Abstract:
:There is currently no known genetic disease linked to prolactin (PRL) or its receptor (PRLR) in humans. Recently, we identified three missense variants of the PRLR in patients presenting with breast tumors. Two of them (named PRLR(I146L) and PRLR(I76V)) had been reported earlier, but failed to draw much attention because the eventual impact of these substitutions on receptor properties remained unknown. In this chapter, we describe the various bioassays (cell types and readouts) that led to the discovery that both variants exhibit gain-of-function properties. Reconstituted cell models involving Ba/F3, HEK293, and MCF-7 cell lines all highlighted the constitutive, PRL-independent potency of PRLR(I146L) to trigger downstream signaling, leading to antiapoptotic and proliferation properties. The lower level of basal activity of PRLR(I76V) could be demonstrated only in the very sensitive Ba/F3 cell assay. While comparative analysis of ligands is a routine procedure in many labs, comparison of receptor variants de facto imposes the use of different cell clones (or population) in which each receptor variant is expressed individually. This is more delicate, as one must ensure that differences in biological responses really reflect differences in the intrinsic properties of receptor variants, and not any feature of cell clones/populations that are used, which could bias the interpretation.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Goffin V,Bogorad RL,Touraine Pdoi
10.1016/B978-0-12-381298-8.00017-4subject
Has Abstractpub_date
2010-01-01 00:00:00pages
329-55eissn
0076-6879issn
1557-7988pii
B978-0-12-381298-8.00017-4journal_volume
484pub_type
杂志文章abstract::Recent work demonstrates that RNA interference (RNAi) can coordinate protein expression. Inhibitory RNAs are expressed naturally in cells as microRNAs (miRNAs) or introduced into cells as small interfering RNAs (siRNAs). Both types of small RNAs can be used at the bench to silence mRNA expression. For many researchers...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(04)92009-5
更新日期:2005-01-01 00:00:00
abstract::Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology. Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and gene...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-801122-5.00019-2
更新日期:2014-01-01 00:00:00
abstract::Osmoregulation is the active control of the cellular water balance and encompasses homeostatic mechanisms crucial for life. The osmoregulatory system in the yeast Saccharomyces cerevisiae is particularly well understood. Key to yeast osmoregulation is the production and accumulation of the compatible solute glycerol, ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)28002-4
更新日期:2007-01-01 00:00:00
abstract::Unfolded protein response (UPR) is a major reaction to intrinsic stress of eukaryotic organisms and is also related to environmental stress reactions. Among yeasts, stress regulation has mainly been investigated in Saccharomyces cerevisiae, while other species with biotechnological or medical interest are less well un...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385116-1.00010-8
更新日期:2011-01-01 00:00:00
abstract::The nitrogen (N) cycle is the only global biogeochemical cycle that is driven by biological functions involving the interaction of many microorganisms. The N cycle has evolved over geological time and its interaction with the oxygen cycle has had profound effects on the evolution and timing of Earth's atmosphere oxyge...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381294-0.00022-5
更新日期:2011-01-01 00:00:00
abstract::Research on circadian biology over the past decade has paid increasing attention to the photoreceptor mechanisms that align the molecular clock to the 24-h light/dark cycle, and some of the results to emerge are surprising. For example, the rods and cones within the mammalian eye are not required for entrainment. A po...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)93037-1
更新日期:2005-01-01 00:00:00
abstract::Microsatellite markers are quickly becoming the molecular marker of choice for studies of parentage and kinship in animals. In this chapter, we review methods and give protocols for screening potential microsatellite markers, as well as protocols for genotyping individuals with useful markers once they have been ident...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)95014-3
更新日期:2005-01-01 00:00:00
abstract::Genetically encodable RNA devices that directly detect small molecules in the cellular environment are of increasing interest for a variety of applications including live cell imaging and synthetic biology. Riboswitches are naturally occurring sensors of intracellular metabolites, primarily found in the bacterial mRNA...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2014.10.031
更新日期:2015-01-01 00:00:00
abstract::Circadian clocks are present in most cells and are essential for maintenance of daily rhythms in physiology, mood, and cognition. Thus, not only neurons of the central circadian pacemaker but also many other peripheral tissues possess the same functional and self-sustained circadian clocks. Surprisingly, however, thei...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2014.10.023
更新日期:2015-01-01 00:00:00
abstract::Reviewing the circumstances that have led to the first monoclonal antibody against the disease-associated form of PrP, we consider the availability of PrP knockout mice and recombinant PrP, as well as a reliable conformational screening protocol as being important prerequisites for a successful immunization approach. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(99)09010-2
更新日期:1999-01-01 00:00:00
abstract::A simple and convenient method is described to determine primary deuterium kinetic isotope effects (1°DKIEs) on reactions where the hydron incorporated into the reaction product is derived from solvent water. The 1°DKIE may be obtained by 1H NMR analyses as the ratio of the yields of H- and D-labeled products from a r...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2017.06.043
更新日期:2017-01-01 00:00:00
abstract::Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylati...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.10.015
更新日期:2017-01-01 00:00:00
abstract::Integrin adhesion receptors mediate cell-cell and cell-substratum adhesion and provide a continuous link for the bidirectional transmission of mechanical force and biochemical signals across the plasma membrane. Integrin-dependent cellular activities such as adhesion, migration, proliferation, and survival rely upon t...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)26004-5
