Springer protocols feed by Pharmacology/Toxicology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Antibody-Based Proteomic Analysis of Apoptosis Signaling
Reagents that assess activation of apoptosis and associated signaling pathways are critical for greater understanding of the molecular basis of programmed cell death. The advent of proteomic technologies to probe these events allows monitoring of hundreds to thousands of proteins, as well as sites of posttranslational modification involved in apoptosis at one time. This view of apoptosis at a network level is a powerful tool in studying known apoptotic pathways, as well as elucidating novel signaling events that affect or are affected by apoptotic signaling. The following is a detailed method for successful proteomic profi...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Automated Ratio Imaging Using Nuclear-Targeted FRET Probe-Expressing Cells for Apoptosis Detection
In recent years, innovative bioassays have been designed to detect intracellular caspase activation as a reliable read-out of apoptotic activity of bioactive compounds. Most anticancer drugs target cells by triggering caspase dependent protein cleavage, culminating in apoptotic cell death. Therefore, detection of caspase activation has been recognized as one of the best approaches for detecting cancer cell death as compared to assaying the ill-defined general cytotoxic activity that often manifests with off-target side effects. Among the available methods of detection, those with cells stably expressing FRET-based fluoresc...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
FRET-Based Measurement of Apoptotic Caspase Activities by High-Throughput Screening Flow Cytometry
Unwanted and excessive apoptosis contributes to various degenerative diseases, and apoptosis-inducing drugs are a mainstay of anticancer treatment regimens. The fields of pharmacology and toxicology consequently have a long history of investigating apoptotic cell death in the context of drug safety and efficacy studies. Canonical apoptotic cell death is crucially dependent on type II cysteinyl aspartate-specific proteases (caspases), and their activation is therefore widely used as a marker for this cell death modality. Here we describe a flow cytometric method for noninvasive, highly sensitive and reproducible FRET-based ...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
A Low-Cost Method for Tracking the Induction of Apoptosis Using FRET-Based Activity Sensors in Suspension Cells
Apoptosis, or programmed cell death, is a tightly regulated cellular event that plays an important role in both normal developmental processes and many pathological states. The induction of apoptosis is tightly regulated through the coordinated action of members of the caspase family of proteases. Here we discuss a relatively inexpensive protocol for monitoring the induction and progression of apoptosis using a genetically encoded fluorescence resonance energy transfer (FRET)-based biosensor of the executioner caspase, caspase-3, in living suspension cells. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Detecting Apoptosis, Autophagy, and Necrosis
There are many commercially available kits to identify specific types of cell death, but at the present time, there is no simple assay that can distinguish apoptosis, necrosis, and autophagy. Autophagy and apoptosis are highly conserved processes that maintain organism and cellular homeostasis. They are also prime targets for the design of tumor therapeutics. Apoptosis is a highly regulated process involved in removing unwanted or unhealthy cells. Autophagy is a metabolic process, in which proteins and organelles are targeted for degradation in the lysosome. Necrosis is initiated by external factors, such as toxins, infect...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Measurement of Apoptosis by Multiparametric Flow Cytometry
Apoptosis remains a critical phenomenon in cell biology, playing a regulatory role in virtually every tissue system. It is particular crucial in the immune system, ranging from immature immune cell development and selection to downregulation of the mature immune response. Apoptosis is a primary mechanism in the action of antitumor drugs, and is thus an important phenomenon in pharmacology, drug discovery, and toxicology. Flow cytometry is the primary technique for measuring apoptosis in suspension cells; many flow cytometry assays have been developed to measure the entire apoptotic process, from the earliest signal transdu...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Determining the Extent of Toxicant-Induced Apoptosis Using Concurrent Phased Apoptosis Assays
Apoptosis is a stage-dependent process exhibiting characteristic biochemical, molecular and morphological features that vary progressively through the apoptosis process. Apoptosis induced by toxicants may activate varied features of apoptosis to different extents and kinetics. Some of the features activated may occur transiently, while others may not occur in a cell system undergoing toxicant-induced apoptosis. Thus, the best approach for quantitating the extent of toxicant-induced apoptosis involves the utilization of a combination of assays focusing on different morphological, biochemical and molecular features of apopto...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Detection of Apoptosis: From Bench Side to Clinical Practice
Apoptosis or programmed cell death is implicated in several pathological conditions, such as cancer and neurodegenerative diseases. An increasing number of therapies are developed by targeting apoptosis signaling components to either induce or inhibit apoptosis in target cells. For these reasons, it is critical to develop appropriate analytical methods for the detection of apoptotic cell death in the context of monitoring relevant disease progression and therapeutic effects of clinical treatments (e.g., chemotherapy in cancer patients). This review provides an overview of the currently used methods for detection of apoptos...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
