers connected to the Internet, often for no purpose other than to exchange electronic mail (e-mail) or to utilize the file-serving capabilities of a Novell network. These investigators are, to a large extent, unaware of the extensive resources that the Internet offers. The aim of this article is to facilitate utilization of the Internet, particularly as a tool to aid in the study of adrenergic receptors, and to guide the readers exploration of this resource. Although the Internet may at first appear to be an unfathomable morass of information, the simple command "help" can often be used to guide one′s path. Wherever possible, the reader is directed to retrieve the latest documentation on each topic directly from the Internet.

肾上腺素能受体属于一个基因超家族，其成员具有大量的序列同源性。迄今为止，这个超家族的60多个成员已经被克隆和测序。随时访问这些丰富的序列信息，以及分析这些信息的软件工具，可以促进实验设计和解释，并加快实验目标的实现。许多调查人员有一台或多台计算机连接到Internet，通常除了交换电子邮件(e-mail)或利用Novell网络的文件服务功能外，没有其他目的。这些调查人员在很大程度上没有意识到互联网提供的广泛资源。这篇文章的目的是促进互联网的利用，特别是作为一种工具来帮助研究肾上腺素能受体，并指导读者探索这一资源。虽然互联网一开始似乎是一个深不可测的信息沼泽，但简单的“帮助”命令往往可以用来引导一个人的路径。在可能的情况下，引导读者直接从Internet检索有关每个主题的最新文档。

引用次数: 0

Adrenergic Receptor Subtypes: Pharmacological Approaches 肾上腺素能受体亚型：药理学方法

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/ncmn.1994.1002

Esbenshade Timothy A., Minneman Kenneth P.

Although the existence of four distinct adrenergic receptor subtypes (α₁, α₂, β₁, β2) has been recognized for more than 15 years, it has recently become clear that the adrenergic receptor family is much larger than previously suspected. Development of more selective agonists and antagonists and careful comparison of pharmacological properties have led to the realization that there are nine or more adrenergic receptor subtypes. Molecular cloning of many of these subtypes, discussed in an accompanying article, supports this conclusion. The adrenergic receptors fall into three major families (α₁, α₂, β) based on pharmacology, structure, and signal transduction, with at least three closely related members within each family. Here, we summarize the known pharmacological differences between these receptors and evaluate the best methods currently available for distinguishing these subtypes using selective drugs.

尽管四种不同的肾上腺素能受体亚型（α1、α2、β1、β2）的存在已经被认识了超过15年，但最近很明显，肾上腺素能受体家族比以前怀疑的要大得多。更具选择性的激动剂和拮抗剂的开发以及药理学性质的仔细比较已经使人们认识到有九种或九种以上的肾上腺素能受体亚型。在随附的文章中讨论了许多这些亚型的分子克隆，支持了这一结论。肾上腺素受体根据药理学、结构和信号转导分为三大家族（α1、α2、β），每个家族中至少有三个密切相关的成员。在此，我们总结了这些受体之间已知的药理学差异，并评估了目前使用选择性药物区分这些亚型的最佳方法。

引用次数: 4

Classification of Adrenergic Receptor Subtypes: Molecular Biologic Approaches 肾上腺素能受体亚型的分类:分子生物学方法

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/NCMN.1994.1003

K. Lynch, J. Harrison, W. Pearson

Abstract Adrenergic receptors have been studied extensively for more than 30 years, first by physiological means, later with pharmacologic and biochemical approaches, and within the past several years by molecular biology. This extensive body of work provided the basis for subdividing the adrenergic receptors into β-, α 1 -, and α 2 -adrenergic receptor types and, subsequently, into β 1 -, β 2 -, α 1A -, α 1B -, α 2A -, and α 2B -adrenergic receptor subtypes. Although the pharmacologic approach indicated that there exist multiple subtypes of each type of adrenergic receptor, it was the molecular cloning of adrenergic receptor cDNAs/genes that demonstrated the existence of three genes encoding each adrenergic receptor type in humans and rats (and therefore probably in all mammals). The nine adrenergic receptor proteins expressed in cultured cells faithfully mimic the basic pharmacologic and biochemical properties ascribed to these receptors. In this article, we review the molecular cloning and characterization of the adrenergic receptors with special emphasis on the α 2 -adrenergic receptors and we discuss a classification scheme based on the hypothetical molecular evolution of the adrenergic receptors.

