Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1177/1087057103252308 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Advances in liver organoids: replicating hepatic complexity for toxicity assessment and disease modeling.
Stem Cell Res Ther
January 2025
Organoid Innovation Center, Suzhou Institute of Nanotech and Nano-bionics, Chinese Academy of Sciences, 398 Ruoshui Rd, Suzhou, Jiangsu, 215123, China.
The lack of in vivo accurate human liver models hinders the investigation of liver-related diseases, injuries, and drug-related toxicity, posing challenges for both basic research and clinical applications. Traditional cellular and animal models, while widely used, have significant limitations in replicating the liver's complex responses to various stressors. Liver organoids derived from human pluripotent stem cells, adult stem cells primary cells, or tissues can mimic diverse liver cell types, major physiological functions, and architectural features.
View Article and Find Full Text PDFA quantitative intracellular peptide binding assay reveals recognition determinants and context dependence of short linear motifs.
J Biol Chem
January 2025
Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA. Electronic address:
Transient protein-protein interactions play key roles in controlling dynamic cellular responses. Many examples involve globular protein domains that bind to peptide sequences known as Short Linear Motifs (SLiMs), which are enriched in intrinsically disordered regions of proteins. Here we describe a novel functional assay for measuring SLiM binding, called Systematic Intracellular Motif Binding Analysis (SIMBA).
View Article and Find Full Text PDFDevelopment and characterization of in vitro inducible immortalization of a murine microglia cell line for high throughput studies.
Sci Rep
January 2025
Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, United States.
There are few in vitro models available to study microglial physiology in a homeostatic context. Recent approaches include the human induced pluripotent stem cell model, but these can be challenging for large-scale assays and may lead to batch variability. To advance our understanding of microglial biology while enabling scalability for high-throughput assays, we developed an inducible immortalized murine microglial cell line using a tetracycline expression system.
View Article and Find Full Text PDFAtmospheric air plasma pre-activation and customizable covalent functionalization of PVDF-membranes of microtiter filter plates.
Sci Rep
January 2025
Department of Pharmaceutical Chemistry, Semmelweis University, Hőgyes Endre U. 9, 1092, Budapest, Hungary.
Microtiter-plate-based systems are unified platforms of high-throughput experimentation (HTE). These polymeric devices are used worldwide on a daily basis-mainly in the pharmaceutical industry-for parallel syntheses, reaction optimization, various preclinical studies and high-throughput screening methods. Accordingly, laboratory automation today aims to handle these commercially available multiwell plates, making developments focused on their modifications a priority area of modern applied research.
View Article and Find Full Text PDFAnthracyclines disaggregate and restore mutant p63 function: a potential therapeutic approach for AEC syndrome.
Cell Death Discov
January 2025
Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129, USA.
Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate (AEC) syndrome is a rare genetic disorder caused by mutations in the TP63 gene, which encodes a transcription factor essential for epidermal gene expression. A key feature of AEC syndrome is chronic skin erosion, for which no effective treatment currently exists. Our previous studies demonstrated that mutations associated with AEC syndrome lead to p63 protein misfolding and aggregation, exerting a dominant-negative effect.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!