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PubMed Journal Database | Methods in molecular biology (Clifton, N.J.) RSS

23:27 EDT 22nd May 2019 | BioPortfolio

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Showing PubMed Articles 1–25 of 3,400+ from Methods in molecular biology (Clifton, N.J.)

Cell Cycle Analysis Using In Vivo Staining of DNA-Synthesizing Cells.

The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters.In the current step-by-step protocol, we present` two approaches optimized for in vivo study of the ce...

Nuclear Receptors: A Historical Perspective.

In this chapter, we summarize the birth of the field of nuclear receptors. These receptors exhibit a multitude of roles in cell biology and hence have attracted a great deal of interest in the drug discovery field. It is not certain whether these receptors evolved independently or an ancestral protein acquired various functions upon binding to preexisting small molecules, ligands. Currently, members of this receptor superfamily are categorized in six groups, including "orphan receptors." Research in the are...

An Optimized Immunoblotting Protocol for Accurate Detection of Endogenous PGC-1α Isoforms in Various Rodent Tissues.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays a central role in the response and adaptation to environmental and nutritional stimuli by initiating tissue-specific transcriptional reprogramming. Since its discovery in 1998, the field of PGC-1α biology has grown exponentially and a large body of research has elucidated the diverse roles of PGC-1α in brown adipose tissue thermogenesis, fatty acid oxidation, muscle fiber type switching, hepatic gluconeogenesis, and circadian cl...

Subcellular Localization of NR4A2 Orphan Nuclear Receptor Expression in Human and Mouse Synovial Joint Tissue.

NR4A1-3 receptors are required in inflammatory disease initiation and progression, where they function as early response regulators, controlling the extent of the inflammatory response and promoting inflammatory resolution. NR4A receptor activity controls inflammatory processes in several diseases characterized by chronic inflammation including rheumatoid arthritis (RA) and atherosclerosis. Studies indicate that cell-type and cellular microenvironment can alter NR4A1-3 receptor activity and influence their ...

In Vivo Quantitation of Estrogen Receptor β Subtype Expression in Ovarian Surface Epithelium Using Immunofluorescence Profiling and Confocal Microscopy.

Immunohistochemistry using formalin-fixed, paraffin-embedded tissue, chromogen label, and light microscopy has traditionally been used to semiquantify estrogen receptor (ER) to guide diagnosis and management of breast cancer. Quantitation of ER for this purpose currently only assesses levels of the ER-alpha subtype. Considerable variability in results reported has been due to protocol and fixation variability, intraobserver and interobserver variability, and different scoring systems and thresholds for scor...

In Vivo ChIP-Seq of Nuclear Receptors: A Rough Guide to Transform Frozen Tissues into High-Confidence Genome-Wide Binding Profiles.

Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) is a powerful tool to map context-dependent genome-wide binding of nuclear hormone receptors and their coregulators. This information can provide important mechanistic insight into where, when and how DNA-protein interactions are linked to target gene regulation. Here we describe a simple, yet reliable ChIP-seq method, including nuclear isolation from frozen tissue samples, cross-linking DNA-protein complexes, chromatin shearing,...

High-Content Analysis of Constitutive Androstane Receptor Nuclear Translocation.

The constitutive androstane receptor (CAR; NR1I3) is a xenobiotic receptor that upregulates metabolism and detoxification mechanisms in the liver in response to chemical stimulation. Drug-induced activation of CAR may result in clinically significant drug-drug interactions and lead to complicated therapeutic outcomes. Accumulating evidence has also suggested that CAR may be a potential drug target for metabolic disorders and liver cancer by modulating cell cycle progression, energy homeostasis, and cell pro...

Antibody Validation Strategy for Nuclear Receptors.

Antibodies are invaluable biological tools that we can use to detect the presence, location, or alteration of nuclear receptors. However, antibodies frequently cross-react with other proteins and their performance can vary from batch to batch, from application to application and from lab to lab. When each lot of antibody is not thoroughly validated for each assay, each sample type, and each lab and user, antibody-based assays can lead to flawed interpretations and reproducibility problems. In this chapter, ...

Immunofluorescence Labeling of Nuclear Receptor Expression in Formalin-Fixed, Paraffin-Embedded Tissue.