肾上腺素能受体的研究已经有30多年的历史，最初是通过生理学手段，后来通过药理学和生化方法，最近几年又通过分子生物学进行了广泛的研究。这项广泛的工作为肾上腺素能受体细分为β-、α 1 -和α 2 -肾上腺素能受体类型以及随后的β 1 -、β 2 -、α 1A -、α 1B -、α 2A -和α 2B -肾上腺素能受体亚型提供了基础。尽管药理学方法表明，每种肾上腺素能受体都存在多种亚型，但通过对肾上腺素能受体cdna /基因的分子克隆，证明了在人类和大鼠(因此可能在所有哺乳动物中)存在三种编码每种肾上腺素能受体的基因。在培养细胞中表达的9种肾上腺素能受体蛋白忠实地模拟了这些受体的基本药理学和生化特性。本文综述了近年来肾上腺素能受体的分子克隆和鉴定，重点介绍了α 2 -肾上腺素能受体，并根据肾上腺素能受体的分子进化假说讨论了肾上腺素能受体的分类方案。

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引用次数: 3

Molecular Biology of 2-Adrenergic Receptors 2-肾上腺素能受体的分子生物学

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/NCMN.1994.1005

A. Strosberg

Abstract Affinity purification of the β2-adrenergic receptor from hamster lung has led to the amino acid sequencing of a few of its peptides, followed by the molecular cloning of the corresponding cDNA. Expression of this cDNA confirmed the catecholamine binding properties of the β2-adrenergic receptor. This initial success, that of the cloning of the turkey erythrocyte β1-like and of the platelet α2A-adrenergic receptors, has rapidly led to the identification and cloning by homology of nine different subtypes of α1, α2, and β receptors. All these belong to the very large family of G-protein-coupled membrane receptors, which may include over 1000 proteins that act as receptors for neurotransmitters, hormones, and sensory signals such as light or odors. While a few of the adrenergic receptors had been characterized previously by pharmacologic means, most were actually not known to exist as individual entitles. The "reverse pharmacology" made possible by molecular biology should now lead to the synthesis of new subtype-selective ligands.

摘要对仓鼠肺β2-肾上腺素能受体进行亲和纯化，对其部分肽进行了氨基酸测序，并进行了相应cDNA的分子克隆。该cDNA的表达证实了β2-肾上腺素能受体的儿茶酚胺结合特性。这一初步成功克隆了火鸡红细胞β1样受体和血小板α 2a -肾上腺素能受体，并通过同源性鉴定和克隆了9种不同亚型的α1、α2和β受体。所有这些都属于非常大的g蛋白偶联膜受体家族，其中可能包括超过1000种蛋白质，这些蛋白质充当神经递质，激素和感觉信号(如光或气味)的受体。虽然少数肾上腺素能受体以前已经通过药理学手段进行了表征，但大多数受体实际上并不知道作为个体存在。分子生物学使“反向药理学”成为可能，现在应该导致新的亚型选择性配体的合成。

引用次数: 1

Site-Directed Mutagenesis of a-Adrenergic Receptors a-肾上腺素能受体的定点诱变

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/NCMN.1994.1004

N. H. Lee

Abstract The application of in vitro site-directed mutagenesis has led to the identification of conserved amino acids that play important roles in receptor structure and function. Precise amino acid substitutions can be obtained and then correlated with changes in receptor phenotype. Here, we describe several techniques commonly employed to Introduce site-specific mutations. The benefits and potential drawbacks of each method are discussed. Site-directed mutagenesis of the human α 2A -adrenergic receptor (α 2A AR) has been successfully employed to identify conserved amino acids involved in agonist binding and receptor activation. Aspartate residues in the second and intracellular side of the third transmembrane domain of the α 2A AR are implicated in receptor/G-protein interactions. Since these aspartate residues are highly conserved among all G-protein-coupled receptors, and elimination of these residues has been shown to abolish the ability of other receptors in this class to activate their respective intracellular signaling pathways, It seems likely that these residues are critical for agonist-induced conformational changes that underlie receptor/G-protein interactions. In contrast to the role played by the conserved residues mentioned above, a conserved aspartate residue situated near the extracellular side of the third transmembrane domain plays a pivotal role in adrenergic ligand binding. Genetic analysis of the fifth transmembrane domain of the α 2A AR suggests that a conserved serine residue in this region participates in hydrogen binding to the meta -hydroxyl group of catecholamines. These findings point to the utility of site-directed mutagenesis in identifying structure-function relationships among G-protein-coupled receptors.