更新日期:2007-01-01 00:00:00
abstract::It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecu...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.01.012
更新日期:2015-01-01 00:00:00
abstract::Since the early days of cell volume regulation research, the role of actin cytoskeleton organization and rearrangement has attracted specific interest. Rapid modifications in actin dynamics and architecture have been described. They were shown to regulate cell volume changes, as well as regulatory volume decrease in a...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)28012-7
更新日期:2007-01-01 00:00:00
abstract::Posttranslational modification of circadian clock proteins by phosphorylation is an essential regulatory process in the control of eukaryotic circadian clocks. In the Neurospora circadian clock, the key clock protein FREQUENCY (FRQ) is progressively phosphorylated. The phosphorylation of FRQ is regulated by both kinas...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)93017-6
更新日期:2005-01-01 00:00:00
abstract::RNA helicases are associated with every aspect of RNA metabolism and function. A diverse range of RNA helicases are encoded by essentially every organism. While RNA helicases alter gene expression, RNA helicase expression is itself regulated, frequently in response to abiotic stress. Photosynthetic cyanobacteria prese...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-396546-2.00018-8
更新日期:2012-01-01 00:00:00
abstract::Concomitant with the rapid development in biomedical knowledge, including the methods of molecular biology and proteomics, and the manufacture of ever more precise optical instruments, powerful lasers, and sophisticated microcomputing hardware and software, laser microdissection systems have emerged which are now ente...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)56935-4
更新日期:2002-01-01 00:00:00
abstract::Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For incre...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)65012-6
更新日期:2009-01-01 00:00:00
abstract::Obtaining patient-specific information through the quantification of small molecules and proteins in bodily fluids is essential for personalized therapies. Point-of-care (POC) diagnostic devices hold the promise of delivering such benefit to a wide range of patients. However, there is a lack of enabling technology, as...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2017.01.005
更新日期:2017-01-01 00:00:00
abstract::The bromodomain (BrD) represents an evolutionarily conserved protein domain whose function mostly is to recognize acetylated lysine residues in histones and nuclear proteins in regulation of gene transcription in chromatin. The highly conserved BrD structure features an unusual left-handed, antiparallel four-helix bun...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.01.018
更新日期:2016-01-01 00:00:00
abstract::The Omega class of cytosolic glutathione transferases was initially recognized by bioinformatic analysis of human sequence databases, and orthologous sequences were subsequently discovered in mouse, rat, pig, Caenorhabditis elegans, Schistosoma mansoni, and Drosophila melanogaster. In humans and mice, two GSTO genes h...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)01005-0
更新日期:2005-01-01 00:00:00
abstract::Anomalous small angle X-ray scattering (ASAXS) exploits contrast variation methods to highlight the scattering from one elemental component in a multielement sample, such as one ion species in an ion-DNA system. The ASAXS method has been applied to measure ions condensed around short nucleic acid duplexes. This chapte...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)69019-4
更新日期:2009-01-01 00:00:00
abstract::Linkers play essential roles in the engineering of fusion proteins, and have been extensively demonstrated to affect protein properties such as expression level, solubility, and biological functions. For linker design and optimization, one of the key factors is the flexibility or rigidity of linkers, which describes t...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.12.004
更新日期:2021-01-01 00:00:00
abstract::Two-component signal transduction systems, composed of histidine kinases and response regulators, enable bacteria to sense, respond, and adapt to changes in their internal and external conditions. The importance of these signaling systems is reflected in their widespread distribution and prevalence in the bacterial ki...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)23026-5
更新日期:2007-01-01 00:00:00
abstract::In this chapter we have discussed the methodology used to identify and characterize three intermediates in protein transport from the ER that represent stages of transport vesicle budding, targeting, and fusion. The intermediates are obtained using a variety of transport inhibitors: low-temperature incubations, additi...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(92)19028-5
更新日期:1992-01-01 00:00:00
abstract::In this chapter we have detailed a method that can be generalized to link virtually any DNA substrate to a chromatography matrix at its ends via an avidin-biotin linkage. We have used this technique to construct a left-handed Z-DNA column for the purpose of identification and purification of Z-DNA-binding proteins. Th...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(90)84290-w
更新日期:1990-01-01 00:00:00
abstract::Nonribosomal peptides are natural products typically of bacterial and fungal origin. These highly complex molecules display a broad spectrum of biological activities, and have been exploited for the development of immunosuppressant, antibiotic, anticancer, and other therapeutic agents. The nonribosomal peptides are as...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)04814-9
更新日期:2009-01-01 00:00:00
abstract::This chapter outlined the methods used to study intra- and extracellular modifications of apolipoproteins. These and other related studies have shown that several of the apolipoproteins undergo a series of intra- and extracellular modifications as follows: All apolipoproteins studied contain an 18-26 long signal pepti...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(86)28100-8
更新日期:1986-01-01 00:00:00
abstract::Mass spectrometry (MS) has gradually replaced classical methods as a major tool in protein sequencing and characterization. However, the sample preparation repertoire has not changed very much; it has just been adjusted to the needs of the new analytical method. In this chapter frequently used in-solution enzymatic di...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)05003-2
更新日期:2005-01-01 00:00:00