Biomedical subjects

Richard G Posner

Publications and source records attributed to Richard G Posner.

5 recordsLinked to original sources

PubMed2006

Rules for modeling signal-transduction systems.

Formalized rules for protein-protein interactions have recently been introduced to represent the binding and enzymatic activities of proteins in cellular signaling. Rules encode an understanding of how a system works in terms of the biomolecules in the system and their possible states and interactions. A set of rules can be as easy to read as a diagrammatic interaction map, but unlike most such maps, rules have precise interpretations. Rules can be processed to automatically generate a mathematical or computational model for a system, which enables explanatory and predictive insights into the system's behavior. Rules are independent units of a model specification that facilitate model revision. Instead of changing a large number of equations or lines of code, as may be required in the case of a conventional mathematical model, a protein interaction can be introduced or modified simply by adding or changing a single rule that represents the interaction of interest. Rules can be defined and visualized by using graphs, so no specialized training in mathematics or computer science is necessary to create models or to take advantage of the representational precision of rules. Rules can be encoded in a machine-readable format to enable electronic storage and exchange of models, as well as basic knowledge about protein-protein interactions. Here, we review the motivation for rule-based modeling; applications of the approach; and issues that arise in model specification, simulation, and testing. We also discuss rule visualization and exchange and the software available for rule-based modeling.

Computer Simulation↗

PubMed2006

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH.

BACKGROUND: Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology. METHODS: Dextran beads suitable for flow cytometry were derivatized with glutathione. A fusion protein of GST and green fluorescent protein was used to define kinetic and equilibrium binding characteristics of GST fusion proteins to glutathione beads. Free glutathione competes with this binding, and this competition was used to measure free glutathione concentration. RESULTS: A 10 microl assay can measure 5 microl of 20 microM glutathione (100 pmol glutathione) in 2 h by flow cytometry. This concentration is two orders of magnitude lower than cellular glutathione concentrations, and three orders of magnitude lower than affinity chromatography eluates. One important result is that by generating high site density, the GST fusion proteins can be constrained to the surface of one bead without hopping to the next bead in multiplex assays. CONCLUSIONS: Glutathione in cellular lysates and GST-fusion protein affinity chromatography eluates can be measured by flow cytometry. Many interactions between GST fusion proteins and their fluorescent binding partners should be quantifiable by flow cytometry. Although a system may have the disadvantage that it has a low affinity and a correspondingly quick off-rate in solution, it may remain on beads if the site density can be increased to offer a slow apparent off rate.

Binding Sites↗

PubMed2004

CO2 transduction mechanisms in avian intrapulmonary chemoreceptors: experiments and models.

Intrapulmonary chemoreceptors (IPC) are neurons that sense tonic and phasic CO2 stimuli in the lungs of birds and diapsid reptiles. IPC are different from most other vertebrate respiratory CO2 receptors because: (1) they are stimulated by low PCO2 and inhibited by high PCO2, (2) they have extremely rapid response characteristics, (3) their CO2 sensitivity is nearly abolished by intracellular inhibitors of carbonic anhydrase, and (4) their CO2 sensitivity is strongly depressed by inhibiting Na+/H+ antiport exchange. Experimental evidence suggests that IPC respond to intracellular pH, not CO2 directly, and that intracellular pH and IPC discharge are determined by a kinetic balance between CO2 hydration/dehydration rates, transmembrane acid/base exchange rates, and intracellular buffering. We review experimental evidence for and against various mechanisms of IPC CO2 chemotransduction, present a conceptual and mathematical model of the proposed mechanisms, and compare this model to CO2 transduction in other respiratory chemoreceptors.

Action Potentials↗

PubMed2004

Interaction of a monoclonal IgE-specific antibody with cell-surface IgE-Fc epsilon RI: characterization of equilibrium binding and secretory response.

Aggregation of FcepsilonRI, the high-affinity cell-surface receptor for IgE antibody, is required for degranulation of basophils and mast cells, but not all receptor aggregates elicit this cellular response. The stereochemical constraints on clusters of FcepsilonRI that are able to signal cellular responses, such as degranulation, have yet to be fully defined. To improve our understanding of the properties of FcepsilonRI aggregates that influence receptor signaling, we have studied the interaction of 23G3, a rat IgG(1)(kappa) IgE-specific monoclonal antibody, with IgE-FcepsilonRI complexes on rat mucosal-type mast cells (RBL-2H3 line). We find that 23G3 is a potent secretagogue. This property and the structural features of 23G3 (two symmetrically arrayed IgE-specific binding sites) make 23G3 a potentially valuable reagent for investigating the relationship between FcepsilonRI clustering and FcepsilonRI-mediated signaling events. To develop a mathematical model of 23G3-induced aggregation of FcepsilonRI, we used fluorimetry and flow cytometry to quantitatively monitor equilibrium binding of FITC-labeled 23G3 intact Ab and its Fab' fragment to cell-surface IgE. The results indicate that IgE bound to FcepsilonRI expresses two epitopes for 23G3 binding; that 23G3 binds IgE resident on the cell surface with negative cooperativity; and that 23G3 appears to induce mostly but not exclusively noncyclic dimeric aggregates of FcepsilonRI. There is no simple relationship between receptor aggregation at equilibrium and the degranulation response. Further studies are needed to establish how 23G3-induced aggregation of IgE-FcepsilonRI correlates with cellular responses.

Animals↗

PubMed2002

A quantitative approach for studying IgE-FcepsilonRI aggregation.

Aggregation of cell surface receptors is a ubiquitous means of initiating signal transduction in many cellular systems. In this manuscript, we describe a combined theoretical and experimental approach based on multiparameter flow cytometry for measuring the time course of ligand induced aggregation of IgE-FcepsilonRI on RBL cells. By fluorescently labeling both the ligand and surface IgE (sIgE), we have developed an assay that permits us to simultaneously measure both occupancy of sIgE combining sites and association of antigen with the cell surface. This allows for a direct calculation of the degree of receptor aggregation present on the cell. By employing new mixing technologies developed for flow cytometry, we are able to look at aggregation in the sub second time domain. To extend our work, we have synthesized a new set of chemically well defined ligands (of valences 1-3) to use as probes in our studies. We show that the magnitude of the cellular response is dramatically increased as the valence of our ligand is raised from two to three.

Animals↗