An Overview of Apoptosis Methods in Toxicological Research: Recent Updates
Apoptosis is the most common form of programmed cell death. Apoptosis plays a critical role in many physiological functions, and its dysregulation is an underlying defect in various diseases, including cancer. In fact, many toxicants and chemotherapeutic drugs exert their mechanisms of action through modulation of the apoptosis process. Thus, interest in the apoptosis process, as well as the methods used to assess and quantify its various aspects has continued to spike. This chapter provides a brief overview of the apoptosis process, the most common apoptosis methods, and the principles upon which these methods function. F...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Liposomes in Apoptosis Induction and Cancer Therapy
Cancer is the leading cause of death with multiple obstacles in therapeutic arsenals employed to date. Apoptosis induction in cancer cells has hitherto been a prominent unresolved obstacle for a few decades. Liposomes with multiple merits were extensively employed to entrap several types of anticancer agents, biomolecules and imaging agents to achieve substantial therapeutic effect for various types of cancers. Multifunctional liposomes with enhanced biocompatible properties were designed to enhance the therapeutic effect. Despite the promising drug delivery strategies and significantly reduced toxicity of the liposomal fo...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Targeting Cancer Cell Death with Small Molecule Agents for Potential Therapeutics
Time has come to switch from morphological to molecular definitions of cell death subroutines, due to substantial progress in biochemical and genetic exploration. Currently, cell death subroutines are defined by a series of precise, measurable biochemical features; these include apoptosis, autophagic cell death and necroptosis. Accumulating evidence has gradually revealed the core molecular machinery of cell death in carcinogenesis; the intricate relationships between cell death subroutines and cancer, however, still need to be clarified. Cancer drug discovery, in particular, has benefitted significantly from a rapid progr...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Microplate-Based Whole Zebrafish Caspase 3/7 Assay for Screening Small Molecule Compounds
In this research, using a commercially available human specific caspase 3/7 chemiluminescent test kit (Caspase 3/7 Glo, Promega, Madison, WI), developed for cell based assays, we describe a microplate-based whole zebrafish assay format to identify potential small molecule caspase inhibitors and activators. Based on the high degree of evolutionary conservation among species, we show that human specific 3/7 substrate cross reacts with zebrafish. Using untreated zebrafish, optimum assay conditions (including substrate concentration, number of zebrafish per microwell, and incubation time to generate a linear reaction) are dete...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Novel Electrochemical Biosensor for Apoptosis Evaluation
Apoptosis evaluation is one of the most important tasks of toxicology. By using a peptide as the recognition element, and assembling apoptotic cells on a solid surface, we have established a novel electrochemical method for the detection of apoptosis levels. Such a peptide-based electrochemical biosensor is simple, cost-effective, convenient, and sensitive. Since the results obtained are well in line with other standard methods, this method holds a great potential towards the analysis of apoptosis and its applications. In this chapter, we introduce a general overview of this technical approach for detecting apoptotic cells...
Source: Springer protocols feed by Pharmacology/Toxicology - December 31, 2015 Category: Drugs & Pharmacology Source Type: news
Experiment-Guided Molecular Modeling of Protein & ndash;Protein Complexes Involving GPCRs
Experimental structure determination for G protein-coupled receptors (GPCRs) and especially their complexes with protein and peptide ligands is at its infancy. In the absence of complex structures, molecular modeling and docking play a large role not only by providing a proper 3D context for interpretation of biochemical and biophysical data, but also by prospectively guiding experiments. Experimentally confirmed restraints may help improve the accuracy and information content of the computational models. Here we present a hybrid molecular modeling protocol that integrates heterogeneous experimental data with force field-b...
Source: Springer protocols feed by Pharmacology/Toxicology - August 13, 2015 Category: Drugs & Pharmacology Source Type: news
The Dynamic Process of Drug & ndash;GPCR Binding at Either Orthosteric or Allosteric Sites Evaluated by Metadynamics
Major advances in G Protein-Coupled Receptor (GPCR) structural biology over the past few years have yielded a significant number of high-resolution crystal structures for several different receptor subtypes. This dramatic increase in GPCR structural information has underscored the use of automated docking algorithms for the discovery of novel ligands that can eventually be developed into improved therapeutics. However, these algorithms are often unable to discriminate between different, yet energetically similar, poses because of their relatively simple scoring functions. Here, we describe a metadynamics-based approach to ...
Source: Springer protocols feed by Pharmacology/Toxicology - August 13, 2015 Category: Drugs & Pharmacology Source Type: news
