细胞功能测定

Antisense therapeutics can be applied to hypertension. Hypertension is a widespread disease, yet in spite of several excellent drugs for the treatment of hypertension, the number of patients with controlled hypertension is about 27% in the United States. In all other countries studied, ev ...

In 1977, percutaneous transluminal coronary angioplasty (PTCA) was first introduced (1), and it has become an effective treatment for limited coronary artery disease (2). PTCA treatment has since become more extensive and gained favor as an alternative treatment for coronary artery by ...

Over a span of more than two decades, antisense strategies for gene therapy have expanded from antisense oligonucleotides (AS-ODNs) solely, to the addition of ribozymes and, more recently, to the inclusion of small interfering RNAs (siRNAs). Antisense therapeutics has also experienced ...

During the past decade, only one antisense-based therapy has received full Food and Drug Administration (FDA) approval. Vitravene™, developed by Isis Pharmaceuticals, was the first drug based on antisense technology to be successfully commercialized and used in treatment (1). The the ...

In this chapter we provide an introduction to statistical methods appropriate in G-protein-coupled receptor research, including examples. Topics covered include the choice of appropriate averages and measures of dispersion to summarize data sets, and the choice of tests of signif ...

Accumulating examples have demonstrated that knock-out and knock-in mice of G-protein-coupled receptors (GPCRs) are useful in elucidating physiological functions of the receptor in vivo. GPCR knock-out and knock-in are achieved by either (1) manipulation of the endogenous locus of ...

Numerous recent studies have suggested that the predicted cytosolic domains of G-protein-coupled receptors (GPCRs) represent a surface for association with proteins that may serve multiple roles in receptor localization, turnover, and signaling beyond the well-characteri ...

Many protein interactions with G-protein-coupled receptors (GPCRs) appear to influence receptor signaling and functional regulation. There is great interest therefore in methods for the identification of novel or unanticipated GPCR binding proteins. A proven method for iden ...

The versatility, sensitivity, and feasibility of fluorescence methods are very attractive to study protein-protein interaction at low levels of protein expression. However, one of the most severe limits in protein chemistry has been the difficulty of introducing site-specific fl ...

Complex networks of protein-protein interactions are key determinants of cellular function, including those regulated by G-protein-coupled receptors (GPCRs). Formation of either stable or transitory complexes are involved in regulating all aspects of receptor function, f ...

This chapter describes three different strategies of receptor mutagenesis with their advantages, disadvantages, and limitations. Oligonucleotide-directed mutagenesis using either the Altered Sites� II in vitro mutagenesis system or the GeneTailor� site-directed mut ...

In this chapter we describe methods for detecting the ubiquitination state of G-protein-coupled receptors (GPCRs). This involves coexpression of a GPCR with an epitope-tagged ubiquitin construct in a heterologous expression system. Modification by ubiquitin of the GPCR resulti ...

Reversible phosphorylation is important for G-protein-coupled receptor (GPCR) signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. This chapter describes a powerful analytical method ...

It is now clear that nearly all G-protein-coupled receptors (GPCRs) are phosphorylated and palmitolyated. The process of receptor phosphorylation has been extensively studied because it offers a regulatory mechanism that is both rapid and dynamic. However, it has recently become cle ...

Internalization of G-protein-coupled receptors (GPCRs) occurs in response to agonist activation of the receptors and causes a redistribution of receptors away from the plasma membrane toward endosomes. Internalization of lower-affinity small molecule GPCRs such as muscari ...

Fusion constructs between G-protein-coupled receptors and G-protein α-subunits have been used to examine a variety of aspects of the functioning of these signaling systems. Here we describe some of the various techniques and methods used in detail. The process of fusing the two components, ...

The demonstration that many intracellular signaling processes are mediated by a family of closely related guanine nucleotide binding proteins (G-proteins) has led to the development of specific techniques that can be used to identify which of these polypeptide(s) is involved on recep ...

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through promotion of guanine nucleotide exchange on the Gα-subunit of heterotrimeric G-proteins to release free Gα-GTP and βγ-subunits. In membrane preparations, GTP can be substituted by 35S ...

The activation of heterotrimeric G-proteins is tightly regulated by the exchange of GTP for GDP in the α-subunit; mostly—but not exclusively—seven-transmembrane receptors function as the guanine nucleotide exchange factors (GEFs). A research goal may be to determine which G-prote ...

Although methods for expressing foreign proteins in clonal cell lines are well established, mature neurons remain a difficult preparation for the introduction of foreign genes. Microinjection is a reliable method for producing robust targeted expression in neurons that has advan ...