体外定点诱变的应用已经鉴定出在受体结构和功能中起重要作用的保守氨基酸。可以获得精确的氨基酸取代，然后与受体表型的变化相关。在这里，我们描述了几种通常用于引入位点特异性突变的技术。讨论了每种方法的优点和潜在的缺点。人α 2A -肾上腺素能受体(α 2A AR)的定点诱变已成功用于鉴定参与激动剂结合和受体激活的保守氨基酸。α 2A AR第三跨膜结构域的第二侧和胞内侧的天冬氨酸残基与受体/ g蛋白相互作用有关。由于这些天冬氨酸残基在所有g蛋白偶联受体中是高度保守的，并且这些残基的消除已被证明会破坏这类其他受体激活其各自细胞内信号通路的能力，因此这些残基似乎对受体/ g蛋白相互作用下激动剂诱导的构象变化至关重要。与上述保守残基所起的作用相反，位于第三跨膜结构域细胞外侧附近的保守天冬氨酸残基在肾上腺素能配体结合中起关键作用。α 2A AR第5跨膜结构域的遗传分析表明，该区域有一个保守的丝氨酸残基参与儿茶酚胺间羟基的氢结合。这些发现指出了位点定向诱变在鉴定g蛋白偶联受体之间结构-功能关系方面的效用。

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引用次数: 7

Molecular Biology of β2-Adrenergic Receptors β2-肾上腺素能受体的分子生物学

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/ncmn.1994.1005

Strosberg A.Donny

Affinity purification of the β₂-adrenergic receptor from hamster lung has led to the amino acid sequencing of a few of its peptides, followed by the molecular cloning of the corresponding cDNA. Expression of this cDNA confirmed the catecholamine binding properties of the β₂-adrenergic receptor. This initial success, that of the cloning of the turkey erythrocyte β₁-like and of the platelet α_2A-adrenergic receptors, has rapidly led to the identification and cloning by homology of nine different subtypes of α₁, α₂, and β receptors. All these belong to the very large family of G-protein-coupled membrane receptors, which may include over 1000 proteins that act as receptors for neurotransmitters, hormones, and sensory signals such as light or odors. While a few of the adrenergic receptors had been characterized previously by pharmacologic means, most were actually not known to exist as individual entitles. The "reverse pharmacology" made possible by molecular biology should now lead to the synthesis of new subtype-selective ligands.

从仓鼠肺中亲和纯化β2-肾上腺素能受体，对其一些肽进行了氨基酸测序，然后克隆了相应的cDNA。该cDNA的表达证实了β2-肾上腺素能受体的儿茶酚胺结合特性。土耳其红细胞β1样受体和血小板α2A肾上腺素能受体的克隆取得了初步成功，并通过同源性快速鉴定和克隆了9种不同亚型的α1、α2和β受体。所有这些都属于G蛋白偶联膜受体的大家族，可能包括1000多种蛋白质，它们作为神经递质、激素和感觉信号（如光或气味）的受体。虽然一些肾上腺素能受体以前已经通过药理学手段进行了表征，但大多数受体实际上并不以个体身份存在。分子生物学使“反向药理学”成为可能，现在应该导致新的亚型选择性配体的合成。

引用次数: 1

Quantitative Analysis of Low-Abundance mRNA: Application to Adrenergic Receptors 低丰度mRNA的定量分析:在肾上腺素能受体中的应用

Neuroprotocols

Pub Date : 1994-02-01 DOI: 10.1006/NCMN.1994.1010

R. J. Hughes

Abstract The adrenergic receptors belong to a family of receptors that is postulated to span the plasma membrane seven times and is linked to regulatory GTP-binding proteins. These receptors mediate a wide variety of physiological responses through activation of several distinct second-messenger systems. Belying the importance of the adrenergic receptors in regulating physiological responses in target cells is their paucity. These receptors are present in very low numbers, in some cases only a thousand copies per cell. In addition, the adrenergic receptors are relatively stable proteins. Thus, cells have no need to synthesize large amounts of these proteins and, in consequence, the level of mRNA coding for these receptors is very low. In this paper, I examine some of the ways in which quantitation of these scarce mRNA species has been approached, with particular emphasis on the use of polymerase chain reaction.

肾上腺素能受体属于一个受体家族，被认为跨越质膜七次，并与调节gtp结合蛋白相连。这些受体通过激活几个不同的第二信使系统介导各种各样的生理反应。掩盖肾上腺素能受体在调节靶细胞生理反应中的重要性的是它们的缺乏。这些受体的数量非常少，在某些情况下每个细胞只有1000个拷贝。此外，肾上腺素能受体是相对稳定的蛋白质。因此，细胞不需要合成大量的这些蛋白质，因此，编码这些受体的mRNA水平非常低。在本文中，我研究了一些方法，其中定量这些稀缺的mRNA物种已经接近，特别强调使用聚合酶链反应。

引用次数: 0

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