Immunofluorescent staining (IF) uses antigen-antibody complexes tagged with fluorochromes to observe the expression of proteins within a tissue sample. Multiple groups have described optimized methods to visualize several proteins simultaneously within the same tissue section using immunofluorescence in both mouse and human FFPE tissues. Our group routinely uses an optimized protocol described here to examine nuclear receptor expression in experimental samples from conditional knockout in vivo studies.

Detection of ADP-Ribosylation of the Androgen Receptor Using the Recombinant Macrodomain AF1521 from Archaeoglobus fulgidus.

ADP-ribosylation is a posttranslational modification generated by members of the superfamily of ADP-ribosyltransferases, known as the Parp enzymes. Depending on the superfamily member, Parp enzymes can mono- or poly-ADP-ribosylate a protein substrate. Parp superfamily members confer regulation to a variety of biological processes that include cell signaling, DNA repair, transcription, and stress responses. Here, we describe biochemical methods for detection of ADP-ribose conjugated to the androgen receptor ...

Reconstitution of the Steroid Receptor Heterocomplex.

Steroid receptors are members of a subfamily of the nuclear receptor superfamily. They play a dual role of steroid hormone receptors and transcription factors. Actually, these receptors are steroid-activated transcription factors. Classical soluble receptors exist as oligomeric complexes with the Hsp90-based chaperone machinery. The steroid receptor field was born and developed along with the molecular chaperone field. Chaperones are not exclusive partners associated to these receptors, but also comprise a ...

High-Throughput Imaging of PPIX Using Confocal Microscopy.

Increases in levels of protoporphyrin IX (PPIX; a heme precursor) may be driven by xenobiotic induction of aminolevulinic acid synthase 1 (ALAS1) expression. ALAS1 is the rate-limiting enzyme of heme biosynthesis and may be upregulated to satisfy the increased need for heme in CYP450 enzymes. Therefore, a high-throughput fluorescence spectroscopy method that detects PPIX would enable the screening of drugs that increase ALAS1 through nuclear hormone receptor-mediated induction of transcription that may caus...

PGC-1α Overexpression via Local In Vivo Transfection in Mouse Skeletal Muscle.

The overexpression of a specific protein is a common method for investigating the specific biological function of the substance and the mechanism of action. In vivo electrotransfer has been confirmed to be one of the most reliable, efficient and cost-effective way to overexpress a protein in a select biological tissue. Typically, this technique involves a physical injection of plasmid DNA followed by electric pulses across the injection site. Here, we introduce this method that we used to transfect green fl...

A Reverse Transfection Method for Screening of Nuclear Receptor Activators.

Reporter assays are useful to study nuclear receptor activation and for example to evaluate the propensity of novel drug candidates to cause induction of drug-metabolizing cytochrome P450 enzymes. Here, we describe a protocol for a reverse transfection system to study the activation of human nuclear receptors constitutive androstane receptor and pregnane X receptor. The system provides long-term stability and uniformity of DNA-carrier complexes, thus avoiding the inherent variation in conventional transfect...

Hybrid Reporter Gene Assays: Versatile In Vitro Tools to Characterize Nuclear Receptor Modulators.

Nuclear receptors act as ligand-activated transcription factors translating ligand signals into changes in gene expression. The 48 members of the superfamily of nuclear receptors identified so far are involved amongst others in maintenance of metabolic balance, inflammation, and cancer. Thus, they hold enormous potential for drug discovery and some nuclear receptors are experiencing considerable academic and industrial interest. Nuclear receptor modulator discovery requires reliable, robust, and economic te...

Analysis of the Transcriptional Activity of Retinoic Acid-Related Orphan Receptors (RORs) and Inhibition by Inverse Agonists.

Here, we describe several assays to analyze the transcriptional activity of retinoic acid-related orphan receptors (RORs) and the effect of inverse agonists on their activity. One assay measures the effect of an inverse agonist on the transcriptional activation of a luciferase reporter by RORs in a Tet-On cell system. A mammalian two-hybrid assay analyzes the interaction of the ROR ligand binding domain with a coactivator peptide. Two additional assays examine the effect of an inverse agonist on the activat...

Examining the Role of Nuclear Receptors During In Vivo Chemical-Mediated Breast Tumorigenesis.

The chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) has been used for many decades to induce skin, mammary, and ovarian tumors in mice. There are however a wide range of doses and treatment regimens in the literature that sometimes confound comparative interpretations of different studies. Here we describe a proven method to generate in vivo DMBA-mediated murine mammary tumors to enable consistent studies of the cell targeted role of genes of interest during this process.

Analysis of IL-4/STAT6 Signaling in Macrophages.

Activation of signal transducer and activator of transcription 6 (STAT6) is a key signaling pathway in macrophage function, and is required for the so-called alternative (M2) activation of macrophages. Interleukin (IL)-4 and IL-13 are important M2 polarizing cytokines that act through STAT6 by inducing its phosphorylation and promoting transcription of STAT6-responsive genes. Inactivation of STAT6 signaling in macrophages has not been fully explored; however, a recent model suggests that inactivation of STA...

Isolation and Characterization of Adipose Tissue Macrophages.

This chapter describes a technique that can be used to isolate adipose tissue macrophages (ATMs) from the visceral white adipose tissue. Nevertheless, this technique can also be used to isolate ATMs from subcutaneous white adipose tissue and brown adipose tissue from mouse, human subcutaneous fat depot, and also from the fat body of the toad Xenopus. We detail the flow-cytometric gating strategy that has been developed to identify ATM population, and we describe the isolation of RNA from this population and...

Assessing Mitochondrial Bioenergetics in Isolated Mitochondria from Mouse Heart Tissues Using Oroboros 2k-Oxygraph.

In this chapter, we describe detailed protocols for measuring high-resolution respirometry on mitochondria extracted from adult whole mouse heart using the Oroboros 2k-Oxygraph system. The method provides detailed procedures for the preparation of mitochondria and measurement of high-resolution respirometry in response to various respiration inhibitions. The method described in this chapter could discern the different respiration rate on mitochondria extracted from two spatially distinct mitochondrial subpo...

Generation of Skeletal Myocytes from Embryonic Stem Cells Through Nuclear Receptor Signaling.

The differentiation of mouse embryonic stem (ES) cells into skeletal myocytes can be enhanced by small molecular inducers, which signal through muscle-specific transcription networks. However, to induce differentiation in vitro, the ES cells must be thrusted through the stage of embryoid body (EB) formation. Here, we describe how to efficiently direct the commitment of ES cells into skeletal muscle lineage by using nuclear receptor agonists in a hanging drop protocol.

Obtaining Crystals of PPARγ Ligand Binding Domain Bound to Small Molecules.

Obtaining high-throughput structural characterization of small molecule drug candidates bound to their protein target has been hampered by the challenges of cocrystallizing protein-ligand complexes. These challenges include poor ligand solubility, excess ligand molecules disrupting the homogeneity of the sample to be crystallized, and inefficiency in preparing individual complexes and crystal screens for each drug candidate. Crystallizing apo protein followed by soaking with a solution containing the ligand...

Molecular Modeling Approach to Study the PPARγ-Ligand Interactions.

The chapter is focused on methods relevant for predictive toxicology and computer-aided drug design (adverse outcome pathway development, pharmacophore modeling, docking, and 3D QSAR analysis) and applied to study interactions between peroxisome proliferator-activated receptor γ (PPARγ) and its ligands. The methods have been combined to develop an integrated in silico approach allowing both to predict potential PPARγ-mediated hepatotoxicity of receptor's full agonists, thus supporting hazard characteriza...

High-Dimensional Data Approaches to Understanding Nuclear Hormone Receptor Signaling.

Bioinformatics applies unbiased approaches to develop statistically robust insight into health and disease. At the global, or "20,000 ft" view bioinformatic analyses of NR signaling can measure how the NRs are implicated in human health and disease through the impact of genome-wide significant genetic variation, family-wide NR expression patterns or considering where NRs are significantly identified in other high-dimensional data analyses. With a more NR-centric, or "2000 ft" view, bioinformatic approache...

Using Bioinformatics and Molecular Biology to Streamline Construction of Effector Libraries for Phytopathogenic Pseudomonas syringae Strains.

The war between plants and their pathogens is endless, with plant resistance genes offering protection against pathogens until the pathogen evolves a way to overcome this resistance. Given how quickly new pathogen strains can arise and defeat plant defenses, it is critical to more rapidly identify and examine the specific genomic characteristics new virulent strains have gained which give them the upper hand. An indispensable tool is bioinformatics. Genome sequencing has advanced rapidly in the last decade,...


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